**Meet the editors**

Dr. Blanca Hernandez-Ledesma is currently working as a Postdoctoral researcher under the Ramón & Cajal Programme at the Institute of Food Science Research (CSIC-UAM) in Spain. She was working (1998 to 2002) at the Institute of Industrial Fermentations (CSIC), earning her Ph.D. in Pharmacy by the Complutense University of Madrid, Spain in 2002. She continue

working as postdoctoral fellow at the CSIC until 2007 when she moved to USA for a postdoctoral work at the University of California, Berkeley. In 2010, she went back to Spain to work as postdoctoral researcher under different contracts. Her work has been focused on the biological activities of peptides derived from food sources, evaluating their mechanisms of actions, bioavailability and technological aspects by in vitro experiments, cell culture and animal models.

Dr. Chia-Chien Hsieh is currently an Assistant Professor of Nutritional Sciences at the Department of Human Development and Family Studies, National Taiwan Normal University in Taiwan. She is an expert in the field of Nutrition Biochemistry with emphasis in Cancer Biology and Immunology. She received her Ph.D. degree from National Taiwan University in 2005.

Her thesis focused on the effect of dietary factors in autoimmunity. Then, she worked as a postdoctoral fellow in the National Health Research Institute in Taiwan for two years. In 2007, she joined the Prof. de Lumen´s laboratory at the University of California Berkeley to work on the chemopreventive properties of lunasin peptide. She investigated its molecular mechanisms and conducted lunasin's efficacy studies in animals. She also studied cyclooxygenase-2 signaling in hepatocarcinoma at the University of Pittsburgh in 2009. Her works provide new knowledge on bioactive food components in diseases prevention and therapy.

Contents

**Preface IX** 

**Section 1 Health Benefits of Food Peptides 1** 

Chapter 2 **Bowman-Birk Inhibitors from Legumes:** 

**Section 2 Foods as Source of Bioactive Peptides 73** 

Tânia G. Tavares and F. Xavier Malcata

**of Plant Protein Derived Peptides 145** 

Anne Pihlanto and Sari Mäkinen

David A. Betancur-Ancona

Chapter 4 **Whey Proteins as Source** 

Chapter 6 **Antihypertensive Properties** 

and Chia-Chien Hsieh

Chapter 1 **1997-2012: Fifteen Years of Research on Peptide Lunasin 3**  Blanca Hernández-Ledesma, Ben O. de Lumen

> **Utilisation in Disease Prevention and Therapy 23**  Alfonso Clemente, Maria del Carmen Marín-Manzano, Maria del Carmen Arques and Claire Domoney

**of Bioactive Peptides Against Hypertension 75** 

Chapter 5 **Functional Proteins and Peptides of Hen's Egg Origin 115**  Adham M. Abdou, Mujo Kim and Kenji Sato

Chapter 7 **Vigna Unguiculata as Source of Angiotensin-I Converting Enzyme Inhibitory and Antioxidant Peptides 183**  Maira R. Segura-Campos, Luis A. Chel-Guerrero and

Chapter 8 **Dipeptidyl Peptidase-IV Inhibitory Activity of Peptides in Porcine Skin Gelatin Hydrolysates 205** 

Kuo-Chiang Hsu, Yu-Shan Tung, Shih-Li Huang and Chia-Ling Jao

Chapter 3 **Antihypertensive Peptides from Food Proteins 45**  Roseanne Norris and Richard J. FitzGerald

## Contents

#### **Preface** XI


## Preface

In recent years, nutrition and food sciences have been focused on biologically active peptides present in the sequences of food proteins. Such peptides are inactive within the sequence of the precursor proteins and can be released by enzymatic proteolysis during gastrointestinal digestion or during food processing. Liberated peptides may act as regulatory compounds with hormone-like activity in the human body. To date, food-derived peptides with antihypertensive, antimicrobial, antioxidant, anticarcinogenic, anti-inflammatory, opioid agonist or antagonist, antiviral, among others, have been characterized. These peptides possess properties that help to prevent and/or treat different disorders, maintaining the well-health status of humans.

**Bioactive Food Peptides in Health and Disease** provides a general overview of foodderived peptides for the promotion of human health and the prevention/management of chronic diseases. The book provides updated and interesting information on bioactive peptides obtained from both animal and plant food sources, emphasizing on important aspects related to their bioactivity, mechanism of action, and bioavailability. Also, the chapters describe the impact of bioactive peptides on the physiological absorption, regulation and disease prevention. The book also covers the recent technological advances for the production of food peptides.

The editors want to thank the authors for their important contribution to the success of this book. They are eminent researchers all over the world that have accepted to share and turn their ideas and work into a book that we hope to be an essential resource and reference for nutritional and food scientists, biochemists, industry producers and consumers.

> **Dr. Blanca Hernández-Ledesma**  Institute of Food Science Research (CIAL, CSIC-UAM), Madrid, Spain

> > **Dr. Chia-Chien Hsieh**  Department of Nutritional Science and Toxicology. University of California, Berkeley, California, USA

**Section 1** 

**Health Benefits of Food Peptides** 

**Section 1** 

**Health Benefits of Food Peptides** 

**Chapter 1** 

© 2013 Hsieh et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Hsieh et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**1997-2012: Fifteen Years** 

Additional information is available at the end of the chapter

**1.1. Chemopreventive role of food peptides** 

through intense alterations in diet regimens.

http://dx.doi.org/10.5772/52368

**1. Introduction** 

anticancer strategy [5].

**of Research on Peptide Lunasin** 

Blanca Hernández-Ledesma, Ben O. de Lumen and Chia-Chien Hsieh

Cancer is a major killer in today's world accounting for around 13% of all deaths according to the World Health Organisation. It has been estimated that by 2020, approximately 17 million new cancer cases will be diagnosed, and 10 million cancer patients will die [1]. Epidemiological evidence has demonstrated that as many as 35% of these cancer cases may be related to dietary factors, and thus modifications of nutritional and lifestyle habits can prevent this disease [2]. Cell experiments, animal models and human trials have revealed that a large number of natural compounds present in the diet could lower cancer risk and even, sensitize tumor cells against anti-cancer therapies [3]. Therefore, knowledge on the effect of diet components on health will bring new opportunities for chemoprevention

In the last few years, food proteins and peptides have become one group of nutraceuticals with demonstrated effects preventing the different stages of cancer, including initiation, promotion, and progression [4]. Certain advantages over alternative chemotherapy molecules, such as their high affinity, strong specificity for targets, low toxicity and good penetration of tissues, have made food proteins and peptides a new and promising

Protease inhibitors are found in plant tissues, particularly from legumes. One of the most extensively studied inhibitors in the field of carcinogenesis is the soybean derived Bowman-Birk protease inhibitor (BBI). It is a 71-amino acids polypeptides which chemopreventive properties have been demonstrated in both *in vitro* and *in vivo* systems [6]. As a result of this evidence, BBI acquired the status of "investigational new drug" from the Food and Drug Administration in 1992, and since then, large-scale human trials are being carried out to

## **Chapter 1**

## **1997-2012: Fifteen Years of Research on Peptide Lunasin**

Blanca Hernández-Ledesma, Ben O. de Lumen and Chia-Chien Hsieh

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52368

## **1. Introduction**

#### **1.1. Chemopreventive role of food peptides**

Cancer is a major killer in today's world accounting for around 13% of all deaths according to the World Health Organisation. It has been estimated that by 2020, approximately 17 million new cancer cases will be diagnosed, and 10 million cancer patients will die [1]. Epidemiological evidence has demonstrated that as many as 35% of these cancer cases may be related to dietary factors, and thus modifications of nutritional and lifestyle habits can prevent this disease [2]. Cell experiments, animal models and human trials have revealed that a large number of natural compounds present in the diet could lower cancer risk and even, sensitize tumor cells against anti-cancer therapies [3]. Therefore, knowledge on the effect of diet components on health will bring new opportunities for chemoprevention through intense alterations in diet regimens.

In the last few years, food proteins and peptides have become one group of nutraceuticals with demonstrated effects preventing the different stages of cancer, including initiation, promotion, and progression [4]. Certain advantages over alternative chemotherapy molecules, such as their high affinity, strong specificity for targets, low toxicity and good penetration of tissues, have made food proteins and peptides a new and promising anticancer strategy [5].

Protease inhibitors are found in plant tissues, particularly from legumes. One of the most extensively studied inhibitors in the field of carcinogenesis is the soybean derived Bowman-Birk protease inhibitor (BBI). It is a 71-amino acids polypeptides which chemopreventive properties have been demonstrated in both *in vitro* and *in vivo* systems [6]. As a result of this evidence, BBI acquired the status of "investigational new drug" from the Food and Drug Administration in 1992, and since then, large-scale human trials are being carried out to

© 2013 Hsieh et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Hsieh et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

evaluate its use as an anticarcinogenic agent in the form of BBI concentrate (BBIC) [7-9]. These studies have shown that BBIC is well-tolerated by the patients and led to promising results for prostate and oral carcinomas.

1997-2012: Fifteen Years of Research on Peptide Lunasin 5

**(mg/100 g product) Reference** 

soy genotype, the environmental factors, the manufacturing process and the storage conditions. Thus, these parameters might be used to control the content of this bioactive

Regular soymilk Soybeans 15.7 1.3 [22] Soybeans 12.3 0.8 [22] Soybeans 11.8 1.3 [22]

> Whole soybeans 9.3 0.3 [23] Soybeans, soybean oil, soy lecithin 9.2 1.7 [23] Soy flour, Stevia sweetened 7.0 0.1 [23] Soy flour, Stevia sweetened 6.3 0.2 [23] Whole soybeans, filtered water 7.9 0.0 [23] Whole soybeans, filtered water 6.3 0.1 [23] Whole soybeans, filtered water 6.1 0.1 [23] Whole soybeans 6.0 0.6 [23] Whole soybeans 2.2 0.1 [23] Soybeans, soy lecithin 5.2 0.7 [23] Soybeans, calcium fortified 1.8 0.3 [23] Aqueous extract of soybeans 2.3 0.4 [23]

> barley extract 18.9 2.6 [22]

barley extract 14.2 1.1 [22] Organic soybeans 13.8 2.6 [22] Organic soybeans 14.4 2.4 [22] Organic soybeans 14.7 0.8 [22] Organic soybeans, rice syrup 13.7 0.9 [22] Organic soybeans, soy protein isolate 13.9 1.0 [22] Organic soybeans, malt syrup 18.3 2.4 [22] Organic soybeans, barley extract 10.7 0.8 [22] Whole organic soybeans, isoflavones 9.1 0.1 [23] Organic soybeans 8.8 0.0 [23] Organic soybeans, calcium fortified 8.2 0.0 [23]

enriched 5.6 0.7 [23]

enriched 5.4 0.6 [23] Organic soybeans 3.8 0.6 [23] Organic soybeans 2.9 0.8 [23]

Corn syrup, soy protein 2.8 0.2 [22] Rice syrup, soy protein concentrate 1.5 0.1 [22] Soy protein isolate, soy oil, iron fortified 8.9 0.4 [23]

**Type of sample Composition-main ingredients Lunasin** 

Organic soybeans, malted wheat and

Whole organic soybeans, calcium

Whole organic soybeans, calcium

Soy formula Corn syrup, soy protein isolate 4.1 0.4 [22]

Organic soymilk Organic soybeans, malted wheat and

peptide.

Milk contains a number of proteins and peptides exhibiting chemopreventive properties. As an example, lactoferrin is a well-known whey protein for its inhibitory action on cancer cells proliferation, as well as for its antimicrobial, anti-inflammatory and antioxidant abilities [10]. The protective effects of orally administered lactoferrin against chemically induced carcinogenesis, tumor growth, and/or metastasis have been demonstrated in an increasing number of animal model studies, thereby suggesting its great potential therapeutic use in cancer disease prevention and/or treatment. Lactoferricin is a cationic peptide produced by acid-pepsin hydrolysis of lactoferrin. Similarly to its source protein, lactoferricin has been demonstrated, by cell culture and animal models, to exert anticarcinogenic properties against different types of cancer, such as leukemia, colon, breast, and lung cancer, among others [11]. This peptide acts through cell proliferation inhibition, apoptosis induction, angiogenesis suppression, and modulation of protein expression involved in different carcinogenesis pathways.

Recent studies have identified and characterized, peptides derived from animal and vegetal sources as promising chemopreventive agents [12-14]. One of these peptides, called lunasin, was identified in soybean and other plants and legumes. Studies performed in the last five years have revealed lunasin's properties in both cell culture and animal models, making it a potential strategy for cancer prevention and/or therapy. The purpose of this chapter is to summarize the evidence reported since lunasin's discovery in 1997 on its possible benefits as a chemopreventive agent as well as its demonstrated mechanisms of action.

## **2. Lunasin: Discovery and beyond**

Lunasin has been described as a 43-amino acid peptide encoded within the soybean 2S albumin. Its sequence is SKWQHQQDSCRKQLQGVNLTPCEKHIMEKIQGRGDDDDD DDDD, containing 9 Asp residues, and an Arg-Gly-Asp cell adhesion motif [15]. Lunasin was first identified in the soybean seed, with variable concentrations ranged from 0.5 to 8.1 mg lunasin/g seed [16,17]. This variation has been found to mainly depend on the soybean genotype, suggesting the possibility of selecting and breeding varieties of soybean with higher lunasin contents [16]. The stages of seed development have also been found to affect lunasin's concentration, and a notable increase occurs during seed maturation [18]. However, sprouting leads to a continuing decrease of lunasin with soaking time. Recent studies have revealed the influence on lunasin content of environmental factors, such as temperature, soil moisture and germination time, as well as of processing conditions [19-21].

Presence of lunasin has been demonstrated in commercial and pilot plant produced soybean products, including soy milk, infant formula, high protein soy shake, tofu, bean curd, soybean cake, tempeh, and su-jae (Table 1) [22,23]. Results from these studies establish the influence on lunasin concentration in the food products of different parameters, such as the soy genotype, the environmental factors, the manufacturing process and the storage conditions. Thus, these parameters might be used to control the content of this bioactive peptide.

4 Bioactive Food Peptides in Health and Disease

results for prostate and oral carcinomas.

carcinogenesis pathways.

**2. Lunasin: Discovery and beyond** 

evaluate its use as an anticarcinogenic agent in the form of BBI concentrate (BBIC) [7-9]. These studies have shown that BBIC is well-tolerated by the patients and led to promising

Milk contains a number of proteins and peptides exhibiting chemopreventive properties. As an example, lactoferrin is a well-known whey protein for its inhibitory action on cancer cells proliferation, as well as for its antimicrobial, anti-inflammatory and antioxidant abilities [10]. The protective effects of orally administered lactoferrin against chemically induced carcinogenesis, tumor growth, and/or metastasis have been demonstrated in an increasing number of animal model studies, thereby suggesting its great potential therapeutic use in cancer disease prevention and/or treatment. Lactoferricin is a cationic peptide produced by acid-pepsin hydrolysis of lactoferrin. Similarly to its source protein, lactoferricin has been demonstrated, by cell culture and animal models, to exert anticarcinogenic properties against different types of cancer, such as leukemia, colon, breast, and lung cancer, among others [11]. This peptide acts through cell proliferation inhibition, apoptosis induction, angiogenesis suppression, and modulation of protein expression involved in different

Recent studies have identified and characterized, peptides derived from animal and vegetal sources as promising chemopreventive agents [12-14]. One of these peptides, called lunasin, was identified in soybean and other plants and legumes. Studies performed in the last five years have revealed lunasin's properties in both cell culture and animal models, making it a potential strategy for cancer prevention and/or therapy. The purpose of this chapter is to summarize the evidence reported since lunasin's discovery in 1997 on its possible benefits as

Lunasin has been described as a 43-amino acid peptide encoded within the soybean 2S albumin. Its sequence is SKWQHQQDSCRKQLQGVNLTPCEKHIMEKIQGRGDDDDD DDDD, containing 9 Asp residues, and an Arg-Gly-Asp cell adhesion motif [15]. Lunasin was first identified in the soybean seed, with variable concentrations ranged from 0.5 to 8.1 mg lunasin/g seed [16,17]. This variation has been found to mainly depend on the soybean genotype, suggesting the possibility of selecting and breeding varieties of soybean with higher lunasin contents [16]. The stages of seed development have also been found to affect lunasin's concentration, and a notable increase occurs during seed maturation [18]. However, sprouting leads to a continuing decrease of lunasin with soaking time. Recent studies have revealed the influence on lunasin content of environmental factors, such as temperature, soil moisture and germination time, as well as of processing conditions [19-21].

Presence of lunasin has been demonstrated in commercial and pilot plant produced soybean products, including soy milk, infant formula, high protein soy shake, tofu, bean curd, soybean cake, tempeh, and su-jae (Table 1) [22,23]. Results from these studies establish the influence on lunasin concentration in the food products of different parameters, such as the

a chemopreventive agent as well as its demonstrated mechanisms of action.



1997-2012: Fifteen Years of Research on Peptide Lunasin 7

revealed the presence of lunasin in different *Lupinus* cultivated and wild species [30]. A more rigorous and systematic search of lunasin and lunasin homologues in different seeds should be need to carry out in order to establish a relation between the presence of this

One of the properties of an ideal cancer preventive agent is that it can be taken orally. This means being able to survive degradation by gastrointestinal and serum proteinases and peptidases, and to reach the target organ or tissue in an active form. Simulation of gastrointestinal digestion of lunasin has demonstrated that, while synthetic pure lunasin is easily hydrolyzed by pepsin and pancreatin, lunasin in soy protein is resistant to the action of these enzymes. Bioavailability studies carried out with animals have confirmed the preliminary results obtained by *in vitro* analysis. First studies carried out in mice and rats fed lunasin-enriched soy protein found that 35% of ingested lunasin reaches the target tissues and organs in an intact and active form [17,28]. Lunasin from rye and barley have also shown stability towards pepsin and pancreatin *in vitro* digestion and the liver, kidney, and blood of rats fed with lunasin-enriched rye or barley, respectively, contained this peptide as detected by Western blot [26,27]. Naturally protease inhibitors, such as Bowman-Birk protease inhibitor and Kunitz trypsin inhibitor have been demonstrated to exert a protective effect on lunasin against digestion by gastrointestinal enzymes, playing this protection a key role in making lunasin bioavailable [31]. These authors reported that lunasin is bioavailable after its oral administration to mice, reaching different tissues, including lung, mammary gland, prostate, and brain, where this peptide might exert is chemopreventive effects. These authors also found that lunasin extracted from the blood and liver of lunasin-enriched soy flour-fed rats was bioactive and able to suppress foci

A clinical trial focused on evaluating lunasin's bioavailability has demonstrated that in healthy volunteer men, 4.5% of lunasin ingested in the form of soy protein reaches plasma [32]. Results from this study are relevant in supporting future clinical trials to demonstrate

Peptide lunasin has demonstrated to exert promising chemopreventive properties against different types of cancers by both cell culture and animal experiments (Table 2). First studies performed with mammalian cells revealed that lunasin did not affect their morphology and proliferation. However, this peptide acted preventing their transformation induced by chemical carcinogens-7,12-dimethylbenz[a]anthracene (DMBA) and 3-methylcholanthrene (MCA) [33,34], viral and ras-oncogenes [33,35,36]. These experiments made lunasin be considered a "watchdog" agent in the cell nucleus that once the transformation event occurs, it acts as a surrogate tumor suppressor that tightly binds to deacetylated core histones disrupting the balance between acetylation-deacetylation, which is perceived by the cell as abnormal and leads to cell death [37]. This first mechanism of action involving

peptide and the taxonomic properties of the plants.

formation in the same concentration as synthetic lunasin.

**3. Lunasin's role as chemopreventive peptide** 

cancer preventive properties of lunasin.

**2.1. Bioavailability of lunasin** 

#### 6 Bioactive Food Peptides in Health and Disease

**Table 1.** Type, composition, and lunasin content of soybean-derived foods

In search of natural sources of lunasin besides soybean, a first screening has been carried out using different beans, grains and herbal plants. Lunasin has been found in cereal grains known for its health effects, such as barley, wheat, and rye [24-27]. Several seeds of oriental herbal and medicinal plants have been analyzed, finding that lunasin is present in all of the *Solanaceae* family, except *L. Chinensis*, but not in any of the *Phaseolus* beans [28]. These findings suggested the presence of lunasin or lunasin-like peptides in other grains and plants. This peptide has been identified in *Amaranth*, a plant well-known and used by the Aztecs for its high nutritional value and its biological properties [29]. A recent study has revealed the presence of lunasin in different *Lupinus* cultivated and wild species [30]. A more rigorous and systematic search of lunasin and lunasin homologues in different seeds should be need to carry out in order to establish a relation between the presence of this peptide and the taxonomic properties of the plants.

#### **2.1. Bioavailability of lunasin**

6 Bioactive Food Peptides in Health and Disease

Silken Tofu

Organic Medium

Extra firm tofu

Deep fried soybean

**Type of sample Composition-main ingredients Lunasin** 

Soft Tofu Soybeans 9.6 0.9 [22] Soft Tofu Soybeans 7.3 1.0 [22]

Kinugoshi Soybeans 9.6 0.7 [22] Silken Tofu Soybeans 4.4 0.5 [22] Silken Tofu Soybeans 3.7 0.5 [22] Medium firm Tofu Soybeans 14.3 1.8 [22]

firm Tofu Soybeans 6.7 1.3 [22] Firm Tofu Soybeans 3.5 0.2 [22]

Chinese style Soybeans 3.7 0.1 [22] Baked tofu Soybeans, soy sauce (wheat) 5.5 0.3 [22] Fried tofu Soybean, soybean oil, soy sauce 0.4 0.1 [22] Dry tofu Soybeans 2.5 0.3 [22] Soy shake Soy and milk, chocolate flavored 1.3 0.01 [23]

Organic tempeh Soybeans, *Rhizopus oligosporus* n.d. [22]

Marinated bean curd Soybeans, soy sauce 9.5 1.0 [22] Soybean curd noodle Soybeans n.d. [22]

cake Soybeans, soybean oil 1.9 0.3 [22] Baked soybean cake Soybeans, soy sauce, sesame oil 1.1 0.2 [22]

In search of natural sources of lunasin besides soybean, a first screening has been carried out using different beans, grains and herbal plants. Lunasin has been found in cereal grains known for its health effects, such as barley, wheat, and rye [24-27]. Several seeds of oriental herbal and medicinal plants have been analyzed, finding that lunasin is present in all of the *Solanaceae* family, except *L. Chinensis*, but not in any of the *Phaseolus* beans [28]. These findings suggested the presence of lunasin or lunasin-like peptides in other grains and plants. This peptide has been identified in *Amaranth*, a plant well-known and used by the Aztecs for its high nutritional value and its biological properties [29]. A recent study has

Soy protein isolate, milk, dark chocolate

Soy, sor protein isolate, chocolate

Soybeans, flaxseed, brown rice, *R.* 

**Table 1.** Type, composition, and lunasin content of soybean-derived foods

Soy protein isolate, soy oil, iron fortified 8.1 0.4 [23] Soy protein isolate, iron fortified 7.3 0.4 [23]

Soy, milk, vanilla flavored 1.3 0.01 [23]

flavored 2.0 0.04 [23]

flavored 3.6 0.02 [23]

Soybeans, brown rice, *R. oligosporus* 8.2 0.4 [22]

*oligosporus* 6.1 0.4 [22] Soybeans, brown rice, *R. oligosporus* n.d. [22]

**(mg/100 g product) Reference** 

One of the properties of an ideal cancer preventive agent is that it can be taken orally. This means being able to survive degradation by gastrointestinal and serum proteinases and peptidases, and to reach the target organ or tissue in an active form. Simulation of gastrointestinal digestion of lunasin has demonstrated that, while synthetic pure lunasin is easily hydrolyzed by pepsin and pancreatin, lunasin in soy protein is resistant to the action of these enzymes. Bioavailability studies carried out with animals have confirmed the preliminary results obtained by *in vitro* analysis. First studies carried out in mice and rats fed lunasin-enriched soy protein found that 35% of ingested lunasin reaches the target tissues and organs in an intact and active form [17,28]. Lunasin from rye and barley have also shown stability towards pepsin and pancreatin *in vitro* digestion and the liver, kidney, and blood of rats fed with lunasin-enriched rye or barley, respectively, contained this peptide as detected by Western blot [26,27]. Naturally protease inhibitors, such as Bowman-Birk protease inhibitor and Kunitz trypsin inhibitor have been demonstrated to exert a protective effect on lunasin against digestion by gastrointestinal enzymes, playing this protection a key role in making lunasin bioavailable [31]. These authors reported that lunasin is bioavailable after its oral administration to mice, reaching different tissues, including lung, mammary gland, prostate, and brain, where this peptide might exert is chemopreventive effects. These authors also found that lunasin extracted from the blood and liver of lunasin-enriched soy flour-fed rats was bioactive and able to suppress foci formation in the same concentration as synthetic lunasin.

A clinical trial focused on evaluating lunasin's bioavailability has demonstrated that in healthy volunteer men, 4.5% of lunasin ingested in the form of soy protein reaches plasma [32]. Results from this study are relevant in supporting future clinical trials to demonstrate cancer preventive properties of lunasin.

#### **3. Lunasin's role as chemopreventive peptide**

Peptide lunasin has demonstrated to exert promising chemopreventive properties against different types of cancers by both cell culture and animal experiments (Table 2). First studies performed with mammalian cells revealed that lunasin did not affect their morphology and proliferation. However, this peptide acted preventing their transformation induced by chemical carcinogens-7,12-dimethylbenz[a]anthracene (DMBA) and 3-methylcholanthrene (MCA) [33,34], viral and ras-oncogenes [33,35,36]. These experiments made lunasin be considered a "watchdog" agent in the cell nucleus that once the transformation event occurs, it acts as a surrogate tumor suppressor that tightly binds to deacetylated core histones disrupting the balance between acetylation-deacetylation, which is perceived by the cell as abnormal and leads to cell death [37]. This first mechanism of action involving

histone acetylation inhibition is considered as one of the most important epigenetic modifications acting on signal transduction pathways involved in cancer development [38,39]. When the cells are in the steady-state conditions, the core H3 and H4 histones are mostly deacetylated, as a repressed state. When cells were treated with peptide lunasin and well-known deacetylase inhibitor sodium butyrate, histone acetylation was inhibited in C3H10T1/2 fibroblasts and breast cancer MCF-7 cells [33,36]. Furthermore, lunasin has been demonstrated to compete with different histone acetyltransferase enzymes (HATs), such as yGCN5 and PCAF, inhibiting the acetylation and repressing the cell cycle progression [24,25,28]. Recently, we have reported that lunasin is a potent inhibitor of histones H3 and H4 histone acetylation [40]. Lunasin's inhibitory activity was found to be higher than that demonstrated by other compounds, such as anacardic acid and curcumin, which chemopreventive properties have been already reported [41-43]. Studies focused on elucidating lunasin's structure-activity relationship establish that lunasin's sequence is essential for inhibiting H4 acetylation whereas poly-D sequence is the main active sequence responsible for H3 acetylation inhibition [40] (Table 3).

1997-2012: Fifteen Years of Research on Peptide Lunasin 9

**Table 2.** Biological effects of peptide lunasin demonstrated by cell culture experiments

Although first studies only established lunasin's capacity to act when transformation process happens, studies performed in the last few years have demonstrated that this peptide also acts on established cancer cells lines. This activity against different types of cancer cell lines is summarized in this chapter. Moreover, results obtained from cancer animal models are also included.

#### **3.1. Chemopreventive properties against breast cancer**

With a prevalence of about 4.4 million women and a lethality rate of more than 410,000 cases per year, breast cancer is the most common cancer disease and the leading cause of death in women worldwide [44]. Based on the prevalence of estrogen receptors (ER) within the cell, breast cancer is categorized into the ER–positive type and the ER-negative type. About 70- 80% of all breast cancers are estrogen sensitive and they are treated by conventional procedures including surgery, radiation chemotherapy, and estrogen analogues. However, ER-negative tumors are more aggressive and resistant to treatments [45,46]. Therefore, searching for new preventive and/or curative strategies for this type of breast cancer has centered the interest of current investigations.

#### *3.1.1. Lunasin against breast cancer in vitro*

Up to one third on breast cancers that are initially ER-independent become resistant to endocrine therapy during tumor progression [47]. Due to this emergence of hormoneresistance, it is necessary to search for alternative therapies. Lunasin has been demonstrated to inhibit cell proliferation in ER-negative breast cancer MDA-MB-231 cells in a dosedependent manner, showing an IC50 value of 181 M [48]. Studies carried out to establish a structure/activity relationship showed an IC50 value of 138 M for the 21 amino acid sequence localized at the C-terminus of lunasin, thus being the main responsible for lunasin's inhibitory effect on breast cancer cells proliferation [40].


responsible for H3 acetylation inhibition [40] (Table 3).

**3.1. Chemopreventive properties against breast cancer** 

lunasin's inhibitory effect on breast cancer cells proliferation [40].

animal models are also included.

centered the interest of current investigations.

*3.1.1. Lunasin against breast cancer in vitro* 

histone acetylation inhibition is considered as one of the most important epigenetic modifications acting on signal transduction pathways involved in cancer development [38,39]. When the cells are in the steady-state conditions, the core H3 and H4 histones are mostly deacetylated, as a repressed state. When cells were treated with peptide lunasin and well-known deacetylase inhibitor sodium butyrate, histone acetylation was inhibited in C3H10T1/2 fibroblasts and breast cancer MCF-7 cells [33,36]. Furthermore, lunasin has been demonstrated to compete with different histone acetyltransferase enzymes (HATs), such as yGCN5 and PCAF, inhibiting the acetylation and repressing the cell cycle progression [24,25,28]. Recently, we have reported that lunasin is a potent inhibitor of histones H3 and H4 histone acetylation [40]. Lunasin's inhibitory activity was found to be higher than that demonstrated by other compounds, such as anacardic acid and curcumin, which chemopreventive properties have been already reported [41-43]. Studies focused on elucidating lunasin's structure-activity relationship establish that lunasin's sequence is essential for inhibiting H4 acetylation whereas poly-D sequence is the main active sequence

Although first studies only established lunasin's capacity to act when transformation process happens, studies performed in the last few years have demonstrated that this peptide also acts on established cancer cells lines. This activity against different types of cancer cell lines is summarized in this chapter. Moreover, results obtained from cancer

With a prevalence of about 4.4 million women and a lethality rate of more than 410,000 cases per year, breast cancer is the most common cancer disease and the leading cause of death in women worldwide [44]. Based on the prevalence of estrogen receptors (ER) within the cell, breast cancer is categorized into the ER–positive type and the ER-negative type. About 70- 80% of all breast cancers are estrogen sensitive and they are treated by conventional procedures including surgery, radiation chemotherapy, and estrogen analogues. However, ER-negative tumors are more aggressive and resistant to treatments [45,46]. Therefore, searching for new preventive and/or curative strategies for this type of breast cancer has

Up to one third on breast cancers that are initially ER-independent become resistant to endocrine therapy during tumor progression [47]. Due to this emergence of hormoneresistance, it is necessary to search for alternative therapies. Lunasin has been demonstrated to inhibit cell proliferation in ER-negative breast cancer MDA-MB-231 cells in a dosedependent manner, showing an IC50 value of 181 M [48]. Studies carried out to establish a structure/activity relationship showed an IC50 value of 138 M for the 21 amino acid sequence localized at the C-terminus of lunasin, thus being the main responsible for

**Table 2.** Biological effects of peptide lunasin demonstrated by cell culture experiments


1997-2012: Fifteen Years of Research on Peptide Lunasin 11

A plethora of chromatin alterations appears to be responsible for the development and progression of various types of cancers, including breast cancer. Acetylation of specific lysine residues in histones is generally linked to chromatin disruption and transcriptional activation of genes [49]. In our studies, a dose-dependent inhibitory effect on H4 acetylation at positions H4-Lys8 and H4-Lys12 was observed after treatment of lunasin at 75 M in MDA-MB-231 cells, reaching 17% and 19% inhibition, respectively, compared to control [40]. It should be needed to extensively study the relevance of these results on lunasin's chemopreventive activity to provide data about its molecular mechanism of action on epigenetic alterations. It

We have also demonstrated that lunasin modulates expression of different genes and proteins involved in cell cycle, apoptosis and signal transduction [48]. A pivotal regulatory pathway determining rates of cell cycle transition from G1 to S phase is the cyclin/cyclindependent kinases (CDK)/p16/retinoblastoma protein (RB) pathway. Over-expression of cyclins D1 and D3 is one of the most frequent alterations present in breast tumors. Cyclins D interacts with CDK4 or CDK6 to form a catalytically active complex, which phosphorylates RB to free active E2F [50]. Inhibition of deregulated cell cycle progress in cancer cells is being considered an effective strategy to delay or halt tumor growth. Lunasin up-regulates RB gene expression [48], and inhibits RB phosphorylation [28], suggesting that both transcriptional and post-translational modifications may be responsible for its inhibitory effect on cancer cell cycle progression. Moreover, lunasin has been found to inhibit cell proliferation, arrest the cell cycle in the S phase in 45%, and provokes a down-regulatory effect on the mRNA levels of CDK2, CDK4, CDC25A, Caspase 8, and Ets2, Myc, Erbb2, AKT1, PIK3R1 and Jun signaling genes in MDA-MB-231 cells [48]. Also, lunasin's downregulatory action on levels of proteins, such as cyclin D1, cyclin D3, CDK4 and CDK6, might also contribute on its breast cancer MDA-MB-231 cells cycle arrest effect [40]. The ability of lunasin to modulate expression of genes and proteins involved in cell cycle, apoptosis and signal transduction seems to play a relevant role in its properties against breast cancer. However, further research should be needed to elucidate the complete molecular and

Lunasin's role as chemopreventive agent against breast cancer has also been demonstrated in *in vivo* mouse models. Our first findings showed a relevant inhibitory effect of a lunasinenriched diet on mammary tumors development in DMBA-induced SENCAR mice [34]. Tumor generation and tumor incidence were reduced by 38% and 25%, respectively, in the mice fed with lunasin-enriched diet (containing 0.23% lunasin) compared with control group. Moreover, the tumor sections obtained from the lunasin-enriched group showed slight stromal invasion and degree of morphological aggressiveness due to the effect of this peptide contained in the soy protein preparation. Park and co-works have reported that isoflavone-deprived soy peptides prevent DMBA-induced rat mammary tumorigenesis, as well as inhibit the growth of human breast cancer MCF-7 cells in a dose-dependent manner, and induce cell death [51]. Lunasin might be responsible for the effects reported by these

would be useful to define new prognostic markers and therapeutic targets.

epigenetic mechanism of action in breast cancer.

*3.1.2. Lunasin against breast cancer in vivo* 

authors.

**Table 3.** Structure/activity relationship of lunasin and its derived fragments

A plethora of chromatin alterations appears to be responsible for the development and progression of various types of cancers, including breast cancer. Acetylation of specific lysine residues in histones is generally linked to chromatin disruption and transcriptional activation of genes [49]. In our studies, a dose-dependent inhibitory effect on H4 acetylation at positions H4-Lys8 and H4-Lys12 was observed after treatment of lunasin at 75 M in MDA-MB-231 cells, reaching 17% and 19% inhibition, respectively, compared to control [40]. It should be needed to extensively study the relevance of these results on lunasin's chemopreventive activity to provide data about its molecular mechanism of action on epigenetic alterations. It would be useful to define new prognostic markers and therapeutic targets.

We have also demonstrated that lunasin modulates expression of different genes and proteins involved in cell cycle, apoptosis and signal transduction [48]. A pivotal regulatory pathway determining rates of cell cycle transition from G1 to S phase is the cyclin/cyclindependent kinases (CDK)/p16/retinoblastoma protein (RB) pathway. Over-expression of cyclins D1 and D3 is one of the most frequent alterations present in breast tumors. Cyclins D interacts with CDK4 or CDK6 to form a catalytically active complex, which phosphorylates RB to free active E2F [50]. Inhibition of deregulated cell cycle progress in cancer cells is being considered an effective strategy to delay or halt tumor growth. Lunasin up-regulates RB gene expression [48], and inhibits RB phosphorylation [28], suggesting that both transcriptional and post-translational modifications may be responsible for its inhibitory effect on cancer cell cycle progression. Moreover, lunasin has been found to inhibit cell proliferation, arrest the cell cycle in the S phase in 45%, and provokes a down-regulatory effect on the mRNA levels of CDK2, CDK4, CDC25A, Caspase 8, and Ets2, Myc, Erbb2, AKT1, PIK3R1 and Jun signaling genes in MDA-MB-231 cells [48]. Also, lunasin's downregulatory action on levels of proteins, such as cyclin D1, cyclin D3, CDK4 and CDK6, might also contribute on its breast cancer MDA-MB-231 cells cycle arrest effect [40]. The ability of lunasin to modulate expression of genes and proteins involved in cell cycle, apoptosis and signal transduction seems to play a relevant role in its properties against breast cancer. However, further research should be needed to elucidate the complete molecular and epigenetic mechanism of action in breast cancer.

#### *3.1.2. Lunasin against breast cancer in vivo*

10 Bioactive Food Peptides in Health and Disease

**Table 3.** Structure/activity relationship of lunasin and its derived fragments

Lunasin's role as chemopreventive agent against breast cancer has also been demonstrated in *in vivo* mouse models. Our first findings showed a relevant inhibitory effect of a lunasinenriched diet on mammary tumors development in DMBA-induced SENCAR mice [34]. Tumor generation and tumor incidence were reduced by 38% and 25%, respectively, in the mice fed with lunasin-enriched diet (containing 0.23% lunasin) compared with control group. Moreover, the tumor sections obtained from the lunasin-enriched group showed slight stromal invasion and degree of morphological aggressiveness due to the effect of this peptide contained in the soy protein preparation. Park and co-works have reported that isoflavone-deprived soy peptides prevent DMBA-induced rat mammary tumorigenesis, as well as inhibit the growth of human breast cancer MCF-7 cells in a dose-dependent manner, and induce cell death [51]. Lunasin might be responsible for the effects reported by these authors.

A recent study has shown that lunasin reduces tumor incidence and generation in a xenograft mouse model using human breast cancer MDA-MB-231 cells [31]. Lunasin's inhibitory effect on the tumor weight and volume was also reported by these authors. In contrast, BBI showed no effect on tumor development. The tumor histological sections obtained from the lunasin-treated group showed cell proliferative inhibition and cell apoptosis induction. These first animal models consider lunasin as a new and promising alternative to prevent and/or treat breast cancer.

1997-2012: Fifteen Years of Research on Peptide Lunasin 13

of this disease. In the last years, pathogenesis of colorectal cancer has been elucidated, giving the approach for development of new drugs to combat this malignancy. Accumulating studies have shown the capability of bioactive food components to modulate the risk of developing colon cancer [58]. Recently, lunasin's potential chemopreventive role

It has been demonstrated that lunasin causes cytotoxicity in four different human colon cancer cell lines, KM12L4, RKO, HCT-116, and HT-29 cell, with IC50 values of 13.0 µM, 21.6 µM, 26.3 µM and 61.7 µM, respectively [59]. These values suggest that lunasin is most potent killing the highly metastatic KM12L4 colon cancer cells than any other colon cell lines used in this study. Moreover, lunasin was capable to provoke cytotoxic effects on the oxaliplatin-resistant variants of these colon cancer cells [60]. Studies on mechanism of action of this peptide have revealed that lunasin causes arrest of cell cycle in G2/M phase and induction of the mitochondrial pathway of apoptosis. The cell cycle arrest was attributed with concomitant increase in the expression of the p21 protein in HT-29 colon cancer cells, while both p21 and p27 protein expressions were up-regulated by lunasin treatment in KM12L4 colon cancer cells [59,61]. Moreover, treatment with lunasin decreased the ratio of Bcl-2:Bax by up-regulating the expression of the pro-apoptotic Bax and down-regulating the expression of the anti-apoptotic Bcl-2, also increasing the activity of caspase-3 [61]. This might be attributed to the increase in the expression of the pro-apoptotic form of clusterin which is positively affected by the increase p21 expression in cell nucleus. Treatment of lunasin causes translocation of Bax into the mitochondrial membrane resulting in the release of cytochrome c and the increase of the expression of cytosolic cytochrome c in KM12L4 cells. It was also demonstrated that treatment with lunasin provokes an increase in the activity of caspase-9 and caspase-3 in both HT-29 and KM12L4 cells [59]. Furthermore, lunasin has been showed to modify the expression of human extracellular matrix and adhesion genes [59]. The Arg-Gly-Asp motif present in the lunasin structure is a recognition site for integrin receptors present in the extracellular matrix (ECM). Integrins are heterodimeric receptors associated with cell adhesion, and cancer metastasis [62]. Treatment of KM12L4 cells with lunasin resulted in the modification on the expression of 62 genes associated with ECM and cell adhesion [59]. These authors also reported that lunasin downregulated the gene expression of collagen type VII 1, integrin 2, matrix metalloproteinase 10, selectin E and integrin 5 by 10.1-, 8.2-, 7.7-, 6.5- and 5.0-fold, respectively, compared to the untreated colorectal cancer cells. On the other hand, the expression of collagen type XIV 1 was up-regulated upon lunasin treatment by 11.6-fold. These results suggest a potential role of peptide lunasin as an agent to combat metastatic colon cancer particularly in cases

Colon cancer liver metastasis is a widely used model to study the effects of different markers and chemotherapy on colon cancer metastasis. Recently, Dia & de Mejia (2011b)

has been also reported.

*3.2.1. Lunasin against colon cancer in vitro* 

where resistance to chemotherapy develops.

*3.2.2. Lunasin against colon cancer in vivo* 

#### *3.1.3. Lunasin's combinations as a novel strategy against breast cancer*

Cancer chemotherapeutic strategies commonly require multiple agents to prevent and/or treat cancer because of its ability to achieve greater inhibitory effects on cancer cells with lower toxicity potential on normal cells [3]. In the last two decades, it has been recognized the aspirin's chemopreventive role against different types of cancer. However, aspirin use has been associated with undesirable side effects, peptic ulcer complications, particularly bleeding and mucosal injury [52,53]. Studies are searching new agents to be combined to aspirin, increasing its effectiveness or decreasing its side effects. Our findings revealed that lunasin potentiates aspirin's cell proliferation inhibitory and apoptosis inducing properties in MDA-MB-231 cells [48]. This combination regulates the genes expression encoding G1 and S-phase regulatory proteins and the extrinsic-apoptosis dependent pathway, at least partially, through synergistic down-regulatory effects were observed for ERBB2, AKT1, PIK3R1, FOS and JUN signaling genes. Moreover, additional studies have demonstrated that lunasin/aspirin combination inhibits foci formation and cell proliferation in chemical carcinogens DMBA and MCA induced-NIH/3T3 cells [54]. The effect was notably higher than that observed when compounds of the combination acted as a single agent.

Anacardic acid is a natural compound found in the shell of the cashew nut. It has been linked to anti-oxidative, anti-microbial, anti-inflammatory and anti-carcinogenic activities [55,56]. Our findings revealed that lunasin/anacardic acid combination arrests cell cycle in Sphase and induces apoptosis at higher levels than that observed when each compound is used individually. This combination also promotes the inhibition of ERBB2, AKT1, JUN and RAF1 signaling genes expression. Synergistic effects have also been observed when lunasin was combined with anacardic acid to treat breast cancer cells and chemical-induced fibroblast cells [57].

The safety and efficacy of chronic use of these combinations should be further tested in animal models and human studies to establish the optimal dose and duration of treatment. Moreover, studies derived from these findings about mechanisms of action of these lunasin's combinations would open a new vision in the development of novel therapies against breast cancer.

#### **3.2. Lunasin's chemopreventive properties against colon cancer**

Colon cancer is the second leading cause of cancer death in the Western world. The high incidence, morbidity and mortality of colon cancer make necessary the effective prevention of this disease. In the last years, pathogenesis of colorectal cancer has been elucidated, giving the approach for development of new drugs to combat this malignancy. Accumulating studies have shown the capability of bioactive food components to modulate the risk of developing colon cancer [58]. Recently, lunasin's potential chemopreventive role has been also reported.

#### *3.2.1. Lunasin against colon cancer in vitro*

12 Bioactive Food Peptides in Health and Disease

fibroblast cells [57].

against breast cancer.

alternative to prevent and/or treat breast cancer.

*3.1.3. Lunasin's combinations as a novel strategy against breast cancer* 

than that observed when compounds of the combination acted as a single agent.

**3.2. Lunasin's chemopreventive properties against colon cancer** 

Anacardic acid is a natural compound found in the shell of the cashew nut. It has been linked to anti-oxidative, anti-microbial, anti-inflammatory and anti-carcinogenic activities [55,56]. Our findings revealed that lunasin/anacardic acid combination arrests cell cycle in Sphase and induces apoptosis at higher levels than that observed when each compound is used individually. This combination also promotes the inhibition of ERBB2, AKT1, JUN and RAF1 signaling genes expression. Synergistic effects have also been observed when lunasin was combined with anacardic acid to treat breast cancer cells and chemical-induced

The safety and efficacy of chronic use of these combinations should be further tested in animal models and human studies to establish the optimal dose and duration of treatment. Moreover, studies derived from these findings about mechanisms of action of these lunasin's combinations would open a new vision in the development of novel therapies

Colon cancer is the second leading cause of cancer death in the Western world. The high incidence, morbidity and mortality of colon cancer make necessary the effective prevention

A recent study has shown that lunasin reduces tumor incidence and generation in a xenograft mouse model using human breast cancer MDA-MB-231 cells [31]. Lunasin's inhibitory effect on the tumor weight and volume was also reported by these authors. In contrast, BBI showed no effect on tumor development. The tumor histological sections obtained from the lunasin-treated group showed cell proliferative inhibition and cell apoptosis induction. These first animal models consider lunasin as a new and promising

Cancer chemotherapeutic strategies commonly require multiple agents to prevent and/or treat cancer because of its ability to achieve greater inhibitory effects on cancer cells with lower toxicity potential on normal cells [3]. In the last two decades, it has been recognized the aspirin's chemopreventive role against different types of cancer. However, aspirin use has been associated with undesirable side effects, peptic ulcer complications, particularly bleeding and mucosal injury [52,53]. Studies are searching new agents to be combined to aspirin, increasing its effectiveness or decreasing its side effects. Our findings revealed that lunasin potentiates aspirin's cell proliferation inhibitory and apoptosis inducing properties in MDA-MB-231 cells [48]. This combination regulates the genes expression encoding G1 and S-phase regulatory proteins and the extrinsic-apoptosis dependent pathway, at least partially, through synergistic down-regulatory effects were observed for ERBB2, AKT1, PIK3R1, FOS and JUN signaling genes. Moreover, additional studies have demonstrated that lunasin/aspirin combination inhibits foci formation and cell proliferation in chemical carcinogens DMBA and MCA induced-NIH/3T3 cells [54]. The effect was notably higher It has been demonstrated that lunasin causes cytotoxicity in four different human colon cancer cell lines, KM12L4, RKO, HCT-116, and HT-29 cell, with IC50 values of 13.0 µM, 21.6 µM, 26.3 µM and 61.7 µM, respectively [59]. These values suggest that lunasin is most potent killing the highly metastatic KM12L4 colon cancer cells than any other colon cell lines used in this study. Moreover, lunasin was capable to provoke cytotoxic effects on the oxaliplatin-resistant variants of these colon cancer cells [60]. Studies on mechanism of action of this peptide have revealed that lunasin causes arrest of cell cycle in G2/M phase and induction of the mitochondrial pathway of apoptosis. The cell cycle arrest was attributed with concomitant increase in the expression of the p21 protein in HT-29 colon cancer cells, while both p21 and p27 protein expressions were up-regulated by lunasin treatment in KM12L4 colon cancer cells [59,61]. Moreover, treatment with lunasin decreased the ratio of Bcl-2:Bax by up-regulating the expression of the pro-apoptotic Bax and down-regulating the expression of the anti-apoptotic Bcl-2, also increasing the activity of caspase-3 [61]. This might be attributed to the increase in the expression of the pro-apoptotic form of clusterin which is positively affected by the increase p21 expression in cell nucleus. Treatment of lunasin causes translocation of Bax into the mitochondrial membrane resulting in the release of cytochrome c and the increase of the expression of cytosolic cytochrome c in KM12L4 cells. It was also demonstrated that treatment with lunasin provokes an increase in the activity of caspase-9 and caspase-3 in both HT-29 and KM12L4 cells [59]. Furthermore, lunasin has been showed to modify the expression of human extracellular matrix and adhesion genes [59]. The Arg-Gly-Asp motif present in the lunasin structure is a recognition site for integrin receptors present in the extracellular matrix (ECM). Integrins are heterodimeric receptors associated with cell adhesion, and cancer metastasis [62]. Treatment of KM12L4 cells with lunasin resulted in the modification on the expression of 62 genes associated with ECM and cell adhesion [59]. These authors also reported that lunasin downregulated the gene expression of collagen type VII 1, integrin 2, matrix metalloproteinase 10, selectin E and integrin 5 by 10.1-, 8.2-, 7.7-, 6.5- and 5.0-fold, respectively, compared to the untreated colorectal cancer cells. On the other hand, the expression of collagen type XIV 1 was up-regulated upon lunasin treatment by 11.6-fold. These results suggest a potential role of peptide lunasin as an agent to combat metastatic colon cancer particularly in cases where resistance to chemotherapy develops.

#### *3.2.2. Lunasin against colon cancer in vivo*

Colon cancer liver metastasis is a widely used model to study the effects of different markers and chemotherapy on colon cancer metastasis. Recently, Dia & de Mejia (2011b)

have reported that lunasin acts as chemopreventive agent against this type of metastasis using colon cancer KM12L4 cells directly injected into the spleen of athymic mice [60]. Lunasin administered at concentration of 4 mg/kg body weight resulted in a significant inhibition of liver metastasis of colon cancer cells, potentially because of its binding to 51 integrin and subsequent suppression of FAK/ERK/NF-B signalling. Lunasin was also capable to potentiate the effect of oxaliplatin in preventing the outgrowth of metastasis. Moreover, lunasin potentiated the effect of oxaliplatin in modifying expression of proteins involved in apoptosis and metastasis including Bax, Bcl-2, IKK- and P65 [60]. These results suggest that lunasin can be used as a potential integrin antagonist thereby preventing the attachment and extravasation of colon cancer cells leading to its anti-metastatic effect. These results open a new vision about the lunasin used in metastasis that might benefit to prolong the survival of mice with metastatic colon cancer.

1997-2012: Fifteen Years of Research on Peptide Lunasin 15

malignancies worldwide [67], being clearly associated with increased cancer risk and progression [68]. Lunasin has been found to exert anti-inflammatory activity that might contribute to its chemopreventive properties. First studies demonstrated that lunasin potently inhibits lipopolysaccharide (LPS)-induced production of pro-inflammatory mediators interleuquine-6 (IL-6), tumor necrosis factor-α (TNF-α), and prostaglandin (PG) E2 (PGE2) in macrophage RAW 264.7 cells [69], through modulation of cyclooxygenase-2 (COX-2)/PGE2 and inducible nitric oxide synthase/nitric oxide pathways, and suppression of NF-B pathways [70,71]. Larkins and co-workers (2006) have demonstrated that COX-2 inhibition can decrease breast cancer cells motility, invasion and matrix metalloproteinase expression [72]. Abnormally up-regulated COX and PGs expression are features in human breast tumors, thus lunasin might have a role in treatment and prevention of this kind of cancer. Moreover, the same biological activity was observed for lunasin-like peptides purified from defatted soybean flour by combination of ion-exchange chromatography and size exclusion chromatography. These peptides showed potent anti-inflammatory activity by inhibiting LPS-induced RAW 264.7 cells through suppression of NF-B pathways [70,71]. Interestingly, Liu and Pan (2010) used *E. coli* as a host to produce valuable bioactive lunasin that was also showed its antiinflammatory properties. The purified recombinant lunasin form *E.coli* expressed system inhibits histone acetylation, and inhibits the production of pro-inflammatory cytokines, such

as TNF-α, interleukin-1β and nitric oxide in LPS-stimulated RAW 264.7 cells [73].

chemopreventive role against cancer and other oxidative stress-related disorders.

**5. Production of lunasin** 

Large amounts of reactive oxygen species (ROS) have been shown to participate in the etiology of several human degenerative diseases, including inflammation, cardiovascular and neurodegenerative disorders, and cancer [74]. It is believed that persistent inflammatory cells recruitment, repeated generation of ROS and pro-inflammatory mediators, as well as continued proliferation of genomically unstable cells contribute to neoplasic transformation and ultimately result in tumor invasion and metastasis [75]. Restoration/activation of improperly working or repressed antioxidant machinery or suppression of abnormally amplified inflammatory signaling can provide important strategies for chemoprevention.

Lunasin has been found to exert potent antioxidant properties, inhibiting linoleic acid oxidation and acting as a potent free radical scavenger, and reducing LPS-induced production of ROS by RAW 264.7 macrophage cells at a dose-dependent manner [69]. Recently, lunasin purified from *Solanum nigrum L.* has been found to protect DNA from oxidative damage by scavenging the generation of hydroxyl radical, as well as reducing Fe3+ to Fe2+ through blocking fenton reaction and inhibiting linoleic acid oxidation [76]. Moreover, these authors demonstrate lunasin's suppresive effects on the production of intracellular ROS and glutathione. Preliminary results indicate a similar inhibitory effect of ROS and GSH productions was also observed in Caco2 cells [77]. This activity might contribute on lunasin's

Although the potential anticancer effect of lunasin has been demonstrated for over a decade, little progress has been made to test *in vivo* efficacy of purified lunasin in large-scale animal

#### **3.3. Lunasin's chemopreventive properties against other type of cancers**

Leukemia is considered to be the most common type of cancer in children. Leukemia disrupts the normal reproduction and repair processes of white blood cells causing them to divide too quickly before they mature and resulting in the arrest on the proper production of all blood cells [63]. Chemopreventive properties of peptide lunasin have also been shown in human leukaemia L1210 cells, with an IC50 value of 14 M [64]. Cell cycle analysis performed by these authors showed that lunasin caused a dose-dependent G2 cell cycle arrest and induction of apoptosis. The expressions of caspases-3, -8 and -9 were significantly up-regulated by 12-, 6- and 6-fold, respectively, which resulted in the increase of percentage of L1210 leukemia cells undergoing apoptosis from 2 to 40% [64].

Prostate cancer is one of the leading causes of cancer death in worldwide men. The multistage, genetic, and epigenetic alterations nature of prostate cancer during disease progression and the response to therapy, represent fundamental challenges in our quest to understand and control this prevalent disease [65]. Recently, Galvez and co-workers have studied lunasin's effects on tumorigenic RWPE-1 and non-tumorigenic RWPE-2 human prostate epithelial cells [66]. These authors observed that HIF1A, PRKAR1A, TOB1, and THBS1 genes were up-regulated by lunasin in RWPE-1 but not in RWPE-2 cells, confirming lunasin's capacity to selectively act on cancer cells without affecting non-cancerous cells. Moreover, lunasin specifically inhibited H4-Lys8 acetylation while enhanced H4-Lys16 acetylation catalyzed by HAT enzymes p300, PCAF, and HAT1A [66]. As a dietary peptide capable of up-regulate gene expression by specific epigenetic modifications of the human genome, lunasin is suggested to represent a novel food bioactive peptide with the potential to reduce cancer risk.

## **4. Anti-inflammatory and antioxidant activities of lunasin**

Inflammation and oxidative stress are two of the most critical factors implicated in carcinogenesis and other degenerative disorders. Accumulating evidences have revealed that chronic inflammation is involved in the development of approximately 15–20% of malignancies worldwide [67], being clearly associated with increased cancer risk and progression [68]. Lunasin has been found to exert anti-inflammatory activity that might contribute to its chemopreventive properties. First studies demonstrated that lunasin potently inhibits lipopolysaccharide (LPS)-induced production of pro-inflammatory mediators interleuquine-6 (IL-6), tumor necrosis factor-α (TNF-α), and prostaglandin (PG) E2 (PGE2) in macrophage RAW 264.7 cells [69], through modulation of cyclooxygenase-2 (COX-2)/PGE2 and inducible nitric oxide synthase/nitric oxide pathways, and suppression of NF-B pathways [70,71]. Larkins and co-workers (2006) have demonstrated that COX-2 inhibition can decrease breast cancer cells motility, invasion and matrix metalloproteinase expression [72]. Abnormally up-regulated COX and PGs expression are features in human breast tumors, thus lunasin might have a role in treatment and prevention of this kind of cancer. Moreover, the same biological activity was observed for lunasin-like peptides purified from defatted soybean flour by combination of ion-exchange chromatography and size exclusion chromatography. These peptides showed potent anti-inflammatory activity by inhibiting LPS-induced RAW 264.7 cells through suppression of NF-B pathways [70,71]. Interestingly, Liu and Pan (2010) used *E. coli* as a host to produce valuable bioactive lunasin that was also showed its antiinflammatory properties. The purified recombinant lunasin form *E.coli* expressed system inhibits histone acetylation, and inhibits the production of pro-inflammatory cytokines, such as TNF-α, interleukin-1β and nitric oxide in LPS-stimulated RAW 264.7 cells [73].

Large amounts of reactive oxygen species (ROS) have been shown to participate in the etiology of several human degenerative diseases, including inflammation, cardiovascular and neurodegenerative disorders, and cancer [74]. It is believed that persistent inflammatory cells recruitment, repeated generation of ROS and pro-inflammatory mediators, as well as continued proliferation of genomically unstable cells contribute to neoplasic transformation and ultimately result in tumor invasion and metastasis [75]. Restoration/activation of improperly working or repressed antioxidant machinery or suppression of abnormally amplified inflammatory signaling can provide important strategies for chemoprevention.

Lunasin has been found to exert potent antioxidant properties, inhibiting linoleic acid oxidation and acting as a potent free radical scavenger, and reducing LPS-induced production of ROS by RAW 264.7 macrophage cells at a dose-dependent manner [69]. Recently, lunasin purified from *Solanum nigrum L.* has been found to protect DNA from oxidative damage by scavenging the generation of hydroxyl radical, as well as reducing Fe3+ to Fe2+ through blocking fenton reaction and inhibiting linoleic acid oxidation [76]. Moreover, these authors demonstrate lunasin's suppresive effects on the production of intracellular ROS and glutathione. Preliminary results indicate a similar inhibitory effect of ROS and GSH productions was also observed in Caco2 cells [77]. This activity might contribute on lunasin's chemopreventive role against cancer and other oxidative stress-related disorders.

#### **5. Production of lunasin**

14 Bioactive Food Peptides in Health and Disease

the survival of mice with metastatic colon cancer.

to reduce cancer risk.

have reported that lunasin acts as chemopreventive agent against this type of metastasis using colon cancer KM12L4 cells directly injected into the spleen of athymic mice [60]. Lunasin administered at concentration of 4 mg/kg body weight resulted in a significant inhibition of liver metastasis of colon cancer cells, potentially because of its binding to 51 integrin and subsequent suppression of FAK/ERK/NF-B signalling. Lunasin was also capable to potentiate the effect of oxaliplatin in preventing the outgrowth of metastasis. Moreover, lunasin potentiated the effect of oxaliplatin in modifying expression of proteins involved in apoptosis and metastasis including Bax, Bcl-2, IKK- and P65 [60]. These results suggest that lunasin can be used as a potential integrin antagonist thereby preventing the attachment and extravasation of colon cancer cells leading to its anti-metastatic effect. These results open a new vision about the lunasin used in metastasis that might benefit to prolong

**3.3. Lunasin's chemopreventive properties against other type of cancers** 

of L1210 leukemia cells undergoing apoptosis from 2 to 40% [64].

**4. Anti-inflammatory and antioxidant activities of lunasin** 

Leukemia is considered to be the most common type of cancer in children. Leukemia disrupts the normal reproduction and repair processes of white blood cells causing them to divide too quickly before they mature and resulting in the arrest on the proper production of all blood cells [63]. Chemopreventive properties of peptide lunasin have also been shown in human leukaemia L1210 cells, with an IC50 value of 14 M [64]. Cell cycle analysis performed by these authors showed that lunasin caused a dose-dependent G2 cell cycle arrest and induction of apoptosis. The expressions of caspases-3, -8 and -9 were significantly up-regulated by 12-, 6- and 6-fold, respectively, which resulted in the increase of percentage

Prostate cancer is one of the leading causes of cancer death in worldwide men. The multistage, genetic, and epigenetic alterations nature of prostate cancer during disease progression and the response to therapy, represent fundamental challenges in our quest to understand and control this prevalent disease [65]. Recently, Galvez and co-workers have studied lunasin's effects on tumorigenic RWPE-1 and non-tumorigenic RWPE-2 human prostate epithelial cells [66]. These authors observed that HIF1A, PRKAR1A, TOB1, and THBS1 genes were up-regulated by lunasin in RWPE-1 but not in RWPE-2 cells, confirming lunasin's capacity to selectively act on cancer cells without affecting non-cancerous cells. Moreover, lunasin specifically inhibited H4-Lys8 acetylation while enhanced H4-Lys16 acetylation catalyzed by HAT enzymes p300, PCAF, and HAT1A [66]. As a dietary peptide capable of up-regulate gene expression by specific epigenetic modifications of the human genome, lunasin is suggested to represent a novel food bioactive peptide with the potential

Inflammation and oxidative stress are two of the most critical factors implicated in carcinogenesis and other degenerative disorders. Accumulating evidences have revealed that chronic inflammation is involved in the development of approximately 15–20% of

Although the potential anticancer effect of lunasin has been demonstrated for over a decade, little progress has been made to test *in vivo* efficacy of purified lunasin in large-scale animal

studies or human clinical trials. The main limitations of these studies have been the lack of a method for obtaining gram quantities of highly purified lunasin from plant sources needed to perform such studies. Chemical synthesis is a rapid and effective method to produce lunasin in small quantities but the high cost and difficulties of the scale-up process makes lunasin's synthesis an economically impractical alternative. In addition, the process employs chemicals that are potential environmental hazards. To date, the reported methods to isolate and purify lunasin from soybean only allowed obtaining small quantities of this peptide at 80% purity [70]. However, recently, Cavazos and co-workers (2012) have developed an improved method to isolate and purify lunasin from defatted soy flour, resulting in at least 95% purity [23]. Simultaneously, a large-scale method to generate highly purified lunasin from defatted soy flour has been developed by Seber and co-workers (2012) [78]. This method is based on the sequential application of anion-exchange chromatography, ultrafiltration, and reversed-phase chromatography, obtaining preparations of > 99% purity with a yield of 442 mg/kg of defatted soy flour. Moreover, these preparations show the same biological activity than that reported for synthetic lunasin although the sequence contains Asn as an additional C-terminal amino acid residue.

1997-2012: Fifteen Years of Research on Peptide Lunasin 17

from natural seeds. However, there is still much to be learned about the effects and mechanisms of lunasin on cancer prevention/therapy. The major challenge on the use of lunasin in treating cancer would be the conversion of existing results into clinical outcomes. The next step should be to design clinical trials to confirm lunasin's chemopreventive properties against different types of cancer. Moreover, genomics, proteomics and biochemical tools should be applied to complete elucidate its molecular mechanism of action. Other aspects, such as searching for lunasin in other seeds, optimization of techniques to enrich products with this peptide and studying lunasin's interactions with

other food constituents affecting its activity should also be conducted.

*Institute of Food Science Research (CIAL, CSIC-UAM, CEI UAM+CSIC), Madrid, Spain* 

*Department of Nutritional Science and Toxicology, University of California Berkeley, CA, USA* 

[1] Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. GLOBOCAN 2008, Cancer incidence and mortality worldwide: IARC Cancer Base No. 10 [Internet]. Lyon, France: International Agency for Research on Cancer; 2010. Available from:

[2] Manson M. Cancer prevention – the potential for diet to modulate molecular signaling.

[3] de Kok TM, van Breda SG, Manson MM. Mechanisms of combined action of different chemopreventive dietary compounds. European Journal Nutrition 2008;47:51-9. [4] de Mejia EG, Dia VP. The role of nutraceutical proteins and peptides in apoptosis, angiogenesis, and metastasis of cancer cells. Cancer Metastasis Review 2010; 29:511-28. [5] Bhutia SK, Maiti TK. Targeting tumors with peptides from natural sources. Trends in

[6] Losso JN. The biochemical and functional food properties of the Bowman-Birk

[7] Armstrong WB, Kennedy AR, Wan XS, Atiba J, McLaren E, Meyskens FL. Single-dose administration of Bowman-Birk inhibitor concentrate in patients with oral leukoplakia.

[8] Armstrong WB, Wan XS, Kennedy AR, Taylor TH, Meyskens FL. Development of the Bowman-Birk inhibitor for oral cancer chemoprevention and analysis of neu immunohistochemical staining intensity with Bowman-Birk inhibitor concentrate

[9] Meyskens FL. Development of Bowman-Birk inhibitor for chemoprevention of oral

Inhibitor. Critical Reviews in Food Science & Nutrition 2008;48:94-118.

Cancer Epidemiology Biomarkers & Prevention 2000;9:43-7.

head and neck cancer. Cancer Prevention 2001;952:116-23.

**Author details** 

**7. References** 

 \*

Corresponding Author

Blanca Hernández-Ledesma

http://globocan.iarc.fr.

Biotechnology 2008;26:210-7.

Trends in Molecular Medicine 2003;9:11-8.

treatment. Laryngoscope 2003;113:1687-702.

Ben O. de Lumen and Chia-Chien Hsieh \*

An additional alternative to increase lunasin content in soybean has been recently reported [79]. This strategy aims to exploit the potential of sourdough lactic acid bacteria to release lunasin during fermentation of cereal and non conventional flours. After fermentation, lunasin from the water soluble extracts was increased up to 2-4 times, being *Lactobacillus curvatus* SAL33 and *Lactobacillus brevis* AM7 the strains capable to release higher concentrations of this peptide. This new strategy opens new possibilities for the biological synthesis and for the formulation of functional foods containing bioactive lunasin.

The use of recombinant production by transgenic organisms is widely employed in industry owing to their ease of use, robustness and costs, and has become the most effective system for the production of long peptides and proteins. A recent study has explored efficient recombinant production of lunasin by exploiting the *Clostridium thermocellum* CipB cellulose-binding domain as a fusion partner protein [80]. This system resulted in yields of peptide of up to 210 mg/L, but the authors consider that these yields might be increased in bioreactors where oxygen and nutrients levels are tightly regulated.

#### **6. Conclusions**

Peptides are becoming a group of health-promoting food components with promising chemopreventive and chemotherapeutic properties against cancer. Among them, peptide lunasin, found in soybean and other plants, is turning into one of the most promising. This peptide has been demonstrated its bioavailability after resisting gastrointestinal and serum degradation, and reaching blood and target organs in an intact and active form. Efficacy of lunasin against breast, colon, leukemia and prostate cancer using cell culture experiments and animal models have been revealed in the last decade. These results make lunasin a good candidate for a new generation of chemopreventive/chemotherapeutical agents derived from natural seeds. However, there is still much to be learned about the effects and mechanisms of lunasin on cancer prevention/therapy. The major challenge on the use of lunasin in treating cancer would be the conversion of existing results into clinical outcomes. The next step should be to design clinical trials to confirm lunasin's chemopreventive properties against different types of cancer. Moreover, genomics, proteomics and biochemical tools should be applied to complete elucidate its molecular mechanism of action. Other aspects, such as searching for lunasin in other seeds, optimization of techniques to enrich products with this peptide and studying lunasin's interactions with other food constituents affecting its activity should also be conducted.

## **Author details**

16 Bioactive Food Peptides in Health and Disease

Asn as an additional C-terminal amino acid residue.

studies or human clinical trials. The main limitations of these studies have been the lack of a method for obtaining gram quantities of highly purified lunasin from plant sources needed to perform such studies. Chemical synthesis is a rapid and effective method to produce lunasin in small quantities but the high cost and difficulties of the scale-up process makes lunasin's synthesis an economically impractical alternative. In addition, the process employs chemicals that are potential environmental hazards. To date, the reported methods to isolate and purify lunasin from soybean only allowed obtaining small quantities of this peptide at 80% purity [70]. However, recently, Cavazos and co-workers (2012) have developed an improved method to isolate and purify lunasin from defatted soy flour, resulting in at least 95% purity [23]. Simultaneously, a large-scale method to generate highly purified lunasin from defatted soy flour has been developed by Seber and co-workers (2012) [78]. This method is based on the sequential application of anion-exchange chromatography, ultrafiltration, and reversed-phase chromatography, obtaining preparations of > 99% purity with a yield of 442 mg/kg of defatted soy flour. Moreover, these preparations show the same biological activity than that reported for synthetic lunasin although the sequence contains

An additional alternative to increase lunasin content in soybean has been recently reported [79]. This strategy aims to exploit the potential of sourdough lactic acid bacteria to release lunasin during fermentation of cereal and non conventional flours. After fermentation, lunasin from the water soluble extracts was increased up to 2-4 times, being *Lactobacillus curvatus* SAL33 and *Lactobacillus brevis* AM7 the strains capable to release higher concentrations of this peptide. This new strategy opens new possibilities for the biological

The use of recombinant production by transgenic organisms is widely employed in industry owing to their ease of use, robustness and costs, and has become the most effective system for the production of long peptides and proteins. A recent study has explored efficient recombinant production of lunasin by exploiting the *Clostridium thermocellum* CipB cellulose-binding domain as a fusion partner protein [80]. This system resulted in yields of peptide of up to 210 mg/L, but the authors consider that these yields might be increased in

Peptides are becoming a group of health-promoting food components with promising chemopreventive and chemotherapeutic properties against cancer. Among them, peptide lunasin, found in soybean and other plants, is turning into one of the most promising. This peptide has been demonstrated its bioavailability after resisting gastrointestinal and serum degradation, and reaching blood and target organs in an intact and active form. Efficacy of lunasin against breast, colon, leukemia and prostate cancer using cell culture experiments and animal models have been revealed in the last decade. These results make lunasin a good candidate for a new generation of chemopreventive/chemotherapeutical agents derived

synthesis and for the formulation of functional foods containing bioactive lunasin.

bioreactors where oxygen and nutrients levels are tightly regulated.

**6. Conclusions** 

Blanca Hernández-Ledesma *Institute of Food Science Research (CIAL, CSIC-UAM, CEI UAM+CSIC), Madrid, Spain* 

Ben O. de Lumen and Chia-Chien Hsieh \* *Department of Nutritional Science and Toxicology, University of California Berkeley, CA, USA* 

## **7. References**


<sup>\*</sup> Corresponding Author

	- [10] Rodrigues L, Teixeira J, Schmitt F, Paulsson M, Lindmark Mansson H. Lactoferrin and cancer disease prevention. Critical Reviews in Food Science and Nutrition 2009;49:203–17.

1997-2012: Fifteen Years of Research on Peptide Lunasin 19

[25] Jeong HJ, Jeong JB, Kim DS, Park JH, Lee JB, Kweon DH, Chung GY, Seo EW, de Lumen BO. The cancer preventive peptide lunasin from wheat inhibits core histone acetylation.

[26] Jeong HJ, Lee JR, Jeong JB, Park JH, Cheong YK, de Lumen BO. The cancer preventive seed peptide lunasin from rye is bioavailable and bioactive. Nutrition and Cancer

[27] Jeong HJ, Jeong JB, Hsieh C-C, Hernández-Ledesma B, de Lumen BO. Lunasin is prevalent in barley and is bioavailable and bioactive in *in vivo* and *in vitro* studies.

[28] Jeong JB, Jeong HJ, Park JH, Lee SH, Lee JR, Lee HK, Chung GY, Choi JD, de Lumen BO. Cancer-preventive peptide lunasin from *Solanum nigrum* L. inhibits acetylation of core histones H3 and H4 and phosphorylation of retinoblastoma protein (Rb). Journal of

[29] Silva-Sanchez C, de la Rosa APB, Leon-Galvan MF, de Lumen BO, de Leon-Rodriguez A, de Mejia EG. Bioactive peptides in amaranth (*Amaranthus hypochondriacus*) seed.

[30] Ramos Herrera OJ, Sepúlveda Jiménez G, López Laredo AR, Hernández-Ledesma B, Hsieh C-C, de Lumen BO, Bermúdez Torres K. Identification of chemopreventive peptide lunasin in some *Lupinus* species. Proceeding 13th International Lupin

[31] Hsieh C-C, Hernández-Ledesma B, de Lumen BO. Complementary roles in cancer prevention: Protease inhibitor makes the cancer preventive peptide lunasin

[32] Dia VP, Torres S, de Lumen BO, Erdman JW, de Mejia EG. Presence of lunasin in plasma of men after soy protein consumption. Journal of Agricultural and Food

[33] Galvez AF, Chen N, Macasieb J, de Lumen BO. Chemopreventive property of a soybean peptide (Lunasin) that binds to deacetylated histones and inhibit acetylation. Cancer

[34] Hsieh C-C, Hernández-Ledesma B, de Lumen BO. Soybean peptide lunasin suppresses in vitro and in vivo 7,12-dimethylbenz[a]anthracene-induced tumorigenesis. Journal of

[35] Lam Y, Galvez AF, de Lumen BO. Lunasin suppresses E1A-mediated transformation of mammalian cells but does not inhibit growth of immortalized and established cancer

[36] Jeong HJ, Park JH, Lam Y, de Lumen BO. Characterization of lunasin isolated from

[37] de Lumen BO. Lunasin: A cancer preventive soy peptide. Nutrition Reviews 2005;63:16-21. [38] Dwarakanath BS, Verma A, Bhatt AN, Parmar VS, Raj HG. Targeting protein acetylation for improving cancer therapy. Indian Journal of Medicinal Research

[39] Dalvai M, Bystricky K. The role of histone modifications and variants in regulating gene expression in breast cancer. Journal of Mammary Gland Biology and Neoplasia

soybean. Journal of Agricultural and Food Chemistry 2003;51:7901-6.

Cancer Letters 2007;255:42-8.

Nutrition and Cancer 2010;62:1113-9.

Agricultural and Food Chemistry 2007;55:10707-13.

Conference, June 6-10, 2011, Poznan, Poland.

cell lines. Nutrition and Cancer 2003;47:88-94.

bioavailable. PLoS ONE 2010;5:e8890.

Chemistry 2009;57:1260-6.

Research 2001;61:7473-8.

2008;128:13-21.

2010;15:19-33.

Food Science 2010;75:H311-6.

Journal of Agricultural and Food Chemistry 2008;56:1233-40.

2009;61:680-6.


[25] Jeong HJ, Jeong JB, Kim DS, Park JH, Lee JB, Kweon DH, Chung GY, Seo EW, de Lumen BO. The cancer preventive peptide lunasin from wheat inhibits core histone acetylation. Cancer Letters 2007;255:42-8.

18 Bioactive Food Peptides in Health and Disease

2007;107:327-36.

2010;31:1629-34.

2007;55:632-7.

2005;53:7686-90.

2008;91:936-46.

Food Chemistry 2010;119:636-42.

[10] Rodrigues L, Teixeira J, Schmitt F, Paulsson M, Lindmark Mansson H. Lactoferrin and cancer disease prevention. Critical Reviews in Food Science and Nutrition 2009;49:203–17. [11] Lizzi AR, Carnicelli V, Clarkson MM, Di Giulio A, Oratore A. Lactoferrin derived peptides: mechanisms of action and their perspectives as antimicrobial and antitumoral

[12] Picot L, Bordenave S, Didelot S, Fruitier-Arnaudin I, Sannie F, Thorkelsson G, Berge JP, Guerard F, Chabeaud A, Piot JM. Antiproliferative activity of fish protein hydrolysates

[13] Jang A, Jo C, Kang KS, Lee M. Antimicrobial and human cancer cell cytotoxic effect of synthetic angiotensin-converting enzyme (ACE) inhibitory peptides. Food Chemistry

[14] Kannan A, Hettiarachchy NS, Lay JO, Liyanage R. Human cancer cell proliferation inhibition by a pentapeptide isolated and characterized from rice bran. Peptides

[15] Galvez AF, de Lumen BO. A soybean cDNA encoding a chromatin binding peptide

[16] de Mejia EG, Vasconez M, de Lumen BO, Nelson R. Lunasin concentration in different soybean genotypes, commercial soy protein, and isoflavone products. Journal of

[17] Jeong HJ, Jeong JB, Kim DS, de Lumen BO. Inhibition of core histone acetylation by the cancer preventive peptide lunasin. Journal of Agricultural and Food Chemistry

[18] Park JH, Jeong HJ, de Lumen BO. Contents and bioactivities of lunasin, Bowman-Birk inhibitor, and isoflavones in soybean seed. Journal of Agricultural and Food Chemistry

[19] Wang WY, Dia VP, Vasconez M, de Mejia EG, Nelson RL. Analysis of soybean proteinderived peptides and the effect of cultivar, environmental conditions, and processing on lunasin concentration in soybean and soy products. Journal of AOAC International

[20] Paucar-Menacho LM, Berhow MA, Mandarino JMG, de Mejia EG, Chang YK. Optimisation of germination time and temperature on the concentration of bioactive compounds in Brazilian soybean cultivar BRS 133 using response surface methodology.

[21] Paucar-Menacho LM, Berhow MA, Mandarino JMG, Chang YK, de Mejia EG. Effect of time and temperature on bioactive compounds in germinated Brazilian soybean

[22] Hernandez-Ledesma B, Hsieh C-C, de Lumen BO. Lunasin and Bowman-Birk protease inhibitor (BBI) in US commercial soy foods. Food Chemistry 2009;115:574-80. [23] Cavazos A, Morales El, Dia VP, González de Mejia E. Analysis of lunasin in comercial and pilot plant produced soybean products and an improved method of lunasin

[24] Jeong HJ, Lam Y, de Lumen BO. Barley lunasin suppresses ras-induced colony formation and inhibits core histone acetylation in mammalian cells. Journal of

cultivar BRS 258. Food Research International 2010; 43:1856-65.

purification. Journal of Food Science 2012;77:C539-45.

Agricultural and Food Chemistry 2002;50:5903-8.

inhibits mitosis of mammalian cells. Nature Biotechnology 1999;17:495-500.

on human breast cancer cell lines. Process Biochemistry 2006;41:1217-22.

agents. Mini-reviews in Medicinal Chemistry 2009;9:687-95.

Agricultural and Food Chemistry 2004;52:5882-87.


[40] Hernández-Ledesma, B, Hsieh C-C, de Lumen BO. Relationship between lunasin's sequence and its inhibitory activity of histones H3 and H4 acetylation. Molecular Nutrition and Food Research 2011;55:989-98.

1997-2012: Fifteen Years of Research on Peptide Lunasin 21

[56] Sung B, Pandey MK, Ahn KS, Yi TF, Chaturvedi MM, Liu MY, Aggarwal BB. Anacardic acid (6-nonadecyl salicylic acid), an inhibitor of histone acetyltransferase, suppresses expression of nuclear factor-κB-regulated gene products involved in cell survival, proliferation, invasion, and inflammation through inhibition of the inhibitory subunit of nuclear factor-κB kinase, leading to potentiation of apoptosis. Blood 2008;111:4880-91. [57] Hsieh C-C, Hernández-Ledesma B, de Lumen BO. Cell proliferation inhibitory and apoptosis inducing properties of anacardic acid and lunasin in human breast cancer

[58] Kim YS, Milner JA. Dietary modulation of colon cancer risk. Journal of Nutrition

[59] Dia VP, de Mejia EG. Lunasin induces apoptosis and modifies the expression of genes associated with extracellular matrix and cell adhesion in human metastatic colon cancer

[60] Dia VP, de Mejia EG. Lunasin potentiates the effect of oxaliplatin preventing outgrowth of colon cancer metastasis, binds to a5b1 integrin and suppresses FAK/ERK/NF-B

[61] Dia VP, de Mejia EG. Lunasin promotes apoptosis in human colon cancer cells by mitochondrial pathway activation and induction of nuclear clusterin expression. Cancer

[62] Dittmar T, Heyder C, Gloria-Maercker E, Hatzmann W, Zanker KS. Adhesion molecules and chemokines: the navigation system for circulating tumor (stem) cells to metastasize in an organ-specific manner. Clinical and Experimental Metastasis 2008; 25:11-32. [63] Kasteng F, Sobocki P, Svedman C, Lundkvist J. Economic evaluations of leukemia: a review of the literature. International Journal of Technology Assessment in Health Care

[64] de Mejia EG, Wang W, Dia VP. Lunasin, with an arginine-glycine-aspartic acid motif, causes apoptosis to L1210 leukemia cells by activation of caspase-3. Molecular Nutrition

[65] Strope SA, Andriole GL. Update on chemoprevention for prostate cancer. Current

[66] Galvez AF, Huang L, Magbanua MMJ, Dawson K, Rodriguez R. Differential expression of thrombospondin (THBS1) in tumorigenic and nontumorigenic prostate epithelial cells in response to a chromatin-binding soy peptide. Nutrition Cancer 2011;63:623-6. [67] Kuper H, Adami HO, Trichopoulos D. Infections as a major preventable cause of

[68] Allavena P, Garlanda C, Borrello MG, Sica A, Mantovani A. Pathways connecting inflammation and cancer. Current Opinion in Genetics Development 2008;18:3-10. [69] Hernández-Ledesma B, Hsieh C-C, de Lumen BO. Anti-inflammatory and antioxidant properties of peptide lunasin in RAW 264.7 macrophages. Biochemical and Biophysical

[70] Dia VP, Wang W, Oh VL, de Lumen BO, de Mejia EG. Isolation, purification and characterization of lunasin from defatted soybean flour and in vitro evaluation of its

[71] de Mejia EG., Dia VP. Lunasin and lunasin-like peptides inhibit inflammation through suppression of NF-kB pathway in the macrophage. Peptides 2009;30:2388-98.

human cancer. Journal of International Medicine 2000;248:171-83.

anti-inflammatory activity. Food Chemistry 2009;114:108-15.

MDA-MB-231 cells. Food Chemistry 2010;125:630-6.

signaling. Cancer Letters 2011;313:167–80.

and Food Research 2010;54:406-14.

Opinion in Urology 2010;20:194-7.

Research Communications 2009;390:803-8.

cells. Molecular Nutrition and Food Research 2011;55:623-34.

2007;173:2576S–9S.

Letters 2010;295:44-53.

2007;23:43-53.


[56] Sung B, Pandey MK, Ahn KS, Yi TF, Chaturvedi MM, Liu MY, Aggarwal BB. Anacardic acid (6-nonadecyl salicylic acid), an inhibitor of histone acetyltransferase, suppresses expression of nuclear factor-κB-regulated gene products involved in cell survival, proliferation, invasion, and inflammation through inhibition of the inhibitory subunit of nuclear factor-κB kinase, leading to potentiation of apoptosis. Blood 2008;111:4880-91.

20 Bioactive Food Peptides in Health and Disease

2003;278:19134-40.

2004;279:51163-71.

2000;403:41-5.

Nutrition and Food Research 2011;55:989-98.

[40] Hernández-Ledesma, B, Hsieh C-C, de Lumen BO. Relationship between lunasin's sequence and its inhibitory activity of histones H3 and H4 acetylation. Molecular

[41] Balasubramanyam K, Swaminathan V, Ranganathan A, Kundu TK. Small molecule modulators of histone acetyltransferase p300. Journal of Biological Chemistry

[42] Balasubramanyam K, Altaf M, Varier RA, Swaminathan V, Ravindran A, Sadhale PP, Kundu TK. Polyisoprenylated benzophenone, garcinol, a natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene

[43] Balasubramanyam K, Varier RA, Altaf M, Swaminathan V, Siddappa NB, Ranga U, Kundu TK. Curcumin, a novel p300/CREB-binding protein-specific inhibitor of acetyltransferase, represses the acetylation of histone/nonhistone proteins and histone acetyltransferasedependent chromatin transcription. Journal of Biological Chemistry

[44] Mangiapane S, Blettner M, Schlattmann P. Aspirin use and breast cancer risk: a metaanalysis and meta-regression of observational studies from 2001 to 2005.

[45] Li Y, Brown PH. Translational approaches for the prevention of estrogen receptornegative breast cancer. European Journal of Cancer Prevention 2007;16:203-15.

[47] Im JY, Park H, Kang KW, Choi WS, Kim HS. Modulation of cell cycles and apoptosis by apicidin in estrogen receptor (ER)-positive and-negative human breast cancer cells.

[48] Hsieh C-C, Hernández-Ledesma B, de Lumen BO. Lunasin, a novel seed peptide, sensitizes human breast cancer MDA-MB-231 cells to aspirin-arrested cell cycle and

[49] Strahl BD, Allis CD. The language of covalent histone modifications. Nature

[50] Sutherland RL, Musgrove EA. Cyclins and breast cancer. Journal of Mammary Gland

[51] Park K, Choi K, Kim H, Kim K, Lee MH, Lee JH, Rim JCK. Isoflavone-deprived soy peptide suppresses mammary tumorigenesis by inducing apoptosis. Experimental and

[52] Lanas A, Bajador E, Serrano P, Fuentes J, Carreño S, Guardia J. Nitrovasodilators, lowdose aspirin, other nonsteroidal antiinflammatory drugs, and the risk of upper

[53] Laine L. Review article: Gastrointestinal bleeding with low-dose aspirin: What's the

[54] Hsieh C-C, Hernández-Ledesma B, de Lumen BO. Lunasin-aspirin combination against NIH/3T3 cells transformation induced by chemical carcinogens. Plant Foods for Human

[55] Kubo I, Ochi M, Vieira PC, Komatsu S. Antitumor agents from the cashew (*Anacardium occidentale*) apple juice. Journal of Agricultural and Food Chemistry 1993;41:1012-5.

gastrointestinal bleeding. New England Journal of Medicine 2000;343:834-9.

risk? Alimentation and Pharmacology Therapy 2006;24:897-908.

expression. Journal of Biological Chemistry 2004;279:33716-26.

Pharmacoepidemiology and Drug Safety 2008;12:115-24.

Chemico-Biological Interactions 2008;172:235-44.

Biology and Neoplasia 2004;9:95-104.

Molecular Medicine 2009;41:371-80.

Nutrition 2011;66:107-13.

[46] Cuzick J. Chemoprevention of breast cancer. Breast Cancer 2008;15:10-6.

induced-apoptosis. Chemico-Biological Interactions 2010;186:127-34.

	- [72] Larkins TL, Nowell M, Singh S, Sanford GL. Inhibition of cyclooxygenase-2 decreases breast cancer cell motility, invasion and matrix metalloproteinase expression. BMC Cancer 2006;6:181.

**Chapter 2** 

© 2013 Clemente et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Clemente et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Bowman-Birk Inhibitors from Legumes:** 

Alfonso Clemente, Maria del Carmen Marín-Manzano,

Maria del Carmen Arques and Claire Domoney

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51262

**1. Introduction** 

**Utilisation in Disease Prevention and Therapy** 

Serine proteases have long been recognized as major players in a wide range of biological processes including cell signaling, cell cycle progression, digestion, immune responses, blood coagulation and wound healing. Their role in the physiology of many human diseases, ranging from cancer and inflammatory disorders to degenerative diseases, now represents an increasingly important feature of this family of enzymes. Proteases are tightly controlled through a number of different mechanisms, including regulation of gene expression, recognition of the substrate by the active site, activity regulation by small molecules, changes in cellular location, post-translational modifications, interaction with other proteins and/or through inhibition of proteolysis by protease inhibitors (PI) [1-3]. This last mechanism usually involves competition with substrates for access to the active site of the enzyme and the formation of tight inhibitory complexes. An understanding of the role played by serine proteases and their specific inhibitors in human diseases offers novel and

Within this framework, there is a growing interest in naturally-occurring serine protease inhibitors of the Bowman-Birk family due to their potential chemopreventive and/or therapeutic properties which can impact on several human diseases, including cancer, neurodegenerative diseases and inflammatory disorders. In light of the Food and Drug Administration (FDA) approval for trials of Bowman-Birk inhibitors (BBI) concentrate (BBIC), a protein extract of soybean (*Glycine max*) enriched in BBI, as an 'Investigational New Drug', human trials have been completed in patients with benign prostatic hyperplasia [5], oral leukoplakia [6-8] and ulcerative colitis [9] (**Table 1**). Although, in most of these cases, the intrinsic ability of BBI to inhibit serine proteases has been related to beneficial health properties, the mechanisms of action and the identity of their therapeutic targets are

challenging opportunities for preventive and/or therapeutic intervention [4].


## **Bowman-Birk Inhibitors from Legumes: Utilisation in Disease Prevention and Therapy**

Alfonso Clemente, Maria del Carmen Marín-Manzano, Maria del Carmen Arques and Claire Domoney

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51262

## **1. Introduction**

22 Bioactive Food Peptides in Health and Disease

Cancer 2006;6:181.

1993;90:7915-22.

ONE 2012;7:e35409.

2011;64:111-20.

[72] Larkins TL, Nowell M, Singh S, Sanford GL. Inhibition of cyclooxygenase-2 decreases breast cancer cell motility, invasion and matrix metalloproteinase expression. BMC

[73] Liu CF, Pan TM. Recombinant expression of bioactive peptide lunasin in *Escherichia coli*.

[74] Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proceedings of the National Academy of Sciences of USA

[75] Khan N, Afaq F, Mukhtar H. Cancer chemoprevention through dietary antioxidants:

[76] Jeong JB, de Lumen BO, Jeong HJ. Lunasin peptide purified from *Solanum nigrum* L. protects DNA from oxidative damage by suppressing the generation of hydroxyl

[77] Hernández-Ledesma B, Espartosa DM, Hsieh C-C, de Lumen BO, Recio I Role of peptide lunasin's antioxidant activity on its chemopreventive properties. Annals of

[78] Seber LE, Barnett BW, McConnell EJ, Hume SD, Cai J, Boles K, Davis KR. Scalable purification and characterization of the anticancer lunasin peptide from soybean. PLoS

[79] Rizzello CG, Nionelli L, Coda R, Gobbetti M. Synthesis of the cancer preventive peptide lunasin by lactic acid bacteria during sourdough fermentation. Nutrition and Cancer

[80] Kyle S, James KAR, McPherson MJ. Recombinant production of the therapeutic peptide

progress and promise. Antioxidant & Redox Signaling 2008;10:475-510.

radical via blocking fenton reaction. Cancer Letters 2010;293:58-64.

Nutrition and Metabolism 2011;58:354.

lunasin. Microbial Cell Factories 2012; 11:28.

Applied Microbiology and Biotechnology 2010;88:177-86.

Serine proteases have long been recognized as major players in a wide range of biological processes including cell signaling, cell cycle progression, digestion, immune responses, blood coagulation and wound healing. Their role in the physiology of many human diseases, ranging from cancer and inflammatory disorders to degenerative diseases, now represents an increasingly important feature of this family of enzymes. Proteases are tightly controlled through a number of different mechanisms, including regulation of gene expression, recognition of the substrate by the active site, activity regulation by small molecules, changes in cellular location, post-translational modifications, interaction with other proteins and/or through inhibition of proteolysis by protease inhibitors (PI) [1-3]. This last mechanism usually involves competition with substrates for access to the active site of the enzyme and the formation of tight inhibitory complexes. An understanding of the role played by serine proteases and their specific inhibitors in human diseases offers novel and challenging opportunities for preventive and/or therapeutic intervention [4].

Within this framework, there is a growing interest in naturally-occurring serine protease inhibitors of the Bowman-Birk family due to their potential chemopreventive and/or therapeutic properties which can impact on several human diseases, including cancer, neurodegenerative diseases and inflammatory disorders. In light of the Food and Drug Administration (FDA) approval for trials of Bowman-Birk inhibitors (BBI) concentrate (BBIC), a protein extract of soybean (*Glycine max*) enriched in BBI, as an 'Investigational New Drug', human trials have been completed in patients with benign prostatic hyperplasia [5], oral leukoplakia [6-8] and ulcerative colitis [9] (**Table 1**). Although, in most of these cases, the intrinsic ability of BBI to inhibit serine proteases has been related to beneficial health properties, the mechanisms of action and the identity of their therapeutic targets are

© 2013 Clemente et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Clemente et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

largely unknown. In this chapter, we describe the emerging evidence for the positive contribution of BBI from legumes to disease prevention and therapy.

Bowman-Birk Inhibitors from Legumes: Utilisation in Disease Prevention and Therapy 25

Bowman-Birk family are canonical serine PI of low molecular weight, being particularly abundant in legume seeds. Soybean BBI represent the most extensively studied members of the Bowman-Birk family, but related BBI from other dicotyledonous legumes [including chickpea (*Cicer arietinum*), common bean (*Phaseolus vulgaris*), lentil (*Lens culinaris*) and pea (*Pisum sativum*)] and from monocotyledonous grasses (*Poaceae*) [including wheat (*Triticum aestivum*), rice (*Oryza sativa*) and barley (*Hordeum vulgare*) species], have been identified and

The BBI that are expressed in seeds are the products of multi-gene families. Several isoinhibitors have been identified in seeds of individual species [11, 12]. The expression of distinct genes, together with the post-translational modifications of primary gene products, combine to give rise to the array of BBI-like variants described for many legume species. Variants in overall and active site sequences, size, functional properties and spatial pattern of expression have been described [13]. As a result, qualitative and quantitative differences in protease inhibitory activities have been shown in comparisons of pea genotypes [14, 15]. The close linkage of the genes encoding BBI, demonstrated for a number of legume species [16], allows the development of near-isolines having distinct haplotypes. In pea, the cosegregation of quantitative and qualitative variation has been used to develop a series of near-isolines, which have allowed the biological significance of a five-fold variation in seed protease inhibitory activity to be investigated at the level of ileal digestibility [17, 18]. These lines now facilitate related studies on the positive contribution of seed BBI to the prevention

The occurrence of BBI in soy foods (soymilk, soy infant formula, defatted soy meal, oilcake, tofu, soybean protein isolate and soybean protein concentrate, among others) is noteworthy, where BBI may be present in different amounts. The soy varieties used, the products themselves and the technological processes used in their preparations all contribute to variation in BBI concentration. In order to quantify BBI in soy foods, enzymatic and immunological methods have been developed; however, no comprehensive information on the concentration of BBI in soy foods is available currently. Recently, Hernández-Ledesma *et al*. [19] reported BBI concentrations in 12 soymilk samples ranging from 7.2 to 55.9 mg BBI/100 mL of soymilk, while BBI was not detected in a soy-based infant formula. BBI was also reported in tofu samples, with concentrations ranging between 2.9 and 12.4 mg/100 g product. Since BBI could not be detected in natto and miso samples, it may be assumed that BBI were degraded during the fermentation

The inhibitory activity of BBI is due to the formation of stable complexes between the inhibitor and target proteases. The conformation of the reactive site loop is complementary to the active site of the protease inhibited and allows BBI to bind tightly to proteases in a substrate-like manner [20, 21]; the resulting non-covalent complex renders the target

characterized.

of disease states.

process.

**2.2. Inhibitory properties** 


aCIU: chymotrypsin inhibitory units; bPSA: prostate specific antigen

**Table 1.** Clinical trials utilizing a protein extract of soybean enriched in Bowman-Birk inhibitors (BBIC)

## **2. The Bowman-Birk family**

#### **2.1. Sources and occurrence**

Plant PI can be categorized into at least 12 different families with 10 of these targeting serine proteases and adopting the standard mechanism of inhibition [10]. Members of the Bowman-Birk family are canonical serine PI of low molecular weight, being particularly abundant in legume seeds. Soybean BBI represent the most extensively studied members of the Bowman-Birk family, but related BBI from other dicotyledonous legumes [including chickpea (*Cicer arietinum*), common bean (*Phaseolus vulgaris*), lentil (*Lens culinaris*) and pea (*Pisum sativum*)] and from monocotyledonous grasses (*Poaceae*) [including wheat (*Triticum aestivum*), rice (*Oryza sativa*) and barley (*Hordeum vulgare*) species], have been identified and characterized.

The BBI that are expressed in seeds are the products of multi-gene families. Several isoinhibitors have been identified in seeds of individual species [11, 12]. The expression of distinct genes, together with the post-translational modifications of primary gene products, combine to give rise to the array of BBI-like variants described for many legume species. Variants in overall and active site sequences, size, functional properties and spatial pattern of expression have been described [13]. As a result, qualitative and quantitative differences in protease inhibitory activities have been shown in comparisons of pea genotypes [14, 15]. The close linkage of the genes encoding BBI, demonstrated for a number of legume species [16], allows the development of near-isolines having distinct haplotypes. In pea, the cosegregation of quantitative and qualitative variation has been used to develop a series of near-isolines, which have allowed the biological significance of a five-fold variation in seed protease inhibitory activity to be investigated at the level of ileal digestibility [17, 18]. These lines now facilitate related studies on the positive contribution of seed BBI to the prevention of disease states.

The occurrence of BBI in soy foods (soymilk, soy infant formula, defatted soy meal, oilcake, tofu, soybean protein isolate and soybean protein concentrate, among others) is noteworthy, where BBI may be present in different amounts. The soy varieties used, the products themselves and the technological processes used in their preparations all contribute to variation in BBI concentration. In order to quantify BBI in soy foods, enzymatic and immunological methods have been developed; however, no comprehensive information on the concentration of BBI in soy foods is available currently. Recently, Hernández-Ledesma *et al*. [19] reported BBI concentrations in 12 soymilk samples ranging from 7.2 to 55.9 mg BBI/100 mL of soymilk, while BBI was not detected in a soy-based infant formula. BBI was also reported in tofu samples, with concentrations ranging between 2.9 and 12.4 mg/100 g product. Since BBI could not be detected in natto and miso samples, it may be assumed that BBI were degraded during the fermentation process.

#### **2.2. Inhibitory properties**

24 Bioactive Food Peptides in Health and Disease

Benign prostatic hyperplasia

Oral

leukoplakia

Double-blind

A

**2. The Bowman-Birk family** 

**2.1. Sources and occurrence** 

Ulcerative colitis

randomized, Placebocontrolled phase IIb trial

randomized, double blind, placebocontrolled trial

aCIU: chymotrypsin inhibitory units; bPSA: prostate specific antigen

largely unknown. In this chapter, we describe the emerging evidence for the positive

Disease Type of trial Experimental design Main results Ref.

reaction

Significant decrease (up to 43 %) in

BBI was well tolerated. No clinical evidence of toxicity or any adverse

31 % of patients achieved clinical response and lesion area decreased after treatment. Dose-dependent effect. BBI was non-toxic. The positive clinical effect of BBIC could be due to the inhibition of serine proteases involved in the cleavage of neu-oncogen protein on the cell surface, preventing the release of the extracellular domain of the protein into the bloodstream

Although this study has not been completed yet, preliminary results suggest that BBIC is not fully

BBIC might be associated with the regression of disease without apparent toxicity or adverse side

effective in patients

effects

**Table 1.** Clinical trials utilizing a protein extract of soybean enriched in Bowman-Birk inhibitors (BBIC)

Plant PI can be categorized into at least 12 different families with 10 of these targeting serine proteases and adopting the standard mechanism of inhibition [10]. Members of the

[5]

[6]

[7]

[8]

[9]

PSAb levels after treatment. Decrease of prostate volume in patients. No dose-limiting toxicity

contribution of BBI from legumes to disease prevention and therapy.

19 patients. Daily doses up to 800 CIU a

patients. Single daily

Administration: twice daily, up to 1066 CIU

148 patients. Daily dose: 600 CIU

12 weeks of therapy. 28 patients. Daily dose: 800 CIU

Phase I trial Duration: 6 months.

Phase I trial Duration: 1 month. 24

Plase II trial Duration: 1 month. 32 patients.

dose: 800 CIU

The inhibitory activity of BBI is due to the formation of stable complexes between the inhibitor and target proteases. The conformation of the reactive site loop is complementary to the active site of the protease inhibited and allows BBI to bind tightly to proteases in a substrate-like manner [20, 21]; the resulting non-covalent complex renders the target protease inactive. Upon complex formation, BBI may be cleaved very slowly (low *K*cat). In legumes, the enzyme inhibitory activity is associated with two subdomains of the BBI, located at opposite sides of the molecule; each canonical loop is contained within a nonapeptide region joined via a disulphide bond between flanking cysteine residues. The double-headed BBI have a characteristic highly conserved array of intra-molecular disulphide bridges occurring among 14 Cys residues [22]. The two binding loops can each inhibit independently and simultaneously an enzyme molecule, which may be the same or distinct types of enzyme [13]. The specificity of each reactive site is determined by the identity of the amino acid residue in position P1 [23]. In double-headed BBI, the first active site is usually involved in trypsin inhibition, with the P1 position being occupied by Arg or Lys. The presence of an Ala residue at the P1 position has been reported to be associated with inhibition of elastase [24]. Chymotrypsin is often the target of the second inhibitory domain as it has a hydrophobic amino acid in its P1 position [13, 25]. Additional residues adjacent to the reactive site peptide bond (P1-P1´) can have a significant effect on the affinity of BBI for particular protease targets [13]. BBI from legumes include potent inhibitors of both trypsin and chymotrypsin, with *K*i values within the nanomolar range reported for different members, including soybean [26], pea [27, 28], lentil [29, 30] and lupin (*Lupinus albus*) BBI [31].

Bowman-Birk Inhibitors from Legumes: Utilisation in Disease Prevention and Therapy 27

mice 3 h after oral administration [37]. Labelled BBI was detected in the luminal contents of the small and large intestine; analysis of tissue homogenates revealed also the presence of active BBI in internal organs where soybean BBI have been shown to exert anti-carcinogenic effects (see next section). By using inverted sacs from different sections of the small intestine, it was demonstrated that active BBI could be transported effectively across the gut epithelium. It has been shown that soybean BBI have a serum half-life of 10 h in rats and hamsters, and are excreted in urine and faeces [38]. In humans, BBI are taken up rapidly and can be detected in the urine within 24-48 h [6]. These findings suggest that BBI are absorbed after oral administration and can reach

BBI have potential health-promoting properties within the GIT [22]. *In vivo* studies have demonstrated the presence of active BBI in the small intestine. Hajós *et al.* [39] reported the survival (~ 5 % of total ingested) of soybean BBI in an immunological reactive form in the small intestine of rats; unfortunately, the inhibitory activities of BBI were not evaluated in these experiments. More recently, it has been demonstrated that BBI from chickpea seeds can resist both acidic conditions and the action of digestive enzymes, and transit through the stomach and small intestine of pigs, generally held as a suitable model for human digestive physiology [40]. The presence of active BBI (5-8 % of the total ingested BBI) at the terminal ileum revealed the resistance of at least some, or a significant proportion, of these proteins to the extreme conditions of the GIT *in vivo*. Chromatographic, electrophoretic and enzymatic data obtained from ileal samples suggested that most of the BBI activity is derived from a protein core containing the two binding domains, and resistant to proteolysis. *In vitro* incubation studies of soybean BBI with mixed fecal samples of pigs showed that BBI remained active and their intrinsic ability to inhibit serine proteases was not significantly affected by the enzymatic or metabolic activity of fecal microbiota [41]. All of these results are significant to investigations of the potential uses of BBI in preventive or

**4. Chemopreventive properties of Bowman-Birk inhibitors** 

Chemoprevention is the use of natural agents or synthetic drugs to halt or reverse the carcinogenesis process before the emergence of invasive cancer. The fact that certain dietary constituents can exert chemopreventive properties has major public health implications and the widespread, long-term use of such compounds should be promoted in populations at normal risk, based on understanding the scientific basis of their beneficial effects. In particular, BBI have been linked to a possible protective effect against both inflammatory

Nutritional intervention and/or dietary manipulation have been suggested as key strategies to prevent and/or control colorectal carcinogenesis [42, 43], one of the major causes of

several tissues and organs.

therapeutic medicine.

**4.1. Colorectal cancer** 

disorders and cancer development (**Table 2**).

#### **3. Bioavailability and metabolism of BBI**

In order to exert any local or systemic health benefits, dietary BBI must resist degradation and maintain biological activity, at least to some extent, after food processing and further passage through the gastrointestinal tract (GIT) [22]. BBI from several legume sources have been shown to resist thermal treatment (up to 100 °C), under either neutral or acidic conditions [32]. Most of the heat-resistant trypsin inhibitory activity in processed legumes is attributable to BBI. At temperatures of 80 °C or lower, chickpea BBI were found to be stable and their inhibitory activities to be unaffected by thermal treatment [33]. Soybean BBI do not lose activity at pH values as low as 1.5 in the presence of pepsin at 37 °C for 2 h [34]; these proteins are also stable to both the acidic conditions and the action of digestive enzymes under simulated gastric and intestinal digestion [35]. Such stability is associated with the rigid structure provided by the seven intra-molecular disulphide bridges that maintain the structural and functional features of the binding sites by adding covalent attachment to the inhibitor core [10, 36]. BBI are fully inactivated by autoclaving or reduction of their disulphide bridges followed by alkylation of the cysteinyl sulfhydryl groups [26].

The resistance of BBI to extreme conditions within the GIT may favour the transport of biologically active BBI across the gut epithelium and could allow their distribution to target organs or tissues in order to exert their beneficial effects locally. The uptake and distribution of soybean BBI, following oral administration, has been examined in rodents. By using [125I] BBI, it was demonstrated that BBI becomes widely distributed in mice 3 h after oral administration [37]. Labelled BBI was detected in the luminal contents of the small and large intestine; analysis of tissue homogenates revealed also the presence of active BBI in internal organs where soybean BBI have been shown to exert anti-carcinogenic effects (see next section). By using inverted sacs from different sections of the small intestine, it was demonstrated that active BBI could be transported effectively across the gut epithelium. It has been shown that soybean BBI have a serum half-life of 10 h in rats and hamsters, and are excreted in urine and faeces [38]. In humans, BBI are taken up rapidly and can be detected in the urine within 24-48 h [6]. These findings suggest that BBI are absorbed after oral administration and can reach several tissues and organs.

BBI have potential health-promoting properties within the GIT [22]. *In vivo* studies have demonstrated the presence of active BBI in the small intestine. Hajós *et al.* [39] reported the survival (~ 5 % of total ingested) of soybean BBI in an immunological reactive form in the small intestine of rats; unfortunately, the inhibitory activities of BBI were not evaluated in these experiments. More recently, it has been demonstrated that BBI from chickpea seeds can resist both acidic conditions and the action of digestive enzymes, and transit through the stomach and small intestine of pigs, generally held as a suitable model for human digestive physiology [40]. The presence of active BBI (5-8 % of the total ingested BBI) at the terminal ileum revealed the resistance of at least some, or a significant proportion, of these proteins to the extreme conditions of the GIT *in vivo*. Chromatographic, electrophoretic and enzymatic data obtained from ileal samples suggested that most of the BBI activity is derived from a protein core containing the two binding domains, and resistant to proteolysis. *In vitro* incubation studies of soybean BBI with mixed fecal samples of pigs showed that BBI remained active and their intrinsic ability to inhibit serine proteases was not significantly affected by the enzymatic or metabolic activity of fecal microbiota [41]. All of these results are significant to investigations of the potential uses of BBI in preventive or therapeutic medicine.

#### **4. Chemopreventive properties of Bowman-Birk inhibitors**

Chemoprevention is the use of natural agents or synthetic drugs to halt or reverse the carcinogenesis process before the emergence of invasive cancer. The fact that certain dietary constituents can exert chemopreventive properties has major public health implications and the widespread, long-term use of such compounds should be promoted in populations at normal risk, based on understanding the scientific basis of their beneficial effects. In particular, BBI have been linked to a possible protective effect against both inflammatory disorders and cancer development (**Table 2**).

#### **4.1. Colorectal cancer**

26 Bioactive Food Peptides in Health and Disease

*albus*) BBI [31].

groups [26].

**3. Bioavailability and metabolism of BBI** 

protease inactive. Upon complex formation, BBI may be cleaved very slowly (low *K*cat). In legumes, the enzyme inhibitory activity is associated with two subdomains of the BBI, located at opposite sides of the molecule; each canonical loop is contained within a nonapeptide region joined via a disulphide bond between flanking cysteine residues. The double-headed BBI have a characteristic highly conserved array of intra-molecular disulphide bridges occurring among 14 Cys residues [22]. The two binding loops can each inhibit independently and simultaneously an enzyme molecule, which may be the same or distinct types of enzyme [13]. The specificity of each reactive site is determined by the identity of the amino acid residue in position P1 [23]. In double-headed BBI, the first active site is usually involved in trypsin inhibition, with the P1 position being occupied by Arg or Lys. The presence of an Ala residue at the P1 position has been reported to be associated with inhibition of elastase [24]. Chymotrypsin is often the target of the second inhibitory domain as it has a hydrophobic amino acid in its P1 position [13, 25]. Additional residues adjacent to the reactive site peptide bond (P1-P1´) can have a significant effect on the affinity of BBI for particular protease targets [13]. BBI from legumes include potent inhibitors of both trypsin and chymotrypsin, with *K*i values within the nanomolar range reported for different members, including soybean [26], pea [27, 28], lentil [29, 30] and lupin (*Lupinus* 

In order to exert any local or systemic health benefits, dietary BBI must resist degradation and maintain biological activity, at least to some extent, after food processing and further passage through the gastrointestinal tract (GIT) [22]. BBI from several legume sources have been shown to resist thermal treatment (up to 100 °C), under either neutral or acidic conditions [32]. Most of the heat-resistant trypsin inhibitory activity in processed legumes is attributable to BBI. At temperatures of 80 °C or lower, chickpea BBI were found to be stable and their inhibitory activities to be unaffected by thermal treatment [33]. Soybean BBI do not lose activity at pH values as low as 1.5 in the presence of pepsin at 37 °C for 2 h [34]; these proteins are also stable to both the acidic conditions and the action of digestive enzymes under simulated gastric and intestinal digestion [35]. Such stability is associated with the rigid structure provided by the seven intra-molecular disulphide bridges that maintain the structural and functional features of the binding sites by adding covalent attachment to the inhibitor core [10, 36]. BBI are fully inactivated by autoclaving or reduction of their disulphide bridges followed by alkylation of the cysteinyl sulfhydryl

The resistance of BBI to extreme conditions within the GIT may favour the transport of biologically active BBI across the gut epithelium and could allow their distribution to target organs or tissues in order to exert their beneficial effects locally. The uptake and distribution of soybean BBI, following oral administration, has been examined in rodents. By using [125I] BBI, it was demonstrated that BBI becomes widely distributed in

Nutritional intervention and/or dietary manipulation have been suggested as key strategies to prevent and/or control colorectal carcinogenesis [42, 43], one of the major causes of

cancer-related mortality worldwide in both men and women [44]. There is now robust preclinical evidence to suggest that dietary BBI from several legume sources can prevent or suppress cancer development and associated inflammatory disorders within the GIT [22]. Soybean BBI have been reported to be effective at concentrations as low as 10 mg/100 g diet, in reducing the incidence and frequency of colorectal tumors, in studies based on the dimethylhydrazine (DMH) rat model, where no adverse effect of BBI was documented for animal growth or organ physiology [45]. When the inhibitory activity of BBI is abolished, any suppressive effect on colorectal tumor development disappears, suggesting that the inhibitory properties of BBI against serine proteases may be required for their reported chemopreventive properties. Proteases play a critical role in tumorigenesis, where their activities become dysregulated in colorectal cancer and neoplastic polyps [46]. In particular, serine proteases are key players in several biological functions linked to tumor development, including cell growth (dys)regulation and cell invasion as well as angiogenesis and inflammatory disorders. Some of these proteases have been reported as promising cancer biomarkers [47-49] (**Table 3**). An understanding of the role played by specific serine proteases in the biological processes associated with disease may suggest modes of therapeutic intervention [1, 50]. Successful examples of therapeutic intervention using PI include ubiquitin-proteasome inhibitors in the treatment of multiple myeloma [51]. The ubiquitin-proteasome pathway is essential for most cellular processes, including protein quality control, cell cycle, transcription, signalling pathways, protein transport, DNA repair and stress responses [52]. Inhibition of proteasome activity leads to accumulation of poly-ubiquitinylated and misfolded proteins, endoplastic reticulum stress and eventually apoptosis. Although soybean BBI has been demonstrated to inhibit the proteasomal activity of MCF7 breast cancer cells (see section 4.4), the proteasomal inhibition in colon cancer cells need to be unambiguously demonstrated. Another potential therapeutic target of BBI is matriptase (also known as MT-SP1 or epithin), an epithelial-specific member of the type II transmembrane serine protease family, which plays a critical role in differentiation and function of the epidermis, gastrointestinal epithelium and other epithelial tissues. Several studies suggest that matriptase is overexpressed in a wide variety of malignant tumors including prostate, ovarian, uterine, colon, epithelial-type mesothelioma and cervical cell carcinoma [53]. It has been proposed to have multiple functions, acting as a potential activator of critical molecules associated with tumor invasion and metastasis. MT-SP1 contributes to the upstream activation of tumor growth and its progression through the selective degradation of extracellular matrix proteins and activation of cellular regulatory proteins, such as urokinase-type plasminogen activator, hepatocyte-growth factor/scatter factor and protease-activated receptor [54]. Although the ability of soybean BBI to inhibit a secreted form of recombinant MT-SP1 has been demonstrated [55], the clinical relevance of such inhibition has not been proven yet. The validation of specific serine proteases as BBI targets, together with the identification of natural BBI variants, and the design of specific potent inhibitors of these proteases, will contribute to the assessment of BBI as colorectal chemopreventive agents for preventive and/or therapeutic medicine [22].

Bowman-Birk Inhibitors from Legumes: Utilisation in Disease Prevention and Therapy 29

**action** 

Suppression of adenomatous

Suppression of histological inflammation parameters, lower mortality rate and delayed onset of mortality

Protective role against inflammation and preneoplastic lesions

unaffected

demonstrated

Proliferation of HT29 colon cancer cells was decreased (IC50 = 32 µM), whereas the non- malignant fibroblastic CCD18Co cells were

The anti-proliferative effect of BBI in colon cancer cells are

rTI1B, a major BBI isoinhibitor from pea, having trypsin and chymotrypsin inhibitory activity, affected the

proliferation of colon cancer cells; however, a derived inactive mutant did not show any anti-proliferative

BBI was more effective in prevention than in therapeutic treatment, with activity related to its protease inhibitory ability

Time- and concentrationdependent anti-proliferative effect on HT29 cells, arrest at G0-G1 phase; trypsin- and chymotrypsin-like proteases

are potential targets

effect

Reduction of incidence and frequency of tumors likely via

protease inhibition

tumors of the GIT

**Refs.** 

[45]

[56]

[57]

[58]

[30]

[15]

[26]

[68]

[100]

**BBI source Carcinogen Model system Effect and/or mechanisms of** 

carcinogenesis in rodents

inflammation

carcinogenesis

cells

cells

cells


> Mouse stomach carcinogenesis

and anal inflammation

**Cancer type** 

Colorectal Soybean DMHa Colon

Soybean DMH Mouse colon

Soybean DSSb Mouse colon

Horsegram DMH Colorectal

Lentil - Colon cancer

Pea - Colon cancer

Soybean - Colon cancer

pyrene

 Recombinant proteins

Gastric Field bean Benzo-

cancer-related mortality worldwide in both men and women [44]. There is now robust preclinical evidence to suggest that dietary BBI from several legume sources can prevent or suppress cancer development and associated inflammatory disorders within the GIT [22]. Soybean BBI have been reported to be effective at concentrations as low as 10 mg/100 g diet, in reducing the incidence and frequency of colorectal tumors, in studies based on the dimethylhydrazine (DMH) rat model, where no adverse effect of BBI was documented for animal growth or organ physiology [45]. When the inhibitory activity of BBI is abolished, any suppressive effect on colorectal tumor development disappears, suggesting that the inhibitory properties of BBI against serine proteases may be required for their reported chemopreventive properties. Proteases play a critical role in tumorigenesis, where their activities become dysregulated in colorectal cancer and neoplastic polyps [46]. In particular, serine proteases are key players in several biological functions linked to tumor development, including cell growth (dys)regulation and cell invasion as well as angiogenesis and inflammatory disorders. Some of these proteases have been reported as promising cancer biomarkers [47-49] (**Table 3**). An understanding of the role played by specific serine proteases in the biological processes associated with disease may suggest modes of therapeutic intervention [1, 50]. Successful examples of therapeutic intervention using PI include ubiquitin-proteasome inhibitors in the treatment of multiple myeloma [51]. The ubiquitin-proteasome pathway is essential for most cellular processes, including protein quality control, cell cycle, transcription, signalling pathways, protein transport, DNA repair and stress responses [52]. Inhibition of proteasome activity leads to accumulation of poly-ubiquitinylated and misfolded proteins, endoplastic reticulum stress and eventually apoptosis. Although soybean BBI has been demonstrated to inhibit the proteasomal activity of MCF7 breast cancer cells (see section 4.4), the proteasomal inhibition in colon cancer cells need to be unambiguously demonstrated. Another potential therapeutic target of BBI is matriptase (also known as MT-SP1 or epithin), an epithelial-specific member of the type II transmembrane serine protease family, which plays a critical role in differentiation and function of the epidermis, gastrointestinal epithelium and other epithelial tissues. Several studies suggest that matriptase is overexpressed in a wide variety of malignant tumors including prostate, ovarian, uterine, colon, epithelial-type mesothelioma and cervical cell carcinoma [53]. It has been proposed to have multiple functions, acting as a potential activator of critical molecules associated with tumor invasion and metastasis. MT-SP1 contributes to the upstream activation of tumor growth and its progression through the selective degradation of extracellular matrix proteins and activation of cellular regulatory proteins, such as urokinase-type plasminogen activator, hepatocyte-growth factor/scatter factor and protease-activated receptor [54]. Although the ability of soybean BBI to inhibit a secreted form of recombinant MT-SP1 has been demonstrated [55], the clinical relevance of such inhibition has not been proven yet. The validation of specific serine proteases as BBI targets, together with the identification of natural BBI variants, and the design of specific potent inhibitors of these proteases, will contribute to the assessment of BBI as colorectal

chemopreventive agents for preventive and/or therapeutic medicine [22].




Bowman-Birk Inhibitors from Legumes: Utilisation in Disease Prevention and Therapy 31

**Function Pathological process Refs.** 

progression

gastric cancer

Carcinogenesis,

eneration

Pulmonary disease, inflammation

Inflammation, asthma,

inflammation, neurodeg-

Atherosclerosis, asthma, inflammatory disorders

Pathogenesis of epithelial tissues, tumor growth and

Inflammation, metastasis [62]

[97, 98]

[55]

[62, 99]

[64]

[58, 78]

remission of disease. Several mechanisms have been proposed to explain the antiinflammatory properties of BBI. The ability of BBI to decrease the production and release of superoxide anion radicals, mediators of inflammatory processes, in purified human polymorphonuclear leukocytes [60] and in differentiated HL60 cells [59] has been reported. The decrease in superoxide radical levels may reduce free radical-induced DNA damage and transformation to malignant phenotypes. In addition, superoxide radicals can initiate a wide range of toxic oxidative reactions, including lipid peroxidation. In this regard, it has been demonstrated that BBI can reduce the content of lipid peroxides in irradiated cells *in vitro* [61], a reduction that is presumed to be linked to the anti-inflammatory activity of BBI. The role that certain serine proteases play during proteolysis in acute and chronic inflammatory processes is well recognized. The ability of soybean BBI to inhibit serine proteases involved in inflammatory processes, such as cathepsin G [62, 63], elastase [64] and mast cell chymase [64] has been reported. The last enzyme acts as a chemo-attractant and may play a role in the accumulation of inflammatory cells during development of allergic and non-allergic diseases [65]. The interaction of chymase and BBI may impact on other processes involved in anti-inflammatory responses, such as the regulation of collagenase [66] and interleukin 1 (IL-1) [67]. Nevertheless, a clear correlation between the inhibition of these serine proteases and the anti-inflammatory properties associated with soybean BBI

has not been demonstrated clearly [22].

Tryptase Phagocytosis, degradation of

coagulation

Cathepsin G ECM degradation, migration,

Matriptase Matrix degradation, regulation

metabolism

disorders

Proteasome Protein degradation, cell

Chymase Degradation of ECM

disorders

ECMa compounds, regulation of inflammatory responses, blood

regulation of inflammatory

of intestinal barrier, iron

Pathogen killing, ECM degradation, inflammatory

compounds, regulation of inflammatory responses

proliferation, differentiation, angiogenesis and apoptosis

BBI and related proteins (adapted from Clemente et al., 2011 [22]).

**Table 3.** Serine proteases involved in pathological processes as potential therapeutic targets of soybean

**Serine protease** 

Human elastase

aECM: extracellular matrix

aDMH: dimethylhydrazine; bDSS: dextran sulphate sodium

**Table 2.** *In vitro* and *in vivo* studies showing chemopreventive properties of Bowman-Birk inhibitors

A strong interest exists in investigating the potential of BBI as anti-inflammatory agents within the GIT. In rodents, soybean BBI treatment appears to have a potent suppressive effect on colon and anal gland inflammation, following exposure to carcinogenic agents [56], or when assessed in an acute injury/colitis model [57]. The protective effect of BBI from soybean or those from perennial horsegram (*Macrotymola axillare*) against inflammation and development of pre-neoplastic lesions induced in the DMH mouse model was reported recently [58]. Given the lack of toxicity as well as the reported anti-inflammatory properties in animals, the potential for BBIC to benefit patients with ulcerative colitis has been evaluated. In a randomized double-blind placebo-controlled trial, a dose of 800 chymotrypsin inhibitor units (CIU) per day over a three-month treatment period was associated with a clinical response and induction of remission, as assessed by the Sutherland Disease Activity Index score [59], in patients with ulcerative colitis, without apparent toxicity [9]. Approximately 50 % of patients responded clinically and 36 % showed remission of disease. Several mechanisms have been proposed to explain the antiinflammatory properties of BBI. The ability of BBI to decrease the production and release of superoxide anion radicals, mediators of inflammatory processes, in purified human polymorphonuclear leukocytes [60] and in differentiated HL60 cells [59] has been reported. The decrease in superoxide radical levels may reduce free radical-induced DNA damage and transformation to malignant phenotypes. In addition, superoxide radicals can initiate a wide range of toxic oxidative reactions, including lipid peroxidation. In this regard, it has been demonstrated that BBI can reduce the content of lipid peroxides in irradiated cells *in vitro* [61], a reduction that is presumed to be linked to the anti-inflammatory activity of BBI. The role that certain serine proteases play during proteolysis in acute and chronic inflammatory processes is well recognized. The ability of soybean BBI to inhibit serine proteases involved in inflammatory processes, such as cathepsin G [62, 63], elastase [64] and mast cell chymase [64] has been reported. The last enzyme acts as a chemo-attractant and may play a role in the accumulation of inflammatory cells during development of allergic and non-allergic diseases [65]. The interaction of chymase and BBI may impact on other processes involved in anti-inflammatory responses, such as the regulation of collagenase [66] and interleukin 1 (IL-1) [67]. Nevertheless, a clear correlation between the inhibition of these serine proteases and the anti-inflammatory properties associated with soybean BBI has not been demonstrated clearly [22].


aECM: extracellular matrix

30 Bioactive Food Peptides in Health and Disease

Soybean - Breast cancer

Prostate Soybean - Prostate cancer

Soybean - Prostate cancer

Soybean - Prostate cancer

Oral Soybean - Oral

aDMH: dimethylhydrazine; bDSS: dextran sulphate sodium

**BBI source Carcinogen Model system Effect and/or mechanisms of** 


cells

cells

cells and rat prostate carcinogenesis

xeno-grafts in nude mice

leukoplakia

**Table 2.** *In vitro* and *in vivo* studies showing chemopreventive properties of Bowman-Birk inhibitors

A strong interest exists in investigating the potential of BBI as anti-inflammatory agents within the GIT. In rodents, soybean BBI treatment appears to have a potent suppressive effect on colon and anal gland inflammation, following exposure to carcinogenic agents [56], or when assessed in an acute injury/colitis model [57]. The protective effect of BBI from soybean or those from perennial horsegram (*Macrotymola axillare*) against inflammation and development of pre-neoplastic lesions induced in the DMH mouse model was reported recently [58]. Given the lack of toxicity as well as the reported anti-inflammatory properties in animals, the potential for BBIC to benefit patients with ulcerative colitis has been evaluated. In a randomized double-blind placebo-controlled trial, a dose of 800 chymotrypsin inhibitor units (CIU) per day over a three-month treatment period was associated with a clinical response and induction of remission, as assessed by the Sutherland Disease Activity Index score [59], in patients with ulcerative colitis, without apparent toxicity [9]. Approximately 50 % of patients responded clinically and 36 % showed

**action** 

BBI induced apoptosis, cell death and lysosome

membrane permeabilization

Proteasome was reported as potential therapeutic target in

BBI exerted chemopreventive activity associated with induction of connexin-43 expression and apoptosis

BBI and BBIC inhibited the growth of LNCaP cells

BBI prevented the generation of activated oxygen species and activated DNA repair through a p53-dependent

dependent reduction in oral lesion size in 31% of patients without any adverse effects; modulation of protease activity and *neu* oncogene levels was observed

MCF-7 cells

mechanism

BBIC exerted a dose-

**Refs.** 

[79]

[78]

[76,77]

[72]

[74, 75]

[6,7,69]

**Cancer type** 

Breast Black-eyed pea

> **Table 3.** Serine proteases involved in pathological processes as potential therapeutic targets of soybean BBI and related proteins (adapted from Clemente et al., 2011 [22]).

In previous studies, a significant concentration- and time-dependent decrease in the growth of an array of colon cancer cells (HT29, Caco2, LoVo) has been demonstrated *in vitro*, following treatment with BBI variants from several legume sources, including pea, lentil and soybean; in contrast, the growth of non-malignant colonic fibroblastic CCD18-Co cells was unaffected by BBI [15, 26, 30]. Recently, the anti-proliferative effect of rTI1B, a major pea isoinhibitor expressed heterologously in *Pichia pastoris*, has been evaluated using colon cancer cells grown *in vitro*. Comparisons of the effects of rTI1B with those observed using a related synthetic mutant derivative, showed that the proliferation of HT29 colon cancer cells was inhibited significantly by rTI1B in a dose-dependent manner, whereas the mutant which lacked trypsin and chymotrypsin inhibitory activity did not show any significant effect on colon cancer cell growth (**Figure 1**) [68]. Although the molecular mechanism(s) of this chemopreventive activity remains unknown, the reported data indicate that both trypsin- and chymotrypsin-like serine proteases involved in carcinogenesis are likely primary targets for BBI.

Bowman-Birk Inhibitors from Legumes: Utilisation in Disease Prevention and Therapy 33

examination, diagnostic sampling and evaluation of response to treatment. In a Phase I clinical trial, no clinical evidence of toxicity or any adverse effect was apparent when BBIC was administered as a single oral dose of up to 800 CIU to twenty-four patients with oral leukoplakia over one month-period [6]. The study revealed that BBIC was well-tolerated and no allergic reactions, gastrointestinal side-effects or other clinical symptoms were elicited. In a non-randomized phase IIa clinical trial, treatment with BBIC for one month resulted in a dose-dependent reduction in oral lesion size in 31% of patients [7]. The positive clinical effect of BBIC was associated with modulation of protease activity and *neu* oncogene levels (as the surrogate endpoint biomarker for the trial) in exfoliated oral mucosal cells [7, 69]; however, this evidence is indirect and the specific target proteases inhibited were not reported. A recent phase IIb randomized, double-blind, placebo-controlled trial involving patient treatment with BBIC for six months was performed. Even though this multiinstitutional study has not been completed yet, BBIC does not seem to be fully effective as

Prostate cancer is the second most frequently diagnosed cancer in men although the incidence of cancer varies greatly throughout the world. Dietary habits and lifestyle have been identified as major risk factors in prostate cancer growth and progression, suggesting that prostate cancer might be preventable [70]. Epidemiological studies have shown an inverse association between soy intake and the risk of developing prostate cancer [71]. Preclinical and clinical studies have shown the potential chemopreventive properties of BBI in prostate cancer. Purified soybean BBI and BBIC have been shown to inhibit the growth of LNCaP human prostate cancer xenografts in nude mice [72], and to decrease the growth, invasion and clonogenic survival of several human prostate cancer cells [73]. The effectiveness of soybean BBI in preventing the generation of activated oxygen species in prostate cancer cells [74] and in activating DNA repair through a p53-dependent mechanism has been reported [75]. More recently, BBIC has prevented the growth of prostate tumors in transgenic rats developing adenocarcinoma, most likely as a consequence of its antiproliferative activity via induction of connexin 43 expression [76, 77]. In humans, a doubleblind, randomized, phase I trial was carried out in nineteen male subjects with benign prostatic hyperplasia, which is a precursor condition for prostate cancer, and lower urinary tract symptoms [5]. In this study, the authors demonstrated that BBIC treatment for six months reduced levels of prostate-specific antigen (PSA), a clinical marker for prostate cancer, and prostate volume in patients. Additional clinical studies will be necessary to

chemopreventive agent for the management of oral leukoplakia [8].

determine the potential of BBIC as prostate cancer chemopreventive agent.

Breast cancer is one of the most frequent cancer types and is responsible for the highest mortality rate among women. Novel complementary strategies, including chemoprevention, have been suggested. As 125I-BBI, when orally administrated in rodents, has been

**4.3. Prostate cancer** 

**4.4. Breast cancer** 

**Figure 1.** Dose–response effects of rTI1B (closed bars), a major pea isoinhibitor expressed in *Pichia pastoris*, and the corresponding inactive mutant (open bars), having amino acid substitutions at the P1 positions in the two inhibitory domains, on the *in vitro* growth of HT29 human colorectal adenocarcinoma cells. Growth media were supplemented with rTI1B in the concentration range 15–61 µM and cells harvested after a period of 96 h. Data are means of at least three independent experiments, each having four technical replicates; bars represent standard deviations. Means not sharing superscript letters differ significantly (*p* <0.05; Bonferroni's test) (Adapted from Clemente et al., (2012) [68]).

#### **4.2. Oral leukoplakia**

Leukoplakia in the oral cavity is considered a suitable model for the study of chemoprevention because the precancerous lesions are readily accessible to visual examination, diagnostic sampling and evaluation of response to treatment. In a Phase I clinical trial, no clinical evidence of toxicity or any adverse effect was apparent when BBIC was administered as a single oral dose of up to 800 CIU to twenty-four patients with oral leukoplakia over one month-period [6]. The study revealed that BBIC was well-tolerated and no allergic reactions, gastrointestinal side-effects or other clinical symptoms were elicited. In a non-randomized phase IIa clinical trial, treatment with BBIC for one month resulted in a dose-dependent reduction in oral lesion size in 31% of patients [7]. The positive clinical effect of BBIC was associated with modulation of protease activity and *neu* oncogene levels (as the surrogate endpoint biomarker for the trial) in exfoliated oral mucosal cells [7, 69]; however, this evidence is indirect and the specific target proteases inhibited were not reported. A recent phase IIb randomized, double-blind, placebo-controlled trial involving patient treatment with BBIC for six months was performed. Even though this multiinstitutional study has not been completed yet, BBIC does not seem to be fully effective as chemopreventive agent for the management of oral leukoplakia [8].

#### **4.3. Prostate cancer**

32 Bioactive Food Peptides in Health and Disease

primary targets for BBI.

**4.2. Oral leukoplakia** 

In previous studies, a significant concentration- and time-dependent decrease in the growth of an array of colon cancer cells (HT29, Caco2, LoVo) has been demonstrated *in vitro*, following treatment with BBI variants from several legume sources, including pea, lentil and soybean; in contrast, the growth of non-malignant colonic fibroblastic CCD18-Co cells was unaffected by BBI [15, 26, 30]. Recently, the anti-proliferative effect of rTI1B, a major pea isoinhibitor expressed heterologously in *Pichia pastoris*, has been evaluated using colon cancer cells grown *in vitro*. Comparisons of the effects of rTI1B with those observed using a related synthetic mutant derivative, showed that the proliferation of HT29 colon cancer cells was inhibited significantly by rTI1B in a dose-dependent manner, whereas the mutant which lacked trypsin and chymotrypsin inhibitory activity did not show any significant effect on colon cancer cell growth (**Figure 1**) [68]. Although the molecular mechanism(s) of this chemopreventive activity remains unknown, the reported data indicate that both trypsin- and chymotrypsin-like serine proteases involved in carcinogenesis are likely

**Figure 1.** Dose–response effects of rTI1B (closed bars), a major pea isoinhibitor expressed in *Pichia pastoris*, and the corresponding inactive mutant (open bars), having amino acid substitutions at the P1

adenocarcinoma cells. Growth media were supplemented with rTI1B in the concentration range 15–61 µM and cells harvested after a period of 96 h. Data are means of at least three independent experiments, each having four technical replicates; bars represent standard deviations. Means not sharing superscript letters differ significantly (*p* <0.05; Bonferroni's test) (Adapted from Clemente et al., (2012) [68]).

Leukoplakia in the oral cavity is considered a suitable model for the study of chemoprevention because the precancerous lesions are readily accessible to visual

positions in the two inhibitory domains, on the *in vitro* growth of HT29 human colorectal

Prostate cancer is the second most frequently diagnosed cancer in men although the incidence of cancer varies greatly throughout the world. Dietary habits and lifestyle have been identified as major risk factors in prostate cancer growth and progression, suggesting that prostate cancer might be preventable [70]. Epidemiological studies have shown an inverse association between soy intake and the risk of developing prostate cancer [71]. Preclinical and clinical studies have shown the potential chemopreventive properties of BBI in prostate cancer. Purified soybean BBI and BBIC have been shown to inhibit the growth of LNCaP human prostate cancer xenografts in nude mice [72], and to decrease the growth, invasion and clonogenic survival of several human prostate cancer cells [73]. The effectiveness of soybean BBI in preventing the generation of activated oxygen species in prostate cancer cells [74] and in activating DNA repair through a p53-dependent mechanism has been reported [75]. More recently, BBIC has prevented the growth of prostate tumors in transgenic rats developing adenocarcinoma, most likely as a consequence of its antiproliferative activity via induction of connexin 43 expression [76, 77]. In humans, a doubleblind, randomized, phase I trial was carried out in nineteen male subjects with benign prostatic hyperplasia, which is a precursor condition for prostate cancer, and lower urinary tract symptoms [5]. In this study, the authors demonstrated that BBIC treatment for six months reduced levels of prostate-specific antigen (PSA), a clinical marker for prostate cancer, and prostate volume in patients. Additional clinical studies will be necessary to determine the potential of BBIC as prostate cancer chemopreventive agent.

#### **4.4. Breast cancer**

Breast cancer is one of the most frequent cancer types and is responsible for the highest mortality rate among women. Novel complementary strategies, including chemoprevention, have been suggested. As 125I-BBI, when orally administrated in rodents, has been

demonstrated in the bloodstream and distributed through the body [37-38], its chemopreventive properties could occur in breast tissue. *In vitro* studies have reported the potential of BBI as chemopreventive agents in breast cancer. Soybean BBI has been shown to inhibit, specifically and potently, the chymotrypsin-like proteasomal activity in MCF7 breast cancer cells *in vitro* and *in vivo* [78]. The proteasomal inhibition was associated with an accumulation of ubiquitinylated proteins and the proteasome substrates, p21Cip1/WAF1 and p27Kip1; a down-regulation of cyclin D1 and E was also observed. These authors suggested that soybean BBI abates proteasome function and, dependent on dose and time, up-regulates MAP kinase phosphatase-1 (MKP-1), which in turn suppresses phosphorylation coupled to extracellular signal-related kinase activity in MCF7 treated cells. The ability of soybean BBI to inhibit the proteasomal chymotrypsin-like activity in intact MCF7 cells suggests that the protease inhibitor can penetrate cells and facilitate the inhibition of intracellular target proteases. Recent findings have demonstrated that BBI from black-eye pea (*Vigna unguiculata*) induced apoptotic cell death in MCF7 breast cancer cells associated with severe cell morphological alterations, including the alteration of the nuclear morphology, plasma membrane fragmentation, cytoplasm disorganization, presence of double-membrane vesicles, mitochondrial swelling and lysosome membrane permeabilization [79].

Bowman-Birk Inhibitors from Legumes: Utilisation in Disease Prevention and Therapy 35

radioprotective properties [85]. The radioprotective effect of soybean BBI was mainly associated with the chymotrypsin inhibitory site [86] and could be mimicked using a synthetic linearized nonapeptide (CALSYPAQC), corresponding to the active site for chymotrypsin inhibition, but lacking protease inhibitor activity [85]. These observations provide opportunities for the use of synthetic peptides for protecting against ionizing radiation. BBI, when applied topically, once a day for 5 days, to SKH-1 hairless mice with a high risk of developing UV-induced skin tumors, inhibited the formation and growth of skin tumors [85]. In addition, topical application of nondenatured soymilk, once a day for a period of five days prior to UV irradiation, to mini-swine skin reduced or completely eliminated UVinduced formation of thymine dimers and apoptotic cells. Finally, BBIC appears to play a radioprotective role in radiation-induced cataract formation reducing the prevalence and

severity of the lens opacifications in mice exposed to high-energy protons [88].

elucidate the potential therapeutic role of BBI in muscular dystrophy.

**diseases** 

**5. Beneficial properties of Bowman-Birk inhibitors in non-related cancer** 

The loss of muscle protein due to inactivity, disease or aging is a process known as muscular atrophy or wasting. Skeletal muscular atrophy in response to disuse involves both a decrease in protein synthesis and increased protein degradation, predisposing humans to undergo a substantial loss of muscle mass. In connection with this, complex proteolytic cascades may provide a mechanism for the initiation of protein degradation during atrophy. Dietary intervention suggests possible therapeutic strategies *via* protease inhibition to diminish muscular atrophy and loss of strength following unloading. Skeletal muscular atrophy can be reproduced experimentally in rodents by hind-limb unloading. Dietary supplementation containing 1% BBIC has been reported to inhibit unloading-induced weakness in mice [89], promoting redox homeostasis in muscle fibers and blunting atrophy-induced weakness [90]. Morris *et al*. [89] suggested that inhibiting muscle degrading proteases may provide a new pharmacological strategy in treating skeletal muscular atrophy; however, such proteases remain uncharacterized. More recently, oral administration of BBIC was reported to improve muscle mass and function and to modulate pathological processes in the mouse model of Duchenne muscular dystrophy (mdx mouse) [91]. Chymase, a serine protease that is released from mast cells, is involved in inflammatory processes and is susceptible to inhibition by BBI, and hence has been suggested as a BBI target in mdx mice. Additional studies are necessary to

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by progressive demyelination of the brain and spinal cord. Available therapeutic treatments have only limited efficacy and show significant side effects. The search for novel therapeutic agents that can be administered orally, and act synergistically with existing therapies, would be useful for patients with MS. Purified soybean BBI and BBIC have been shown to be effective in the suppression of experimental autoimmune encephalomyelitis in rodents, a model to study the pathogenic mechanisms of MS and to test potential therapies [92]. The oral administration of BBI in mice caused an improvement

#### **4.5. Radioprotection**

Radiotherapy is used in the treatment of a broad range of malignant tumors with the aim to inflict maximal damage on the tumor tissue. Exposure of surrounding normal tissue to therapeutic radiation should be minimized to avoid side effects that can have a significant impact on general status and quality of life of patients. The use of radioprotective agents to reduce the damage in normal tissue may improve the therapeutic benefit of radiotherapy. The radioprotective properties of BBI have been tested on cell cultures; so far, no data regarding efficacy in humans are available. Soybean BBI have shown potent and selective radioprotection of normal tissue *in vitro* [80] and *in vivo* [81], without protecting tumor tissue. Dittmann *et al.* [81] showed that soybean BBI increased clonogenic survival after irradiation only in cells, either normal or transformed, having a wild-type *p53* tumor suppressor gene. In a cell line with inducible expression of mutated *p53*, the radioprotective effect of BBI was only detected when the expression of the mutated *p53* was switched off [75]. The activation of the DNA–repair machinery, induced by pre-treatment of fibroblastic cells with soybean BBI, suggests a possible BBI-mediated *p53*-dependent mechanism for radioprotection [82, 83]. Since a high number of tumors have lost *p53* function during their development, the clinical application of BBI to protect normal tissue from radiation damage would effectively improve the therapeutic outcome of radiotherapy.

The involvement of BBI in radiation-induced signaling cascades, and their role in stabilizing a specific tyrosine phosphatase that interferes with the activation of an epidermal growth factor receptor in response to radiation exposure, could be responsible for such protection [84]. Experiments carried out with linear forms of BBI demonstrated that the secondary structure of BBI, required for the protease inhibitory activity, was not necessary for its radioprotective properties [85]. The radioprotective effect of soybean BBI was mainly associated with the chymotrypsin inhibitory site [86] and could be mimicked using a synthetic linearized nonapeptide (CALSYPAQC), corresponding to the active site for chymotrypsin inhibition, but lacking protease inhibitor activity [85]. These observations provide opportunities for the use of synthetic peptides for protecting against ionizing radiation. BBI, when applied topically, once a day for 5 days, to SKH-1 hairless mice with a high risk of developing UV-induced skin tumors, inhibited the formation and growth of skin tumors [85]. In addition, topical application of nondenatured soymilk, once a day for a period of five days prior to UV irradiation, to mini-swine skin reduced or completely eliminated UVinduced formation of thymine dimers and apoptotic cells. Finally, BBIC appears to play a radioprotective role in radiation-induced cataract formation reducing the prevalence and severity of the lens opacifications in mice exposed to high-energy protons [88].

34 Bioactive Food Peptides in Health and Disease

**4.5. Radioprotection** 

demonstrated in the bloodstream and distributed through the body [37-38], its chemopreventive properties could occur in breast tissue. *In vitro* studies have reported the potential of BBI as chemopreventive agents in breast cancer. Soybean BBI has been shown to inhibit, specifically and potently, the chymotrypsin-like proteasomal activity in MCF7 breast cancer cells *in vitro* and *in vivo* [78]. The proteasomal inhibition was associated with an accumulation of ubiquitinylated proteins and the proteasome substrates, p21Cip1/WAF1 and p27Kip1; a down-regulation of cyclin D1 and E was also observed. These authors suggested that soybean BBI abates proteasome function and, dependent on dose and time, up-regulates MAP kinase phosphatase-1 (MKP-1), which in turn suppresses phosphorylation coupled to extracellular signal-related kinase activity in MCF7 treated cells. The ability of soybean BBI to inhibit the proteasomal chymotrypsin-like activity in intact MCF7 cells suggests that the protease inhibitor can penetrate cells and facilitate the inhibition of intracellular target proteases. Recent findings have demonstrated that BBI from black-eye pea (*Vigna unguiculata*) induced apoptotic cell death in MCF7 breast cancer cells associated with severe cell morphological alterations, including the alteration of the nuclear morphology, plasma membrane fragmentation, cytoplasm disorganization, presence of double-membrane vesicles,

Radiotherapy is used in the treatment of a broad range of malignant tumors with the aim to inflict maximal damage on the tumor tissue. Exposure of surrounding normal tissue to therapeutic radiation should be minimized to avoid side effects that can have a significant impact on general status and quality of life of patients. The use of radioprotective agents to reduce the damage in normal tissue may improve the therapeutic benefit of radiotherapy. The radioprotective properties of BBI have been tested on cell cultures; so far, no data regarding efficacy in humans are available. Soybean BBI have shown potent and selective radioprotection of normal tissue *in vitro* [80] and *in vivo* [81], without protecting tumor tissue. Dittmann *et al.* [81] showed that soybean BBI increased clonogenic survival after irradiation only in cells, either normal or transformed, having a wild-type *p53* tumor suppressor gene. In a cell line with inducible expression of mutated *p53*, the radioprotective effect of BBI was only detected when the expression of the mutated *p53* was switched off [75]. The activation of the DNA–repair machinery, induced by pre-treatment of fibroblastic cells with soybean BBI, suggests a possible BBI-mediated *p53*-dependent mechanism for radioprotection [82, 83]. Since a high number of tumors have lost *p53* function during their development, the clinical application of BBI to protect normal tissue from radiation damage

The involvement of BBI in radiation-induced signaling cascades, and their role in stabilizing a specific tyrosine phosphatase that interferes with the activation of an epidermal growth factor receptor in response to radiation exposure, could be responsible for such protection [84]. Experiments carried out with linear forms of BBI demonstrated that the secondary structure of BBI, required for the protease inhibitory activity, was not necessary for its

mitochondrial swelling and lysosome membrane permeabilization [79].

would effectively improve the therapeutic outcome of radiotherapy.

## **5. Beneficial properties of Bowman-Birk inhibitors in non-related cancer diseases**

The loss of muscle protein due to inactivity, disease or aging is a process known as muscular atrophy or wasting. Skeletal muscular atrophy in response to disuse involves both a decrease in protein synthesis and increased protein degradation, predisposing humans to undergo a substantial loss of muscle mass. In connection with this, complex proteolytic cascades may provide a mechanism for the initiation of protein degradation during atrophy. Dietary intervention suggests possible therapeutic strategies *via* protease inhibition to diminish muscular atrophy and loss of strength following unloading. Skeletal muscular atrophy can be reproduced experimentally in rodents by hind-limb unloading. Dietary supplementation containing 1% BBIC has been reported to inhibit unloading-induced weakness in mice [89], promoting redox homeostasis in muscle fibers and blunting atrophy-induced weakness [90]. Morris *et al*. [89] suggested that inhibiting muscle degrading proteases may provide a new pharmacological strategy in treating skeletal muscular atrophy; however, such proteases remain uncharacterized. More recently, oral administration of BBIC was reported to improve muscle mass and function and to modulate pathological processes in the mouse model of Duchenne muscular dystrophy (mdx mouse) [91]. Chymase, a serine protease that is released from mast cells, is involved in inflammatory processes and is susceptible to inhibition by BBI, and hence has been suggested as a BBI target in mdx mice. Additional studies are necessary to elucidate the potential therapeutic role of BBI in muscular dystrophy.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by progressive demyelination of the brain and spinal cord. Available therapeutic treatments have only limited efficacy and show significant side effects. The search for novel therapeutic agents that can be administered orally, and act synergistically with existing therapies, would be useful for patients with MS. Purified soybean BBI and BBIC have been shown to be effective in the suppression of experimental autoimmune encephalomyelitis in rodents, a model to study the pathogenic mechanisms of MS and to test potential therapies [92]. The oral administration of BBI in mice caused an improvement of several disease parameters (onset, severity, weight loss, inflammation, neuronal loss and demyelination), with no apparent adverse effects [93, 94]. Interestingly, BBI ameliorated disease, even when treatment was initiated after disease onset, *via* an IL-10-dependent mechanism [94]; the molecular basis for the induction of IL-10 production by BBI remains to be elucidated. Recent studies have demonstrated that BBI is responsible for delayed onset of disease but did not stop disease development, which became similarly severe in treated mice as in control animals [95, 96]. The ability of soybean BBI to delay both inflammatory and neurodegenerative aspects of autoimmune encephalomyelitis suggests that it may be useful for treating acute MS exacerbations and neurological dysfunction.

Bowman-Birk Inhibitors from Legumes: Utilisation in Disease Prevention and Therapy 37

[2] Shen A. Allosteric Regulation of Protease Activity by Small Molecules. Molecular

[3] Drag M, Salvesen GS. Emerging Principles in Protease-based Drug Discovery. Nature

[4] Deu E, Verdoes M, Bogyo M. New Approaches for Dissecting Protease Functions to Improve Probe Development and Drug Discovery. Nature Structural & Molecular

[5] Malkowicz SB, McKenna WG, Vaughn DJ, Wan XS, Propert KJ, Rockwell K, Marks SHF, Wein AJ, Kennedy AR. Effects of Bowman-Birk Inhibitor Concentrate (BBIC) in

[6] Armstrong WB, Kennedy AR, Wan XS, Atiba J, McLaren CE, Meyskens FL. Single-dose Administration of Bowman-Birk Inhibitor Concentrate in Patients with Oral

[7] Armstrong WB, Kennedy AR, Wan XS, Taylor TH, Nguyen QA, Jensen J, Thompson W. Clinical Modulation of Oral Leukoplakia and Protease Activity by Bowman-Birk Inhibitor Concentrate in a Phase IIa Chemoprevention Trial. Clinical Cancer Research

[8] Meyskens FL, Taylor T, Armstrong W, Kong L, Gu M, Gonzalez R, Villa M, Wong V, Garcia A, Perloff M, Kennedy A, Wan S, Ware JH, Messadi D, Lorch J, Wirth L, Jaffe Z, Goodwin J, Civantos F, Sullivan M, Reid M, Merciznu M, Jayaprakash V, Kerr AR, Le A. Phase IIb Randomized Clinical Chemoprevention Trial of a Soybean-derived Compound (Bowman-Birk Inhibitor Concentrate) for Oral Leukoplakia. Cancer

[9] Lichtenstein GR, Deren J, Katz S, Lewis JD, Kennedy AR, Ware JH. Bowman-Birk Inhibitor Concentrate: A Novel Therapeutic Agent for Patients with Active Ulcerative

[10] Bateman KS, James MNG. Plant Proteinase Inhibitors: Structure and Mechanism of

[11] Domoney C, Welham T, Ellis N, Mozzanega P, Turner L. Three Classes of Proteinase Inhibitor Gene Have Distinct but Overlapping Patterns of Expression in *Pisum sativum* 

[12] De Almeida B, Garcia da Silva W, Alves M, Gonzalves E. *In silico* Characterization and Expression Analysis of the Multigene Family Encoding the Bowman-Birk Protease

[13] Clemente A, Domoney C. Biological Significance of Polymorphism in Legume Protease Inhibitors from the Bowman-Birk Family. Current Protein & Peptide Science 2006; 7:

[14] Domoney C, Welham T. Trypsin Inhibitors in *Pisum*: Variation in Amount and Pattern of Accumulation in Developing Seed. Seed Science Research 1992; 2: 147–154. [15] Clemente A, Gee JM, Johnson IT, Domoney C. Pea (*Pisum sativum* L.) Protease Inhibitors from the Bowman-Birk Class Influence the Growth of Human Colorectal Adenocarcinoma HT29 Cells *in vitro*. Journal of Agricultural and Food Chemistry 2005;

Leukoplakia. Cancer Epidemiology, Biomarkers & Prevention 2000; 9: 43-47.

Patients with Benign Prostatic Hyperplasia. Prostate 2001; 48: 16-28.

Biosystems 2010; 6: 1431-1443.

Biology 2012; 19: 9-16.

2000; 6: 4684-4691.

201-216.

53: 8979–8986.

Reviews Drug Discovery 2010; 9: 690-701.

Prevention Research 2010; 3: CN02-05.

Colitis. Digestive Diseases and Sciences 2008; 53: 175-180.

Plants. Plant Molecular Biology 2002; 48*:* 319–329.

Inhibition. Current Protein & Peptide Science 2011; 12: 341-347.

Inhibitor in Soybean. Molecular Biology Reports 2012; 39: 327-334.

## **6. Concluding remarks**

In recent years, much effort has focused on clarifying the potential chemopreventive properties of BBI. Preclinical and clinical studies have clearly demonstrated that BBI uptake is well-tolerated and no side-effects were elicited. This is particularly relevant and lack of toxicity is a major consideration, given the necessity for prolonged duration of administration. Consistently, several studies have shown that serine proteases are potential BBI targets in prevention and therapy; however, these targets have not been proven thus far. The validation of specific serine proteases as BBI targets will contribute to the assessment of BBI as chemopreventive agents that may be used in preventive and/or therapeutic medicine.

## **Author details**

Alfonso Clemente\* , María del Carmen Marín-Manzano and María del Carmen Arques *Department of Physiology and Biochemistry of Nutrition, Estación Experimental del Zaidín, Spanish Council for Scientific Research (CSIC), Granada, Spain* 

Claire Domoney *Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich, UK* 

## **Acknowledgement**

A.C. acknowledges support by ERDF-co-financed grants from the Spanish CICYT (AGL2010-15877AGL and AGL2011-26353). A.C. is involved in COST Action FA1005 INFOGEST on Food Digestion. C.D. acknowledges support from the European Union (Grain Legumes Integrated Project, a Framework Programme 6 project, grant no. FOOD-CT-2004-506223) and from Defra, United Kingdom (grant nos. AR0105 and AR0711).

## **7. References**

[1] Turk B. Targeting Proteases: Successes, Failures and Future Prospects. Nature Reviews Drug Discovery 2006; 5: 785-799.

<sup>\*</sup> Corresponding Autor

[2] Shen A. Allosteric Regulation of Protease Activity by Small Molecules. Molecular Biosystems 2010; 6: 1431-1443.

36 Bioactive Food Peptides in Health and Disease

**6. Concluding remarks** 

**Author details** 

Alfonso Clemente\*

Claire Domoney

**7. References** 

Corresponding Autor

 \*

Drug Discovery 2006; 5: 785-799.

**Acknowledgement** 

of several disease parameters (onset, severity, weight loss, inflammation, neuronal loss and demyelination), with no apparent adverse effects [93, 94]. Interestingly, BBI ameliorated disease, even when treatment was initiated after disease onset, *via* an IL-10-dependent mechanism [94]; the molecular basis for the induction of IL-10 production by BBI remains to be elucidated. Recent studies have demonstrated that BBI is responsible for delayed onset of disease but did not stop disease development, which became similarly severe in treated mice as in control animals [95, 96]. The ability of soybean BBI to delay both inflammatory and neurodegenerative aspects of autoimmune encephalomyelitis suggests that it may be

In recent years, much effort has focused on clarifying the potential chemopreventive properties of BBI. Preclinical and clinical studies have clearly demonstrated that BBI uptake is well-tolerated and no side-effects were elicited. This is particularly relevant and lack of toxicity is a major consideration, given the necessity for prolonged duration of administration. Consistently, several studies have shown that serine proteases are potential BBI targets in prevention and therapy; however, these targets have not been proven thus far. The validation of specific serine proteases as BBI targets will contribute to the assessment of BBI as chemopreventive agents that may be used in preventive and/or therapeutic medicine.

, María del Carmen Marín-Manzano and María del Carmen Arques

*Department of Physiology and Biochemistry of Nutrition, Estación Experimental del Zaidín,* 

*Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich, UK* 

2004-506223) and from Defra, United Kingdom (grant nos. AR0105 and AR0711).

A.C. acknowledges support by ERDF-co-financed grants from the Spanish CICYT (AGL2010-15877AGL and AGL2011-26353). A.C. is involved in COST Action FA1005 INFOGEST on Food Digestion. C.D. acknowledges support from the European Union (Grain Legumes Integrated Project, a Framework Programme 6 project, grant no. FOOD-CT-

[1] Turk B. Targeting Proteases: Successes, Failures and Future Prospects. Nature Reviews

*Spanish Council for Scientific Research (CSIC), Granada, Spain* 

useful for treating acute MS exacerbations and neurological dysfunction.


[16] Page D, Aubert G, Duc G, Welham T, Domoney C. Combinatorial Variation in Coding and Promoter Sequences of Genes at the *Tri* Locus in *Pisum sativum* Accounts for Variation in Trypsin Inhibitor Activity in Seeds. Molecular Genetics and Genomics 2002; 267: 359-369.

Bowman-Birk Inhibitors from Legumes: Utilisation in Disease Prevention and Therapy 39

[30] Caccialupi P, Ceci LR, Siciliano RA, Pignone D, Clemente A, Sonnante G. Bowman-Birk Inhibitors in Lentil: Heterologous Expression, Functional Characterisation and Antiproliferative Properties in Human Colon Cancer Cells. Food Chemistry 2010; 120: 1058-

[31] Scarafoni A, Consonni A, Galbusera V, Negri A, Tedeshi G, Rasmussen P, Magni C, Duranti M. Identification and Characterization of a Bowman-Birk Inhibitor Active Towards Trypsin but not Chymotrypsin in *Lupinus albus* Seeds. Phytochemistry 2008;

[32] Osman MA, Reid PM, Weber CW. Thermal Inactivation of Tepary Bean (*Phaseolus acutifolius*), Soybean and Lima Bean Protease Inhibitors: Effect of Acidic and Basic pH.

[33] Clemente A, Vioque J, Sánchez-Vioque R, Pedroche J, Bautista J, Millán F. Factors Affecting the *in vitro* Protein Digestibility of Chickpea Albumins. Journal of the Science

[34] Weder JK. Inhibition of Human Proteinases by Grain Legumes. Advances in

[35] Park JH, Jeong HJ, Lumen BOD. *In Vitro* Digestibility of the Cancer-Preventive Soy Peptides Lunasin and BBI. Journal of Agricultural and Food Chemistry 2007; 55: 10703-

[36] Trivedi MV, Laurence JS, Siahann TJ. The Role of Thiols and Disulfides on Protein

[37] Billings PC, St Clair WH, Maki PA, Kennedy AR. Distribution of the Bowman-Birk Protease Inhibitor in Mice Following Oral Administration. Cancer Letters 1992; 62: 191-

[38] Kennedy AR. Chemopreventive Agents: Protease Inhibitors. Pharmacology &

[39] Hajós G, Gelencser E, Pustzai A, Grant G, Sakhri M, Bardocz S. Biological Effects and Survival of Trypsin Inhibitors and the Aglutinin from Soybean in the Small Intestine of

[40] Clemente A, Jiménez E, Marín-Manzano MC, Rubio LA. Active Bowman-Birk Inhibitors Survive Gastrointestinal Digestion at the Terminal Ileum of Pigs fed Chickpea-Based

[41] Marín-Manzano MC, Ruiz R, Jiménez E, Rubio LA, Clemente A. Anti-carcinogenic Soyabean Bowman-Birk Inhibitors Survive Fermentation in their Active Form and do not Affect the Microbiota Composition *In Vitro*. The British Journal of Nutrition 2009;

[42] Reddy BS. Novel Approaches in the Prevention of Colon Cancer by Nutritional Manipulation and Chemoprevention. Cancer Epidemiology, Biomarkers & Prevention

[43] Pan MH, Lai CS, Wu JC, Ho CT. Molecular Mechanisms for Chemoprevention of Colorectal Cancer by Natural Dietary Compounds. Molecular Nutrition & Food

the Rat. Journal of Agricultural and Food Chemistry 1995; 43: 165-170.

Diets. Journal of the Science of Food and Agriculture 2008; 88: 513-521.

1066.

10706.

197.

101: 967-971.

2000; 9: 239-247.

Research 2011; 55: 32-45.

69: 1820-1825.

Food Chemistry 2002; 78: 419-423.

Therapeutics 1998; 78: 167-209.

of Food and Agriculture 2000; 80: 79-84.

Experimental Medicine and Biology 1986; 199: 239-279.

Stability. Current Protein & Peptide Science 2009; 10: 614-625.


[30] Caccialupi P, Ceci LR, Siciliano RA, Pignone D, Clemente A, Sonnante G. Bowman-Birk Inhibitors in Lentil: Heterologous Expression, Functional Characterisation and Antiproliferative Properties in Human Colon Cancer Cells. Food Chemistry 2010; 120: 1058- 1066.

38 Bioactive Food Peptides in Health and Disease

Agriculture 2003; 83: 644-651.

2002; 267: 359-369.

86: 436-444.

[16] Page D, Aubert G, Duc G, Welham T, Domoney C. Combinatorial Variation in Coding and Promoter Sequences of Genes at the *Tri* Locus in *Pisum sativum* Accounts for Variation in Trypsin Inhibitor Activity in Seeds. Molecular Genetics and Genomics

[17] Wiseman J, Al-Mazooqi W, Welham T, Domoney C. The Apparent Ileal Digestibility, Determined with Young Broilers, of Amino Acids in Near-isogenic Lines of Peas (*Pisum sativum* L.) Differing in Trypsin Inhibitor Activity. Journal of the Science of Food and

[18] Wiseman J, Al-Marzooqi W, Hedley C, Wang TL, Welham T, Domoney C. The Effects of Genetic Variation at *r*, *rb* and *Tri* Loci in *Pisum sativum* L. on Apparent Ileal Digestibility of Amino Acids in Young Broilers. Journal of the Science of Food and Agriculture 2006;

[19] Hernández-Ledesma B, Hsieh CC, de Lumen BO. Lunasin and Bowman-Birk Protease Inhibitor (BBI) in US Commercial Soy Foods. Food Chemistry 2009; 115: 574-580. [20] Bode W, Huber R. Natural Protein Proteinase-Inhibitors and their Interaction with

[21] Chen P, Rose J, Love R, Wei CH, Wang BC. Reactive Sites of an Anticarcinogenic Bowman-Birk Proteinase Inhibitor are Similar to Other Trypsin Inhibitors. The Journal

[22] Clemente A, Sonnante G, Domoney C. Bowman-Birk Inhibitors from Legumes on Human Gastrointestinal Health: Current Status and Perspectives. Current Protein &

[23] Schechter I, Berger A. On the Size of the Active Site in Proteases. I. Papain. Biochemical

[24] Rocco M, Marloni L, Chambery A, Poerio E, Parente A, Di Maro A. A Bowman-Birk Inhibitor with Anti-elastase Activity from *Lathyrus sativus* L. Seeds. Molecular

[25] Piergiovanni AR, Galasso I. Polymorphism of Trypsin and Chymotrypsin Binding Loops in Bowman-Birk Inhibitors from Common Bean (*Phaseolus vulgaris* L.). Plant

[26] Clemente A, Moreno J, Marín-Manzano MC, Jiménez E, Domoney C. The Cytotoxic Effect of Bowman-Birk Isoinhibitors, IBB1 and IBBD2, from Soybean (*Glycine max*) on HT29 Human Colorectal Cancer Cells is Related to their Intrinsic Ability to Inhibit

[27] Ferrasson E, Quillien L, Gueguen J. Proteinase Inhibitors from Pea Seeds: Purification and Characterization. Journal of Agricultural and Food Chemistry 1997; 45: 127-131. [28] Clemente A, MacKenzie DA, Jeenes DJ, Domoney C. The Effect of Variation within Inhibitory Domains on the Activity of Pea Protease Inhibitors from the Bowman-Birk

[29] Ragg EM, Galbusera V, Scarafoni A, Negri A, Tedeschi G, Consoni A, Sessa F, Duranti M. Inhibitory Properties and Solution Structure of a Potent Bowman-Birk Protease

Inhibitor from Lentil (*Lens culinaris*, L.) Seeds. FEBS Journal 2006; 273: 4024-4039.

Serine Proteases. Molecular Nutrition & Food Research 2010; 54: 396-405.

Class. Protein Expression and Purification 2004; 36: 106-114.

Proteinases. European Journal of Biochemistry 1992; 204: 433-451.

and Biophysical Research Communications 1967; 27: 157-162.

of Biological Chemistry 1992; 267: 1990-1994.

Peptide Science 2011; 12: 358-373.

Biosystems 2011; 7: 2500-2507.

Science 2004; 166: 1525-1531.


[44] Jemal A, Siegel R, Ward E, Hao YP, Xu JQ, Thun MJ. Cancer Statistics, 2009. Cancer Journal for Clinicians 2009; 59: 225-249.

Bowman-Birk Inhibitors from Legumes: Utilisation in Disease Prevention and Therapy 41

Induced by 1,2-dimethylhydrazine in Swiss Mice. Food and Chemical Toxicology 2012;

[59] Sutherland LR, Martin F, Greer S, Robinson M, Greenberger N, Saibil F, Martin T, Sparr J, Prokipchuck E, Borgen L. 5-aminosalicylic Acid Enema in the Treatment of Distal Ulcerative Colitis, Proctosigmoiditis and Proctitis. Gastroenterology 1987; 92: 1894-1898. [60] Frenkel K, Chranzan K, Ryan CA, Wiesner R, Troll W. Chymotrypsin-specific Protease Inhibitors Decrease H2O2 Formation by Activated Human Polymorphonuclear

[61] Baturay NZ, Roque H. *In vitro* Reduction of Peroxidation in UVC Irradiated Cell Cultures by Concurrent Exposure with Bowman-Birk Protease Inhibitor. Teratogenesis,

[62] Larionova NI, Gladysheva IP, Tikhonova TV, Kazanskaya NF. Inhibition of Cathepsin G and Human Granulocyte Elastase by Multiple Forms of Bowman-Birk Type Soybean

[63] Gladysheva IP, Zamolodchikova TS, Sokolova EA, Larionova NI. Interaction Between Duodenase, a Proteinase with Dual Specificity, and Soybean Inhibitors of Bowman-Birk

[64] Ware JH, Wan XS, Rubin H, Schechter NM, Kennedy AR. Soybean Bowman-Birk Protease Inhibitor is a Highly Effective Inhibitor of Human Mast Cell Chymase.

[65] Tani K, Ogushi K, Kido H, Kawano T, Kumori Y, Kamikura T, Cui P, Sone S. Chymase is a Potent Chemoattractant for Human Monocytes and Neutrophils. Journal of

[66] Saarinen J, Kalkkinen N, Welgus HG, Kovanen PT. Activation of Human Interstitial Procollagenase through Direct Cleavage of the Leu83- Thr84 Bond by Mast Cell

[67] Mizutani H, Schechter NM, Lazarus G, Black RA, Kupper TS. Rapid and Specific Conversion of Precursor Interleukin 1beta (1L-beta) to an Active IL-1 Species by Human

[68] Clemente A, Marín-Manzano MC, Jiménez E, Arques MC, Domoney C. The Antiproliferative Effects of TI1B, a Major Bowman-Birk isoinhibitor from Pea (*Pisum sativum* L), on HT29 Colon Cancer Cells are Mediated Through Protease Inhibition. The British

[69] Wan XS, Meyskens FL, Armstrong WB, Taylor TH, Kennedy AR. Relationship Between Protease Activity and *neu* Oncogene Expression in Patients with Oral Leukoplakia Treated with the Bowman-Birk Inhibitor. Cancer Epidemiology, Biomarkers &

[70] Shirai T. Significance of Chemoprevention for Prostate Cancer Development: Experimental *in vivo* Approaches to Chemoprevention. Pathology International 2008;

[71] Yan L, Spitznagel EL. Meta-analysis of Soy Food and Risk of Prostate Cancer in Men.

50: 1405-1412.

Leukocytes. Carcinogenesis 1987; 8: 1207-1212.

Carcinogenesis and Mutagenesis 1991; 11: 195-202.

Inhibitor. Biochemistry-Moscow 1993; 58: 1046-1052.

Leukocyte Biology 2000; 67: 585-589.

Prevention 1999; 8: 601-608.

58: 1-6.

and Kunitz Type. Biochemistry-Moscow 1999; 64: 1244-1249.

Archives of Biochemistry and Biophysics 1997; 344: 133-138.

Chymase. Journal of Biological Chemistry 1994; 269: 18134-18140.

Journal of Nutrition 2012 (doi.10.1017/S000711451200075X).

International Journal of Cancer 2005; 117: 667-669.

Mast Cell Chymase. Journal of Experimental Medicine 1991; 174: 821-825.


Induced by 1,2-dimethylhydrazine in Swiss Mice. Food and Chemical Toxicology 2012; 50: 1405-1412.

[59] Sutherland LR, Martin F, Greer S, Robinson M, Greenberger N, Saibil F, Martin T, Sparr J, Prokipchuck E, Borgen L. 5-aminosalicylic Acid Enema in the Treatment of Distal Ulcerative Colitis, Proctosigmoiditis and Proctitis. Gastroenterology 1987; 92: 1894-1898.

40 Bioactive Food Peptides in Health and Disease

Journal for Clinicians 2009; 59: 225-249.

Journal of Immunology 2009; 183: 8148-8156.

Research and Practice 2012; 208: 104-108.

Biological Chemistry 2009; 284: 23177-23181.

Biochimie 2010; 92: 1681-1688.

1083-1086.

Annals of Surgical Oncology 2010; 17: 3037-3042.

Altering Ubiquitin Availability. Oncogene 2011; 30: 790-805.

Protease. Journal of Biological Chemistry 2000; 275: 36720-36725.

Nutritional Science and Vitaminology 2003; 49: 27-32.

Digestive Diseases and Sciences 1999; 44: 986-990.

[44] Jemal A, Siegel R, Ward E, Hao YP, Xu JQ, Thun MJ. Cancer Statistics, 2009. Cancer

[45] Kennedy AR, Billings OC, Wan XS, Newberne PM. Effects of Bowman-Birk Inhibitor on

[46] Chan AT, Baba Y, Sima K, Nosho K, Chung DC, Hung KE, Mahmood U, Madden K, Poss K, Ranieri A, Shue D, Kucherlapati R, Fuch CS, Ogino S. Cathepsin B Expression and Survival in Colon Cancer: Implications for Molecular Detection of Neoplasia.

[47] Weldon S, McNally P, McElvaney NG, Elborn JS, McAuley DF, Wartelle J, Belaaouaj A, Levine RJ, Taggart CC. Decrease Levels of Secretory Leukoprotease Inhibitor in the *Pseudomonas*-Infected Cystic Fibrosis Lung are Due to Neutrophil Elastase Degradation.

[48] Inoue Y, Yokobori T, Yokoe T, Toiyama Y, Miki C, Mimori K, Mori M, Kusunoki M. Clinical Significance of Human Kallikrein7 Gene Expression in Colorectal Cancer. The

[49] Petraki C, Dubinski W, Scorilas A, Saleh C, Pasic MD, Komborozo V, Khalil B, Gabril MY, Streutker C, Diamandis EP, Yousef GM. Evaluation and Prognostic Significance of Human Tissue Kallikrein-related Peptidase 6 (KLK6) in Colorectal Cancer. Pathology

[50] Scott CJ, Taggart CC. Biologic Protease Inhibitors as Novel Therapeutic Agents.

[51] Wu WKK, Cho CH, Lee CW, Wu K, Fan D, Yu J, Sung JJY. Proteasome Inhibition: a New Therapeutic Strategy to Cancer Treatment. Cancer Letters 2010; 293: 15-22. [52] Latonen L, Moore HM, Bai B, Jaamaa S, Laiho M. Proteasome Inhibitors Induce Nucleolar Aggregation of Proteasome Target Proteins and Polyadenylated RNA by

[53] Bugge TH, Antalis TM, Wu Q. Type II Transmembrane Serine Proteases. Journal of

[54] Lee SL, Dickson RB, Lin CY. Activation of Hepatocyte Growth Factor and Urokinase/Plasminogen Activator by Matriptase, an Epithelial Membrane Serine

[55] Yamasaki Y, Satomi S, Murai N, Tsuzuki S, Fushiki T. Inhibition of Membrane-Type Serine Protease 1/Matriptase by Natural and Synthetic Protease Inhibitors. Journal of

[56] Billings PC, Newberne P, Kennedy AR. Protease Inhibitor Suppression of Colon and Anal Gland Carcinogenesis Induced by Dimethylhydrazine. Carcinogenesis 1990; 11:

[57] Ware HW, Wan S, Newberne P, Kennedy AR. Bowman-Birk Concentrate Reduces Colon Inflammation in Mice with Dextran Sulphate Sodium-Induced Ulcerative Colitis.

[58] de Paula A, de Abreu P, Santos KT, Guerra R, Martins C, Castro-Borges W, Guerra MH. Bowman-Birk Inhibitors, Proteasome Peptidase Activities and Colorectal Pre-neoplasias

Rat Colon Carcinogenesis. Nutrition and Cancer 2002; 43: 174-186.

Cancer Epidemiology, Biomarkers & Prevention 2010; 19: 2777-2785.


[72] Wan XS, Ware JH, Zhang L, Newberne PM, Evans SM, Clark CL, Kennedy AR. Treatment with Soybean-derived Bowman Birk Inhibitor Increases Serum Prostatespecific Antigen Concentration while Suppressing Growth of Human Prostate Cancer Xenografts in Nude Mice. Prostate 1999b; 41: 243-252.

Bowman-Birk Inhibitors from Legumes: Utilisation in Disease Prevention and Therapy 43

[85] Gueven N, Dittmann K, Mayer C, Rodemann HP. The Radioprotective Potential of the Bowman-Birk Protease Inhibitor is Independent of its Secondary Structure. Cancer

[86] Yavelow J, Collins M, Birk Y, Troll W, Kennedy AR. Nanomolar Concentrations of Bowman-Birk Soybean Protease Inhibitor Suppress X-ray Induced Transformation *In Vitro.* Proceedings of the National Academy of Sciences of the United States of America

[87] Huang MT, Xie JG, Lin CB, Kizoulis M, Seiberg M, Shapiro S, Conney A. Inhibitory Effect of Topical Applications of Non-denatured Soymilk on the Formation and Growth

[88] Davis JG, Wan XS, Ware JH, Kennedy AR Dietary Supplements Reduce the Cataractogenic Potential of Proton and HZE-Particle Radiation in Mice. Radiation

[89] Morris CA, Morris LD, Kennedy AR, Sweeney HL. Attenuation of Skeletal Muscle Atrophy via Protease Inhibition. Journal of Applied Physiology 2005; 99: 1719-1727. [90] Arbogast S, Smith J, Matuszczak Y, Hardin BJ, Moylan JS, Smith JD, Ware J, Kennedy AR, Reid MB. Bowman-Birk Inhibitor Concentrate Prevents Atrophy, Weakness, and Oxidative Stress in Soleus Muscle of Hindlimb-Unloaded Mice. Journal of Applied

[91] Morris CA, Selsby JT, Morris LD, Pendrak K, Sweeney HL. Bowman-Birk Inhibitor Attenuates Dystrophic Pathology in mdx Mice. Journal of Applied Physiology 2010;

[92] Cruz-Orengo L, Holman DW, Dorsey D, Zhou L, Zhang P, Wright M, McCandless EE, Patel JR, Luker GD, Littmann DR, Rusell JH, Klein RS. CXCR7 Influences Leukocyte Entry into the CNS Parenchyma by Controlling Abluminal CXCL12 Abundance During

[93] Gran B, Tabibzadeh N, Martin A, Ventura ES, Ware JH, Zhang GX, Parr JL, Kennedy AR, Rostami AM. The Protease Inhibitor, Bowman-Birk inhibitor, Suppresses Experimental Autoimmune Encephalomyelitis: a Potential Oral Therapy for Multiple

[94] Touil T, Ciric B, Ventura E, Shindler KS, Gran B, Tostami A. Bowman-Birk Inhibitor Suppresses Inflammation and Neuronal Loss in a Mouse Model of Multiple Sclerosis.

[95] Dai H, Ciric B, Zhang GX, Rostami A. Bowman-Birk Inhibitor Attenuates Experimental Autoimmune Encephalomyelitis by Delaying Infiltration of Inflammatory Cells into the

[96] Dai H, Ciric B, Zhang GX, Rostami A. Interleukin-10 Plays a Crucial Role in Suppression of Experimental Autoimmune Encephalomyelitis by Bowman-Birk

[97] Scarpi D, McBride JD, Leatherbarrow RJ. Inhibition of Human Beta-Tryptase by Bowman-Birk Inhibitor Derived Peptides: Creation of a New Tri-Functional Inhibitor.

Autoimmunity. Journal of Experimental Medicine 2011; 208: 327-339.

Sclerosis. Multiple Sclerosis 2006; 12: 688-697.

CNS. Immunologic Research 2011; 51: 145-152.

Journal of the Neurological Sciences 2008; 271: 191-202.

Inhibitor. Journal of Neuroimmunology 2012; 245: 1-7.

Bioorganic & Medicinal Chemistry 2004; 12: 6045-6052.

of UVB-Induced Skin Tumors. Oncology Research 2004; 14: 387-397.

Letters 1998; 125: 77-82.

1985; 82: 5395-5399.

Research 2010; 173: 353-361.

Physiology 2007; 102: 956-964.

109: 1492-1499.


[85] Gueven N, Dittmann K, Mayer C, Rodemann HP. The Radioprotective Potential of the Bowman-Birk Protease Inhibitor is Independent of its Secondary Structure. Cancer Letters 1998; 125: 77-82.

42 Bioactive Food Peptides in Health and Disease

Prostate 2002; 50: 125-133.

and Cancer 2007; 57: 184-193.

International 2009; 59: 790-796.

Und Onkologie 2005; 181: 191-196.

22: 1775-1780.

[72] Wan XS, Ware JH, Zhang L, Newberne PM, Evans SM, Clark CL, Kennedy AR. Treatment with Soybean-derived Bowman Birk Inhibitor Increases Serum Prostatespecific Antigen Concentration while Suppressing Growth of Human Prostate Cancer

[73] Kennedy AR, Wan XS. Effects of the Bowman-Birk Inhibitor on Growth, Invasion, and Clonogenic Survival of Human Prostate Epithelial Cells and Prostate Cancer Cells.

[74] Sun XY, Donald SP, Phang JM. Testosterone and Prostate Specific Antigen Stimulate Generation of Reactive Oxygen Species in Prostate Cancer Cells. Carcinogenesis 2001;

[75] Dittmann K, Virsik-Kopp P, Mayer C, Rave-Frank M, Rodemann HP. The Radioprotective Effect of BBI Is Associated with the Activation of DNA Repair-Relevant

[76] McCormick DL, Johnson WD, Bosland MC, Lubet RA, Steele VE. Chemoprevention of Rat Prostate Carcinogenesis by Soy Isoflavones and Bowman-Birk Inhibitor. Nutrition

[77] Tang MX, Asamoto M, Ogawa K, Naiki-Ito A, Sato S, Takahashi S, Shirai T. Induction of Apoptosis in the LNCaP Human Prostate Carcinoma Cell Line and Prostate Adenocarcinomas of SV40T Antigen Transgenic Rats by the Bowman-Birk. Pathology

[78] Chen YW, Huang SC, Lin-Shiau SY, Lin JK. Bowman-Birk Inhibitor Abates Proteasome Function and Suppresses the Proliferation of MCF7 Breast Cancer Cells Through Accumulation of MAP Kinase Phosphatase-1. Carcinogenesis 2005; 26: 1296-1305. [79] Joanitti GA, Azevedo RB, Freitas SM. Apoptosis and Lysosome Membrane Permeabilization Induction on breast Cancer Cells by an Anticarcinogenic Bowman-

[80] Dittmann K, Löffler H, Bamberg M, Rodemann HP. Bowman-Birk Proteinase Inhibitor (BBI) Modulates Radiosensitivity and Radiation-Induced Differentiation of Human

[81] Dittmann K, Toulany M, Classen J, Heinrich V, Milas L. Selective Radioprotection of Normal Tissues by Bowman-Birk Proteinase Inhibitor (BBI) in Mice. Strahlentherapie

[82] Dittmann KH, Gueven N, Mayer C, Ohneseit P, Zell P, Begg AC, Rodemann HP. The Presence of Wild-Type TP53 is Necessary for the Radioprotective Effect of the Bowman-Birk Proteinase Inhibitor in Normal Fibroblasts. Radiation Research 1998; 150: 648-655. [83] Dittmann K, Mayer C, Kehlbach R, Rodemann HP. The Radioprotector Bowman-Birk Proteinase Inhibitor Stimulates DNA Repair via Epidermal Growth Factor Receptor Phosphorylation and Nuclear Transport. Radiotherapy and Oncology 2008; 86: 375-382. [84] Gueven N, Dittmann K, Mayer C, Rodemann HP. Bowman-Birk Protease Inhibitor Reduces the Radiation-Induced Activation of the EGF Receptor and Induces Tyrosine Phosphatase Activity. International Journal of Radiation Oncology 1998; 73: 157-162.

Birk Inhibitor from *Vigna unguiculata* Seeds. Cancer Letters 2010; 293: 73-81.

Fibroblasts in Culture. Radiotherapy and Oncology 1995; 34: 137-143.

Genes. International Journal of Radiation Oncology 2003; 79: 801-808.

Xenografts in Nude Mice. Prostate 1999b; 41: 243-252.

	- [98] Muricken DG, Gowda LR Molecular Engineering of a Small Trypsin Inhibitor Based on the Binding Loop of Horsegram Seed Bowman-Birk Inhibitor. Journal of Enzyme Inhibition and Medicinal Chemistry 2011; 26: 553-560.

**Chapter 3** 

© 2013 Norris and FitzGerald, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Norris and FitzGerald, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Antihypertensive Peptides from Food Proteins** 

Hypertension or elevated blood pressure (BP) is a global health concern, thought to affect up to 30 % of the adult population in developed and developing countries. It is defined by a BP measurement of 140/90 mmHg or above. Hypertension is a major risk factor concomitant with cardiovascular disease (CVD) states such as coronary heart disease, peripheral artery disease and stroke, and kidney disease. Essential hypertension, the most common type of hypertension and to which 90-95% of cases belong, is manifested as an increase in an individual's BP due to an unknown cause. This class of hypertension can be improved with lifestyle choices such as regular exercise, heart-healthy eating, non smoking, reducing sodium intake and reducing the level of stress [1]. For these reasons it is defined as a controllable risk factor of CVD. At present there is a range of synthetic drugs on the market for treatment of hypertension including diuretics, adrenergic inhibitors such as α- and βblockers, direct vasodilators, calcium channel blockers, angiotensin II (Ang II) receptor blockers and angiotensin converting enzyme (ACE) inhibitors. However, although hypertension can be controlled by pharmacological agents, it represents a major burden on annual global healthcare costs. According to the Centre for Disease Control and Prevention (CDC) [2], it was estimated that hypertension-related costs reached \$76.6 billion in the USA in 2010. It is thought that prevention through lifestyle choices and early treatment for

individuals with mild hypertension can significantly reduce global health-care costs.

Food proteins contain numerous biologically-active peptides (BAPs). These BAPs can exert positive physiological responses in the body beyond their basic nutritional roles in the provision of nitrogen and essential amino acids. Many bioactivities have been found including peptides with antihypertensive capabilities. This has led to significant research on the discovery and generation of peptides with antihypertensive properties *in vivo.* Food proteins such as the casein and whey protein components of milk, meat, egg, marine and meat proteins have all been found to contain peptides with potential antihypertensive properties within their primary sequences. These peptides may become active when

Roseanne Norris and Richard J. FitzGerald

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51710

**1. Introduction** 


## **Antihypertensive Peptides from Food Proteins**

Roseanne Norris and Richard J. FitzGerald

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51710

## **1. Introduction**

44 Bioactive Food Peptides in Health and Disease

Biosystems 2011; 7: 2500-2507.

[98] Muricken DG, Gowda LR Molecular Engineering of a Small Trypsin Inhibitor Based on the Binding Loop of Horsegram Seed Bowman-Birk Inhibitor. Journal of Enzyme

[99] Rocco M, Malorni L, Chambery A, Poerio E, Parente A, Di Maro A. A Bowman-Birk Inhibitor with Anti-Elastase Activity from Lathyrus sativus L. Seeds. Molecular

[100] Fernandes AO, Banerji AP. Inhibition of Benzopyrene-Induced Forestomach Tumors

by Field Bean Protease Inhibitor. Carcinogenesis 1995; 16: 1843-1846.

Inhibition and Medicinal Chemistry 2011; 26: 553-560.

Hypertension or elevated blood pressure (BP) is a global health concern, thought to affect up to 30 % of the adult population in developed and developing countries. It is defined by a BP measurement of 140/90 mmHg or above. Hypertension is a major risk factor concomitant with cardiovascular disease (CVD) states such as coronary heart disease, peripheral artery disease and stroke, and kidney disease. Essential hypertension, the most common type of hypertension and to which 90-95% of cases belong, is manifested as an increase in an individual's BP due to an unknown cause. This class of hypertension can be improved with lifestyle choices such as regular exercise, heart-healthy eating, non smoking, reducing sodium intake and reducing the level of stress [1]. For these reasons it is defined as a controllable risk factor of CVD. At present there is a range of synthetic drugs on the market for treatment of hypertension including diuretics, adrenergic inhibitors such as α- and βblockers, direct vasodilators, calcium channel blockers, angiotensin II (Ang II) receptor blockers and angiotensin converting enzyme (ACE) inhibitors. However, although hypertension can be controlled by pharmacological agents, it represents a major burden on annual global healthcare costs. According to the Centre for Disease Control and Prevention (CDC) [2], it was estimated that hypertension-related costs reached \$76.6 billion in the USA in 2010. It is thought that prevention through lifestyle choices and early treatment for individuals with mild hypertension can significantly reduce global health-care costs.

Food proteins contain numerous biologically-active peptides (BAPs). These BAPs can exert positive physiological responses in the body beyond their basic nutritional roles in the provision of nitrogen and essential amino acids. Many bioactivities have been found including peptides with antihypertensive capabilities. This has led to significant research on the discovery and generation of peptides with antihypertensive properties *in vivo.* Food proteins such as the casein and whey protein components of milk, meat, egg, marine and meat proteins have all been found to contain peptides with potential antihypertensive properties within their primary sequences. These peptides may become active when

© 2013 Norris and FitzGerald, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Norris and FitzGerald, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

released through enzymatic/bacterial hydrolysis [3]. The food industry has recognised the potential of these *natural* antihypertensive agents as possible future functional ingredients, aiding in the primary prevention and/or management of hypertension.

Antihypertensive Peptides from Food Proteins 47

ACE-inhibitory peptides have been identified in a range of food proteins including casein, whey, ovalbumin, red algae, wakame, soy, gelatin, chicken muscle, dried bonito, corn, sardines, rapeseed, potato, chick pea, tuna muscle, pea albumin, garlic, wheat germ, sake, porcine haemoglobin and squid. The ACE inhibitory peptides found in different food proteins has been extensively reviewed (for review see [9-12; 3; 131]. Examples of recently reported food protein ACE inhibitory peptide sources include loach (*Misgurnus anguillicaudatus*) [13], pork meat [14], lima bean (*Phaseolus lunatus)* [15], skate skin [16] and boneless chicken leg meat [17]. ACE inhibitory peptides have been generated in a number of different ways. They can be produced naturally during gastrointestinal (GI) digestion by the hydrolytic action of the proteinases pepsin, trypsin, chymotrypsin and by brush border peptidases [18]. Simulated GI digestion has been carried out on a range of protein sources to assess the effect of GI digestion on ACEinhibitory peptides [19-24]. More commonly, ACE-inhibitory peptides are produced through enzymatic hydrolysis with GI enzymes such as pepsin and trypsin or with enzyme combinations such as Alcalase™ [25]. ACE-inhibitory peptides have also been produced during the fermentation of milk during cheese production. *Lactobacillus* and *Lactococcus lactis* strains have been shown to produce ACE inhibitory peptides. Furthermore, fermented soy products such as soy paste, soy sauce, natto and tempeh

ACE inhibitory peptides can work in three ways and are classed as inhibitor-type, substratetype or prodrug-type based on changes in ACE inhibitory activity after hydrolysis of peptides by ACE [30]. Inhibitor-type peptides are ACE inhibitory peptides whose activity is not significantly altered as the peptides are resistant to cleavage by ACE. Substrate-type ACE inhibitors show a decrease in ACE activity due to cleavage by ACE. Prodrug type refers to the conversion to potent ACE inhibitors following hydrolysis of larger peptide fragments by ACE itself. The resulting peptides tend to produce long-lasting hypotensive effects *in vivo* [30]. A prodrug type ACE inhibitor was isolated from a thermolysin-digest of Katsuo-bushi, a Japanese traditional food processed from dried bonito. The study reported an 8-fold increase in ACE-inhibitory activity when the peptide Leu-Lys-Pro-Asn-Met (IC50=2.4 μM) was hydrolyzed by ACE to produce Leu-Lys-Pro [IC50=0.32 μM; 30]. When Leu-Lys-Pro-Asn-Met and Leu-Lys-Pro were orally administered to spontaneously hypertensive rats (SHR), Leu-Lys-Pro-Asn-Met showed a maximal decrease of BP after 4 and 6 h, results which are comparable to that of Captopril inhibition. However, the maximal

Inhibition of ACE is by far the most studied mechanism of BP control with regard to foodderived biologically-active peptides. Most peptides have been found to inhibit ACE to some degree. However, in most cases, it has yet to be answered whether this is the BP mechanism being employed *in vivo*. There are other regulatory pathways of BP control, independent of ACE, that are also potential targets for the action of antihypertensive peptides (see Figure 1

have been found to produce ACE-inhibitory peptides [26-29].

hypotensive effect of Leu-Lys-Pro was seen at 2 h [30].

for vasorelaxative peptides and molecules).

## **2. Hypotensive mechanisms of action**

The regulation of BP is complex, involving a variety of intertwining metabolic pathways. By far, the most studied BP control pathways with regard to food-derived peptides involve those shown to inhibit ACE *in vitro*. This enzyme is one of the main regulators of BP and is involved in two main systems, the renin-angiotensin system (RAS) and the kinin-nitric oxide system (KNOS). Inhibition of ACE in these systems leads to dilation of the artery walls or vasodilation and subsequent lowering of BP. However, it is not yet known whether this is the main mechanism followed *in vivo* or whether there are a number of other BP control mechanisms involved [4].

## **2.1. ACE inhibition**

ACE inhibition is an excellent physiological target for clinical hypertensive treatment due to its involvement in two BP related systems, the RAS and the KNOS. The RAS is thought to be one of the predominant pressor systems in BP control. In the RAS the N-terminus of the prohormone angiotensinogen, which is derived from the liver, is cleaved by renal renin to produce the decapeptide angiotensin I (Ang I). ACE then removes the C-terminal dipeptide HL to form Ang II, a potent vasoconstrictory peptide which acts directly on vascular smooth muscle cells. Thus, inhibition of ACE consequentially leads to BP reduction. Ang II binds to AT1 and AT2 receptors which are located in peripheral tissues around the body and in the brain. The vasocontriction produced by Ang II is mediated by the AT1 receptor. [5-7]. In the KNOS, ACE inactivates the vasodilatory peptides bradykinin and kallidin. Kallidin is synthesised from kininogen by kallikrein, and its further action on kallidin leads to the formation of bradykinin among other vasoactive peptides. Bradykinin binds to β-receptors which lead to an eventual increase in intracellular Ca2+ level. The binding of bradykinin to βreceptors and the increase in Ca2+ stimulates nitric oxide synthase (NOS) to convert Larginine to nitric oxide (NO), a potent vasodilator. ACE can therefore, indirectly inhibit the production of NO as it hydrolyses bradykinin into inactive fragments [7].

There are a number of widely-used synthetic ACE inhibitors currently on the market that serve as the first line of approach for the treatment of hypertension. Such inhibitors include Captopril, Enalapril and Lisinopril. However, their use is associated with a range of sideeffects including cough, skin rashes, hypotension, loss of taste, angiodema reduced renal function and fetal abnormalities [8]. Natural ACE inhibitory peptides from food are not associated with the side-effects brought about by the synthetic drugs. They are not as potent inhibitors of ACE as the synthetic inhibitors which can have IC50 values in the nM region. As they inhibit ACE to a lesser extent, this potentially allows for safer levels of bradykinin in the body. Thus, for this reason, ACE-inhibitory peptides have gained interest as potential preventative agents for hypertension control.

ACE-inhibitory peptides have been identified in a range of food proteins including casein, whey, ovalbumin, red algae, wakame, soy, gelatin, chicken muscle, dried bonito, corn, sardines, rapeseed, potato, chick pea, tuna muscle, pea albumin, garlic, wheat germ, sake, porcine haemoglobin and squid. The ACE inhibitory peptides found in different food proteins has been extensively reviewed (for review see [9-12; 3; 131]. Examples of recently reported food protein ACE inhibitory peptide sources include loach (*Misgurnus anguillicaudatus*) [13], pork meat [14], lima bean (*Phaseolus lunatus)* [15], skate skin [16] and boneless chicken leg meat [17]. ACE inhibitory peptides have been generated in a number of different ways. They can be produced naturally during gastrointestinal (GI) digestion by the hydrolytic action of the proteinases pepsin, trypsin, chymotrypsin and by brush border peptidases [18]. Simulated GI digestion has been carried out on a range of protein sources to assess the effect of GI digestion on ACEinhibitory peptides [19-24]. More commonly, ACE-inhibitory peptides are produced through enzymatic hydrolysis with GI enzymes such as pepsin and trypsin or with enzyme combinations such as Alcalase™ [25]. ACE-inhibitory peptides have also been produced during the fermentation of milk during cheese production. *Lactobacillus* and *Lactococcus lactis* strains have been shown to produce ACE inhibitory peptides. Furthermore, fermented soy products such as soy paste, soy sauce, natto and tempeh have been found to produce ACE-inhibitory peptides [26-29].

46 Bioactive Food Peptides in Health and Disease

control mechanisms involved [4].

**2.1. ACE inhibition** 

**2. Hypotensive mechanisms of action** 

released through enzymatic/bacterial hydrolysis [3]. The food industry has recognised the potential of these *natural* antihypertensive agents as possible future functional ingredients,

The regulation of BP is complex, involving a variety of intertwining metabolic pathways. By far, the most studied BP control pathways with regard to food-derived peptides involve those shown to inhibit ACE *in vitro*. This enzyme is one of the main regulators of BP and is involved in two main systems, the renin-angiotensin system (RAS) and the kinin-nitric oxide system (KNOS). Inhibition of ACE in these systems leads to dilation of the artery walls or vasodilation and subsequent lowering of BP. However, it is not yet known whether this is the main mechanism followed *in vivo* or whether there are a number of other BP

ACE inhibition is an excellent physiological target for clinical hypertensive treatment due to its involvement in two BP related systems, the RAS and the KNOS. The RAS is thought to be one of the predominant pressor systems in BP control. In the RAS the N-terminus of the prohormone angiotensinogen, which is derived from the liver, is cleaved by renal renin to produce the decapeptide angiotensin I (Ang I). ACE then removes the C-terminal dipeptide HL to form Ang II, a potent vasoconstrictory peptide which acts directly on vascular smooth muscle cells. Thus, inhibition of ACE consequentially leads to BP reduction. Ang II binds to AT1 and AT2 receptors which are located in peripheral tissues around the body and in the brain. The vasocontriction produced by Ang II is mediated by the AT1 receptor. [5-7]. In the KNOS, ACE inactivates the vasodilatory peptides bradykinin and kallidin. Kallidin is synthesised from kininogen by kallikrein, and its further action on kallidin leads to the formation of bradykinin among other vasoactive peptides. Bradykinin binds to β-receptors which lead to an eventual increase in intracellular Ca2+ level. The binding of bradykinin to βreceptors and the increase in Ca2+ stimulates nitric oxide synthase (NOS) to convert Larginine to nitric oxide (NO), a potent vasodilator. ACE can therefore, indirectly inhibit the

There are a number of widely-used synthetic ACE inhibitors currently on the market that serve as the first line of approach for the treatment of hypertension. Such inhibitors include Captopril, Enalapril and Lisinopril. However, their use is associated with a range of sideeffects including cough, skin rashes, hypotension, loss of taste, angiodema reduced renal function and fetal abnormalities [8]. Natural ACE inhibitory peptides from food are not associated with the side-effects brought about by the synthetic drugs. They are not as potent inhibitors of ACE as the synthetic inhibitors which can have IC50 values in the nM region. As they inhibit ACE to a lesser extent, this potentially allows for safer levels of bradykinin in the body. Thus, for this reason, ACE-inhibitory peptides have gained interest as potential

production of NO as it hydrolyses bradykinin into inactive fragments [7].

preventative agents for hypertension control.

aiding in the primary prevention and/or management of hypertension.

ACE inhibitory peptides can work in three ways and are classed as inhibitor-type, substratetype or prodrug-type based on changes in ACE inhibitory activity after hydrolysis of peptides by ACE [30]. Inhibitor-type peptides are ACE inhibitory peptides whose activity is not significantly altered as the peptides are resistant to cleavage by ACE. Substrate-type ACE inhibitors show a decrease in ACE activity due to cleavage by ACE. Prodrug type refers to the conversion to potent ACE inhibitors following hydrolysis of larger peptide fragments by ACE itself. The resulting peptides tend to produce long-lasting hypotensive effects *in vivo* [30]. A prodrug type ACE inhibitor was isolated from a thermolysin-digest of Katsuo-bushi, a Japanese traditional food processed from dried bonito. The study reported an 8-fold increase in ACE-inhibitory activity when the peptide Leu-Lys-Pro-Asn-Met (IC50=2.4 μM) was hydrolyzed by ACE to produce Leu-Lys-Pro [IC50=0.32 μM; 30]. When Leu-Lys-Pro-Asn-Met and Leu-Lys-Pro were orally administered to spontaneously hypertensive rats (SHR), Leu-Lys-Pro-Asn-Met showed a maximal decrease of BP after 4 and 6 h, results which are comparable to that of Captopril inhibition. However, the maximal hypotensive effect of Leu-Lys-Pro was seen at 2 h [30].

Inhibition of ACE is by far the most studied mechanism of BP control with regard to foodderived biologically-active peptides. Most peptides have been found to inhibit ACE to some degree. However, in most cases, it has yet to be answered whether this is the BP mechanism being employed *in vivo*. There are other regulatory pathways of BP control, independent of ACE, that are also potential targets for the action of antihypertensive peptides (see Figure 1 for vasorelaxative peptides and molecules).

Antihypertensive Peptides from Food Proteins 49

vasorelaxative effect in the phenylephrine-contracted thoracic aorta. It was also shown that His-Arg-Trp, at a concentration of 100 μM, caused a significant reduction in intracellular Ca2+ concentration. The increase intracellular [Ca2+], brought about by the action of Bay K8644 or Ang II, was significantly inhibited by His-Arg-Trp (>30%). It was proposed that His-Arg-Trp may have supressed extracellular Ca2+ influx through voltage-gated L-type Ca2+ channels [35]. Another recent study reported a similar result with Trp-His which was also found to block L-type Ca2+ channels. Trp-His at 300 μM elicited an intracellular Ca2+ reduction of 23 % in 8 week-old male Wistar rat thoracic aortae smooth muscle cells. In addition, the reduction in [Ca2+] brought about by Trp-His was eliminated by verapamil indicating that Trp-His specifically works on L-type Ca2+

Food-derived peptides have also been found to be sources of opioid like-activities. These peptides bind to opioid receptors to produce morphine-like effects. Natural opioid peptides include endorphins, enkephalins and dynorphins. In humans opioid receptors are found in the nervous, endocrine and immune systems, and in the intestinal tract. These receptors may be involved in various regulatory processes in the body including the regulation of circulation which can affect BP [37; 38]. Nurminen *et al* [39] found an antihypertensive effect on oral administration of the tetrapeptide, α-lactorphin (Tyr-Gly-Leu-Phe), to SHR and to normotensive Wistar Kyoto rats (WKY). Maximum BP reductions were found in SHR, with a decrease of 23 ± 4 and 17 ± 4 mm Hg in systolic BP (SBP) and diastolic BP (DBP), respectively. However, the α-lactophin-induced reduction in BP was not found after administration of the specific opioid receptor antagonist, Naloxone. Therefore, the antihypertensive effect was considered to be a result of interaction with opioid receptors. A follow-up study looked at the effects of α-lactophin along with a second milk-derived peptide β-lactorphin (Tyr-Leu-Leu-Phe) on mesenteric arterial function to demonstrate the regulatory mechanisms of action. It was shown with the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) that α-lactophin produced an endothelium-dependant vasorelaxation, whereas, β-lactorphin also enhanced endothelium-independent vasorelaxation. The study concluded that α-lactophin may stimulate opioid receptors which in turn releases NO causing the vasorelaxative effect [40]. The casein-derived peptide casoxin D (Tyr-Val-Pro-Phe-Pro-Pro-Phe) has also been reported to have an hypotensive effect via opioid receptors. The peptide was found to have an endothelium-dependent relaxation in canine mesenteric artery strips. Anti-opioid and vasorelaxing effects were mediated by the opioid μ-receptor and BK B1-receptor, respectively [41-42]. Furthermore, it has been suggested that opioid-induced BP regulation by such peptides may act upon receptors in the intestinal tract. Interestingly, this would mean that the peptide would not need to be absorbed into the blood stream at the brush border membrane [43]. It could very well be that opioid-mediated reduction in BP may be the principal mechanism for

channels [36].

antihypertensive peptides.

**2.4. Opioid peptide vasorelaxive effects** 

**Figure 1.** Vasorelaxative peptides and molecules in blood pressure control systems.

#### **2.2. Renin Inhibition**

Renin inhibition is another potential target for BP control. It is thought that inhibition of renin could provide a more effective treatment for hypertension it prevents the formation of Ang-I, which can be converted to Ang-II in some cells independent of ACE, by the enzyme chymase [31]. In addition, unlike ACE which acts on a number of substrates in various biochemical pathways, angiotensinogen is the only known substrate of renin. Therefore, renin inhibitors could ensure a higher specificity in antihypertensive treatment compared to ACE inhibitors [31-32]. Food peptides have recently been found to be inhibitors of renin. Peptides from enzymatic flaxseed fractions were found to inhibit both human recombinant renin and ACE. The study concluded that such peptides with the ability to inhibit both ACE and renin may potentially provide better antihypertensive effects *in vivo* in comparison to peptides that only inhibit ACE [33]. A similar outcome was seen in a study carried out by Li & Aluko [34] where fractions of pea protein isolates inhibited both ACE and renin to a high degree with IC50 values <25 mM.

#### **2.3. Calcium channel blocking effects**

Calcium channel blockers interact with voltage-gated calcium channels (VGCCs) in cardiac muscle and blood vessel walls, reducing intracellular calcium and consequently lowering vasoconstriction. It has been shown in various studies that peptides can have the ability to act as calcium channel blockers. Fifteen synthetic peptides based on Trp-His skeleton analogues were tested for their vasodilatory effects in 1.0 μM phenylephrinecontracted thoracic aortic rings from Sprague-Dawley rats. It was previously reported that Trp-His induced the most potent vasodilation among 67 synthetic di-and tripeptides. The study demonstrated that His-Arg-Trp had an endothelium-independent vasorelaxative effect in the phenylephrine-contracted thoracic aorta. It was also shown that His-Arg-Trp, at a concentration of 100 μM, caused a significant reduction in intracellular Ca2+ concentration. The increase intracellular [Ca2+], brought about by the action of Bay K8644 or Ang II, was significantly inhibited by His-Arg-Trp (>30%). It was proposed that His-Arg-Trp may have supressed extracellular Ca2+ influx through voltage-gated L-type Ca2+ channels [35]. Another recent study reported a similar result with Trp-His which was also found to block L-type Ca2+ channels. Trp-His at 300 μM elicited an intracellular Ca2+ reduction of 23 % in 8 week-old male Wistar rat thoracic aortae smooth muscle cells. In addition, the reduction in [Ca2+] brought about by Trp-His was eliminated by verapamil indicating that Trp-His specifically works on L-type Ca2+ channels [36].

#### **2.4. Opioid peptide vasorelaxive effects**

48 Bioactive Food Peptides in Health and Disease

**2.2. Renin Inhibition** 

degree with IC50 values <25 mM.

**2.3. Calcium channel blocking effects** 

**Figure 1.** Vasorelaxative peptides and molecules in blood pressure control systems.

Renin inhibition is another potential target for BP control. It is thought that inhibition of renin could provide a more effective treatment for hypertension it prevents the formation of Ang-I, which can be converted to Ang-II in some cells independent of ACE, by the enzyme chymase [31]. In addition, unlike ACE which acts on a number of substrates in various biochemical pathways, angiotensinogen is the only known substrate of renin. Therefore, renin inhibitors could ensure a higher specificity in antihypertensive treatment compared to ACE inhibitors [31-32]. Food peptides have recently been found to be inhibitors of renin. Peptides from enzymatic flaxseed fractions were found to inhibit both human recombinant renin and ACE. The study concluded that such peptides with the ability to inhibit both ACE and renin may potentially provide better antihypertensive effects *in vivo* in comparison to peptides that only inhibit ACE [33]. A similar outcome was seen in a study carried out by Li & Aluko [34] where fractions of pea protein isolates inhibited both ACE and renin to a high

Calcium channel blockers interact with voltage-gated calcium channels (VGCCs) in cardiac muscle and blood vessel walls, reducing intracellular calcium and consequently lowering vasoconstriction. It has been shown in various studies that peptides can have the ability to act as calcium channel blockers. Fifteen synthetic peptides based on Trp-His skeleton analogues were tested for their vasodilatory effects in 1.0 μM phenylephrinecontracted thoracic aortic rings from Sprague-Dawley rats. It was previously reported that Trp-His induced the most potent vasodilation among 67 synthetic di-and tripeptides. The study demonstrated that His-Arg-Trp had an endothelium-independent Food-derived peptides have also been found to be sources of opioid like-activities. These peptides bind to opioid receptors to produce morphine-like effects. Natural opioid peptides include endorphins, enkephalins and dynorphins. In humans opioid receptors are found in the nervous, endocrine and immune systems, and in the intestinal tract. These receptors may be involved in various regulatory processes in the body including the regulation of circulation which can affect BP [37; 38]. Nurminen *et al* [39] found an antihypertensive effect on oral administration of the tetrapeptide, α-lactorphin (Tyr-Gly-Leu-Phe), to SHR and to normotensive Wistar Kyoto rats (WKY). Maximum BP reductions were found in SHR, with a decrease of 23 ± 4 and 17 ± 4 mm Hg in systolic BP (SBP) and diastolic BP (DBP), respectively. However, the α-lactophin-induced reduction in BP was not found after administration of the specific opioid receptor antagonist, Naloxone. Therefore, the antihypertensive effect was considered to be a result of interaction with opioid receptors. A follow-up study looked at the effects of α-lactophin along with a second milk-derived peptide β-lactorphin (Tyr-Leu-Leu-Phe) on mesenteric arterial function to demonstrate the regulatory mechanisms of action. It was shown with the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) that α-lactophin produced an endothelium-dependant vasorelaxation, whereas, β-lactorphin also enhanced endothelium-independent vasorelaxation. The study concluded that α-lactophin may stimulate opioid receptors which in turn releases NO causing the vasorelaxative effect [40]. The casein-derived peptide casoxin D (Tyr-Val-Pro-Phe-Pro-Pro-Phe) has also been reported to have an hypotensive effect via opioid receptors. The peptide was found to have an endothelium-dependent relaxation in canine mesenteric artery strips. Anti-opioid and vasorelaxing effects were mediated by the opioid μ-receptor and BK B1-receptor, respectively [41-42]. Furthermore, it has been suggested that opioid-induced BP regulation by such peptides may act upon receptors in the intestinal tract. Interestingly, this would mean that the peptide would not need to be absorbed into the blood stream at the brush border membrane [43]. It could very well be that opioid-mediated reduction in BP may be the principal mechanism for antihypertensive peptides.

#### **2.5. Endothelin-1 and endothelin converting enzyme (ECE) inhibition**

The vasoconstrictory peptide endothelin-1 (ET-1) is released from big endothelin-1 (big ET-1) by the action of endothelin-converting enzyme (ECE). ET-1 mediates vasoconstriction via 2 receptors, ETa and. ETb. Both receptors mediate contractions on smooth muscle, but ETb also induces relaxation of endothelial cells by the production of nitric oxide. ET-1 is known to have a greater vasocontrictive effect than Ang II [44; 7]. Endothelial-dependent release of NOS was found to be the mechanism of action for the antihypertensive egg protein derived ovokinin (f2-7) peptide (Arg-Ala-Asp-His-Pro-Phe). Dilation of isolated SHR mesenteric arteries was found to be inhibited by L-NAME but not by indomethacin, demonstrating NO release from the endothelial cells [45]. A later study showed that ovokinin (2–7) modulates a hypotensive effect through interaction via B2 bradykinin receptors [46].

Antihypertensive Peptides from Food Proteins 51

C-terminal tripeptide Bulky hydrophobic

Aromatic or branched

Proline at one or more

Positively charged residues in position two,

Tyr, Phe, Trp, Leu L-configured residue in

position three

residues

side chains

positions

Arg, Lys

Tyr, Phe and Trp residues are also present at the C-terminus of many potent ACE inhibitors, especially with di- and tripeptide inhibitors [9]. It has been suggested that Leu residues may also contribute to ACE inhibition [49]. Furthermore, the positive charge on the side chains of Arg and Lys residues at the C-terminus have been noted to contribute to the ACE inhibitory potential of a peptide [50-51; 9]. An L-configured amino acid at position three at the Cterminus of the inhibitory peptide may be a requirement for potent inhibition. A study showed that the IC50 for the tripeptide D-Val-Ala-Pro (2 μM) increased to 550 μM with L-Val-Ala-Pro, yet only a slight increase in IC50 was seen for the peptide L-Phe-Val-Ala-Pro (17 μM; Maruyama *et al*., 1987). It is thought that conformation contributes to the ACE

N-Terminus--------------------------------------------------------------------------------------C-Terminus

Peptide conformation important

**Table 1.** Some structural features of potent angiotensin converting enzyme (ACE) inhibitory peptides.

Both domains of ACE (C- and N-domains) contain an active site containing the sequence His-Glu-XX-His. These active sites are located within the cleft of the two domains, and are protected by an N-terminal 'lid'. This 'lid' blocks access of large polypeptides to the active site. This is thought to explain why small peptides are more effective in inhibiting ACE. In addition, ACE inhibition may include inhibitor interaction with subsites on the enzyme that are not generally occupied by substrates or with an anionic inhibitor binding site that is different for the catalytic site of the enzyme. With the catalytic sites of ACE having different conformational requirements, this could indicate that for a more complete inhibition of ACE, there may be a need to use a variety of peptide inhibitors each with slightly different

Quantitative computational tools are increasingly been applied in medicinal and pharmaceutical drug discovery. Recently it has been acknowledged that such models could be adapted to food-derived bioactive peptide sequences. Quantitative structure-activity relationship modelling (QSAR) and substrate docking can be used as an effective tool to assess *in silico* numerous peptide structures for their bioactivity potential. Thus, this work allows for a molecular understanding of peptide structure and bioactivity. QSAR studies are

for longer peptides

inhibitory potential of long-chain peptide inhibitors [3].

Hydrophobic residues 2-12 amino acids in length

conformational features [52-53].

It has been found that food proteins have the ability to act as inhibitors of ECE. Okitsu *et al* [47] found ECE inhibitory peptides in pepsin digests of beef and bonito pyrolic appendix. Up to 45 and 40 % of ECE activity could be inhibited with the beef and bonito peptides, respectively. A second study showed that the ACE-inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg, released through tryptic digestion of bovine β-lactoglobulin, can inhibit the release of ET-1 in cultured porcine aortic endothelial cells (PAECs). At a concentration of 1 mM Ala-Leu-Pro-Met-His-Ile-Arg, ET-1 release was reduced by 29 %. The study concluded that the ET-1 reduction may be due to indirect reduction of ET-release by ACE inhibition through the BK pathway, rather than direct action on ET-1 by the peptide [48]. ACE breaks down BK into inactive fragments in the KNOS. Subsequent accumulation of BK (vasodilator) due to ACE inhibition leads to increased release of the vasodilator NO, and antagonises the release of the ET-1 by endothelial cells.

#### **3. Structure activity relationships**

An understanding of the relationship between a peptide and its bioactivity allows for the targeted release of potentially potent peptide sequences. This would eliminate the need for the time-consuming conventional peptide discovery strategy. There is limited knowledge on the structure-activity relationship of hypotensive peptides. To date, the main focus with regard to bioactive peptide research has been on the generation and characterisation of these peptides. ACE inhibition is by far the most widely studied biomarker with regard to antihypertensive effects of bioactive food peptides. ACE can work on a wide range of peptide substrates, and appears to have a broad specificity. Some structural features that influence the binding of a peptide to the ACE active site have been recognised (Table 1). However, potent inhibitory peptides of ACE are generally short sequences, i.e., 2-12 amino acids in length. However, some larger inhibitory sequences have been identified. Studies have indicated that binding to ACE is strongly influenced by the substrate's C-terminal tripeptide sequence. Hydrophobic amino acid residues with aromatic or branched side chains at each of the C-terminal tripeptide positions are common features among potent inhibitors. The presence of hydrophobic Pro residues at one or more positions in the Cterminal tripeptide region seems to positively influence a peptide's ACE inhibitory activity. Tyr, Phe and Trp residues are also present at the C-terminus of many potent ACE inhibitors, especially with di- and tripeptide inhibitors [9]. It has been suggested that Leu residues may also contribute to ACE inhibition [49]. Furthermore, the positive charge on the side chains of Arg and Lys residues at the C-terminus have been noted to contribute to the ACE inhibitory potential of a peptide [50-51; 9]. An L-configured amino acid at position three at the Cterminus of the inhibitory peptide may be a requirement for potent inhibition. A study showed that the IC50 for the tripeptide D-Val-Ala-Pro (2 μM) increased to 550 μM with L-Val-Ala-Pro, yet only a slight increase in IC50 was seen for the peptide L-Phe-Val-Ala-Pro (17 μM; Maruyama *et al*., 1987). It is thought that conformation contributes to the ACE inhibitory potential of long-chain peptide inhibitors [3].

50 Bioactive Food Peptides in Health and Disease

of the ET-1 by endothelial cells.

**3. Structure activity relationships** 

**2.5. Endothelin-1 and endothelin converting enzyme (ECE) inhibition** 

hypotensive effect through interaction via B2 bradykinin receptors [46].

The vasoconstrictory peptide endothelin-1 (ET-1) is released from big endothelin-1 (big ET-1) by the action of endothelin-converting enzyme (ECE). ET-1 mediates vasoconstriction via 2 receptors, ETa and. ETb. Both receptors mediate contractions on smooth muscle, but ETb also induces relaxation of endothelial cells by the production of nitric oxide. ET-1 is known to have a greater vasocontrictive effect than Ang II [44; 7]. Endothelial-dependent release of NOS was found to be the mechanism of action for the antihypertensive egg protein derived ovokinin (f2-7) peptide (Arg-Ala-Asp-His-Pro-Phe). Dilation of isolated SHR mesenteric arteries was found to be inhibited by L-NAME but not by indomethacin, demonstrating NO release from the endothelial cells [45]. A later study showed that ovokinin (2–7) modulates a

It has been found that food proteins have the ability to act as inhibitors of ECE. Okitsu *et al* [47] found ECE inhibitory peptides in pepsin digests of beef and bonito pyrolic appendix. Up to 45 and 40 % of ECE activity could be inhibited with the beef and bonito peptides, respectively. A second study showed that the ACE-inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg, released through tryptic digestion of bovine β-lactoglobulin, can inhibit the release of ET-1 in cultured porcine aortic endothelial cells (PAECs). At a concentration of 1 mM Ala-Leu-Pro-Met-His-Ile-Arg, ET-1 release was reduced by 29 %. The study concluded that the ET-1 reduction may be due to indirect reduction of ET-release by ACE inhibition through the BK pathway, rather than direct action on ET-1 by the peptide [48]. ACE breaks down BK into inactive fragments in the KNOS. Subsequent accumulation of BK (vasodilator) due to ACE inhibition leads to increased release of the vasodilator NO, and antagonises the release

An understanding of the relationship between a peptide and its bioactivity allows for the targeted release of potentially potent peptide sequences. This would eliminate the need for the time-consuming conventional peptide discovery strategy. There is limited knowledge on the structure-activity relationship of hypotensive peptides. To date, the main focus with regard to bioactive peptide research has been on the generation and characterisation of these peptides. ACE inhibition is by far the most widely studied biomarker with regard to antihypertensive effects of bioactive food peptides. ACE can work on a wide range of peptide substrates, and appears to have a broad specificity. Some structural features that influence the binding of a peptide to the ACE active site have been recognised (Table 1). However, potent inhibitory peptides of ACE are generally short sequences, i.e., 2-12 amino acids in length. However, some larger inhibitory sequences have been identified. Studies have indicated that binding to ACE is strongly influenced by the substrate's C-terminal tripeptide sequence. Hydrophobic amino acid residues with aromatic or branched side chains at each of the C-terminal tripeptide positions are common features among potent inhibitors. The presence of hydrophobic Pro residues at one or more positions in the Cterminal tripeptide region seems to positively influence a peptide's ACE inhibitory activity.


**Table 1.** Some structural features of potent angiotensin converting enzyme (ACE) inhibitory peptides.

Both domains of ACE (C- and N-domains) contain an active site containing the sequence His-Glu-XX-His. These active sites are located within the cleft of the two domains, and are protected by an N-terminal 'lid'. This 'lid' blocks access of large polypeptides to the active site. This is thought to explain why small peptides are more effective in inhibiting ACE. In addition, ACE inhibition may include inhibitor interaction with subsites on the enzyme that are not generally occupied by substrates or with an anionic inhibitor binding site that is different for the catalytic site of the enzyme. With the catalytic sites of ACE having different conformational requirements, this could indicate that for a more complete inhibition of ACE, there may be a need to use a variety of peptide inhibitors each with slightly different conformational features [52-53].

Quantitative computational tools are increasingly been applied in medicinal and pharmaceutical drug discovery. Recently it has been acknowledged that such models could be adapted to food-derived bioactive peptide sequences. Quantitative structure-activity relationship modelling (QSAR) and substrate docking can be used as an effective tool to assess *in silico* numerous peptide structures for their bioactivity potential. Thus, this work allows for a molecular understanding of peptide structure and bioactivity. QSAR studies are

based on the relationship between chemical structure of ligands and receptors, and biological activity. Physicochemical variables or descriptor variables of a ligand such as steric properties, hydrophobicity and electronic properties, molecular mass and shape are used to quantitatively correlate the ligand's chemical structure with bioactivity [54]. A small number of QSAR studies have been carried out on ACE-inhibitory peptides. The structureactivity relationship of di-and tri-peptides using partial least square analysis (PLS) QSAR was assessed by constructing a database of known ACE-inhibitory peptides. Using a 3-z scale descriptor approach, two models were developed for the amino acid components of the peptide datasets. The dipeptide model had a predictive power of 71.1 % while the tripeptide model had a predictive power of 43.4 %. The dipeptide model indicated that amino acids with bulky and hydrophobic side chains were favoured by ACE while the tripeptide model suggested that C-terminal aromatic residues, positively charged residues in position two and hydrophobic residues at the amino terminus were preferred [55]. Another study by the same authors used a 5-z scale model to assess peptides of 4-10 amino acids in length. The study concluded that the tetrapeptide residue at the C-terminus has a large influence on the potency of peptide's 4-10 amino acids in length [56].

Antihypertensive Peptides from Food Proteins 53

in the way of this outcome. Antihypertensive peptides must be resistant to digestive proteinases and peptidases; they must be able to be transported through the bush border membrane intact and must be resistant to serum peptidases. With regard to ACE inhibition, while there have been many studies focusing on the production, isolation and characterisation of ACE-inhibitory peptides, to date little attention has been placed on their bioavailability. It is therefore difficult to determine the relationship between *in vitro* ACEinhibitory activity and an *in vivo* hypotensive effect. This is made even more difficult with the utilisation of several different *in vitro* assays and assay conditions for the determination of ACE-inhibition [59]. Furthermore, variations in *in vivo* experimental design such as administration by intravenous subcutaneous or oral administration, and the use of animal models or hypertensive patients, all hinder the ability to compare results among different

Bioactive peptides when taken orally may be inactived by several digestive proteinases and peptidases including pepsin in the stomach, and the pancreatic enzymes trypsin, elastase, αchymotrypsin and carboxypeptidase A and B in the small intestine. A number of studies have been carried out investigating ACE-inhibitory peptides and their ability to resist gastrointestinal digestion by these enzymes. These studies involve simulating the gastrointestinal process by sequential hydrolysis of ACE inhibitory peptides with pepsin and Pancreatin™, each concluding the importance of gastrointestinal digestion analysis in the ACE inhibitory activity of the peptide [61-66; 49; 21; 20] . It has been noted that certain protein/peptide structures are resistant to gastrointestinal digestion due to the composition and position of amino acids in their primary chains. The rate of hydrolysis of a peptide is also dependent on the peptide's amino acid composition. Peptides containing Pro and hydroxy Pro residues have been found to be resistant to hydrolysis. Furthermore, glycosylated peptides and peptides which have undergone changes during food processing such as during the formation of Maillard reaction products have been shown to be resistant to GI tract enzyme cleavage [67]. Once the peptides reach the brush border membrane of the large intestine, they may also be subjected to further cleavage by a variety of membrane anchored epithelial cell intestinal peptidases. These include a number of aminotripeptidases and several dipeptidases, each with varying specificities [60]. However, it has been found that certain free amino acids released during gastrointestinal breakdown may in turn serve as inhibitors of the brush border membrane dipeptidases, Moreover, it has been reported that during gastrointestinal proteolysis at the brush border membrane, the large variety and high concentration of peptides present would exceed the apparent *V*max for hydrolysis, allowing for safe passage of many di- and tripeptides through the membrane wall. Absorption through the membrane is possible for both di- and tripeptides with the help of a peptide transporter termed PepT1. PepT1 operates as an electrogenic proton/peptide symporter having wide substrate specificity [67]. There is an increasing body of research that shows the presence of the lactotripeptides (LTPs) Ile-Pro-Pro and Val-Pro-Pro in human and animal circulatory systems after oral administration, suggesting the resistance of these peptides to gastrointestinal degradation and their absorption intact across the brush border membrane [68-72]. However, it has been suggested that intestinal absorption of Val-Pro-Pro

studies [60].

Substrate docking involves the docking of molecules (ligands) to a receptor or into a protein target such as an enzyme. All possible docking or binding conformations are assessed for their binding affinity to a molecule, and their potential as high affinity binding ligands is estimated by use of a scoring function. An integrated QSAR and Artificial Neural Network (ANN) approach was used to assess the ACE-inhibitory potential of 58 dipeptides present in the sequence of defatted wheat germ protein. The model was used to investigate preferred structural characteristics of ACE-inhibitory dipeptides and following this, appropriate proteases were successfully selected to produce the dipeptides predicted to be potent inhibitors by the QSAR-ANN model. The QSAR model predicted that the C-terminal of the peptide had principal importance on ACE inhibitory activity, with hydrophobic C-terminal residues being essential for high potency. Furthermore, proteins with a high abundance of hydrophobic residues were considered to be good substrates for the production of potent ACE inhibitory peptides [57]. Recently, the ability of docking to predict ACE inhibitory dipeptide sequences was assessed using the molecular docking program AutoDock Vina. All potential dipeptides and phospho-dipeptides were docked and scored. Phosphodipeptides were predicted by the program to be good inhibitors of ACE. However, the experimentally determined IC50 results for selected phospho-dipeptides did not correlate and the study concluded that phospho-dipeptides may not be potent inhibitors of ACE *in vivo*. Furthermore, LIGPLOT analysis, a program to plot schematic diagrams of proteinligand interactions, carried out on two newly identified ACE inhibitory dipeptides Asp-Trp and Trp-Pro (ACE IC50 values of 258 and 217 μM, respectively) interestingly showed no zinc interaction with the ACE active site [58].

### **4. Peptide bioavailability**

The potential antihypertensive effect of a peptide depends on the peptides ability to reach their target organ intact and in an active form. However, there are several barriers which lie in the way of this outcome. Antihypertensive peptides must be resistant to digestive proteinases and peptidases; they must be able to be transported through the bush border membrane intact and must be resistant to serum peptidases. With regard to ACE inhibition, while there have been many studies focusing on the production, isolation and characterisation of ACE-inhibitory peptides, to date little attention has been placed on their bioavailability. It is therefore difficult to determine the relationship between *in vitro* ACEinhibitory activity and an *in vivo* hypotensive effect. This is made even more difficult with the utilisation of several different *in vitro* assays and assay conditions for the determination of ACE-inhibition [59]. Furthermore, variations in *in vivo* experimental design such as administration by intravenous subcutaneous or oral administration, and the use of animal models or hypertensive patients, all hinder the ability to compare results among different studies [60].

52 Bioactive Food Peptides in Health and Disease

interaction with the ACE active site [58].

**4. Peptide bioavailability** 

based on the relationship between chemical structure of ligands and receptors, and biological activity. Physicochemical variables or descriptor variables of a ligand such as steric properties, hydrophobicity and electronic properties, molecular mass and shape are used to quantitatively correlate the ligand's chemical structure with bioactivity [54]. A small number of QSAR studies have been carried out on ACE-inhibitory peptides. The structureactivity relationship of di-and tri-peptides using partial least square analysis (PLS) QSAR was assessed by constructing a database of known ACE-inhibitory peptides. Using a 3-z scale descriptor approach, two models were developed for the amino acid components of the peptide datasets. The dipeptide model had a predictive power of 71.1 % while the tripeptide model had a predictive power of 43.4 %. The dipeptide model indicated that amino acids with bulky and hydrophobic side chains were favoured by ACE while the tripeptide model suggested that C-terminal aromatic residues, positively charged residues in position two and hydrophobic residues at the amino terminus were preferred [55]. Another study by the same authors used a 5-z scale model to assess peptides of 4-10 amino acids in length. The study concluded that the tetrapeptide residue at the C-terminus has a

large influence on the potency of peptide's 4-10 amino acids in length [56].

Substrate docking involves the docking of molecules (ligands) to a receptor or into a protein target such as an enzyme. All possible docking or binding conformations are assessed for their binding affinity to a molecule, and their potential as high affinity binding ligands is estimated by use of a scoring function. An integrated QSAR and Artificial Neural Network (ANN) approach was used to assess the ACE-inhibitory potential of 58 dipeptides present in the sequence of defatted wheat germ protein. The model was used to investigate preferred structural characteristics of ACE-inhibitory dipeptides and following this, appropriate proteases were successfully selected to produce the dipeptides predicted to be potent inhibitors by the QSAR-ANN model. The QSAR model predicted that the C-terminal of the peptide had principal importance on ACE inhibitory activity, with hydrophobic C-terminal residues being essential for high potency. Furthermore, proteins with a high abundance of hydrophobic residues were considered to be good substrates for the production of potent ACE inhibitory peptides [57]. Recently, the ability of docking to predict ACE inhibitory dipeptide sequences was assessed using the molecular docking program AutoDock Vina. All potential dipeptides and phospho-dipeptides were docked and scored. Phosphodipeptides were predicted by the program to be good inhibitors of ACE. However, the experimentally determined IC50 results for selected phospho-dipeptides did not correlate and the study concluded that phospho-dipeptides may not be potent inhibitors of ACE *in vivo*. Furthermore, LIGPLOT analysis, a program to plot schematic diagrams of proteinligand interactions, carried out on two newly identified ACE inhibitory dipeptides Asp-Trp and Trp-Pro (ACE IC50 values of 258 and 217 μM, respectively) interestingly showed no zinc

The potential antihypertensive effect of a peptide depends on the peptides ability to reach their target organ intact and in an active form. However, there are several barriers which lie Bioactive peptides when taken orally may be inactived by several digestive proteinases and peptidases including pepsin in the stomach, and the pancreatic enzymes trypsin, elastase, αchymotrypsin and carboxypeptidase A and B in the small intestine. A number of studies have been carried out investigating ACE-inhibitory peptides and their ability to resist gastrointestinal digestion by these enzymes. These studies involve simulating the gastrointestinal process by sequential hydrolysis of ACE inhibitory peptides with pepsin and Pancreatin™, each concluding the importance of gastrointestinal digestion analysis in the ACE inhibitory activity of the peptide [61-66; 49; 21; 20] . It has been noted that certain protein/peptide structures are resistant to gastrointestinal digestion due to the composition and position of amino acids in their primary chains. The rate of hydrolysis of a peptide is also dependent on the peptide's amino acid composition. Peptides containing Pro and hydroxy Pro residues have been found to be resistant to hydrolysis. Furthermore, glycosylated peptides and peptides which have undergone changes during food processing such as during the formation of Maillard reaction products have been shown to be resistant to GI tract enzyme cleavage [67]. Once the peptides reach the brush border membrane of the large intestine, they may also be subjected to further cleavage by a variety of membrane anchored epithelial cell intestinal peptidases. These include a number of aminotripeptidases and several dipeptidases, each with varying specificities [60]. However, it has been found that certain free amino acids released during gastrointestinal breakdown may in turn serve as inhibitors of the brush border membrane dipeptidases, Moreover, it has been reported that during gastrointestinal proteolysis at the brush border membrane, the large variety and high concentration of peptides present would exceed the apparent *V*max for hydrolysis, allowing for safe passage of many di- and tripeptides through the membrane wall. Absorption through the membrane is possible for both di- and tripeptides with the help of a peptide transporter termed PepT1. PepT1 operates as an electrogenic proton/peptide symporter having wide substrate specificity [67]. There is an increasing body of research that shows the presence of the lactotripeptides (LTPs) Ile-Pro-Pro and Val-Pro-Pro in human and animal circulatory systems after oral administration, suggesting the resistance of these peptides to gastrointestinal degradation and their absorption intact across the brush border membrane [68-72]. However, it has been suggested that intestinal absorption of Val-Pro-Pro

may operate via paracellular transport, rather than with the help of PepT1 [73]. Larger Prorich peptides have also been found to be transported intact across the brush border membrane. A study found that the ACE-inhibitory and antihypertensive peptide Leu-His-Leu-Pro-Leu-Pro, β-casein (f133-138), was resistant to gastrointestinal digestion. However, this peptide was hydrolysed to the pentapeptide His-Leu-Pro-Leu-Pro by cellular peptidases before transportation across the intestinal epithelium. The study concluded by use of a Caco-2 monolayer model that the likely mechanism of transport was via paracellular passive diffusion [74]. An earlier study quantifying ACE-inhibitory peptides in human plasma found the pentapeptide to be present in human plasma after oral administration which demonstrates the ability of the peptide to be absorbed through the human brush border membrane [75].

Antihypertensive Peptides from Food Proteins 55

Thus, the bioavailability of ACE-inhibitory peptides is essential for their activity. Several approaches to aid in peptide delivery are been considered. Peptides may be chemically modified in order to reduce the rate of enzymatic degradation and to increase bioavailability while also in some cases enhancing bioactivity. The half-life of unmodified peptides in the blood is in most cases very short. They also generally have poor bioavailability in tissues and organs, limiting their ability as preventative therapeutic agents [79; 80]. Modifications such as end changes, glycosylation, alkylation, and conformational changes to amino acids within the peptide may therefore have potential for ACE inhibitory peptides [80; 81]. These approaches have already been adapted to opioid peptides [81]. There is significant scope for these modifications to also be applied to ACE inhibitory and antihypertensive peptides. Encapsulation via nanoparticles and liposomes is also a strategy previously employed for opioid peptides that has possibility for adaption to for ACE inhibitory peptides. These approaches may aid in the passage of a peptide through the GI tract and may enhance the plasma half-life of the peptides. Furthermore, there is potential for bioactive peptides to be produced by microorganisms through genetic engineering to be delivered to target organs *in situ* [60]. Lastly, there is also the possibility to cross-link BAP to protein transduction domains that have been found to be able to cross biological membranes thus promoting peptide and protein delivery into cells [60; 82]. Morris *et al* [82] also devised a similar

strategy using the peptide transporter PepT-1 to carry target peptides into cells.

animals), plant and macroalgae have recently been reviewed [4; 25; 83-86].

The first step employed to determine if a peptide is hypotensive is to conduct trials with small animals such as SHRs, the accepted model for human essential hypertension. A bioactive peptide can only be referred to as 'antihypertensive' after a significant decrease in BP is observed in trials with SHR. There have been many studies carried out in animals to elucidate whether food-derived ACE-inhibitory peptides can lead to an antihypertensive effect *in vivo.* Antihypertensive peptides from milk, egg, animal (including meat and marine

Ile-Pro-Pro (β-casein f74-76; κ-casein f108-110) and Val-Pro-Pro (β-casein f84-86) were among the first dietary peptides found to have a hypotensive effect in SHR. The peptides were first isolated from milk fermented with *Lactobacillus helveticus* and *Saccharomyces cerevisiae* (Ameal S) and their ACE-inhibitory IC50 values were obtained (Val-Pro-Pro and Ile-Pro-Pro having IC50s of 9 and 5 μM, respectively [87]). The antihypertensive effect was first demonstrated when SHR were administered with a single oral dose of the LTPs which resulted in a significant decrease SBP between 6 to 8 h after administration [88]. Thereafter, several studies have been conducted to further characterise the *in vivo* effect of the LTPs. Their long-term effects (12-20 weeks) of administration have been assessed [89-93]. Administration of the peptides via a peptide supplement and via a sour milk drink to SHR resulted in a decrease in SBP of 12 and 17 mm Hg, respectively, compared to the control (water) after 12 weeks [90]. Endothelial function protective effects of Ile-Pro-Pro and Val-Pro-Pro were investigated using isolated SHR mesenteric arteries stored in solutions of

**5.** *Ex-vivo* **and** *in vivo* **animal studies** 

Absorption of peptides across the brush border membrane can be studied by Caco-2 cell monolayers, the representative model for human intestinal epithelial cell barrier. The intestinal transport of pea and whey ACE inhibitory peptides was also studied using a Caco-2 monolayer. It was found that only minor ACE inhibitory activity crossed the Caco-2 cell monolayer in 1 h. However, it was concluded that the extent of ACE inhibitory peptides that may be transported *in vivo* would be higher, as the Caco-2 model is tighter than intestinal mammalian tissue [76]. The transepithelial transport of oligopeptides across the intestinal wall was assessed using a Caco-2 cell monolayer [133]. The study showed that the hydrolysis of peptides by brush-border peptidases is the rate-limiting step for the transepithelial transport of oligopeptides (≥4 residues in length). Bradykinin and Gly-Gly-Tyr-Arg, which were found to be resistant to cellular peptidases, were investigated for their apical-to-basolateral transport mechanism. Bradykinin and its analogues were found to be transported by the intracellular pathway, probably the adsorptive transcytosis. The transport rate was found to be dependent on the hydrophobic properties of the peptides. Gly-Gly-Tyr-Arg was suggested to be transported mainly via the paracellular pathway [133]. Foltz *et al*., [77] devised a predictive *in silico* amino acid clustering model for dipeptides which can predict a dipeptide's ability to withstand small intestinal digestion. Dipeptides (220 in total) were tested for small intestinal stability by simulated digestion and their relative stability (% of initial dipeptide concentration) was plotted against time. Using the area under the curve (AUC) approach, the contribution of N- and C-terminal amino acids were calculated, based on the average AUC of all peptides containing the amino acid of interest. Data clustering allowed for ranking of the N- and C-terminal amino acid residues and they were grouped by their average AUC values. Correlations with experimentally measured stability allowed for classification of dipeptides as intestinally 'stable', 'neutral' or 'instable' using the clustering model.

Following absorption of a peptide into the blood stream, it may undergo hydrolysis by serum peptidases. The ACE inhibitory peptide may need to be able to with-stand hydrolysis in order to reach their target organs intact and yield their antihypertensive effect. It has been suggested that potent ACE inhibitors may be produced in circulation by the action of serum peptidases on less potent inhibitors of ACE and by the action of ACE itself. These peptides have been referred to pro-drug type inhibitors of ACE [78; 60].

Thus, the bioavailability of ACE-inhibitory peptides is essential for their activity. Several approaches to aid in peptide delivery are been considered. Peptides may be chemically modified in order to reduce the rate of enzymatic degradation and to increase bioavailability while also in some cases enhancing bioactivity. The half-life of unmodified peptides in the blood is in most cases very short. They also generally have poor bioavailability in tissues and organs, limiting their ability as preventative therapeutic agents [79; 80]. Modifications such as end changes, glycosylation, alkylation, and conformational changes to amino acids within the peptide may therefore have potential for ACE inhibitory peptides [80; 81]. These approaches have already been adapted to opioid peptides [81]. There is significant scope for these modifications to also be applied to ACE inhibitory and antihypertensive peptides. Encapsulation via nanoparticles and liposomes is also a strategy previously employed for opioid peptides that has possibility for adaption to for ACE inhibitory peptides. These approaches may aid in the passage of a peptide through the GI tract and may enhance the plasma half-life of the peptides. Furthermore, there is potential for bioactive peptides to be produced by microorganisms through genetic engineering to be delivered to target organs *in situ* [60]. Lastly, there is also the possibility to cross-link BAP to protein transduction domains that have been found to be able to cross biological membranes thus promoting peptide and protein delivery into cells [60; 82]. Morris *et al* [82] also devised a similar strategy using the peptide transporter PepT-1 to carry target peptides into cells.

## **5.** *Ex-vivo* **and** *in vivo* **animal studies**

54 Bioactive Food Peptides in Health and Disease

membrane [75].

may operate via paracellular transport, rather than with the help of PepT1 [73]. Larger Prorich peptides have also been found to be transported intact across the brush border membrane. A study found that the ACE-inhibitory and antihypertensive peptide Leu-His-Leu-Pro-Leu-Pro, β-casein (f133-138), was resistant to gastrointestinal digestion. However, this peptide was hydrolysed to the pentapeptide His-Leu-Pro-Leu-Pro by cellular peptidases before transportation across the intestinal epithelium. The study concluded by use of a Caco-2 monolayer model that the likely mechanism of transport was via paracellular passive diffusion [74]. An earlier study quantifying ACE-inhibitory peptides in human plasma found the pentapeptide to be present in human plasma after oral administration which demonstrates the ability of the peptide to be absorbed through the human brush border

Absorption of peptides across the brush border membrane can be studied by Caco-2 cell monolayers, the representative model for human intestinal epithelial cell barrier. The intestinal transport of pea and whey ACE inhibitory peptides was also studied using a Caco-2 monolayer. It was found that only minor ACE inhibitory activity crossed the Caco-2 cell monolayer in 1 h. However, it was concluded that the extent of ACE inhibitory peptides that may be transported *in vivo* would be higher, as the Caco-2 model is tighter than intestinal mammalian tissue [76]. The transepithelial transport of oligopeptides across the intestinal wall was assessed using a Caco-2 cell monolayer [133]. The study showed that the hydrolysis of peptides by brush-border peptidases is the rate-limiting step for the transepithelial transport of oligopeptides (≥4 residues in length). Bradykinin and Gly-Gly-Tyr-Arg, which were found to be resistant to cellular peptidases, were investigated for their apical-to-basolateral transport mechanism. Bradykinin and its analogues were found to be transported by the intracellular pathway, probably the adsorptive transcytosis. The transport rate was found to be dependent on the hydrophobic properties of the peptides. Gly-Gly-Tyr-Arg was suggested to be transported mainly via the paracellular pathway [133]. Foltz *et al*., [77] devised a predictive *in silico* amino acid clustering model for dipeptides which can predict a dipeptide's ability to withstand small intestinal digestion. Dipeptides (220 in total) were tested for small intestinal stability by simulated digestion and their relative stability (% of initial dipeptide concentration) was plotted against time. Using the area under the curve (AUC) approach, the contribution of N- and C-terminal amino acids were calculated, based on the average AUC of all peptides containing the amino acid of interest. Data clustering allowed for ranking of the N- and C-terminal amino acid residues and they were grouped by their average AUC values. Correlations with experimentally measured stability allowed for classification of dipeptides as intestinally

Following absorption of a peptide into the blood stream, it may undergo hydrolysis by serum peptidases. The ACE inhibitory peptide may need to be able to with-stand hydrolysis in order to reach their target organs intact and yield their antihypertensive effect. It has been suggested that potent ACE inhibitors may be produced in circulation by the action of serum peptidases on less potent inhibitors of ACE and by the action of ACE itself. These peptides

'stable', 'neutral' or 'instable' using the clustering model.

have been referred to pro-drug type inhibitors of ACE [78; 60].

The first step employed to determine if a peptide is hypotensive is to conduct trials with small animals such as SHRs, the accepted model for human essential hypertension. A bioactive peptide can only be referred to as 'antihypertensive' after a significant decrease in BP is observed in trials with SHR. There have been many studies carried out in animals to elucidate whether food-derived ACE-inhibitory peptides can lead to an antihypertensive effect *in vivo.* Antihypertensive peptides from milk, egg, animal (including meat and marine animals), plant and macroalgae have recently been reviewed [4; 25; 83-86].

Ile-Pro-Pro (β-casein f74-76; κ-casein f108-110) and Val-Pro-Pro (β-casein f84-86) were among the first dietary peptides found to have a hypotensive effect in SHR. The peptides were first isolated from milk fermented with *Lactobacillus helveticus* and *Saccharomyces cerevisiae* (Ameal S) and their ACE-inhibitory IC50 values were obtained (Val-Pro-Pro and Ile-Pro-Pro having IC50s of 9 and 5 μM, respectively [87]). The antihypertensive effect was first demonstrated when SHR were administered with a single oral dose of the LTPs which resulted in a significant decrease SBP between 6 to 8 h after administration [88]. Thereafter, several studies have been conducted to further characterise the *in vivo* effect of the LTPs. Their long-term effects (12-20 weeks) of administration have been assessed [89-93]. Administration of the peptides via a peptide supplement and via a sour milk drink to SHR resulted in a decrease in SBP of 12 and 17 mm Hg, respectively, compared to the control (water) after 12 weeks [90]. Endothelial function protective effects of Ile-Pro-Pro and Val-Pro-Pro were investigated using isolated SHR mesenteric arteries stored in solutions of

Krebs containing Ile-Pro-Pro and Val-Pro-Pro (1 mM), when mounted in an organ bath. Vascular reactivity measurements demonstrated better preservation of endotheliumdependent relaxation in arteries stored with the LTPs compared to controls [94]. Their bioactive effect in double transgenic rats (dTGR) harbouring human renin and angiotensinogen genes was also assessed. These transgenic rats develop malignant hypertension, cardiac hypertrophy, renal damage, and endothelial dysfunction due to increased Ang II formation. A decrease of 19 mm Hg in SBP was seen in rats administered with fermented milk supplemented with the peptides (Ile-Pro-Pro (1.8 mg/100 ml) and Val-Pro-Pro (1.8 mg/100 ml) compared to the control group Thus, it was concluded that the supplemented fermented milk product can aid in preventing the development of malignant hypertension. There was no effect on BP reported from a group receiving the peptides dissolved in water, despite the higher intake level of peptides. The authors concluded that the reported antihypertensive effect of the fermented milk product can not be explained solely by the Ile-Pro-Pro and Val-Pro-Pro supplements and suggested that a combination of factors such as calcium and potassium content, and less sodium may have contributed to the observed hypotensive effect [95].

Antihypertensive Peptides from Food Proteins 57

Furthermore, it must be noted that although dietary peptides have lower ACE IC50 *in vitro* in comparison to the synthetic ACE drug inhibitor Captopril (IC50 in nM range), in most cases they display higher *in vivo* hypotensive effects than are expected with respect to their *in vitro* results. It has been suggested that this may be due to a higher affinity of dietary peptides to the tissues and a slower elimination in comparison to Captopril. Moreover, it is possible that several BP mechanisms of action are being employed [30; 98]. It was demonstrated that neither the egg-protein derived peptide ovokinin (2-7) (Arg-Ala-Asp-His-Pro-Phe) or Arg-Pro-Leu-Lys-Pro-Trp, the most potent derivative obtained from the structural modification of ovokinin,, inhibit ACE *in vitro*. IC50 values obtained were >1000 μmol/L, despite having a significant effect on BP when orally administered to SHR [99]. Thus, it must be acknowledged that *in vitro* ACE inhibitory determination may not be the best approach to assess the potential of a peptide as an antihypertensive agent. In a study by da Costa *et al* [100] it was found that the most potent *in vitro* ACE inhibitory peptides from whey did not have a significant effect on BP when orally administered to SHR. However, whey peptides with relatively low *in vitro* ACE-inhibitory activity in comparison achieved significant

The majority of the clinical trials regarding the antihypertensive effects of milk-derived peptides to date have been carried out on the LTPs, Ile-Pro-Pro and Val-Pro-Pro. Although some conflicting results exist, the majority of these trials have reported a significant decrease in BP. Their effect on office BP has been well documented (for reviews see 101; 3; 25). It is essential that the BP of test subjects is evaluated in comparison to placebo values and not to baseline values of the test product. As with test products, placebo groups have been found to often decrease in BP over the test period [101]. Furthermore, the 24-h ambulatory BP monitoring (ABPM), of patients BP is thought to be a more reliable method for evaluation of BP as it reduces the 'white coat effect'. Recent clinical trials with LTPs have employed ABPM [102-104; 91]. ABPM was used to assess the effects of LTP administration on dipper (where BP decreases at night time) and non-dipper (where BP does not decrease at night time) hypertensive subjects. Non-dippers are thought to have a higher cardiac vascular risk and BP monitoring in the morning and night can help predict cardiac events such as stroke and myocardial infarction. Twelve patients received a fermented milk product containing Ile-Pro-Pro (1.52 mg) and Val-Pro-Pro (2.53 mg) daily for 4 weeks. The study reported a significant reduction in night-time and early-morning SBP for nondipper subjects but not for dipper subjects [105]. A range of hemodynamic parameters was recently evaluated for 52 human subjects with high-normal BP or first-degree hypertension. These included office BP and ABPM, stress-induced BP increase and cardiac output-related parameters. Subjects were treated with LTPs (3 mg/day) for 6 weeks. The study reported a reduction in office SBP as well as an improvement in pulse wave velocity (an instrumental biomarker for vascular rigidity), stroke volume and stroke volume index (markers of cardiac flow) and acceleration and velocity index (markers of cardiac contractility). No effect on ABPM and BP reaction to stress was observed [106]. LTPs have also been reported to reduce arterial stiffness in

reductions in BP.

**6. Human studies** 

Other rat models, such as the normotensive WKY rat, have been used to evaluate the effect of food peptides on arterial BP. However, a significant hypotensive effect is not always observed in WKY. Single oral administration of Ameal S containing the LTPs decreased BP from 6 to 8 h after administration in SHR. However, no change in SBP was observed in normotensive WKYs [88]. Similarly, the ACE inhibitory peptide Leu-Arg-Pro-Val-Ala-Ala from bovine lactoferrin was found to have a significant antihypertensive effect in SHR but no change in BP was found when the peptide was administered via intravenous injection to WKY rat [96]. Thus, the hypotensive effect of some food-derived peptides may be specific to the hypertensive state of the animal. The effect of fermented milk with LTPs on BP and vascular function in salt-loaded type II diabetic Goto–Kakizaki rats has also been assessed. GK rats are characterized by impaired glucose-induced insulin secretion, abnormal glucose regulation, insulin resistance and polyuria. They are normotensive but when on a high-salt diet can develop hypertension. The study showed a significant decrease in BP and enhanced endothelium-dependent relaxation of mesenteric arteries [97].

There are wide variations in BP responses from different food proteins. These variations may be due to the different food sources themselves but also may be due to differences in experimental models such as the type of animal used, the dosage of peptide required for a significant decrease in BP, duration of administration and administration route, i.e., oral versus intravenous administration. In general, it has been found that peptides administered intravenously have a higher decrease in BP than peptides administered orally. This may be due to lower bioavailability of these peptides in the blood stream as transport of the peptides across the brush border membrane in an intact state may not be possible. Hydrolysis or partial hydrolysis of the peptides by GI and serum enzymes may lead to inactive or less active hypotensive peptide forms. Thus, bioavailability studies are essential to assess the antihypertensive potential of a peptide [3].

Furthermore, it must be noted that although dietary peptides have lower ACE IC50 *in vitro* in comparison to the synthetic ACE drug inhibitor Captopril (IC50 in nM range), in most cases they display higher *in vivo* hypotensive effects than are expected with respect to their *in vitro* results. It has been suggested that this may be due to a higher affinity of dietary peptides to the tissues and a slower elimination in comparison to Captopril. Moreover, it is possible that several BP mechanisms of action are being employed [30; 98]. It was demonstrated that neither the egg-protein derived peptide ovokinin (2-7) (Arg-Ala-Asp-His-Pro-Phe) or Arg-Pro-Leu-Lys-Pro-Trp, the most potent derivative obtained from the structural modification of ovokinin,, inhibit ACE *in vitro*. IC50 values obtained were >1000 μmol/L, despite having a significant effect on BP when orally administered to SHR [99]. Thus, it must be acknowledged that *in vitro* ACE inhibitory determination may not be the best approach to assess the potential of a peptide as an antihypertensive agent. In a study by da Costa *et al* [100] it was found that the most potent *in vitro* ACE inhibitory peptides from whey did not have a significant effect on BP when orally administered to SHR. However, whey peptides with relatively low *in vitro* ACE-inhibitory activity in comparison achieved significant reductions in BP.

#### **6. Human studies**

56 Bioactive Food Peptides in Health and Disease

observed hypotensive effect [95].

endothelium-dependent relaxation of mesenteric arteries [97].

to assess the antihypertensive potential of a peptide [3].

Krebs containing Ile-Pro-Pro and Val-Pro-Pro (1 mM), when mounted in an organ bath. Vascular reactivity measurements demonstrated better preservation of endotheliumdependent relaxation in arteries stored with the LTPs compared to controls [94]. Their bioactive effect in double transgenic rats (dTGR) harbouring human renin and angiotensinogen genes was also assessed. These transgenic rats develop malignant hypertension, cardiac hypertrophy, renal damage, and endothelial dysfunction due to increased Ang II formation. A decrease of 19 mm Hg in SBP was seen in rats administered with fermented milk supplemented with the peptides (Ile-Pro-Pro (1.8 mg/100 ml) and Val-Pro-Pro (1.8 mg/100 ml) compared to the control group Thus, it was concluded that the supplemented fermented milk product can aid in preventing the development of malignant hypertension. There was no effect on BP reported from a group receiving the peptides dissolved in water, despite the higher intake level of peptides. The authors concluded that the reported antihypertensive effect of the fermented milk product can not be explained solely by the Ile-Pro-Pro and Val-Pro-Pro supplements and suggested that a combination of factors such as calcium and potassium content, and less sodium may have contributed to the

Other rat models, such as the normotensive WKY rat, have been used to evaluate the effect of food peptides on arterial BP. However, a significant hypotensive effect is not always observed in WKY. Single oral administration of Ameal S containing the LTPs decreased BP from 6 to 8 h after administration in SHR. However, no change in SBP was observed in normotensive WKYs [88]. Similarly, the ACE inhibitory peptide Leu-Arg-Pro-Val-Ala-Ala from bovine lactoferrin was found to have a significant antihypertensive effect in SHR but no change in BP was found when the peptide was administered via intravenous injection to WKY rat [96]. Thus, the hypotensive effect of some food-derived peptides may be specific to the hypertensive state of the animal. The effect of fermented milk with LTPs on BP and vascular function in salt-loaded type II diabetic Goto–Kakizaki rats has also been assessed. GK rats are characterized by impaired glucose-induced insulin secretion, abnormal glucose regulation, insulin resistance and polyuria. They are normotensive but when on a high-salt diet can develop hypertension. The study showed a significant decrease in BP and enhanced

There are wide variations in BP responses from different food proteins. These variations may be due to the different food sources themselves but also may be due to differences in experimental models such as the type of animal used, the dosage of peptide required for a significant decrease in BP, duration of administration and administration route, i.e., oral versus intravenous administration. In general, it has been found that peptides administered intravenously have a higher decrease in BP than peptides administered orally. This may be due to lower bioavailability of these peptides in the blood stream as transport of the peptides across the brush border membrane in an intact state may not be possible. Hydrolysis or partial hydrolysis of the peptides by GI and serum enzymes may lead to inactive or less active hypotensive peptide forms. Thus, bioavailability studies are essential The majority of the clinical trials regarding the antihypertensive effects of milk-derived peptides to date have been carried out on the LTPs, Ile-Pro-Pro and Val-Pro-Pro. Although some conflicting results exist, the majority of these trials have reported a significant decrease in BP. Their effect on office BP has been well documented (for reviews see 101; 3; 25). It is essential that the BP of test subjects is evaluated in comparison to placebo values and not to baseline values of the test product. As with test products, placebo groups have been found to often decrease in BP over the test period [101]. Furthermore, the 24-h ambulatory BP monitoring (ABPM), of patients BP is thought to be a more reliable method for evaluation of BP as it reduces the 'white coat effect'. Recent clinical trials with LTPs have employed ABPM [102-104; 91]. ABPM was used to assess the effects of LTP administration on dipper (where BP decreases at night time) and non-dipper (where BP does not decrease at night time) hypertensive subjects. Non-dippers are thought to have a higher cardiac vascular risk and BP monitoring in the morning and night can help predict cardiac events such as stroke and myocardial infarction. Twelve patients received a fermented milk product containing Ile-Pro-Pro (1.52 mg) and Val-Pro-Pro (2.53 mg) daily for 4 weeks. The study reported a significant reduction in night-time and early-morning SBP for nondipper subjects but not for dipper subjects [105]. A range of hemodynamic parameters was recently evaluated for 52 human subjects with high-normal BP or first-degree hypertension. These included office BP and ABPM, stress-induced BP increase and cardiac output-related parameters. Subjects were treated with LTPs (3 mg/day) for 6 weeks. The study reported a reduction in office SBP as well as an improvement in pulse wave velocity (an instrumental biomarker for vascular rigidity), stroke volume and stroke volume index (markers of cardiac flow) and acceleration and velocity index (markers of cardiac contractility). No effect on ABPM and BP reaction to stress was observed [106]. LTPs have also been reported to reduce arterial stiffness in

humans. In a double-blind parallel group intervention study, 89 hypertensive subjects received daily milk containing a low dose of 5 mg/day of Ile-Pro-Pro and Val-Pro-Pro for 12 weeks and a dose of 50 mg/day for the following 12 weeks. Arterial stiffness, measured by the augmentation index (AI), decreased in the peptide group by -1.53% compared to 1.20% in the placebo group at the end of the second intervention period [107]. A similar result was seen in a study by Nakamura *et al* [108]. Twelve hypertensive subjects were administered four tablets containing Val-Pro-Pro (2.05 mg) and Ile-Pro-Pro (1.13 mg) daily for 9 weeks and were monitored for various hemodynamic parameters. A significant reduction in AI as well as peripheral SBP and DBP along with central SBP (cSBP) was observed. Furthermore, it has been suggested that LTPs may also have a positive effect on vascular endothelial function in subjects with stage-I hypertension [109] and may improve arterial compliance in postmenopausal women [110].

Antihypertensive Peptides from Food Proteins 59

and retain their bioactivity during multi-step processing including pasteurisation, homogenisation, pressure-driven membrane-based processing such as ultrafiltration and nanofiltration, dehydration by spray-drying or freeze-drying, and peptides must also be stable during long-term storage. Little data exists on the effects of different processing techniques on BAPs in food products. Dehydration via spray drying has been found to produce changes in peptide confirmation, a reduction in amino acid content and may also lead to non-enzymatic browning reactions [43]. The industrial-scale production of a casein hydrolysate containing the antihypertensive peptides Arg-Tyr-Leu-Gly-Tyr (αs1-CN f90- 94) and Ala-Tyr-Phe-Tyr-Pro-Glu-Leu (αs1-CN f143-149) and the stability of the hydrolysate incorporated in a yoghurt to processing conditions, i.e. drying, homogenisation and pasteurisation, and to storage at 4°C was recently investigated. The study showed the hydrolysate to be stable after processing as both *in vitro* ACE-inhibitory activity and the *in vivo* antihypertensive properties in SHR were maintained. Analysis by reverse phase-high pressure liquid chromatography-mass spectrometry (RP-HPLC-MS) showed that the integrity of the antihypertensive peptides was also maintained during storage at 4 °C for 1 month [120]. Similar studies are required for other antihypertensive peptides in order to evaluate the optimal processing conditions required for retention of

It has been shown that heat treatments and mechanical damage can reduce peptides bioactivity. As a result of changes in protein structure, the profile of peptides released may differ as digestive enzymes may be capable of digesting these regions of the protein that were previously inaccessible to the enzyme. This has been previously shown to be the case for whey protein [121-122]. Furthermore, ACE inhibitory peptides from whey protein isolates (WPI) pretreated at 65 °C were shown to have greater inhibitory activity than peptides from WPI pretreated at 95 °C. This result can be explained by the formation of

Optimisation of the hydrolytic process should also be considered when planning to up-scale the production of BAPs. Bioactive peptides may be produced by enzymatic or microbial hydrolysis. However, it is thought that enzymatic hydrolysis is more suited for food-grade BAP production over microbial fermentation [123]. Enzyme immobilisation offers several advantages over the addition of soluble enzymes directly to the product. They can be recycled and the use of immobilized enzymes potentially avoids the generation of interfering metabolite products due to autolysis of the enzymes. Furthermore, protein hydrolysis using immobilized enzymes can also be carried out in milder more controlled conditions and does not need to be inactivated by heat or acidification, which may be damaging for the product [124; 123]. The use of membrane bioreactors may be a substitute for the development of functional materials from food proteins. This system integrates a reaction vessel with a membrane separation system allowing for the recycling of the enzyme, separation, fractionation and/or concentration of the bioactive compound. The use of membrane bioreactors for the development of functional materials from sea-food

bioactivity.

whey aggregates [100].

processing wastes has been recently reviewed [125].

Other hypotensive peptides and food preparations used in human trials include peptides from casein [111], whey [112], dried bonito [113], fermented milk containing gammaaminobutyric acid (GABA; 114) sardine muscle [115] and wakame (*Undaria pinnatifida;* 116)*.*  Recently, a number of meta-analyses on antihypertensive peptides have been carried out. Pripp *et al* [117] performed a meta-analysis on antihypertensive peptides from milk and fish proteins which included 15 human trials. A pooled decrease in SBP of -5.13 mm Hg and a decrease of -2.42 mm Hg for DBP were found. A similar result was found with a metaanalysis of 12 trials with LTPs, (623 participants in total) when pooled data in forest plots found a decrease of -4.8 mm Hg and 2.2 mm Hg in SBP and DBP, respectively. The observed hypotensive effects also seemed to be greater in hypertensive patients than in patients with pre-hypertension [118]. Another meta-analysis carried out on data from LTPs trials interestingly found that the effect of LTPs on BP was more evident in Asian subjects (SBP = - 6.93 mm Hg; DBP=-3.98 mm Hg) than in Caucasians (SBP=-1.17 mm Hg; DBP = -0.52 mm Hg). The study also found that the LTP-induced hypotensive effects were not related to subject age, baseline BP value, administered dose or length of treatment [119]. Conflicting results however, were reported in a recent meta-analysis by Usinger *et al* [132]. Data from 15 controlled trials (1232 subjects in total) that observed the effect of fermented milk or similar products produced by *Lactobacilli* fermentation of milk proteins were used in the metaanalysis. The study reported a pooled decrease in SBP of just -2.45 mm Hg and found no significant decrease for pooled DBP data. Furthermore, the authors stated that the included studies were of variable quality and when excluding the studies with a high risk of bias no significant decrease in SBP or DBP were found.

## **7. Hypotensive peptides as functional food ingredients**

The main considerations which need to be taken into account in the utilisation of antihypertensive peptides as functional ingredients in food products include characterisation of their organoleptic and physicochemical properties. In the first instance, establishment of the optimal method for peptide release via protein hydrolysis is required. Industrial scale processing of BAPs requires that peptides be able to withstand and retain their bioactivity during multi-step processing including pasteurisation, homogenisation, pressure-driven membrane-based processing such as ultrafiltration and nanofiltration, dehydration by spray-drying or freeze-drying, and peptides must also be stable during long-term storage. Little data exists on the effects of different processing techniques on BAPs in food products. Dehydration via spray drying has been found to produce changes in peptide confirmation, a reduction in amino acid content and may also lead to non-enzymatic browning reactions [43]. The industrial-scale production of a casein hydrolysate containing the antihypertensive peptides Arg-Tyr-Leu-Gly-Tyr (αs1-CN f90- 94) and Ala-Tyr-Phe-Tyr-Pro-Glu-Leu (αs1-CN f143-149) and the stability of the hydrolysate incorporated in a yoghurt to processing conditions, i.e. drying, homogenisation and pasteurisation, and to storage at 4°C was recently investigated. The study showed the hydrolysate to be stable after processing as both *in vitro* ACE-inhibitory activity and the *in vivo* antihypertensive properties in SHR were maintained. Analysis by reverse phase-high pressure liquid chromatography-mass spectrometry (RP-HPLC-MS) showed that the integrity of the antihypertensive peptides was also maintained during storage at 4 °C for 1 month [120]. Similar studies are required for other antihypertensive peptides in order to evaluate the optimal processing conditions required for retention of bioactivity.

58 Bioactive Food Peptides in Health and Disease

postmenopausal women [110].

significant decrease in SBP or DBP were found.

**7. Hypotensive peptides as functional food ingredients** 

The main considerations which need to be taken into account in the utilisation of antihypertensive peptides as functional ingredients in food products include characterisation of their organoleptic and physicochemical properties. In the first instance, establishment of the optimal method for peptide release via protein hydrolysis is required. Industrial scale processing of BAPs requires that peptides be able to withstand

humans. In a double-blind parallel group intervention study, 89 hypertensive subjects received daily milk containing a low dose of 5 mg/day of Ile-Pro-Pro and Val-Pro-Pro for 12 weeks and a dose of 50 mg/day for the following 12 weeks. Arterial stiffness, measured by the augmentation index (AI), decreased in the peptide group by -1.53% compared to 1.20% in the placebo group at the end of the second intervention period [107]. A similar result was seen in a study by Nakamura *et al* [108]. Twelve hypertensive subjects were administered four tablets containing Val-Pro-Pro (2.05 mg) and Ile-Pro-Pro (1.13 mg) daily for 9 weeks and were monitored for various hemodynamic parameters. A significant reduction in AI as well as peripheral SBP and DBP along with central SBP (cSBP) was observed. Furthermore, it has been suggested that LTPs may also have a positive effect on vascular endothelial function in subjects with stage-I hypertension [109] and may improve arterial compliance in

Other hypotensive peptides and food preparations used in human trials include peptides from casein [111], whey [112], dried bonito [113], fermented milk containing gammaaminobutyric acid (GABA; 114) sardine muscle [115] and wakame (*Undaria pinnatifida;* 116)*.*  Recently, a number of meta-analyses on antihypertensive peptides have been carried out. Pripp *et al* [117] performed a meta-analysis on antihypertensive peptides from milk and fish proteins which included 15 human trials. A pooled decrease in SBP of -5.13 mm Hg and a decrease of -2.42 mm Hg for DBP were found. A similar result was found with a metaanalysis of 12 trials with LTPs, (623 participants in total) when pooled data in forest plots found a decrease of -4.8 mm Hg and 2.2 mm Hg in SBP and DBP, respectively. The observed hypotensive effects also seemed to be greater in hypertensive patients than in patients with pre-hypertension [118]. Another meta-analysis carried out on data from LTPs trials interestingly found that the effect of LTPs on BP was more evident in Asian subjects (SBP = - 6.93 mm Hg; DBP=-3.98 mm Hg) than in Caucasians (SBP=-1.17 mm Hg; DBP = -0.52 mm Hg). The study also found that the LTP-induced hypotensive effects were not related to subject age, baseline BP value, administered dose or length of treatment [119]. Conflicting results however, were reported in a recent meta-analysis by Usinger *et al* [132]. Data from 15 controlled trials (1232 subjects in total) that observed the effect of fermented milk or similar products produced by *Lactobacilli* fermentation of milk proteins were used in the metaanalysis. The study reported a pooled decrease in SBP of just -2.45 mm Hg and found no significant decrease for pooled DBP data. Furthermore, the authors stated that the included studies were of variable quality and when excluding the studies with a high risk of bias no

It has been shown that heat treatments and mechanical damage can reduce peptides bioactivity. As a result of changes in protein structure, the profile of peptides released may differ as digestive enzymes may be capable of digesting these regions of the protein that were previously inaccessible to the enzyme. This has been previously shown to be the case for whey protein [121-122]. Furthermore, ACE inhibitory peptides from whey protein isolates (WPI) pretreated at 65 °C were shown to have greater inhibitory activity than peptides from WPI pretreated at 95 °C. This result can be explained by the formation of whey aggregates [100].

Optimisation of the hydrolytic process should also be considered when planning to up-scale the production of BAPs. Bioactive peptides may be produced by enzymatic or microbial hydrolysis. However, it is thought that enzymatic hydrolysis is more suited for food-grade BAP production over microbial fermentation [123]. Enzyme immobilisation offers several advantages over the addition of soluble enzymes directly to the product. They can be recycled and the use of immobilized enzymes potentially avoids the generation of interfering metabolite products due to autolysis of the enzymes. Furthermore, protein hydrolysis using immobilized enzymes can also be carried out in milder more controlled conditions and does not need to be inactivated by heat or acidification, which may be damaging for the product [124; 123]. The use of membrane bioreactors may be a substitute for the development of functional materials from food proteins. This system integrates a reaction vessel with a membrane separation system allowing for the recycling of the enzyme, separation, fractionation and/or concentration of the bioactive compound. The use of membrane bioreactors for the development of functional materials from sea-food processing wastes has been recently reviewed [125].

BAP fractionation and enrichment steps include membrane processing incorporating ultrafiltration and liquid chromatography, ion exchange, gel filtration and reverse phase matrices. Electro-membrane filtration (EMF), a combination of conventional membrane filtration and electrophoresis, may be a consideration for industrial scale isolation of BAPs. EMF is more selective than conventional membrane filtration (ultrafiltration) and is less costly than chromatography [123].

Antihypertensive Peptides from Food Proteins 61

Therefore, industrial manufacturers of functional food products need to provide a significant amount of scientific evidence that satisfies the legislative governing body in the specific market region before any new food products can be put on the market claiming to

Antihypertensive peptides have major potential as functional ingredients aiding in the prevention and management of hypertension. Although these peptides have been found to be less potent than antihypertensive synthetic drugs, as part of the daily diet they could play an important part as natural and safe BP control agents. Further detailed mechanistic studies on food protein-derived antihypertensive peptides must be carried out to elucidate the BP mechanism(s) involved. With regard to ACE-inhibitory peptides, a better understanding of the interactions involved in the binding of peptides to the active site of ACE is required such that more effective food peptide-based inhibitors of ACE can be discovered. The use of bioinformatics and *in silico* methods for identification of potential bioactive sequences may allow for more substrates to be assessed in a shorter time scale. Cost effective production methods including enrichment, isolation and purification procedures must first be considered and ease of scalability must be achieved. Before advancement of functional hypotensive products onto the market, moreover, the physicochemical, technofunctional and sensory properties must be considered prior to production of new antihypertensive

Financial support for this work was provided by the Irish Research Council for Science,

[1] Kearney PM, Whelton M, Reynolds K, Whelton PK, He J. Worldwide prevalence of

[2] Centre for Disease Control and Prevention: High Blood Pressure Frequently Asked

http://webcache.googleusercontent.com/search?q=cache:jGRR07v0WXIJ:www.cc.gov/bl

Engineering and Technology (IRCSET) in the form of a studentship to author Norris.

hypertension: a systematic review. Journal of Hypertension 2004;22 11-19.

have hypotensive effects.

**8. Conclusion** 

food products.

**Author details** 

**Acknowledgement** 

**9. References** 

Questions.

Corresponding Author

 \*

Roseanne Norris and Richard J. FitzGerald\*

*Department of Life Sciences, University of Limerick, Limerick, Ireland* 

oodpressure/faqs.htm (Accessed 22 September 2011).

As mentioned earlier, a large portion of antihypertensive peptides are of low molecular weight and many contain hydrophobic residues, attributes which have been classically associated with bitterness in foods. Hence, this is notably an obstacle that must be resolved during the processing of antihypertensive food products. A number of strategies have been applied with the aim of debittering protein hydrolysates including absorption of bitter peptides on activated carbon, selective extraction with alcohols and chromatographic removal using different matrices. Peptidase-mediated debittering has also been applied. This involves the concomitant or sequential incubation of the protein hydrolysates with exopeptidases, with priority cleavage at hydrophobic residues [126]. However, these debittering strategies may lead to the loss of some amino acid residues from hydrolysates. As bioactivity relies greatly on peptide sequence, these debittering methods may not therefore be suitable for debittering of BAPs including antihypertensive peptides, as hydrolysis may result in loss of activity. Changes in peptide structure may also have implications for absorption at the brush border membrane. Therefore, enzymatic debittering strategies need to be approached on a case-by-case basis.

The widespread commercialisation of antihypertensive food products is dependent on the availability of scientific data from *in vivo* animal and human models that positively demonstrates their contribution in reducing BP. Furthermore, legislation which governs health claims in relation to functional foods needs to be taken into account. In Japan, the FOSHU (food for specified health use) licensing system was put in place whereby foods claiming health benefits must first be approved by the system before been allowed to be put on the market [127]. Since then a number of antihypertensive products currently on the market in Japan have been granted FOSHU approval. 'Ameal-S' which is manufactured by Calpis Co., Ltd. is a fermented sour milk containing the LTPs Ile-Pro-Pro and Val-Pro-Pro. The soft drink Casein DP 'Peptio' manufactured by Kanebo Co., Ltd. contains the antihypertensive peptide Phe-Phe-Val-Ala-Pro-Phe-Pro-Gln-Val-Phe-Gly-Phe (αs1-casein f23–34) and is also FOSHU approved. There has been a new European Regulation on nutrition and health claims in the EU since 2007 (Regulation 1924/2006). Advised by the European Commission (EC), the European Food Safety Authority (EFSA) reviews evidence of health claims made by food companies. Interestingly, EFSA has not allowed/approved any peptide related hypotensive claim to date [128-129]. For both the C12-peptide and the bonito protein-derived peptide Leu-Lys-Pro-Asn-Met, it was concluded that a cause and effect relationship has not been established between the consumption of the peptides and maintenance of normal BP [128-129]. In the US, the Food and Drug Administration (FDA) assesses the scientific evidence for health claims under the 1990 Nutrition Labelling and Education Act [130] and the 1994 Dietary Supplement Health and Education Act [131]. Therefore, industrial manufacturers of functional food products need to provide a significant amount of scientific evidence that satisfies the legislative governing body in the specific market region before any new food products can be put on the market claiming to have hypotensive effects.

## **8. Conclusion**

60 Bioactive Food Peptides in Health and Disease

costly than chromatography [123].

strategies need to be approached on a case-by-case basis.

BAP fractionation and enrichment steps include membrane processing incorporating ultrafiltration and liquid chromatography, ion exchange, gel filtration and reverse phase matrices. Electro-membrane filtration (EMF), a combination of conventional membrane filtration and electrophoresis, may be a consideration for industrial scale isolation of BAPs. EMF is more selective than conventional membrane filtration (ultrafiltration) and is less

As mentioned earlier, a large portion of antihypertensive peptides are of low molecular weight and many contain hydrophobic residues, attributes which have been classically associated with bitterness in foods. Hence, this is notably an obstacle that must be resolved during the processing of antihypertensive food products. A number of strategies have been applied with the aim of debittering protein hydrolysates including absorption of bitter peptides on activated carbon, selective extraction with alcohols and chromatographic removal using different matrices. Peptidase-mediated debittering has also been applied. This involves the concomitant or sequential incubation of the protein hydrolysates with exopeptidases, with priority cleavage at hydrophobic residues [126]. However, these debittering strategies may lead to the loss of some amino acid residues from hydrolysates. As bioactivity relies greatly on peptide sequence, these debittering methods may not therefore be suitable for debittering of BAPs including antihypertensive peptides, as hydrolysis may result in loss of activity. Changes in peptide structure may also have implications for absorption at the brush border membrane. Therefore, enzymatic debittering

The widespread commercialisation of antihypertensive food products is dependent on the availability of scientific data from *in vivo* animal and human models that positively demonstrates their contribution in reducing BP. Furthermore, legislation which governs health claims in relation to functional foods needs to be taken into account. In Japan, the FOSHU (food for specified health use) licensing system was put in place whereby foods claiming health benefits must first be approved by the system before been allowed to be put on the market [127]. Since then a number of antihypertensive products currently on the market in Japan have been granted FOSHU approval. 'Ameal-S' which is manufactured by Calpis Co., Ltd. is a fermented sour milk containing the LTPs Ile-Pro-Pro and Val-Pro-Pro. The soft drink Casein DP 'Peptio' manufactured by Kanebo Co., Ltd. contains the antihypertensive peptide Phe-Phe-Val-Ala-Pro-Phe-Pro-Gln-Val-Phe-Gly-Phe (αs1-casein f23–34) and is also FOSHU approved. There has been a new European Regulation on nutrition and health claims in the EU since 2007 (Regulation 1924/2006). Advised by the European Commission (EC), the European Food Safety Authority (EFSA) reviews evidence of health claims made by food companies. Interestingly, EFSA has not allowed/approved any peptide related hypotensive claim to date [128-129]. For both the C12-peptide and the bonito protein-derived peptide Leu-Lys-Pro-Asn-Met, it was concluded that a cause and effect relationship has not been established between the consumption of the peptides and maintenance of normal BP [128-129]. In the US, the Food and Drug Administration (FDA) assesses the scientific evidence for health claims under the 1990 Nutrition Labelling and Education Act [130] and the 1994 Dietary Supplement Health and Education Act [131]. Antihypertensive peptides have major potential as functional ingredients aiding in the prevention and management of hypertension. Although these peptides have been found to be less potent than antihypertensive synthetic drugs, as part of the daily diet they could play an important part as natural and safe BP control agents. Further detailed mechanistic studies on food protein-derived antihypertensive peptides must be carried out to elucidate the BP mechanism(s) involved. With regard to ACE-inhibitory peptides, a better understanding of the interactions involved in the binding of peptides to the active site of ACE is required such that more effective food peptide-based inhibitors of ACE can be discovered. The use of bioinformatics and *in silico* methods for identification of potential bioactive sequences may allow for more substrates to be assessed in a shorter time scale. Cost effective production methods including enrichment, isolation and purification procedures must first be considered and ease of scalability must be achieved. Before advancement of functional hypotensive products onto the market, moreover, the physicochemical, technofunctional and sensory properties must be considered prior to production of new antihypertensive food products.

## **Author details**

Roseanne Norris and Richard J. FitzGerald\* *Department of Life Sciences, University of Limerick, Limerick, Ireland* 

## **Acknowledgement**

Financial support for this work was provided by the Irish Research Council for Science, Engineering and Technology (IRCSET) in the form of a studentship to author Norris.

### **9. References**


http://webcache.googleusercontent.com/search?q=cache:jGRR07v0WXIJ:www.cc.gov/bl oodpressure/faqs.htm (Accessed 22 September 2011).

<sup>\*</sup> Corresponding Author

[3] Murray BA, FitzGerald RJ. Angiotensin converting enzyme inhibitory peptides derived from proteins: biochemistry, bioactivity, and production. Current Pharmaceutical Design 2007;13(8) 773-91.

Antihypertensive Peptides from Food Proteins 63

experiment to semi-continuous model. Journal of Agricultural Food Chemistry 2003;51

[20] Hernandez-Ledesma B, Amigo L, Ramos M, Recio I. Angiotensin converting enzyme inhibitory activity in commercial fermented products. Formation of peptides under simulated gastrointestinal digestion. Journal of Agricultural Food Chemistry 2004;52(6)

[21] Walsh DJ, Bernard H, Murray BA, MacDonald J, Pentzien AK, Wright GA, Wal JM, Struthers AD, Meisel H, FitzGerald RJ. *In vitro* generation and stability of the lactokinin

[23] Cinq-Mars CD, Hu C, Kitts DD, Li-Chan ECY. Investigations into inhibitor type and mode, simulated gastrointestinal digestion, and cell transport of angiotensin Iconverting enzyme-inhibitory peptides in pacific hake (*Meriuccius productus*) fillet

[24] Akillioglu HG, Karakaya S. Effects of heat treatment and *in vitro* digestion on the angiotensin converting enzyme inhibitory activity of some legume species. European

[25] Jäkälä P, Vapaatalo H. Antihypertensive peptides from milk proteins. Pharmaceuticals

[26] Shin AI, Yu R, Park SA, Chung DK, Ahn CW, Nam HS, Kim HS, Lee HJ. His-His-Leu, an angiotensin converting enzyme inhibitory peptide derived from Korean soybean paste, exerts antihypertensive activity *in vivo.* Journal of Agricultural Food Chemistry

[27] Okamoto A, Hanagata H, Matsumoto E, Kawamura Y, Koizumi Y, Yanagiga F. Angiotensin I converting enzyme inhibitory activities of various fermented foods.

[28] Nakahara T, Sano T, Yamaguchi H, Sugimoto KRI, Chikata H, Kinoshita E, Uchida R. Antihypertensive effect of peptide-enriched soy sauce-like seasoning and identification of its angiotensin I-converting enzyme inhibitory substances. Journal of Agricultural

[29] Gibbs BF, Zoygman A, Masse R, Mulligan C. Production and characterisation of bioactive peptides from soy hydrolysates and soy-fermented food. Food Research

[30] Fujita H, Yoshikawa M. LKPNM: a prodrug-type ACE-inhibitory peptide derived from

[32] Udenigwe CC, Li H, Aluko RE. Quantitative structure-activity relationship modelling

[31] Staessen JA, Li Y, Richart T. Oral renin inhibitors. Lancet 2006;368 1449-1456.

of renin-inhibiting dipeptides. Amino Acids 2011;42(4) 1379-1386.

Bioscience, Biotechnology, & Biochemistry 1995;59(6) 1147-1149.

fish protein. Immunopharmacology 1999;44(1-2) 123-127.

hydrolysate. Journal of Agricultural Food Chemistry 2008;56(2) 410-419.

Food Research and Technology 2009;229(6) 915-921.

*β*-lactoglobulin fragment (142-148). Journal of Dairy Science 2004;87 3845–3857. [22] Jang A, Jo C, Lee M. Storage stability of the synthetic angiotensin converting enzyme (ACE) inhibitory peptides separated from beef sarcoplasmic protein extracts at different pH, temperature, and gastric digestion. Food Science and Biotechnology 2007;16(4) 572-

5680-5687.

1504-1510.

575.

2010;3 251-272.

2001;49(6) 3004-3009.

Food Chemistry 2010;58(2) 821-827.

International 2004;37(2) 123-131.


experiment to semi-continuous model. Journal of Agricultural Food Chemistry 2003;51 5680-5687.

[20] Hernandez-Ledesma B, Amigo L, Ramos M, Recio I. Angiotensin converting enzyme inhibitory activity in commercial fermented products. Formation of peptides under simulated gastrointestinal digestion. Journal of Agricultural Food Chemistry 2004;52(6) 1504-1510.

62 Bioactive Food Peptides in Health and Disease

Design 2007;13(8) 773-91.

Journal of Nutrition 2004;134(4) 980S-988S.

Meat Science 2010;86 110-118.

1320–1325.

[3] Murray BA, FitzGerald RJ. Angiotensin converting enzyme inhibitory peptides derived from proteins: biochemistry, bioactivity, and production. Current Pharmaceutical

[4] Martίnez-Maqueda D, Miralles B, Recio I, Hernández-Ledesma B. Antihypertensive

[7] FitzGerald RJ, Murray BA, Walsh DJ. Hypotensive peptides from milk proteins. The

[8] Libby P, Bonow RO, Mann DL, Zipes DP. Braunwald's Heart Disease: A textbook of

[9] Meisel H, Walsh DJ, Murray BA, FitzGerald RJ. ACE Inhibitory Peptides. In: Mine Y, Shahidi F. (ed.) Nutraceutical proteins and peptides in health and disease. New York:

[10] Guang C, Phillips RD. Plant food-derived angiotensin I converting enzyme inhibitory

[11] Wijesekara I, Kim SK. Angiotensin-I-converting enzyme (ACE) inhibitors from marine resources: prospects in the pharmaceutical industry. Marine Drugs 2010;8(4) 1080–1093. [12] Ahhmed AM, Muguruma M. A review of meat protein hydrolysates and hypertension.

[13] Li Y, Zhou J, Huang K, Sun Y, Zeng X. Purification of a Novel Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptide with an Antihypertensive Effect from Loach (*Misgurnus anguillicaudatus*). Journal of Agricultural and Food Chemistry 2012;60(5)

[14] Escudero E, Sentandreu MA, Arihara K, Toldrá F. Angiotensin I-converting enzyme inhibitory peptides generated from *in vitro* gastrointestinal digestion of pork meat.

[15] Chel-Guerrero L, Domínguez-Magaña M, Martínez-Ayala A, Dávila-Ortiz G, Betancur-Ancona D. Lima bean (*Phaseolus lunatus*) protein hydrolysates with ACE-I inhibitory

[16] Lee JK, Jeon JK, Byun HG. Effect of angiotensin I converting enzyme inhibitory peptide

[17] Terashima M, Bara T, Ikemoto N, Katayama M, Morimoto T, Matsumura S. Novel angiotensin converting enzyme (ACE) inhibitory peptides derived from boneless

[18] Chabance B, Marteau P, Rambaud JC, Migliore-Samour D, Bynard M, Perrotin P, Guillet R, Jollés P, & Fiat AM. Casein peptide release and passage to the blood in

[19] Vermeirssen V, Van Camp J, Devos L, Verstraete W. Release of angiotensin I converting enzyme inhibitory activity during *in vitro* gastrointestinal digestion: from batch

purified from skate skin hydrolysate. Food Chemistry 2011;125(2) 495-499.

chicken leg meat. Agricultural and Food Chemistry 2010;58 7432-7436.

humans during digestion of milk and yoghurt*.* Biochimie 1998;80 155-165.

peptides. Journal of Agricultural Food Chemistry 2009;57(12) 5113–5120.

peptides from food proteins: a review. Food & Function 2012;3 350-361. [5] Inagami T. The renin-angiotensin system. Essays in Biochemistry 1994;28 147-164. [6] Turner AJ, Hooper NM. The angiotensin converting enzyme gene family, genomics and

pharmacology. Trends in Pharmacological Science 2002;23 177-183.

cardiovascular Medicine (8th ed.). Philadelphia: Saunders; 2008.

Journal of Agricultural and Food Chemistry 2010;58 2895–2901.

activity. Food and Nutrition Science 2012;3 511-521.

CRC Press, Taylor and Francis Group; 2006. p269-315.


[33] Udenigwe CC, Lin YS, Hou WC, Aluko RE. Kinetics of the inhibition of renin and angiotensin I-converting enzyme by flaxseed protein hydrolysate fractions. Journal of Functional Foods 2009;1(2) 199–207.

Antihypertensive Peptides from Food Proteins 65

[47] Okitsu M, Morita A, Kakitani M, Okada M, Yokogoshi H. Inhibition of the endothelinconverting enzyme by pepsin digests of food proteins. Bioscience, Biotechnology, &

[48] Maes W, Van Camp J, Vermeirssen V, Hemeryck M, Ketelslegers JM, Schrezenmeir J, Van Oostveldt P, Huyghebaert A. Influence of the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR) on the release of endothelin-1 by endothelial cells. Regulatory peptides

[49] Gomez-Ruiz JA, Ramos M, Recio I. Angiotensin converting enzyme inhibitory activity of peptides isolated from Manchego cheese. Stability under simulated gastrointestinal

[50] Ondetti MA, Rubin B, Cushman DW. Design of specific inhibitors of angiotensinconverting enzyme: new class of orally active antihypertensive agents. Science

[51] Cheung HS, Wang FL, Ondetti MA, Sabo EF, Cushman DW. Binding of peptide substrates and inhibtiors of angiotensin-converting enzyme: importance of the COOHterminal dipeptide sequence. Journal of Biological Chemistry 1980;255(2) 401-407. [52] Gobbetti M, Stepaniak L, De Angelis M, Corsetti A, Cagno RD. Latent bioactive peptides in milk proteins: proteolytic activation and significance in dairy processing.

[53] López-Fandiño R, Otte J, van Camp J. Physiological, chemical and technological aspects of milk-protein-derived peptides with antihypertensive and ACE-inhibitory activity.

[54] Pripp, AH, Isakasson T, Stepaniak L, Sorhaug T, Ardo Y. Quantitative structure activity relationship modelling of peptides and proteins as a tool in food science. Trends in

[55] Wu J, Aluko RE, Nakai S. Structural requirements of angiotensin 1-converting enzyme inhibitory peptides: quantitative structure-activity relationship study of di- and

[56] Wu J, Aluko RE, Nakai S. Structural requirements of angiotensin 1-converting enzyme inhibitory peptides: quantitative structure-activity relationship modelling of peptides containing 4-10 amino acid residues. QSAR and Combinatorial Science 2006;25 873-880. [57] He R, Ma H, Zhao W, Qu W, Zhao J, Luo L, Zhu W. Modelling the QSAR of ACEinhibitory peptides with ANN and its applied illustration. International Journal of

[58] Norris R, Casey F, FitzGerald RJ, Shields D, Mooney C. Predictive modelling of angiotensin converting enzyme inhibitory dipeptides. Food Chemistry 2012;133(4),

[59] Murray BA, Walsh DJ, FitzGerald RJ. Modification of the furanacryloyl-Lphenylalanylglycylglycine assay for determination of angiotensin-I-converting enzyme inhibitory activity. Journal of Biochemical and Biophysical Methods 2004;59(2) 127-137.

tripeptides. Journal of Agricultural & Food Chemistry 2006;54 732-738.

digestion. International Dairy Journal 2004;14 1075-1080.

Critical Reviews in Food Science and Nutrition 2002;42 223-239.

International Dairy Journal 2006;16 1277-1293.

Food Science and Technology 2005;16 484-494.

Biochemistry 1995;59(2) 325-326.

2004;118(1-2) 105-109.

1977;196(4288) 441-444.

Peptides 2011;2012 1-9.

1349–1354.


[47] Okitsu M, Morita A, Kakitani M, Okada M, Yokogoshi H. Inhibition of the endothelinconverting enzyme by pepsin digests of food proteins. Bioscience, Biotechnology, & Biochemistry 1995;59(2) 325-326.

64 Bioactive Food Peptides in Health and Disease

11471–11476.

2007;137 825S-829S.

Sciences 2000;66 1535-1543.

Interface Science 2011;165 23-35.

Biochemistry 1995;59 325–326.

mesenteric artery. *FEBS Letters* 1999;452(3) 181-184.

Functional Foods 2009;1(2) 199–207.

British Journal of Nutrition 2000;1 S27-S31.

[33] Udenigwe CC, Lin YS, Hou WC, Aluko RE. Kinetics of the inhibition of renin and angiotensin I-converting enzyme by flaxseed protein hydrolysate fractions. Journal of

[34] Li H, Aluko RE. Identification and inhibitory properties of multifunctional peptides from pea protein hydrolysate. Journal of Agricultural and Food Chemistry 2010;58

[35] Tanaka M, Watanabe S, Wang Z, Matsumoto K, Matsui T. His-Arg-Trp potently attenuates contracted tension of thoracic aorta of Sprague-Dawley rats through the

[36] Wang Z, Watanabe S, Kobayashi Y, Tanaka M, Matsui T. Trp-His, a vasorelaxant dipeptide, can inhibit extracellular Ca2+ entry to rat vascular smooth muscle cells through blockade of dihydropyridine-like L-type Ca2+ channels. Peptides 2010;31(11) 2060-2066. [37] Meisel H, FitzGerald RJ. Opioid peptides encrypted in intact milk protein sequences.

[38] Jauhiainen T, Korpela R. Milk peptides and blood pressure. The Journal of Nutrition

[39] Nurminen ML, Sipola M, Kaarto H, Pihlanto-Leppala A, Piilola K, Korpela R, Tossavainen O, Korhonen H, Vapaatalo H. Alpha-lactorphin lowers blood pressure measured by radiotelemetry in normotensive and spontaneously hypertensive rats. Life

[40] Sipola M, Finckenberg P, Vapaatalo H, Pihlanto-Leppälä A, Korhonen H, Korpela R, Nurminen ML. Alpha-lactorphin and beta-lactorphin improve arterial function in

[41] Yoshikawa M., Tani F., Shiota A., Suganuma H., Usui H., Kurahashi K., Chiba H. Casoxin D, an opioid antagonist/ileum-contracting/vasorelaxing peptide derived from human αs1-casein. In: Brantl V, Teschemacher H. (eds.) β-Casomorphins and related

[42] Kato M, Fujiwara Y, Okamoto A, Yoshikawa M, Chiba H, Shigezo U. Efficient production of Casokin D, a bradykinin agonist peptide derived from human casein, by

[44] Okitsu M, Morita A, Kakitani M, Okada M, Yokogoshi H. Inhibition of the endothelinconverting enzyme by pepsin digests of food proteins. Bioscience Biotechnology

[45] Matoba N, Usui H, Fujita H, Yoshikawa M. A novel anti-hypertensive peptide derived from ovalbumin induces nitric oxide-mediated vasorelaxation in an isolated SHR

[46] Scruggs P, Filipeanu CM, Yang J, Chang JK, Dun NJ. Interaction of ovokinin(2–7) with

vascular bradykinin 2 receptors. *Regulatory Peptides* 2004;120(1-3) 85-91.

*Bacillus brevis*. Bioscience, Biotechnology, & Biochemistry 1995;59(11) 2056-2059. [43] Hernández-Ledesma B, del Mar Contreras M, Recio I. Antihypertensive peptides: production, bioavailability and incorporation into foods. Advances in Colloid and

spontaneously hypertensive rats. Life Sciences 2002;71(11) 1245-1253.

peptides: Recent developments. Weinheim: VCH; 1994. p43–48.

suppression of extracellular Ca2+ influx. Peptides 2009;30(8) 1502-1507.


[60] Vermeirssen V, Van Camp J, Verstraete W. Bioavailability of angiotensin I converting enzyme inhibitory peptide. British Journal of Nutrition 2004;92 357-366.

Antihypertensive Peptides from Food Proteins 67

gastrointestinal tract: analyses using an in vitro model of mammalian gastrointestinal

[73] Satake M, Enjoh M, Nakamura Y, Takano T, Kawamura Y, Arai S, Shimizu M. Transepithelial transport of the bioactive tripeptide, Val-Pro-Pro, in human intestinal Caco-2 cell monolayers. Bioscience, Biotechnology & Biochemistry 2002;66 378-384. [74] Quirós A, Dávalos A, Lasunción MA, Ramos M, Recio I. Bioavailability of the antihypertensive peptide LHLPLP: Transepithelial flux of HLPLP. International Dairy

[75] van Platerink CJ, Janssen HGM, Horsten R, Haverkamp J. Quantification of ACE inhibiting peptides in human plasma using high performance liquid chromatography–

[76] Vermeirssen V, Augustijns P, Morel N, van Camp J, Opsomer A, Verstraete W. *In vitro* intestinal transport and antihypertensive activity of ACE inhibitory pea and whey

[78] Fujita H, Yokoyama K, Yoshikawa M. Classification and antihypertensive activity of angiotensin I-converting enzyme inhibitory peptides derived from food proteins.

[79] Gardener MLG. Transmucosal Passage of Intact Peptides. In: Grimble GK, Backwell FRC. (eds.) Peptides in Mammalian Metabolism. Tissue Utilisation and Clinical

[80] Adessi C, Soto C. Converting a peptide into a drug: Strategies to improve stability and

[81] Witt KA, Davis TP. CNS drug delivery: opioid peptides and the blood-brain barrier. In: Rapaka RS, Sadée W. (eds.) Drug Addiction. New York: Springer Science and Business

[82] Morris MC, Depollier J, Mery J, Heitz F, Divita G. A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nature Biotechnology 2001;19 1173-

[83] Miguel M, Aleixandre MA. Antihypertensive peptides derived from egg proteins. The

[84] Harnedy PA, FitzGerald RJ. Bioactive proteins, peptides, and amino acids from

[85] Harnedy PA, FitzGerald RJ. Bioactive peptides from marine processing waste and

[86] Ryan JT, Ross RP, Bolton D, Fitzgerald GF, Stanton C. Bioactive peptides from muscle

digests. International Journal of Food Sciences and Nutrition 2005;56(6) 415-430. [77] Foltz M, van Buren L, Klaffke W, Duchateau GS. Modeling of the relationship between dipeptide structure and dipeptide stability, permeability, and ACE inhibitory actvitiy.

digestion. Journal of Agricultural Food Chemistry 2008;56(3) 854-858.

mass spectrometry. Journal of Chromatography B 2006;830 151-157.

Journal of Food Science 2009;74 H243-H251.

Journal of Food Science 2000;65 564-569.

Targeting. London: Portland Press Ltd; 1998.

Journal of Nutrition 2006;136(6) 1457-1460.

macroalgae, Journal of Phycology 2011;47 218–232.

shellfish: a review. Journal of Functional Foods 2011;4 6-24.

sources: meat and fish. *Nutrients* 2011;74(7) H243-H251.

bioavailability.,Current Medicinal Chemistry, 2002;9 963-978.

Journal 2008;18 279-286.

Media; 2008.

1176.


gastrointestinal tract: analyses using an in vitro model of mammalian gastrointestinal digestion. Journal of Agricultural Food Chemistry 2008;56(3) 854-858.

[73] Satake M, Enjoh M, Nakamura Y, Takano T, Kawamura Y, Arai S, Shimizu M. Transepithelial transport of the bioactive tripeptide, Val-Pro-Pro, in human intestinal Caco-2 cell monolayers. Bioscience, Biotechnology & Biochemistry 2002;66 378-384.

66 Bioactive Food Peptides in Health and Disease

Chemistry 2003;51(19) 5680-5687.

Journal of Peptide Science 1999;5 289-297.

Annual Reviews in Physiology 2004;66 361–384.

Biotechnology and Biochemistry 1996;60 488–489.

absorption models. Peptides 2008;29(8) 1312-1320.

hypertensive rats, Journal of Nutrition 1996;126 3063-3068.

429-438.

731.

2002;35 367-375.

[60] Vermeirssen V, Van Camp J, Verstraete W. Bioavailability of angiotensin I converting

[61] Vermeirssen V, Van Camp J, Decroos K, Van Wijmelbeke V, Verstraete W. The impact of fermentation and the *in vitro* digestion on the formation of angiotensin-I-converting enzyme inhibitory activity from pea and whey protein. Journal of Dairy Science 2003;86

[62] Vermeirssen V, Van Camp J, Devos J, Verstraete W. Release of Angiotensin I Converting Enzyme (ACE) inhibitory activity during in vitro gastrointestinal digestion: from batch experiment to semi-continuous model. Journal of Agricultural and Food

[63] Matsui T, Li CH, Osajima Y. Preparation and characterisation of novel bioactive peptides responsible for angiotensin I-converting enzyme inhibition from wheat germ.

[64] Miguel M, Aleixandre MA, Ramos M, Lόpez-Fandiño R. Effect of simulated gastrointestinal digestion on the antihypertensive properties of ACE-inhibitory peptides derived from ovalbumin. Journal of Agricultural Food Chemistry 2006;54 726-

[65] Wu J, Ding X. Characterization of inhibition and stability of soy-protein-derived angiotensin I-converting enzyme inhibitory peptides. Food Research International

[66] Tavares T, del Mar Contreras M, Amorim M, Pintado M, Recio I, Malcata FX. Novel whey-derived peptides with inhibitory effect against angiotensin-converting enzyme: In

[68] Nakamura Y, Masuda O, Takano T. Decrease in tissue angiotensin I-converting enzyme activity upon feeding sour milk in spontaneously hypertensive rats. Bioscience,

[69] Masuda O, Nakamura Y, Takano T. Antihypertensive peptides are present in aorta after oral administration of sour milk containing these peptides to spontaneously

[70] Foltz M, Meynen EM, Bianco V, van Platerink C, Koning TM, Kloek J. Angiotensin converting enzyme inhibitory peptides from a lactotripeptide-enriched milk beverage

[72] Ohsawa K, Satsu H, Ohki K, Enjoh M, Takano T, Shimizu, M. Producibility and digestibility of antihypertensive β-casein tripeptides, Val-Pro-Pro and Ile-Pro-Pro, in the

are absorbed intact into the circulation. Journal of Nutrition 2007;137(4) 953-958. [71] Foltz M, Cerstiaens A, van Meensel A, Mols R, van der Pijl PC, Duchateau GS, Augustijns P. The angiotensin converting enzyme inhibitory tripeptides Ile-Pro-Pro and Val-Pro-Pro show increasing permeabilities with increasing physiological relevance of

vitro effect and stability to gastrointestinal enzymes. Peptides 2011;32 1013-1019. [67] Hannelore D. Molecular and integrative physiology of intestinal peptide transport.

enzyme inhibitory peptide. British Journal of Nutrition 2004;92 357-366.


[87] Nakamura Y, Yamamoto N, Sakai K, Okubo A, Yamazaki S, Takano T. Purificiation and characterisation of angiotensin I-converting enzyme inhibitors from sour milk. Journal of Dairy Science 1995;78 777-783.

Antihypertensive Peptides from Food Proteins 69

[99] Yamada Y, Matoba M, Usui H, Onishi K, Yoshikawa M. Design of a highly potent antihypertensive peptide based on ovokinin(2-7). Bioscience, Biotechnology and

[100] da Costa EL, da Rocha Gontijo JA, Netto FM. Effect of heat and enzymatic treatment on the antihypertensive activity of whey protein hydrolysates. International Dairy

[101] Boelsma E, Kloek J. Lactotripeptides and antihypertensive effects:a critical review.

[102] Jauhiainen T, Vapaatalo H, Poussa T, Kyrönpalo S, Rasmussen S, Korpela R. *Lactobacillus helveticus* fermented milk lowers blood pressure in hypertensives subjects in 24-h ambulatory blood pressure measurement. American Journal of Hypertension

[103] Yamasue K, Morikawa N, Mizushima S, Tochikubo O. The blood pressure lowering effect of lactotripeptides and salt intake in 24-h ambulatory blood pressure

[104] Germino FW, Neutel J, Nonaka M, Hendler SS. The impact of lactotripeptides on blood pressure response in stage 1 and stage 2 hypertensives. Journal of Clinical

[105] Kurosawa MT, Nakamura Y, Yamamoto N, Yamada K, Iketani T. Effects of Val-Pro-Pro and Ile-Pro-Pro on nondipper patients: a preliminary study. Journal of Medicinal

[106] Cicero AFG, Rosticci M, Gerocarni B, Bacchelli S, Veronesi M, Strocchi E, Borghi C. Lactotripeptides effect on office and 24-h ambulatory blood pressure, blood pressure stress response, pulse wave velocity and cardiac output in patients with high-normal blood pressure or first-degree hypertension: a randomised double-blind clinical trial.

[107] Jauhiainen T, Rönnback M, Vapaatalo H, Wuolle K, Kautiainen H, Groop PH, Korpela R. Long-term intervention with *Lactobacillus helveticus* fermented milk reduces augmentation index in hypertensive subjects. European Journal of Clinical Nutrition

[108] Nakamura T, Mizutani J, Sasaki K, Yamamoto N, Takazawa K. Beneficial potential of casein hydrolysate containing Val-Pro-Pro and Ile-Pro-Pro on central blood pressure and hemodynamic index: A preliminary study. Journal of Medicinal Food,

[109] Hirota T, Ohki K, Kawagishi R, Kajimoto Y, Mizuno S, Nakamura Y, Kitakaze M. Casein hydrolysate containing the antihypertensive tripeptides Val-Pro-Pro and Ile-Pro-Pro improves vascular endothelial function independent of blood pressure-lowering effects: contribution of the inhibitory action of angiotensin-converting enzyme.

[110] Yoshizawa M, Maeda S, Miyaki A, Misono M, Choi Y, Shimojo N, Ajisaka R, Tanaka H. Additive beneficial effects of lactotripeptides and aerobic exercise on arterial

measurements. Clinical and Experimental Hypertension 2010;32(4) 214-220.

Biochemistry 2002;66(6) 1213-1217.

British Journal of Nutrition 2008;101(6) 776-786.

Journal 2007;17(6) 632-640.

2005;18(12 Pt 1) 1600-1605.

Hypertension 2010;12 153-159.

Hypertension Research 2011;34 1035-1040.

Hypertension Research 2007;30 489-496.

Food, 2011;14(5) 538-542.

2010;64 424–431.

2009;12 1-6.


[99] Yamada Y, Matoba M, Usui H, Onishi K, Yoshikawa M. Design of a highly potent antihypertensive peptide based on ovokinin(2-7). Bioscience, Biotechnology and Biochemistry 2002;66(6) 1213-1217.

68 Bioactive Food Peptides in Health and Disease

Research 2002;69 103-111.

of Dairy Science 1995;78 777-783.

Journal of Dairy Science 1995;78 1253-1257.

Journal of Clinical Nutrition 2003;77 326-330.

*Chinese Journal of Physiology* 2006;49(2) 67-73.

Foods 2009;1(4) 366-374.

211.

[87] Nakamura Y, Yamamoto N, Sakai K, Okubo A, Yamazaki S, Takano T. Purificiation and characterisation of angiotensin I-converting enzyme inhibitors from sour milk. Journal

[88] Nakamura Y, Yamamoto N, Sakai K, Takano T. Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors of angiotensin I- converting enzyme.

[89] Sipola M, Finckenberg P, Korpela R, Vapaatalo H, Nurinen ML. Effect of long-term intake of milk products on blood pressure in hypertensive rats. Journal of Dairy

[90] Sipola M, Finckenberg P, Santisteban J, Korpela R, Vapaatalo H, Nurminen ML. Longterm intake of milk peptides attenuates development of hypertension in spontaneously

[91] Seppo L, Jauhiainen T, Poussa P, Korpela R. A fermented milk high in bioactive peptides has a blood pressure–lowering effect in hypertensive subjects. The American

[92] Tuomilehto J, Lindström J, Hyyrynen J, Korpela R, Karhunen ML, Mikkola L, Jauhiainen T, Seppo L, Nissinen A. Effect of ingesting sour milk fermented using *Lactobacillus helveticus* bacteria producing tripeptides on blood pressure in subjects with

[93] Jauhiainen T, Collin M, Narva M, Cheng ZJ, Poussa T, Vapaatalo H, Korpela R. Effect of long-term intake of milk peptides and minerals on blood pressure and arterial function

[94] Jäkälä P, Jauhiainen T, Korpela R, Vapaatalo H. Milk protein-derived bioactive tripeptides Ile-Pro-Pro and Val-Pro-Pro protect endothelial function *in vitro* in

[95] Jauhiainen T, Pilvi T, Cheng ZJ, Kautuaunen H, Müller DN, Vapaatalo H, Korpela R, Mervaala E. Milk products containing bioactive tripeptides have an antihypertensive effect in double transgenic rats (dTGR) habouring human renin and human

[96] Lee NY, Cheng JT, Enomoto T, Nakamura I. The antihypertensive activity of angiotensin-converting enzyme inhibitory peptide containing in bovine lactoferrin.

[97] Jäkälä P, Hakal A, Turpeinen AM, Korpela R, Vapaatalo H. Casein-derived bioactive tripeptides Ile-Pro-Pro and Val-Pro-Pro attenuate the development of hypertension and improve endothelial function in salt-loaded Goto–Kakizaki rats. Journal of Functional

[98] López-Fandiño R, Recio I, Ramos M. Egg-Protein-Derived Peptides with Antihypertensive Activity. In: Huopalahti R, López-Fandiño R, Anton M, Schade R. (eds.) Bioactive egg compounds. New York; Springer-Verlag Heidelberg; 2007. p199-

angiotensinogen genes. Journal of Nutrition and Metabolism, 2009;2010 1-6.

hypertensive rats. Journal of Physiology and Pharmacology 2001;52 745-754.

mild hypertension. Journal of Human Hypertension 2004;18 795-802.

in spontaneously hypertensive rats. Milchwissenschaft 2005;60 358-362.

hypertensive rats. Journal of Functional Foods 2009;1(3) 266-273.


compliance in postmenopausal women. American Journal of Physiology. Heart and Circulatory Physiology 2009;297 H1899-903.

Antihypertensive Peptides from Food Proteins 71

[123] Agyei D, Danquah MK. Industrial-scale manufacturing of pharmaceutical-grade

[124] Pedroche J, Yust MM, Lqari H, Megias C, Girón-Calle J, Alaiz M, Vioque J, Millán F. Obtaining of *Brassica carinata* protein hydrolysates enriched in bioactive peptides using immobilized digestive proteases. Food Research International 2007;40(7)

[125] Kim SK, Senevirathne M. Membrane bioreactor technology for the development of functional materials from sea-food processing wastes and their potential health benefits.

[126] FitzGerald RJ, Cuinn GO. Enzymatic debittering of food protein hydrolysates.

[127] Ministry of Health, Labour and Welfare: Food for Specified Health Uses (FOSHU).

[128] European Food Safety Authority: Scientific Opinion on the Substantiation of Health Claims Related to a C12 Peptide (Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys) and Maintenance of Normal Blood Pressure (ID 1483, 3130) Pursuant to Article 13(1) of

http://www.efsa.europa.eu/en/efsajournal/doc/1478.pdf (assessed 15 May 2012). [129] European Food Safety Authority: Scientific Opinion on the Substantiation of Health Claims Related to Bonito Protein Peptide and Maintenance of Normal Blood Pressure (ID 1716) Pursuant to Article 13(1) of Regulation (EC) No 1924/2006. http://www.efsa.europa.eu/en/efsajournal/doc/1730.pdf [accessed 15 May 2012]. U.S. Food and Drug Administration. Nutritional Labeling and Education Act (NLEA)

http://www.fda.gov/ICECI/Inspections/InspectionGuides/ucm074948.htm?utm\_campai gn=Google2&utm\_source=fdaSearch&utm\_medium=website&utm\_term=nutrition

[130] U.S. Food and Drug Administration: Dietary Supplement Health and Education Act of

utm\_campaign=Google2&utm\_source=fdaSearch&utm\_medium=website&utm\_term=D

[131] Phelan, M. & Kerins, D. The potential of milk-derived peptides in cardiovascular

[132] Usinger L, Reimer C, Ibsen H. Fermented milk for hypertension. Cochrane Database of

http://www.fda.gov/RegulatoryInformation/Legislation/FederalFoodDrugand CosmeticActFDCAct/SignificantAmendmentstotheFDCAct/ucm148003.htm?

labelling and education act&utm\_content=1 (accessed 15 May 2012).

ietarySupplement Health and Education Act&utm\_content=1

bioactive peptides. Biotechnology Advances 2011; 29(3) 272–277.

http://www.mhlw.go.jp/english/topics/foodsafety/fhc/02html

931–938.

1994.

(accessed 15 May 2012).

disease. Food & Function 2011; 2,153-167.

10.1002/14651858.CD008118.pub2.

Systematic Reviews 2012;4 Art. No.: CD008118. DOI:

Membranes 2011;1(4) 327-344.

(accessed 16 May 2012).

Regulation (EC) No 1924/2006.

Biotechnology Advances 2006;24 234– 237.

Requirements (8/94 - 2/95). [Online], available:


[123] Agyei D, Danquah MK. Industrial-scale manufacturing of pharmaceutical-grade bioactive peptides. Biotechnology Advances 2011; 29(3) 272–277.

70 Bioactive Food Peptides in Health and Disease

490-5.

450-454.

2000;14(8) 519-523.

10.3402/fnr.v52i0.1641.

Science 1996;79(8) 1454-1459.

Chemistry 2012;60(19) 4895-4904.

24(10) 933-940.

470-476.

Circulatory Physiology 2009;297 H1899-903.

American Journal of Hypertension 2007;20(1) 1-5.

compliance in postmenopausal women. American Journal of Physiology. Heart and

[111] Cadée JA, Chang CY, Chen CW, Huang CN, Chen SL, Wang CK. Bovine casein hydrolysate (C12 Peptide) reduces blood pressure in prehypertensive subjects.

[112] Pins JJ, Keenan JM. The antihypertensive effects of a hydrolysed whey protein isolate

[113] Fujita H, Yamagami T, Ohshima K. Effects of an ACE-inhibitory agent, katsuobushi oligopeptide, in the spontaneously hypertensive rat and in borderline and mildly

[114] Inoue K, Shirai T, Ochiai H, Kasao M, Hayakawa K, Kimura M, Sansawa H. Bloodpressure-lowering effect of a novel fermented milk containing gamma-aminobutyric acid (GABA) in mild hypertensives. European Journal of Clinical Nutrition, 2003;57(3)

[115] Kawasaki T, Seki E, Osajima K, Yoshida M, Asada K, Matsui T, Osajima Y. Antihypertensive effect of valyl-tyrosine, a short chain peptide derived from sardine muscle hydrolyzate, on mild hypertensive subjects. Journal of Human Hypertension

[116] Suetsuna K, Nakano T. Identification of an antihypertensive peptide from a peptic digest of wakame (*Undaria pinnatifida*). *Journal of Nutritional* Biochemistry 2000;11(9)

[117] Pripp AH. Effect of peptides derived from food proteins on blood pressure: a metaanalysis of randomized controlled trials. Food & Nutrition Research 2008;52 doi:

[118] Xu, J-Y., Qin, L.Q., M.S.M., Wang, P.Y., Li, W. & Chang, C. Effect of milk tripeptides on blood pressure: a meta-analysis of randomized controlled trials. Nutrition 2008;

[119] Cicero AFG, Gerocarni B, Laghi L, Borghi C. Blood pressure lowering effect of lactotripeptides assumed as functional foods: a meta-analysis of current available

[120] Contreras MM, Sevilla MA, Monroy-Ruix J, Amigo L, Gómez-Sala B, Molina E, Ramos M, Recio I. Food-grade production of an antihypertensive casein hydrolysate and resistance of active peptides to drying and storage. International Dairy Journal 2011;21

[121] Smithers GW, Ballard FJ, Copeland AD, DeSilva KJ, Dionysius DS, Francis GL, Goddard C, Grieve PA, McIntosh GH, Mitchell IR, Pearce RJ, Regester GO. New opportunities from the isolation and utilization of whey proteins. Journal of Dairy

[122] O' Loughlin IB, Murray BA, Kelly PM, FitzGerald RJ, Brodkorb A. Enzymatic hydrolysis of heat-induced aggregates of whey protein isolate. Agricultural & Food

clinical trials. Journal of Human Hypertension 2011;1 1-12.

supplement (BioZate® 1): a pilot study. FASEB Journal 2003;17 A1110.

hypertensive subjects. Nutrition Research 2001;21(8) 1149-1158.


http://www.efsa.europa.eu/en/efsajournal/doc/1478.pdf (assessed 15 May 2012).

[129] European Food Safety Authority: Scientific Opinion on the Substantiation of Health Claims Related to Bonito Protein Peptide and Maintenance of Normal Blood Pressure (ID 1716) Pursuant to Article 13(1) of Regulation (EC) No 1924/2006. http://www.efsa.europa.eu/en/efsajournal/doc/1730.pdf [accessed 15 May 2012]. U.S. Food and Drug Administration. Nutritional Labeling and Education Act (NLEA) Requirements (8/94 - 2/95). [Online], available:

http://www.fda.gov/ICECI/Inspections/InspectionGuides/ucm074948.htm?utm\_campai gn=Google2&utm\_source=fdaSearch&utm\_medium=website&utm\_term=nutrition labelling and education act&utm\_content=1 (accessed 15 May 2012).

[130] U.S. Food and Drug Administration: Dietary Supplement Health and Education Act of 1994.

http://www.fda.gov/RegulatoryInformation/Legislation/FederalFoodDrugand CosmeticActFDCAct/SignificantAmendmentstotheFDCAct/ucm148003.htm? utm\_campaign=Google2&utm\_source=fdaSearch&utm\_medium=website&utm\_term=D ietarySupplement Health and Education Act&utm\_content=1 (accessed 15 May 2012).

	- [133] Shimizu M, Tsunogai M, Arai S. Transepithelial transport of oligopeptides in the human intestinal cell, caco-2. Peptides 1997;18(5) 681-687.

**Section 2** 

**Foods as Source of Bioactive Peptides** 

**Foods as Source of Bioactive Peptides** 

72 Bioactive Food Peptides in Health and Disease

[133] Shimizu M, Tsunogai M, Arai S. Transepithelial transport of oligopeptides in the

human intestinal cell, caco-2. Peptides 1997;18(5) 681-687.

**Chapter 4** 

© 2013 Tavares and Malcata, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Tavares and Malcata, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Whey Proteins as Source of Bioactive Peptides** 

A food can be considered as functional if, beyond its nutritional outcomes, it provides benefits upon one or more physiological functions, thus improving health while reducing the risk of illness [1]. This definition – originally proposed by the European Commission Concerted Action on Functional Food Science in Europe (FuFoSE), should be refined in that: (i) the functional effect is different from the nutritional one; and (ii) the benefit provided requires scientific consubstantiation in terms of improvement of physiological functions, or reduction of occurrence of pathological conditions. The concept of functional food emerged in Japan during the 80's, chiefly because of the need to improve the quality of life of a growing elderly population – who typically incurs in much higher health costs [2]. Nowadays, a growing consumer awareness of the relationship between nutrition and health

Bioactive peptides can be commercially sold as nutraceuticals; a nutraceutical is an edible substance possessing health benefits that may accordingly be used to prevent or treat a disease. However, a distinction should be made between nutraceuticals taken to prevent diseases – and which are present as natural ingredients of functional foods consumed as part of the daily diet, and nutraceuticals used as adjuvants for treatment of diseases – which

Milk and dairy products have been concluded to be functional foods; a number of studies have indeed shown that many peptides from milk proteins play a role in several metabolic processes, so a considerable interest has arisen from the part of the dairy industry towards large-scale production of dairy proteins in general, and bioactive peptides in particular. Manufacture of bioactive peptides is usually carried out through hydrolysis using digestive, microbial, plant or animal enzymes, or by fermentation with lactic starter cultures. In some cases, a combination of these processes has proven crucial to obtain functional peptides of

**Against Hypertension** 

Tânia G. Tavares and F. Xavier Malcata

http://dx.doi.org/10.5772/52680

**1. Introduction** 

Additional information is available at the end of the chapter

has made the market of functional foods to boom.

require pharmacologically active compounds.

## **Whey Proteins as Source of Bioactive Peptides Against Hypertension**

Tânia G. Tavares and F. Xavier Malcata

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52680

## **1. Introduction**

A food can be considered as functional if, beyond its nutritional outcomes, it provides benefits upon one or more physiological functions, thus improving health while reducing the risk of illness [1]. This definition – originally proposed by the European Commission Concerted Action on Functional Food Science in Europe (FuFoSE), should be refined in that: (i) the functional effect is different from the nutritional one; and (ii) the benefit provided requires scientific consubstantiation in terms of improvement of physiological functions, or reduction of occurrence of pathological conditions. The concept of functional food emerged in Japan during the 80's, chiefly because of the need to improve the quality of life of a growing elderly population – who typically incurs in much higher health costs [2]. Nowadays, a growing consumer awareness of the relationship between nutrition and health has made the market of functional foods to boom.

Bioactive peptides can be commercially sold as nutraceuticals; a nutraceutical is an edible substance possessing health benefits that may accordingly be used to prevent or treat a disease. However, a distinction should be made between nutraceuticals taken to prevent diseases – and which are present as natural ingredients of functional foods consumed as part of the daily diet, and nutraceuticals used as adjuvants for treatment of diseases – which require pharmacologically active compounds.

Milk and dairy products have been concluded to be functional foods; a number of studies have indeed shown that many peptides from milk proteins play a role in several metabolic processes, so a considerable interest has arisen from the part of the dairy industry towards large-scale production of dairy proteins in general, and bioactive peptides in particular. Manufacture of bioactive peptides is usually carried out through hydrolysis using digestive, microbial, plant or animal enzymes, or by fermentation with lactic starter cultures. In some cases, a combination of these processes has proven crucial to obtain functional peptides of

© 2013 Tavares and Malcata, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Tavares and Malcata, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

small size [3,4]. Proteins recovered from whey released by cheese manufacture already found a role as current ingredients on industrial scale. Use of these proteins (concentrated or isolated), and mainly of biologically active peptides derived therefrom as dietary supplements, pharmaceutical preparations or functional ingredients is of the utmost interest for the pharmaceutical and food industries – while helping circumventing the pollution problems associated with plain whey disposal.

Whey Proteins as Source of Bioactive Peptides Against Hypertension 77

Despite containing ca. 93 % water, whey is a reservoir of milk components of a high value: it indeed contains ca. half of the nutrients found in whole milk. Said composition depends obviously on how the cheese is produced and the milk source; the compound found to higher level is lactose (4.5-5 %, w/v), followed by soluble proteins (0.6-0.8 %, w/v), lipids (0.4-0.5 %, w/v) and minerals (8-10 %, w/wdry extract) – particularly calcium, and vitamins such as thiamine, riboflavin and pyridoxin [11-13]. In fact, whey is now considered as a coproduct rather than a by-product of cheese production, in view of its wide range of potential

Milk has been recognized as one of the main sources of protein [16] in feed for young animals and food for humans of all ages [17]. Bovine milk contains ca. 3 % protein [9] – of which 80 % is caseins and 20 % is whey proteins [18]. Whey comprises a heterogeneous group of proteins that remain in the supernatant after precipitation of caseins; they are characterized by genetic polymorphisms that usually translate into replacement of one or

Two major types of proteinaceous material can be found in whey: -lactoglobulin (-Lg) and -lactalbumin (-La); and proteose-peptone (derived from hydrolysis of -casein, - CN), small amounts of blood-borne proteins (including bovine serum albumin, BSA, and immunoglobulins, Igs), and low molecular weight (MW) peptides derived from casein hydrolysis (soluble at pH 4.6 and 20 °C) [16, 19]. Whey proteins have a compact globular structure that accounts for their solubility (unlike caseins that exist as a micellar suspension, with a relatively uniform distribution of non-polar, polar and charged groups). These proteins have amino acid profiles quite different from caseins: they have a smaller fraction of Glu and Pro, but a greater fraction of sulfur-containing amino acid residues (i.e. Cys and Met). These proteins are dephosphorylated, easily denatured by heat, insensitive to Ca2+, and susceptible to intramolecular bond formation via disulfide bridges between Cys sulfhydryl groups. Selected physicochemical parameters typical of whey proteins are

**Proteins Concentration (gL-1) MW (kDa) Isoelectric point (pI)** 


Protease-peptone 0.5 4 – 20 Caseinomacropeptide 7

**Table 2.** Characteristics of major whey proteins (adapted from Zydney [186])

more amino acid residues in their original peptide sequence.

applications [13-15].

tabulated in Table 2.

**2.2. Protein composition** 

## **2. Cheese whey**

Despite having been labeled over the years as polluting waste owing to its high lactose and protein contents [5], whey is a popular protein supplement in various functional foods and the like [6]. In fact, whey compounds exhibit a number of functional, physiological and nutritional features that make them potentially useful for a wide range of applications (Table 1).


**Table 1.** Major features associated with use of whey (adapted from Alais [62])

Whey can be converted into lactose-free whey powder, condensed whey, whey protein concentrates and whey protein isolates [7] – all of which are commercially available at present. In the case of bovine milk, ca. 9 L of whey is produced from 10 L of milk during cheesemaking; estimates of worldwide production of cheese in 2011 point at ca. 15 million tonnes (United States Department of Agriculture – Foreign Agricultural Service). For environmental reasons, whey cannot be dumped as such into rivers due to its high chemical and biological oxygen demands. On the other hand, whey can be hardly used as animal feed or fertilizer due to economic unfeasibility.

#### **2.1. Physicochemical composition**

There are two types of whey, depending on how it is obtained; when removal of casein is via acid precipitation at its isoelectric point (pH 4.6 at room temperature) [8], it is called acid whey; however, the most common procedure is coagulation via enzymatic action, so the product obtained is called sweet whey [9-10].

Despite containing ca. 93 % water, whey is a reservoir of milk components of a high value: it indeed contains ca. half of the nutrients found in whole milk. Said composition depends obviously on how the cheese is produced and the milk source; the compound found to higher level is lactose (4.5-5 %, w/v), followed by soluble proteins (0.6-0.8 %, w/v), lipids (0.4-0.5 %, w/v) and minerals (8-10 %, w/wdry extract) – particularly calcium, and vitamins such as thiamine, riboflavin and pyridoxin [11-13]. In fact, whey is now considered as a coproduct rather than a by-product of cheese production, in view of its wide range of potential applications [13-15].

#### **2.2. Protein composition**

76 Bioactive Food Peptides in Health and Disease

High nutritional value of protein fraction in terms of amino acid residues

Possibility of lactose production in

Reduction in pollution owing to

biochemical oxygen demand of proteins

or fertilizer due to economic unfeasibility.

product obtained is called sweet whey [9-10].

**2.1. Physicochemical composition** 

(e.g. Lys, Thr, Leu, Ser)

**2. Cheese whey** 

(Table 1).

parallel

problems associated with plain whey disposal.

small size [3,4]. Proteins recovered from whey released by cheese manufacture already found a role as current ingredients on industrial scale. Use of these proteins (concentrated or isolated), and mainly of biologically active peptides derived therefrom as dietary supplements, pharmaceutical preparations or functional ingredients is of the utmost interest for the pharmaceutical and food industries – while helping circumventing the pollution

Despite having been labeled over the years as polluting waste owing to its high lactose and protein contents [5], whey is a popular protein supplement in various functional foods and the like [6]. In fact, whey compounds exhibit a number of functional, physiological and nutritional features that make them potentially useful for a wide range of applications

Whey can be converted into lactose-free whey powder, condensed whey, whey protein concentrates and whey protein isolates [7] – all of which are commercially available at present. In the case of bovine milk, ca. 9 L of whey is produced from 10 L of milk during cheesemaking; estimates of worldwide production of cheese in 2011 point at ca. 15 million tonnes (United States Department of Agriculture – Foreign Agricultural Service). For environmental reasons, whey cannot be dumped as such into rivers due to its high chemical and biological oxygen demands. On the other hand, whey can be hardly used as animal feed

There are two types of whey, depending on how it is obtained; when removal of casein is via acid precipitation at its isoelectric point (pH 4.6 at room temperature) [8], it is called acid whey; however, the most common procedure is coagulation via enzymatic action, so the

High dilution requiring costly dehydration High salt content (ca. 10 % of dry matter) High sugar content requiring delactosation

Widely dispersed cheese production facilities Technical innovation needed in separation (e.g. ultrafiltration and diafiltration)

Highly perishable raw material

**Advantageous features Disadvantageous features** 

**Table 1.** Major features associated with use of whey (adapted from Alais [62])

Milk has been recognized as one of the main sources of protein [16] in feed for young animals and food for humans of all ages [17]. Bovine milk contains ca. 3 % protein [9] – of which 80 % is caseins and 20 % is whey proteins [18]. Whey comprises a heterogeneous group of proteins that remain in the supernatant after precipitation of caseins; they are characterized by genetic polymorphisms that usually translate into replacement of one or more amino acid residues in their original peptide sequence.

Two major types of proteinaceous material can be found in whey: -lactoglobulin (-Lg) and -lactalbumin (-La); and proteose-peptone (derived from hydrolysis of -casein, - CN), small amounts of blood-borne proteins (including bovine serum albumin, BSA, and immunoglobulins, Igs), and low molecular weight (MW) peptides derived from casein hydrolysis (soluble at pH 4.6 and 20 °C) [16, 19]. Whey proteins have a compact globular structure that accounts for their solubility (unlike caseins that exist as a micellar suspension, with a relatively uniform distribution of non-polar, polar and charged groups). These proteins have amino acid profiles quite different from caseins: they have a smaller fraction of Glu and Pro, but a greater fraction of sulfur-containing amino acid residues (i.e. Cys and Met). These proteins are dephosphorylated, easily denatured by heat, insensitive to Ca2+, and susceptible to intramolecular bond formation via disulfide bridges between Cys sulfhydryl groups. Selected physicochemical parameters typical of whey proteins are tabulated in Table 2.


**Table 2.** Characteristics of major whey proteins (adapted from Zydney [186])

#### *2.2.1. -Lactoglobulin (-Lg)*

The major protein in ruminant whey is -Lg, which represents ca. 50 % of the total whey protein inventory in cow's milk and 12 % of the total milk proteins [9, 20-21]. Although it can be found in the milk of many other mammals, it is essentially absent in human milk [22]. This is a globular protein, with 162 amino acid residues in its primary structure and a MW of 18.4 kDa. There are at least twelve genetic variants of -Lg (A, B, C, D, DR, DYAK/E, F, G, H, I, W and X) – of which A is the most common.

Whey Proteins as Source of Bioactive Peptides Against Hypertension 79

14 % -sheet and 60 % other motifs; it is also very similar to lysozyme [9]. This protein is one of the most studied proteins with regard to understanding the mechanism of protein stability and folding/unfolding [42]; at low pH [43], high temperature [44] or moderate concentrations of denaturants – e.g. guanidine hydrochloride [45], -La adopts a conformational structure called molten globule. A partially unfolded state, the apo-state, is formed at neutral pH upon removal of Ca2+ by ethylenediamine tetracetic acid (EDTA) [46-

The molten globule state of -La retains a high fraction of its native secondary structure, as well as a flexible tertiary structure [45, 48-49]; it accordingly appears as an intermediate in the balance between native and unfolded states [50-51]. This structure of -La is highly heterogeneous, with proeminence of -helix driven mainly by weak hydrophobic

CMP is a heterogeneous polypeptide fraction derived from cleavage of Phe105-Met106 in casein (-CN). When milk is hydrolyzed with chymosin during cheesemaking, -CN is hydrolyzed into two portions: one remains in the cheese (*para*--CN) and the other (CMP) is lost in whey; the latter is relatively small, with 63 residues and a MW of ca. 8 kDa [52]. Further to its polymorphisms, CMP may exist in various forms depending on the extent of post-transcriptional changes: it glycosylates through an *O*-glycoside bridge, and phosphorylates via a Ser residue. Note that post-transcriptional modifications of -CN

The amino acid sequence of CMP is well-known; it lacks aromatic amino acid residues (Phe, Trp and Tyr) and Arg, but several acidic and hydroxyl amino acids are present [53]. CMP from cow is soluble at pH in the range 1-10, with a minimum solubility (88 %) between pH 1 and 5 [54-55]. CMP appears to remain essentially soluble following heat treatment at 80-95 °C for 15 min at pH 4 and 7 [55]. Its emulsifying activity exhibits a minimum near the isoelectric point [54]. Dziuba and Minkiewicz [56] showed that a decrease in pH leads to a decrease in CMP volume, owing to reduction of internal electrostatic forces and steric repulsion; this apparently has a significant influence upon

BSA is derived directly from the blood, and represents 0.7-1.3 % of all whey proteins [8]. Its molecule has 582 amino acid residues and a MW of 69 kDa – and contains 17 disulfide bonds and one free sulphydryl group [9]. Because of its size and higher levels of structure, BSA can bind free fatty acids and other lipids, as well as flavor compounds [57] – but this feature is severely hampered upon denaturation. Its heat-induced gelation at pH 6.5 is initiated by intermolecular thiol-disulphide interchange – similar to what happens with -

47]; this state preserves its secondary, but not its tertiary structure [48].

interactions – while the -sheet domain is significantly unfolded.

occur exclusively in the CMP portion of the molecule.

*2.2.3. Caseinomacropeptide (CMP)* 

its emulsifying capacity.

Lg [58].

*2.2.4. Bovine serum albumin (BSA)* 

The monomer of -Lg has a free thiol group and two disulfide bridges – which makes -Lg exhibit a rigid spacial structure [8]; however, its conformation is pH-dependent [23] – and at temperatures above 65 °C (at pH 6.5), -Lg denatures, thus giving rise to aggregate formation [24]. Between pH values 5.2 and 7.2, that protein appears as a dimmer – with a MW of 36.0 kDa [8]. At low pH, association of monomers leads to octamer formation; and, at high temperatures, the dimer dissociates into its monomers. The solubility of -Lg depends on pH and ion strength – but it does not precipitate during milk acidification [25].

A number of useful nutritional and functional features have made -Lg become an ingredient of choice for food and beverage formulation: in fact, it holds excellent heatgelling [26] and foaming features – which can be used as structuring and stabilizer agents in such dairy products as yogurts and cheese spreads. This protein is resistant to gastric digestion, as is stable in the presence of acids and proteolytic enzymes [22, 27-30]; hence, it tends to remain intact during passage through the stomach. It is also a rich source of Cys, an amino acid bearing a key role in stimulating synthesis of glutathione (GSH) – composed by three amino acids, Glu, Cys and Gly [31].

Many techniques have been developed for purification of β-Lg – which normally rely on its precipitation [32-35]; when large scale purification is intended, precipitation is usually complemented by ion exchange [35-36].

#### *2.2.2. -Lactalbumin (-La)*


The molten globule state of -La retains a high fraction of its native secondary structure, as well as a flexible tertiary structure [45, 48-49]; it accordingly appears as an intermediate in the balance between native and unfolded states [50-51]. This structure of -La is highly heterogeneous, with proeminence of -helix driven mainly by weak hydrophobic interactions – while the -sheet domain is significantly unfolded.

## *2.2.3. Caseinomacropeptide (CMP)*

78 Bioactive Food Peptides in Health and Disease

*-Lg)* 

I, W and X) – of which A is the most common.

three amino acids, Glu, Cys and Gly [31].

complemented by ion exchange [35-36].

*-La)* 

*-Lactalbumin (*

The major protein in ruminant whey is -Lg, which represents ca. 50 % of the total whey protein inventory in cow's milk and 12 % of the total milk proteins [9, 20-21]. Although it can be found in the milk of many other mammals, it is essentially absent in human milk [22]. This is a globular protein, with 162 amino acid residues in its primary structure and a MW of 18.4 kDa. There are at least twelve genetic variants of -Lg (A, B, C, D, DR, DYAK/E, F, G, H,

The monomer of -Lg has a free thiol group and two disulfide bridges – which makes -Lg exhibit a rigid spacial structure [8]; however, its conformation is pH-dependent [23] – and at temperatures above 65 °C (at pH 6.5), -Lg denatures, thus giving rise to aggregate formation [24]. Between pH values 5.2 and 7.2, that protein appears as a dimmer – with a MW of 36.0 kDa [8]. At low pH, association of monomers leads to octamer formation; and, at high temperatures, the dimer dissociates into its monomers. The solubility of -Lg depends

A number of useful nutritional and functional features have made -Lg become an ingredient of choice for food and beverage formulation: in fact, it holds excellent heatgelling [26] and foaming features – which can be used as structuring and stabilizer agents in such dairy products as yogurts and cheese spreads. This protein is resistant to gastric digestion, as is stable in the presence of acids and proteolytic enzymes [22, 27-30]; hence, it tends to remain intact during passage through the stomach. It is also a rich source of Cys, an amino acid bearing a key role in stimulating synthesis of glutathione (GSH) – composed by

Many techniques have been developed for purification of β-Lg – which normally rely on its precipitation [32-35]; when large scale purification is intended, precipitation is usually


on pH and ion strength – but it does not precipitate during milk acidification [25].

*-Lactoglobulin (*

*2.2.1.* 

*2.2.2.*  CMP is a heterogeneous polypeptide fraction derived from cleavage of Phe105-Met106 in casein (-CN). When milk is hydrolyzed with chymosin during cheesemaking, -CN is hydrolyzed into two portions: one remains in the cheese (*para*--CN) and the other (CMP) is lost in whey; the latter is relatively small, with 63 residues and a MW of ca. 8 kDa [52]. Further to its polymorphisms, CMP may exist in various forms depending on the extent of post-transcriptional changes: it glycosylates through an *O*-glycoside bridge, and phosphorylates via a Ser residue. Note that post-transcriptional modifications of -CN occur exclusively in the CMP portion of the molecule.

The amino acid sequence of CMP is well-known; it lacks aromatic amino acid residues (Phe, Trp and Tyr) and Arg, but several acidic and hydroxyl amino acids are present [53]. CMP from cow is soluble at pH in the range 1-10, with a minimum solubility (88 %) between pH 1 and 5 [54-55]. CMP appears to remain essentially soluble following heat treatment at 80-95 °C for 15 min at pH 4 and 7 [55]. Its emulsifying activity exhibits a minimum near the isoelectric point [54]. Dziuba and Minkiewicz [56] showed that a decrease in pH leads to a decrease in CMP volume, owing to reduction of internal electrostatic forces and steric repulsion; this apparently has a significant influence upon its emulsifying capacity.

#### *2.2.4. Bovine serum albumin (BSA)*

BSA is derived directly from the blood, and represents 0.7-1.3 % of all whey proteins [8]. Its molecule has 582 amino acid residues and a MW of 69 kDa – and contains 17 disulfide bonds and one free sulphydryl group [9]. Because of its size and higher levels of structure, BSA can bind free fatty acids and other lipids, as well as flavor compounds [57] – but this feature is severely hampered upon denaturation. Its heat-induced gelation at pH 6.5 is initiated by intermolecular thiol-disulphide interchange – similar to what happens with - Lg [58].

## *2.2.5. Immunoglobulins (IGs)*

IGs represent 1.9-3.3 % of the total milk proteins, and are derived from blood serum [8]; they constitute a complex group, the elements of which are produced by β-lymphocytes. Igs encompass three distinct classes: IgM, IgA and IgG (IgG1 and IgG2) – with IgG1 being the major Ig present in bovine milk and colostrum [8], whereas IgA is predominant in human milk. The physiological function of Igs is to provide various types of immunity to the body; they consist of two heavy (53 kDa) and two light (23 kDa) polypeptide chains, linked by disulfide bridges [9]. The complete Ig, or antibody molecule has a MW of about 180 kDa [59]. Igs are partially resistant to proteolytic enzymes, and are in particular not inactivated by gastric acids [59].

Whey Proteins as Source of Bioactive Peptides Against Hypertension 81

 Breast and intestinal cancer; [14, 75] Chemically-induced cancer [76-77] Increment of gluthatione levels [64] Increase of tumour cell vulnerability [78-79] Antimicrobial activities [80]

Prostaglandin production [83-85]

 Palmitate [89] Fatty acids [90] Cellular defence against oxidative stress

Transfer of passive immunity [95]

**ACEa-inhibitor** [97-107]

Retinol [9, 41, 87, 88]

[81]

[31, 65, 91-93]

[94]

[96]

[108-111]

Increment of satiety response Increment in plasma amino acids, cholecystokinin and glucagon-like

Enzyme hydrolysis **ACEa-inhibitor** [82]

and detoxification

activity

Enzyme hydrolysis Antimicrobial against several grampositive bacteria

Enzyme hydrolysis Antiulcerative [83, 86]

Enhancement of pregastic esterase

Regulation of mammary gland phosphorus metabolism

Enzyme hydrolysis Antimicrobial (bactericidal) [112-113] Enzyme hydrolysis Hypocholesterolemic [113-114]

mainly such biological activities as anticarcinogenic [68] and immunomodulatory [69]. It was observed that whey proteins trigger immune responses that are significantly higher than those by diets containing casein or soy protein. Antimicrobial and antiviral actions, immune system stimulation and anticarcinogenic activity (among other metabolic features) have indeed been associated with ingestion of -Lg and -La, as well as LF, LP, BSA and

With regard to bioactive peptides, research has undergone a notable intensification during the past decade [4, 70]. Advances in nutritional biochemistry and biomedical research have in fact helped unravel the complex relationships between nutrition and disease, thus suggesting that food proteins and peptides originated during digestion (or from *in vitro* proteolysis) may play important roles in preventing or treating diseases associated with

**Protein/Peptide Treatment Biological function Reference**  *Whole whey protein* Prevention of cancer [74]

peptide

Antiulcerative

*-Lactoglobulin* Transporter

Enzyme hydrolysis; Fermentation

CMP; the main biological activities of whey proteins are listed in Table 3.

malnutrition, pathogens and injuries [71-72].

### *2.2.6. Lactoferrin (LSs)*

LFs are single-chain polypeptides of ca. 80 kDa, containing 1-4 glycans depending on the species. Bovine and human LFs consist of 989 and 691 amino acids, respectively [60]: the former is present to a concentration of ca. 0.1 mg mL-1 [25, 61], and is an iron-binding glycoprotein - so it is thought to play a role in iron transport and absorption in the gut of young people.

#### *2.2.7. Proteose-peptones (PPs)*

The total PP fraction (TPP) of bovine milk represents ca. 10 % of the whole whey protein content; it is accounted for by the whey protein fraction soluble after heating at 95 °C for 30 min, followed by acidification to pH 4.6 [62]. The TPP fraction is often divided in two main groups: the first one includes PPs originated from casein hydrolysis; its principal components have been labeled as 5 (PP5), 8 fast (PP8 fast) and 8 slow (PP8 slow), according to their electrophoretic mobility [62, 63]. PP3 constitutes the second group, and it is not derived from casein (it is actually found only in whey); it is known for its extreme hydrophobicity.

#### **2.3. Functional ingredients from whey proteins**

Whey proteins have unique characteristics [64] beyond their great importance in nutrition; they exhibit chemical, physical, physiological, functional and technological features also useful for food processing [14]. Based on these properties, more and more individual proteins and protein concentrates of whey have been incorporated in food at industrial scale. Therefore, whey proteins address two major issues in practice: nutritionally, they supply energy and essential amino acids, besides being important for growth and cellular repair; in terms of functionality, they play important roles upon texture, structure and overall appearance of food – e.g. gel formation, foam stability and water retention.

A few physiological properties useful in therapies have been found [65]: a number of reviews have accordingly examined to some length the bioactive properties of whey proteins in general [66-67], or of -Lg and -La in particular [26]; other authors have covered mainly such biological activities as anticarcinogenic [68] and immunomodulatory [69]. It was observed that whey proteins trigger immune responses that are significantly higher than those by diets containing casein or soy protein. Antimicrobial and antiviral actions, immune system stimulation and anticarcinogenic activity (among other metabolic features) have indeed been associated with ingestion of -Lg and -La, as well as LF, LP, BSA and CMP; the main biological activities of whey proteins are listed in Table 3.

80 Bioactive Food Peptides in Health and Disease

IGs represent 1.9-3.3 % of the total milk proteins, and are derived from blood serum [8]; they constitute a complex group, the elements of which are produced by β-lymphocytes. Igs encompass three distinct classes: IgM, IgA and IgG (IgG1 and IgG2) – with IgG1 being the major Ig present in bovine milk and colostrum [8], whereas IgA is predominant in human milk. The physiological function of Igs is to provide various types of immunity to the body; they consist of two heavy (53 kDa) and two light (23 kDa) polypeptide chains, linked by disulfide bridges [9]. The complete Ig, or antibody molecule has a MW of about 180 kDa [59]. Igs are partially resistant to proteolytic enzymes, and are in particular not inactivated

LFs are single-chain polypeptides of ca. 80 kDa, containing 1-4 glycans depending on the species. Bovine and human LFs consist of 989 and 691 amino acids, respectively [60]: the former is present to a concentration of ca. 0.1 mg mL-1 [25, 61], and is an iron-binding glycoprotein - so it is thought to play a role in iron transport and absorption in the gut of

The total PP fraction (TPP) of bovine milk represents ca. 10 % of the whole whey protein content; it is accounted for by the whey protein fraction soluble after heating at 95 °C for 30 min, followed by acidification to pH 4.6 [62]. The TPP fraction is often divided in two main groups: the first one includes PPs originated from casein hydrolysis; its principal components have been labeled as 5 (PP5), 8 fast (PP8 fast) and 8 slow (PP8 slow), according to their electrophoretic mobility [62, 63]. PP3 constitutes the second group, and it is not derived from casein (it is actually found only in whey); it is known for its extreme

Whey proteins have unique characteristics [64] beyond their great importance in nutrition; they exhibit chemical, physical, physiological, functional and technological features also useful for food processing [14]. Based on these properties, more and more individual proteins and protein concentrates of whey have been incorporated in food at industrial scale. Therefore, whey proteins address two major issues in practice: nutritionally, they supply energy and essential amino acids, besides being important for growth and cellular repair; in terms of functionality, they play important roles upon texture, structure and

A few physiological properties useful in therapies have been found [65]: a number of reviews have accordingly examined to some length the bioactive properties of whey proteins in general [66-67], or of -Lg and -La in particular [26]; other authors have covered

overall appearance of food – e.g. gel formation, foam stability and water retention.

*2.2.5. Immunoglobulins (IGs)* 

by gastric acids [59].

young people.

hydrophobicity.

*2.2.6. Lactoferrin (LSs)* 

*2.2.7. Proteose-peptones (PPs)* 

**2.3. Functional ingredients from whey proteins** 

With regard to bioactive peptides, research has undergone a notable intensification during the past decade [4, 70]. Advances in nutritional biochemistry and biomedical research have in fact helped unravel the complex relationships between nutrition and disease, thus suggesting that food proteins and peptides originated during digestion (or from *in vitro* proteolysis) may play important roles in preventing or treating diseases associated with malnutrition, pathogens and injuries [71-72].



Whey Proteins as Source of Bioactive Peptides Against Hypertension 83

Although inactive within the primary structure of their source proteins, hydrolysis (e.g. mediated by a protease) may release peptides with specific amino acid sequences possessing biological activity. A number of chemical and biological methods of screening have accordingly been developed to aid in search for specific health effects; however, only some of those found *in vitro* have eventually been confirmed in studies encompassing human

Scientific evidence has shown that whey proteins contain a wide range of peptides that can play crucial physiological functions and modulate some regulatory processes (see Table 3). Due to its high biological value, coupled with excellent functional properties and clean flavor, whey has earned the status of a recommended source of functional ingredients [71] – designed to reduce or control chronic diseases and promote health, thus eventually reducing

Favorable health effects have indeed been claimed for some peptides derived from food proteins – being able to affect the cardiovascular, nervous, digestive or immune systems; these encompass antimicrobial properties, blood pressure-lowering (or angiotensin-converting enzyme (ACE)-inhibitory) effects, cholesterol-lowering ability, antithrombotic and antioxidant activities, enhancement of mineral absorption and/or bioavailability thereof, cyto- or immunomodulatory effects, and opioid features. With regard to the mechanisms underlying the physiological roles of bioactive peptides, a few involve action only upon certain receptors, whereas others are enzyme inhibitors; they may also regulate intestinal absorption, and exhibit antimicrobial or antioxidant activities. Recall that oxidative metabolism is essential for survival of cells, but it generates free radicals (and other reactive oxygen species) as side effect – which may cause oxidative damage. Antioxidant activity has been found specifically in whey proteins, probably via scavenging of such radicals via Tyr and Cys amino acid residues – which is predominantly based on proton-coupled single electron or hydrogen atom transfer

On the other hand, bioactive peptides derived from food proteins differ in general from endogenous bioactive peptides in that they can entail multifunctional features [98]. Furthermore, bioactive peptides that cannot be absorbed though the gastrointestinal tract may exert a direct role upon the intestinal lumen, or through interaction with receptors in the intestinal wall itself; some of these receptors have been implicated in such diseases as

Bioactive peptides derived from whey proteins constitute a new concept, and have open up a wide range of possibilities within the market for functional foods [4, 169]; of special interest are those released via enzymatic action – as happens during clotting in

The enzymes used to bring about milk coagulation are selected protein preparations that provide in general a high clotting activity – i.e. a considerable, but selective proteolytic

cancer, diabetes, osteoporosis, stress, obesity and cardiovascular complications.

volunteers [73].

cheesemaking.

the costs of health care [3, 166].

mechanisms; or else chelation of transition metals [167-168].

**3. Production of bioactive peptides in whey** 

ACEa- angiotensin-converting enzyme

**Table 3.** Biological functions of whey proteins/peptides (adapted from Madureira *et al.* [87])

Although inactive within the primary structure of their source proteins, hydrolysis (e.g. mediated by a protease) may release peptides with specific amino acid sequences possessing biological activity. A number of chemical and biological methods of screening have accordingly been developed to aid in search for specific health effects; however, only some of those found *in vitro* have eventually been confirmed in studies encompassing human volunteers [73].

82 Bioactive Food Peptides in Health and Disease

ACEa- angiotensin-converting enzyme

**Protein/Peptide Treatment Biological function Reference** 

*-Lactalbumin* Prevention of cancer [119]

disease

Antiulcerative

*Bovine serum albumin* Fatty acid binding [13]

*Immunoglobulins* Immunomodulation [140]

immunity

*Caseinomacropeptide* Antithrombotic [148-153]

**Table 3.** Biological functions of whey proteins/peptides (adapted from Madureira *et al.* [87])

Enzyme hydrolysis Antimicrobial against several grampositive bacteria

Enzyme hydrolysis Opioid agonist [73, 97, 115] Enzyme hydrolysis **Antihypertensive** [99, 116-117] Enzyme hydrolysis Ileum contracting [97, 99] Enzyme hydrolysis Antinociceptive [118] Prevention of cancer Enzyme hydrolysis Intestinal cancer [14]

Treatment of chronic stress-induced

Enzyme hydrolysis Opioid agonist [97, 115, 128] Enzyme hydrolysis **ACEa-inhibitor** [26, 97-98, 101,

Enzyme hydrolysis **Antihypertensive** [117, 129] Enzyme hydrolysis Ileum contracting [97]

Enzyme hydrolysis **ACEa-inhibitor** [136-137] Enzyme hydrolysis Ileum contracting [138] Enzyme hydrolysis Opioid agonist [97, 128, 139]

Disease protection through passive

Enzyme hydrolysis Prebiotic [161]

cholecystokinin peptide

Increment in plasma amino acids and

Antimicrobial (bactericidal)

 Apoptosis of tumoral cells [120-122] Lactose synthesis [25, 123]

 *Streptococcus pneumonia* [125] Stress reduction [123, 126] Immunomodulation [127]

Prostaglandin production [130-132]

Antioxidant [133-134] Prevention of cancer [135]

Antibacterial [143-145] Antifungal [146] Opioid agonist [147]

**ACEa-inhibitor** [154-156] Antimicrobial [56, 111, 157-

[124]

[108-110]

[141-142]

160]

[162-165]

107]

Scientific evidence has shown that whey proteins contain a wide range of peptides that can play crucial physiological functions and modulate some regulatory processes (see Table 3). Due to its high biological value, coupled with excellent functional properties and clean flavor, whey has earned the status of a recommended source of functional ingredients [71] – designed to reduce or control chronic diseases and promote health, thus eventually reducing the costs of health care [3, 166].

Favorable health effects have indeed been claimed for some peptides derived from food proteins – being able to affect the cardiovascular, nervous, digestive or immune systems; these encompass antimicrobial properties, blood pressure-lowering (or angiotensin-converting enzyme (ACE)-inhibitory) effects, cholesterol-lowering ability, antithrombotic and antioxidant activities, enhancement of mineral absorption and/or bioavailability thereof, cyto- or immunomodulatory effects, and opioid features. With regard to the mechanisms underlying the physiological roles of bioactive peptides, a few involve action only upon certain receptors, whereas others are enzyme inhibitors; they may also regulate intestinal absorption, and exhibit antimicrobial or antioxidant activities. Recall that oxidative metabolism is essential for survival of cells, but it generates free radicals (and other reactive oxygen species) as side effect – which may cause oxidative damage. Antioxidant activity has been found specifically in whey proteins, probably via scavenging of such radicals via Tyr and Cys amino acid residues – which is predominantly based on proton-coupled single electron or hydrogen atom transfer mechanisms; or else chelation of transition metals [167-168].

On the other hand, bioactive peptides derived from food proteins differ in general from endogenous bioactive peptides in that they can entail multifunctional features [98]. Furthermore, bioactive peptides that cannot be absorbed though the gastrointestinal tract may exert a direct role upon the intestinal lumen, or through interaction with receptors in the intestinal wall itself; some of these receptors have been implicated in such diseases as cancer, diabetes, osteoporosis, stress, obesity and cardiovascular complications.

## **3. Production of bioactive peptides in whey**

Bioactive peptides derived from whey proteins constitute a new concept, and have open up a wide range of possibilities within the market for functional foods [4, 169]; of special interest are those released via enzymatic action – as happens during clotting in cheesemaking.

The enzymes used to bring about milk coagulation are selected protein preparations that provide in general a high clotting activity – i.e. a considerable, but selective proteolytic

activity. Animal rennet obtained from the calf stomach, composed by 88-94 % and 6-12 % chymosin and pepsin, respectively, has been the coagulant of choice for cheesemaking. However, due to increased world production of cheese, the supply of animal rennet has lied below its demand; the increased prices have driven a search for alternative coagulants (including plant and microbial sources). With regard to animal rennet substitutes, pig pepsin has enjoyed a remarkable commercial success; with regard to rennet from microbial origin, the proteases from *Mucor miehei*, *Mucor pusillus* and *Endothia parasitica* are the most successful [170]. Recombinant bovine chymosin is, nowadays, one of the proteinases with greater commercial expression – even though its use is still prohibited in certain countries [171].

Whey Proteins as Source of Bioactive Peptides Against Hypertension 85

chromatographic techniques [41]. Furthermore, the absence of heat treatment allows the bioactive components to remain intact (or become only slightly affected) during processing. Recall that membrane separation allows differential concentration of a liquid, provided that the solute of interest is larger in molecular diameter than the membrane pores – so the liquid that percolates the membrane (filtrate) contains only components smaller than that size

The dairy industry has pioneered development of equipment and techniques for membrane filtration, which recovers whey proteins in a non-denatured state. Typical procedures include: (i) basic membrane separation, e.g. reverse osmosis, ultrafiltration and diafiltration [180-186], that permits fractionation of proteins, as well as concentration and purification thereof; (ii) nanofiltration (or ultraosmosis) that allows removal of salts or low MW contaminants; and (iii) microfiltration to remove suspended solid particles or microorganisms [179, 187]. Note that isolation of individual whey proteins on laboratory scale has resorted chiefly to salting out, ion exchange chromatography and/or crystallization [188]; such a fractionation allows fundamental studies of their immunological properties to be carried out, which are necessary to establish and support industrial interest [189-190].

Hypertension is a major public health issue worldwide that affects nearly one fourth of the population; and it is usually associated with such other disorders as obesity, pre-diabetes, renal disease, atherosclerosis and heart stroke [191-194]. Its specific treatment will likely reduce the risk of incidence of cardiovascular diseases, which currently account for 30 % of

Blood pressure can be regulated through diet changes and physical exercise, as well as administration of calcium T channel antagonists, angiotensin II receptor antagonists, diuretics and ACE inhibitors [104]. A few mechanisms have been described that rationalize how peptides lower blood pressure. Traditionally, control of hypertension has focused on the renin-angiotensin system, via inhibition of ACE [173]. Captopril, enalapril and lisinopril have accordingly been used as antihypertensive drugs that act essentially as ACE inhibitors; they found a widespread application in treatment of patients with hypertension, heart failure or diabetic nephropathies [193, 196-197]. However, they bring about undesirable side

In fact, increasing evidence has been provided that mechanisms other than ACE inhibition may be involved in blood pressure decrease arising from consumption of many foodderived peptides [200]; although there are few studies to date with antihypertensive peptides obtained from whey. One of them corresponds to interaction with opioid receptors that are present in the central nervous system and in peripheral tissues, while another is based on release of nitric oxide (NO) that causes vasodilatation and thus affects blood pressure. Those peptides hold the advantage of no side effects, unlike happens with such other opiates as morphine [102]. One example is α-lactorfin, a tetrapeptide derived from α-La [129, 201], for which studies showed that antihypertensive effects are mediated through

effects, so safer (and, hopefully, less expensive) alternatives are urged [198-199].

**5. Activity of peptides from whey upon hypertension** 

threshold [179].

all causes of death [195].

Chymosin and the other rennet substitutes are aspartic proteases, with optimal activity at acidic pH, and possessing high degree of homology in primary and 3-dimensional structures, 3-dimensional structure and catalytic mechanism. The specificity towards the substrate is, however, rather variable; although they have a greater tendency to break peptide bonds between hydrophobic amino acids having bulky side residues, they hydrolyze a large number of bond types [172]. Of particular interest is vegetable rennet, which – with few exceptions, enjoys a still limited use worldwide. Many plant enzyme preparations proved indeed to be excessively proteolytic for manufacture of cheese, causing defects in terms of flavor and texture of the final product. These difficulties arise from the presence of non-specific enzymes that belong to complex enzyme systems (which, as such, are difficult to control). An exception to the poor suitability of vegetable coagulants is the proteinases in aqueous extracts of plants of the *Cynara* genus – which have been employed for traditional cheesemaking in Portugal and Spain since the Roman period.

Bioactive peptides derived from whey proteins can be released at industrial scale via enzyme-mediated hydrolysis with digestive enzymes – and pepsin, trypsin and chymotrypsin have been the most frequent vectors therefor [4, 169, 173]. However, whey proteins are not easily broken down by proteases in general – a realization that also explains their tendency to cause allergies upon ingestion [174]. Hence, less conventional sources of proteolytic enzymes have been sought that can cleave the whey protein backbone at specific and usual sites. This is the case of aspartic proteinases present in the flowers of *Cynara cardunculus* – a plant related to the (common) globe artichoke. They can cleave the whey protein backbone next to hydrophobic amino acid residues, especially Phe, Leu, Thr and Tyr [82, 175], and act mainly on α-La, either in whole whey or following concentration to whey protein concentrate (WPC) [176-178]; conversely β-Lg appears not to be hydrolyzed thereby to a significant extent [82, 175].

#### **4. Recovery of proteins/peptides from whey**

The relatively low concentration of proteins in whey requires concentration processes to assure high hydrolysis productivity. Development of membrane separation techniques has been essential toward this endeavor – and food industry has taken advantage of its relatively easy scale-up, as well as its being inexpensive when compared with preparative chromatographic techniques [41]. Furthermore, the absence of heat treatment allows the bioactive components to remain intact (or become only slightly affected) during processing. Recall that membrane separation allows differential concentration of a liquid, provided that the solute of interest is larger in molecular diameter than the membrane pores – so the liquid that percolates the membrane (filtrate) contains only components smaller than that size threshold [179].

The dairy industry has pioneered development of equipment and techniques for membrane filtration, which recovers whey proteins in a non-denatured state. Typical procedures include: (i) basic membrane separation, e.g. reverse osmosis, ultrafiltration and diafiltration [180-186], that permits fractionation of proteins, as well as concentration and purification thereof; (ii) nanofiltration (or ultraosmosis) that allows removal of salts or low MW contaminants; and (iii) microfiltration to remove suspended solid particles or microorganisms [179, 187]. Note that isolation of individual whey proteins on laboratory scale has resorted chiefly to salting out, ion exchange chromatography and/or crystallization [188]; such a fractionation allows fundamental studies of their immunological properties to be carried out, which are necessary to establish and support industrial interest [189-190].

## **5. Activity of peptides from whey upon hypertension**

84 Bioactive Food Peptides in Health and Disease

to a significant extent [82, 175].

**4. Recovery of proteins/peptides from whey** 

[171].

activity. Animal rennet obtained from the calf stomach, composed by 88-94 % and 6-12 % chymosin and pepsin, respectively, has been the coagulant of choice for cheesemaking. However, due to increased world production of cheese, the supply of animal rennet has lied below its demand; the increased prices have driven a search for alternative coagulants (including plant and microbial sources). With regard to animal rennet substitutes, pig pepsin has enjoyed a remarkable commercial success; with regard to rennet from microbial origin, the proteases from *Mucor miehei*, *Mucor pusillus* and *Endothia parasitica* are the most successful [170]. Recombinant bovine chymosin is, nowadays, one of the proteinases with greater commercial expression – even though its use is still prohibited in certain countries

Chymosin and the other rennet substitutes are aspartic proteases, with optimal activity at acidic pH, and possessing high degree of homology in primary and 3-dimensional structures, 3-dimensional structure and catalytic mechanism. The specificity towards the substrate is, however, rather variable; although they have a greater tendency to break peptide bonds between hydrophobic amino acids having bulky side residues, they hydrolyze a large number of bond types [172]. Of particular interest is vegetable rennet, which – with few exceptions, enjoys a still limited use worldwide. Many plant enzyme preparations proved indeed to be excessively proteolytic for manufacture of cheese, causing defects in terms of flavor and texture of the final product. These difficulties arise from the presence of non-specific enzymes that belong to complex enzyme systems (which, as such, are difficult to control). An exception to the poor suitability of vegetable coagulants is the proteinases in aqueous extracts of plants of the *Cynara* genus – which have been employed

Bioactive peptides derived from whey proteins can be released at industrial scale via enzyme-mediated hydrolysis with digestive enzymes – and pepsin, trypsin and chymotrypsin have been the most frequent vectors therefor [4, 169, 173]. However, whey proteins are not easily broken down by proteases in general – a realization that also explains their tendency to cause allergies upon ingestion [174]. Hence, less conventional sources of proteolytic enzymes have been sought that can cleave the whey protein backbone at specific and usual sites. This is the case of aspartic proteinases present in the flowers of *Cynara cardunculus* – a plant related to the (common) globe artichoke. They can cleave the whey protein backbone next to hydrophobic amino acid residues, especially Phe, Leu, Thr and Tyr [82, 175], and act mainly on α-La, either in whole whey or following concentration to whey protein concentrate (WPC) [176-178]; conversely β-Lg appears not to be hydrolyzed thereby

The relatively low concentration of proteins in whey requires concentration processes to assure high hydrolysis productivity. Development of membrane separation techniques has been essential toward this endeavor – and food industry has taken advantage of its relatively easy scale-up, as well as its being inexpensive when compared with preparative

for traditional cheesemaking in Portugal and Spain since the Roman period.

Hypertension is a major public health issue worldwide that affects nearly one fourth of the population; and it is usually associated with such other disorders as obesity, pre-diabetes, renal disease, atherosclerosis and heart stroke [191-194]. Its specific treatment will likely reduce the risk of incidence of cardiovascular diseases, which currently account for 30 % of all causes of death [195].

Blood pressure can be regulated through diet changes and physical exercise, as well as administration of calcium T channel antagonists, angiotensin II receptor antagonists, diuretics and ACE inhibitors [104]. A few mechanisms have been described that rationalize how peptides lower blood pressure. Traditionally, control of hypertension has focused on the renin-angiotensin system, via inhibition of ACE [173]. Captopril, enalapril and lisinopril have accordingly been used as antihypertensive drugs that act essentially as ACE inhibitors; they found a widespread application in treatment of patients with hypertension, heart failure or diabetic nephropathies [193, 196-197]. However, they bring about undesirable side effects, so safer (and, hopefully, less expensive) alternatives are urged [198-199].

In fact, increasing evidence has been provided that mechanisms other than ACE inhibition may be involved in blood pressure decrease arising from consumption of many foodderived peptides [200]; although there are few studies to date with antihypertensive peptides obtained from whey. One of them corresponds to interaction with opioid receptors that are present in the central nervous system and in peripheral tissues, while another is based on release of nitric oxide (NO) that causes vasodilatation and thus affects blood pressure. Those peptides hold the advantage of no side effects, unlike happens with such other opiates as morphine [102]. One example is α-lactorfin, a tetrapeptide derived from α-La [129, 201], for which studies showed that antihypertensive effects are mediated through the vasodilatory action of binding to opioid receptors. Furthermore, endotheliumdependent relaxation of mesenteric arteries in spontaneously hypertensive rats (SHR, which is the animal model normally accepted to study human hypertension) that was inhibited by an endothelial nitric oxide synthase (eNOS) inhibitor was also observed [202]. That peptide may even chelate minerals, and thus facilitates calcium absorption [200].

Whey Proteins as Source of Bioactive Peptides Against Hypertension 87

[214], Korhonen and Pihlanto [4], Silva and Malcata [215], Vermeirssen [216], and Martínez-Maqueda [208] have comprehensively reviewed this subject. In general, it has been claimed that a diet rich in foods containing antihypertensive peptides is effective toward prevention

ACE-inhibitory peptides may be obtained from precursor food proteins via enzymatic hydrolysis, using viable or lysed microorganisms or specific proteases [3, 73, 137, 169]. Although *in vitro* studies are useful at screening stages, the efficacy and safety of such peptides requires *in vivo* testing – first in animals, and then in human volunteers [217]. This issue is particularly relevant because *in vitro* ACE inhibition does not necessarily correlate with *in vivo* antihypertensive features, as peptides often undergo breakdown during gastrointestinal digestion that hampers manifestation of their potential physiological function. Conversely, antihypertensive activity may be promoted after long-chain peptide

In the latest two decades, various active peptides have been identified from animal proteins, including some with antihypertensive effects in animals (e.g. SHR) and even in humans [3, 73, 137, 169, 173, 201, 208, 212, 217, 299]: bovine plasma proteins [218], egg proteins [203, 219] and tuna proteins [220]; but also plant proteins, e.g. from soy [221], wine [222] and maize [223]. Nevertheless, milk proteins still appear to be the best source of ACE-inhibitory

Recall that caseins are the most abundant proteins in milk, and have an open and flexible structure that makes them susceptible to attack by proteases; hence, many ACE-inhibitors have been obtained via enzyme-mediated approaches [224-225] – e.g. casokinins. Studies on peptides with ACE-inhibitory activity obtained from whey proteins (called lactokinins) are more limited – which may be due to the rigid structure of -Lg (the major whey protein) that makes it particularly resistant to digestive enzymes. However, bioactive protein fragments with ACE-inhibitory activity have been found in whey protein hydrolyzates [107, 217, 226-228]; and Manso and López-Fandiño [155] also identified this activity in CMP hydrolyzates. Characterization of hydrolyzates of the main whey proteins – including the amino acid sequences of peptides therein that exhibit *in vitro* ACE-inhibiting activity, is

The ACE-inhibitory activity depends on the protein substrate and the proteolytic enzymes used to break it down. ACE (i.e. a dipeptidyl carboxypeptidase) is an enzyme ubiquitous in tissues and biological fluids – where it plays an important physiological role upon regulation of the cardiovascular function, including a basic role in regulation of peripheral blood pressure via the renin-angiotensin system [229-230]. ACE inhibitors and angiotensin II receptor blockers [231-232] have been therapeutically important, since they act as efficient

ACE-inhibitor peptide can reduce blood pressure in a process regulated (in part) by the renin-angiotensin system: renin — a protease secreted in response to various physiological stimuli, cleaves the protein angiotensinogen to produce the inactive decapeptide

precursors release bioactive fragments by gastrointestinal enzymes [73].

and treatment of hypertension [173, 201].

peptides.

provided in Table 4.

drugs and bring about very few collateral effects.

Alternative mechanisms are other routes of vasoregulator substance synthesis – e.g. kallikrein-kinin, neutral endopeptidase and endothelin-converting enzyme systems. The release of vasodilator substances like prostaglandin I2 or carbon monoxide could be implied in dependent and independent mechanisms of ACE inhibition responsible for antihypertensive effects [203-205]; an example is the peptide ALPMHIR, which inhibits release of an endothelial factor (ET-1) that causes contractions in smooth muscle cells [206].

In the last decade, production of antioxidant peptides from whey has been reported [207]. Experimental evidence – including SHR and human studies, claimed that oxidative stress is one of the causes of hypertension and several vascular diseases, via increase production of reactive oxygen species and reduction of NO synthesis and bioavailability of antioxidants [208].

Nevertheless, the most intensively studied peptides – i.e. VPP and IPP derived from caseins, showed possible mechanisms of action that could be found also in other peptides. In studies performed with rats, VPP and IPP increased plasma renin levels and activity [202]; and decreased ACE activity in the serum; they also showed endothelial function protection in mesenteric arteries [208]. The influence of VPP and IPP on gene expression of SHR abdominal aorta unfolded a significant increase of genes related with blood pressure regulation – the eNOS and connexin 40 genes [208]. Other studies have highlighted the peptide effects on the vasculature itself, showing that the antihypertensive activity of the peptide rapakinin is induced mainly by CCK1 and IP-receptor-dependent vasorelaxation; this peptide relaxes the mesenteric artery of SHR via prostaglandin I2-IP receptor, followed by CCK-CCK1 receptor pathway; other peptides improve aorta and mesenteric acetylcholine relaxation, and decrease left ventricular hypertrophy, accompanied by significant decrease in interstitial fibrosis [209]. In order to prevent hypertension, two alternative enzyme inhibitors were suggested: renin (a protease recognized as the initial compound of the renin–angiotensin system) and plateletactivating factor acetylhydrolase (PAF-AH) (a circulating enzyme secreted by inflammatory cells and involved in atherosclerosis) [208].

#### **5.1. Inhibition of angiotensin-converting enzyme (ACE)**

Since diet has a direct relationship to hypertension, the food industry (in association with research and public health institutions) has promoted development of novel functional ingredients that can contribute to keep a normal blood pressure – thus avoiding the need to take antihypertensive drugs [73, 173, 209-212]. Various investigators have accordingly hypothesized that certain peptides formed through hydrolysis of food proteins have the ability to inhibit ACE; López-Fandiño [173], FitzGerald [104, 137], Gobetti [213], Meisel [214], Korhonen and Pihlanto [4], Silva and Malcata [215], Vermeirssen [216], and Martínez-Maqueda [208] have comprehensively reviewed this subject. In general, it has been claimed that a diet rich in foods containing antihypertensive peptides is effective toward prevention and treatment of hypertension [173, 201].

86 Bioactive Food Peptides in Health and Disease

[208].

the vasodilatory action of binding to opioid receptors. Furthermore, endotheliumdependent relaxation of mesenteric arteries in spontaneously hypertensive rats (SHR, which is the animal model normally accepted to study human hypertension) that was inhibited by an endothelial nitric oxide synthase (eNOS) inhibitor was also observed [202]. That peptide

Alternative mechanisms are other routes of vasoregulator substance synthesis – e.g. kallikrein-kinin, neutral endopeptidase and endothelin-converting enzyme systems. The release of vasodilator substances like prostaglandin I2 or carbon monoxide could be implied in dependent and independent mechanisms of ACE inhibition responsible for antihypertensive effects [203-205]; an example is the peptide ALPMHIR, which inhibits release of an endothelial factor (ET-1) that causes contractions in smooth muscle cells [206].

In the last decade, production of antioxidant peptides from whey has been reported [207]. Experimental evidence – including SHR and human studies, claimed that oxidative stress is one of the causes of hypertension and several vascular diseases, via increase production of reactive oxygen species and reduction of NO synthesis and bioavailability of antioxidants

Nevertheless, the most intensively studied peptides – i.e. VPP and IPP derived from caseins, showed possible mechanisms of action that could be found also in other peptides. In studies performed with rats, VPP and IPP increased plasma renin levels and activity [202]; and decreased ACE activity in the serum; they also showed endothelial function protection in mesenteric arteries [208]. The influence of VPP and IPP on gene expression of SHR abdominal aorta unfolded a significant increase of genes related with blood pressure regulation – the eNOS and connexin 40 genes [208]. Other studies have highlighted the peptide effects on the vasculature itself, showing that the antihypertensive activity of the peptide rapakinin is induced mainly by CCK1 and IP-receptor-dependent vasorelaxation; this peptide relaxes the mesenteric artery of SHR via prostaglandin I2-IP receptor, followed by CCK-CCK1 receptor pathway; other peptides improve aorta and mesenteric acetylcholine relaxation, and decrease left ventricular hypertrophy, accompanied by significant decrease in interstitial fibrosis [209]. In order to prevent hypertension, two alternative enzyme inhibitors were suggested: renin (a protease recognized as the initial compound of the renin–angiotensin system) and plateletactivating factor acetylhydrolase (PAF-AH) (a circulating enzyme secreted by

Since diet has a direct relationship to hypertension, the food industry (in association with research and public health institutions) has promoted development of novel functional ingredients that can contribute to keep a normal blood pressure – thus avoiding the need to take antihypertensive drugs [73, 173, 209-212]. Various investigators have accordingly hypothesized that certain peptides formed through hydrolysis of food proteins have the ability to inhibit ACE; López-Fandiño [173], FitzGerald [104, 137], Gobetti [213], Meisel

may even chelate minerals, and thus facilitates calcium absorption [200].

inflammatory cells and involved in atherosclerosis) [208].

**5.1. Inhibition of angiotensin-converting enzyme (ACE)** 

ACE-inhibitory peptides may be obtained from precursor food proteins via enzymatic hydrolysis, using viable or lysed microorganisms or specific proteases [3, 73, 137, 169]. Although *in vitro* studies are useful at screening stages, the efficacy and safety of such peptides requires *in vivo* testing – first in animals, and then in human volunteers [217]. This issue is particularly relevant because *in vitro* ACE inhibition does not necessarily correlate with *in vivo* antihypertensive features, as peptides often undergo breakdown during gastrointestinal digestion that hampers manifestation of their potential physiological function. Conversely, antihypertensive activity may be promoted after long-chain peptide precursors release bioactive fragments by gastrointestinal enzymes [73].

In the latest two decades, various active peptides have been identified from animal proteins, including some with antihypertensive effects in animals (e.g. SHR) and even in humans [3, 73, 137, 169, 173, 201, 208, 212, 217, 299]: bovine plasma proteins [218], egg proteins [203, 219] and tuna proteins [220]; but also plant proteins, e.g. from soy [221], wine [222] and maize [223]. Nevertheless, milk proteins still appear to be the best source of ACE-inhibitory peptides.

Recall that caseins are the most abundant proteins in milk, and have an open and flexible structure that makes them susceptible to attack by proteases; hence, many ACE-inhibitors have been obtained via enzyme-mediated approaches [224-225] – e.g. casokinins. Studies on peptides with ACE-inhibitory activity obtained from whey proteins (called lactokinins) are more limited – which may be due to the rigid structure of -Lg (the major whey protein) that makes it particularly resistant to digestive enzymes. However, bioactive protein fragments with ACE-inhibitory activity have been found in whey protein hydrolyzates [107, 217, 226-228]; and Manso and López-Fandiño [155] also identified this activity in CMP hydrolyzates. Characterization of hydrolyzates of the main whey proteins – including the amino acid sequences of peptides therein that exhibit *in vitro* ACE-inhibiting activity, is provided in Table 4.

The ACE-inhibitory activity depends on the protein substrate and the proteolytic enzymes used to break it down. ACE (i.e. a dipeptidyl carboxypeptidase) is an enzyme ubiquitous in tissues and biological fluids – where it plays an important physiological role upon regulation of the cardiovascular function, including a basic role in regulation of peripheral blood pressure via the renin-angiotensin system [229-230]. ACE inhibitors and angiotensin II receptor blockers [231-232] have been therapeutically important, since they act as efficient drugs and bring about very few collateral effects.

ACE-inhibitor peptide can reduce blood pressure in a process regulated (in part) by the renin-angiotensin system: renin — a protease secreted in response to various physiological stimuli, cleaves the protein angiotensinogen to produce the inactive decapeptide


Whey Proteins as Source of Bioactive Peptides Against Hypertension 89

**Reduction in SBPb (mm Hg) (Dose (mg kg-1bw))** 

733 23.4 (0.1) [129, 233]

327 [233]

3 [238]

349 [26]

**References** 

**Source protein** 

*Bovine serum albumin*

*Caseinomacrop eptide* 

b Systolic blood pressure. c Synhetic peptides used.

patients [217].

**Enzyme Peptide** 

Pepsin + trypsin + chymotrypsin

**fragment** 

19

Trypsin f99-108 VGINYWLA

Proteinase K f208-216 ALKAWSV

Pepsin f50-53 YGLFc

Synthetic f52-53 LFc

a Concentration of peptide needed to inhibit 50 % of original ACE activity.

**Amino acid sequence** 

HK

ARc

*Lactoferrin* Pepsin f20-25 RRWQWR 16.7 (10) [240]

**Table 4.** Primary structural characteristics of whey peptides with ACE-inhibitory activity and antihypertensive activity in spontaneously hypertensive rats, and vectors of generation thereof.

angiotensin I. Cleavage of angiotensin I – via removal of two amino acid residues from the C-terminal end by ACE, produces the active octapeptide angiotensin II that is a potent vasoconstrictor; however, there are alternative routes to generate angiotensin II [198, 241- 242]. Angiotensin II activates angiotensin II type 1 (AT1) receptor — a member of the Gprotein-coupled-receptor superfamily, which plays various roles, e.g. vasoconstriction, as well as stimulation of aldosterone synthesis and release (which leads to sodium retention, and thus increases blood pressure) [198, 217, 242]. In addition, ACE acts on the kallikreinkinin system, catalyzing degradation of the nonapeptide bradykinin – which is a vasodilator [241]. ACE-inhibitor peptides exert a hypotensive effect by preventing angiotensin II formation and degradation of bradykinin, thus reducing blood pressure in hypertensive

Several tests on SHRs – probably the best experimental model for antihypertensive studies because they exhibit vascular reactivity and renal function similar to those in human beings [243], have been described that prove control of arterial blood pressure following a single oral administration of known ACE-inhibitory hydrolyzates or/and peptides derived from whey proteins. The antihypertensive effect associated with some of those peptides is comparable to that exhibited by VPP – an antihypertensive peptide included in functional foods that is already available in the market [117, 129, 137, 154, 201, 210-212, 242, 244-247]. To measure ACE-inhibitory activity, distinct biological, radiochromatographic, colorimetric and radioimmunologic methods have been employed – using angiotensin I as substrate. Chemical methods are sensitive, and resort to a tripeptide with a substituted aminoterminus, Z-Phe-His-Leu, as ACE-substrate – from which the dipeptide His-Leu is released

Trypsin f104-108 WLAHK 77 [233]

Proteinase K f221-222 FP 315 27 (8) [136]

Trypsin f106-112 MAIPPKK 28 (10) [239]

Pepsin f22-23 WQ 11.4 (10) [240]

**IC50 (µM)a**

f50-52 YGL 409 [233]


a Concentration of peptide needed to inhibit 50 % of original ACE activity.

b Systolic blood pressure.

c Synhetic peptides used.

88 Bioactive Food Peptides in Health and Disease

**Enzyme Peptide** 

Fermentation + trypsin + chymotrypsin

Yogurt starter + trypsin + pepsin

Fermentation with lactic acid bacteria + prozyme 6

Fermentation + trypsin + chymotrypsin

Fermentation by cheese microflora

Pepsin + trypsin + chymotrypsin

Pepsin + trypsin + chymotrypsin

Pepsin + trypsin + chymotrypsin

Pepsin + trypsin + chymotrypsin

Pepsin + trypsin + chymotrypsin

Pepsin + trypsin + chymotrypsin

Pepsin + trypsin + chymotrypsin

**fragment** 

Cardosins -Lg f33-42 DAQSAPLR

Cardosins -La f16-26 KGYGGVSL

Cardosins -La f97-103 DKVGINYc

Cardosins -La f97-104 DKVGINY

Trypsin f32-40 LDAQSAPL

Thermolysin f58-61 LQKWc

Thermolysin f103-105 LLFc

Trypsin f142-148 ALPMHIRc

Thermolysin f18-26 YGGVSLPE

Synthetic f50-51 or f18- YGc

*-Lactalbumin* Thermolysin f15-26 LKGYGGVS

Proteinase K -Lg f78-80 IPAc

**Amino acid sequence** 

VYc

PEWc

Wc

Neutrase -La f105-110 INYWL 11 [234]

Trypsin f7-9 MKG 71.8 [103] Trypsin f10-14 LDIQK 27.6 [103]

Trypsin f22-25 LAMA 556 [233]

Protease N Amano f36-42 SAPLRVY 8 [235]

Trypsin f81-83 VKF 1029 [233]

R

f102-103 YLc

f102-105 YLLFc

f142-145 ALPMc

f142-146 ALPMHc

LPEW

Thermolysin f20-26 GVSLPEW 30 [237] Thermolysin f21-26 VSLPEW 57 [237]

W

**IC50 (µM)a**






f15-19 VAGTW 1054 [233]

f94-100 VLDTDYK 946 [106, 233]

f106-111 CMENSA 788 [233]

**Reduction in SBPb (mm Hg) (Dose (mg kg-1bw))** 

12.2 10 (5) [107, 117]

0.7 20 (5) [107, 117]

25.4 15 (5) [107, 117]

635 [233]

34.7 18.1 (10) [103, 236]

122 [214]

172 [113]

79.8 29 (10) [113, 236]

928 21.4 (8) [116]

521 [233]

43 [226]

83 [237]

16 [237]

1522 [98]

99.9 [107]

141 31 (8) [136]

**References** 

**Source protein** 

*- Lactoglobulin*

*Whole whey protein*

> **Table 4.** Primary structural characteristics of whey peptides with ACE-inhibitory activity and antihypertensive activity in spontaneously hypertensive rats, and vectors of generation thereof.

angiotensin I. Cleavage of angiotensin I – via removal of two amino acid residues from the C-terminal end by ACE, produces the active octapeptide angiotensin II that is a potent vasoconstrictor; however, there are alternative routes to generate angiotensin II [198, 241- 242]. Angiotensin II activates angiotensin II type 1 (AT1) receptor — a member of the Gprotein-coupled-receptor superfamily, which plays various roles, e.g. vasoconstriction, as well as stimulation of aldosterone synthesis and release (which leads to sodium retention, and thus increases blood pressure) [198, 217, 242]. In addition, ACE acts on the kallikreinkinin system, catalyzing degradation of the nonapeptide bradykinin – which is a vasodilator [241]. ACE-inhibitor peptides exert a hypotensive effect by preventing angiotensin II formation and degradation of bradykinin, thus reducing blood pressure in hypertensive patients [217].

Several tests on SHRs – probably the best experimental model for antihypertensive studies because they exhibit vascular reactivity and renal function similar to those in human beings [243], have been described that prove control of arterial blood pressure following a single oral administration of known ACE-inhibitory hydrolyzates or/and peptides derived from whey proteins. The antihypertensive effect associated with some of those peptides is comparable to that exhibited by VPP – an antihypertensive peptide included in functional foods that is already available in the market [117, 129, 137, 154, 201, 210-212, 242, 244-247]. To measure ACE-inhibitory activity, distinct biological, radiochromatographic, colorimetric and radioimmunologic methods have been employed – using angiotensin I as substrate. Chemical methods are sensitive, and resort to a tripeptide with a substituted aminoterminus, Z-Phe-His-Leu, as ACE-substrate – from which the dipeptide His-Leu is released

and quantified by specific fluorometric procedures. A similar tripeptide used as substrate of ACE is Bz-Gly-His-Leu, or Hippuryl-His-Leu (HHL); upon incubation with the enzyme, hippuric acid is formed and the dipeptide His-Leu is released, which is subsequently measured by one of several colorimetric [248] or fluorometric methods [249], or even by capillary electrophoresis [250].

Whey Proteins as Source of Bioactive Peptides Against Hypertension 91

Although the relationships between structure and activity have not been fully elucidated, ACE-inhibitory peptides possess a number of analogies with each other. The tripeptide at the C-terminus is crucial – because this is where the peptide binds to the active site of the enzyme [256]. ACE prefers substrates (or competitive inhibitors) with hydrophobic residues (e.g. Trp, Tyr, Phe and Pro) at the C-terminus, and shows poorer affinity to substrates containing dicarboxylic amino acids in the final position, or those that have a Pro residue in the one before the last position. However, presence of Pro as the last residue [258], or in the third position from the terminus [259] favors binding of peptide to enzyme, in much the

Bioinformatics has been used more recently to find the structural requirements of ACEinhibitor peptides; these are termed quantitative structure/activity relationship (QSAR) models. Through a QSAR model, Pripp [262] concluded – for milk-derived peptides up to six amino acids in length, that there is a relationship between ACE-inhibitory activity and presence of a hydrophobic (or positively charged) amino acid residue in the last position of the sequence; however, no special relation was found with the structure of the N-terminus. Based on the QSAR model for peptides containing between 4 and 10 amino acid residues, Wu [263] claimed that the residue of the C-terminal tetrapeptide may determine the potency of ACE inhibition – with preference for Tyr and Cys in the first C-terminal position; His, Trp and Met in the second; Ile, Leu, Val and Met in the third; and Trp in the fourth position. Results from other QSAR-based studies aimed at finding ACE-inhibitory activity of di- and tripeptides derived from food proteins have shown that dipeptides with hydrophobic chains, as well as tripeptides with an aromatic amino acid residue at the C-terminus, a positively charged residue at the intermediate position and a hydrophobic amino acid

same way as when Leu appears in the last position [260,261].

residue at the N-terminus are likely to exhibit ACE-inhibitory power [263].

bradykinin [266].

On the other hand, a biopeptide may adopt a different configuration depending on the prevailing environmental conditions; but the final structural conformation may be crucial for its ACE-inhibitory activity. The fact that the catalytic center of ACE has different structural requirements may unfold the need to develop complex mixtures of peptides, with different structural conformations, so as to produce more complete inhibition than a single peptide [264]. Meisel [265] postulated that the mechanism of ACE inhibition may involve interaction of inhibitor with the subunits that are not normally occupied by substrate, or with the anionic bond site that is different from the enzyme catalytic center. Moreover, somatic ACE has two homologous domains – each of which has an active site with distinct biochemical characteristics. *In vitro* ACE-inhibition studies showed that it is necessary to block the two active centers for complete inhibition of its action upon angiotensin I and bradykinin. Nevertheless, *in vivo* studies in rats showed that the selective inhibition of the N- or C-terminal domains of ACE prevents conversion of angiotensin I to II, but not of

Despite the importance arising from the three amino acids in their C-terminus, it was shown that peptides with identical sequences at the C-terminus may exhibit quite different ACEinhibitory activities from each other. One example is VRYL and VPSERYL, both identified in

One of the most performing methods to measure ACE-inhibitory activity was developed by Cushman and Cheung [251], and is based on spectrophotometric measurement at 228 nm of hippuric acid formed by incubating the substrate HHL with ACE – in the presence of selected inhibitory substances. More recently, a modified tripeptide, furanacriloil Gly-Phe-Gly, has been chosen as substrate for a spectrophotometric method [252]. The ACEinhibitory activity is usually measured in terms of IC50 (i.e. the concentration of inhibitory substance required to inhibit 50 % of ACE activity); a low IC50 value means that a small concentration of inhibitory substance is required to produce enzyme inhibition, so that substance displays a potent inhibitory activity.

As shown in Table 4, ACE-inhibitor peptides are produced mainly by enzymatic hydrolysis, but active sequences have also been obtained via chemical synthesis [253]. Starter and nonstarter bacteria are commonly used in cheese manufacture – taking advantage namely of their proteolytic system, which contains at least 16 different peptidases that have already been characterized. Some of these bacteria were found to have ACE-inhibitory activity, or release peptides with this activity. For instance, *Lactobacillus helveticus* is able to release ACEinhibitory peptides; the best-known ACE-inhibitory peptides – *viz.* VPP and IPP, have indeed been identified in milk fermented with *L. helveticus* strains [154, 244, 254]. More recently, an ACE-inhibitory peptide derived from -CN – FFVAPFPEVFGK, was successfully expressed by genetic engineering in *Escherichia coli* [255].

#### *5.1.1. Structure/activity relationships*

ACE-inhibitor peptides contain usually between 2 and 12 amino acid residues – even though larger peptides may also exhibit such an activity [173]. Ondetti [229] rationalized the interaction of competitive inhibitors for the ACE active site based on enzyme homology with carboxypeptidase A; the first ACE-inhibitor (i.e. captopril), which is one of the oral drugs widely used to treat hypertension, was designed based on this model. Recently, this model was reviewed and used to design even more potent ACE inhibitors [229, 256-257]. The base model proposes that residues of the carboxy-terminal (C-terminal) tripeptide interact with the S1, S'1 and S'2 subunits of the enzyme active site. One of the subunits has a positively charged group that forms an ionic bond with the C-terminal peptide group. The following subunit contains a group capable of interacting with the peptidic bond of the Cterminal amino acid – probably through hydrogen bonding. The third subunit has a Zn2+ atom able to carry the carbonyl group of the peptidic bond between the one before the last and the last amino acid residue of the substrate – thus making it more susceptible to hydrolysis [256].

Although the relationships between structure and activity have not been fully elucidated, ACE-inhibitory peptides possess a number of analogies with each other. The tripeptide at the C-terminus is crucial – because this is where the peptide binds to the active site of the enzyme [256]. ACE prefers substrates (or competitive inhibitors) with hydrophobic residues (e.g. Trp, Tyr, Phe and Pro) at the C-terminus, and shows poorer affinity to substrates containing dicarboxylic amino acids in the final position, or those that have a Pro residue in the one before the last position. However, presence of Pro as the last residue [258], or in the third position from the terminus [259] favors binding of peptide to enzyme, in much the same way as when Leu appears in the last position [260,261].

90 Bioactive Food Peptides in Health and Disease

capillary electrophoresis [250].

substance displays a potent inhibitory activity.

*5.1.1. Structure/activity relationships* 

hydrolysis [256].

and quantified by specific fluorometric procedures. A similar tripeptide used as substrate of ACE is Bz-Gly-His-Leu, or Hippuryl-His-Leu (HHL); upon incubation with the enzyme, hippuric acid is formed and the dipeptide His-Leu is released, which is subsequently measured by one of several colorimetric [248] or fluorometric methods [249], or even by

One of the most performing methods to measure ACE-inhibitory activity was developed by Cushman and Cheung [251], and is based on spectrophotometric measurement at 228 nm of hippuric acid formed by incubating the substrate HHL with ACE – in the presence of selected inhibitory substances. More recently, a modified tripeptide, furanacriloil Gly-Phe-Gly, has been chosen as substrate for a spectrophotometric method [252]. The ACEinhibitory activity is usually measured in terms of IC50 (i.e. the concentration of inhibitory substance required to inhibit 50 % of ACE activity); a low IC50 value means that a small concentration of inhibitory substance is required to produce enzyme inhibition, so that

As shown in Table 4, ACE-inhibitor peptides are produced mainly by enzymatic hydrolysis, but active sequences have also been obtained via chemical synthesis [253]. Starter and nonstarter bacteria are commonly used in cheese manufacture – taking advantage namely of their proteolytic system, which contains at least 16 different peptidases that have already been characterized. Some of these bacteria were found to have ACE-inhibitory activity, or release peptides with this activity. For instance, *Lactobacillus helveticus* is able to release ACEinhibitory peptides; the best-known ACE-inhibitory peptides – *viz.* VPP and IPP, have indeed been identified in milk fermented with *L. helveticus* strains [154, 244, 254]. More recently, an ACE-inhibitory peptide derived from -CN – FFVAPFPEVFGK, was

ACE-inhibitor peptides contain usually between 2 and 12 amino acid residues – even though larger peptides may also exhibit such an activity [173]. Ondetti [229] rationalized the interaction of competitive inhibitors for the ACE active site based on enzyme homology with carboxypeptidase A; the first ACE-inhibitor (i.e. captopril), which is one of the oral drugs widely used to treat hypertension, was designed based on this model. Recently, this model was reviewed and used to design even more potent ACE inhibitors [229, 256-257]. The base model proposes that residues of the carboxy-terminal (C-terminal) tripeptide interact with the S1, S'1 and S'2 subunits of the enzyme active site. One of the subunits has a positively charged group that forms an ionic bond with the C-terminal peptide group. The following subunit contains a group capable of interacting with the peptidic bond of the Cterminal amino acid – probably through hydrogen bonding. The third subunit has a Zn2+ atom able to carry the carbonyl group of the peptidic bond between the one before the last and the last amino acid residue of the substrate – thus making it more susceptible to

successfully expressed by genetic engineering in *Escherichia coli* [255].

Bioinformatics has been used more recently to find the structural requirements of ACEinhibitor peptides; these are termed quantitative structure/activity relationship (QSAR) models. Through a QSAR model, Pripp [262] concluded – for milk-derived peptides up to six amino acids in length, that there is a relationship between ACE-inhibitory activity and presence of a hydrophobic (or positively charged) amino acid residue in the last position of the sequence; however, no special relation was found with the structure of the N-terminus. Based on the QSAR model for peptides containing between 4 and 10 amino acid residues, Wu [263] claimed that the residue of the C-terminal tetrapeptide may determine the potency of ACE inhibition – with preference for Tyr and Cys in the first C-terminal position; His, Trp and Met in the second; Ile, Leu, Val and Met in the third; and Trp in the fourth position. Results from other QSAR-based studies aimed at finding ACE-inhibitory activity of di- and tripeptides derived from food proteins have shown that dipeptides with hydrophobic chains, as well as tripeptides with an aromatic amino acid residue at the C-terminus, a positively charged residue at the intermediate position and a hydrophobic amino acid residue at the N-terminus are likely to exhibit ACE-inhibitory power [263].

On the other hand, a biopeptide may adopt a different configuration depending on the prevailing environmental conditions; but the final structural conformation may be crucial for its ACE-inhibitory activity. The fact that the catalytic center of ACE has different structural requirements may unfold the need to develop complex mixtures of peptides, with different structural conformations, so as to produce more complete inhibition than a single peptide [264]. Meisel [265] postulated that the mechanism of ACE inhibition may involve interaction of inhibitor with the subunits that are not normally occupied by substrate, or with the anionic bond site that is different from the enzyme catalytic center. Moreover, somatic ACE has two homologous domains – each of which has an active site with distinct biochemical characteristics. *In vitro* ACE-inhibition studies showed that it is necessary to block the two active centers for complete inhibition of its action upon angiotensin I and bradykinin. Nevertheless, *in vivo* studies in rats showed that the selective inhibition of the N- or C-terminal domains of ACE prevents conversion of angiotensin I to II, but not of bradykinin [266].

Despite the importance arising from the three amino acids in their C-terminus, it was shown that peptides with identical sequences at the C-terminus may exhibit quite different ACEinhibitory activities from each other. One example is VRYL and VPSERYL, both identified in

Manchego cheese; despite having the same C-terminal tripeptide sequence, they exhibit IC50 values of 24.1 M and 249.5 M, respectively – i.e. the latter is 10-fold less active than the former. If Val were replaced by a dicarboxylic amino acid at the fourth position of the Cterminus, e.g. via synthesis of ERVL, the IC50 measured would be 200.3 M, which corresponds to an ACE-inhibitory activity 8-fold lower than VRYL – hence demonstrating the crucial role of Val in that position for the intended bioactivity [261].

Whey Proteins as Source of Bioactive Peptides Against Hypertension 93

be absorbed from the digestive tract may still exert their function directly in the intestinal

Besides carrying out protein degradation to varying extents, gastrointestinal digestion plays a key role in formation of ACE-inhibitory peptides [216, 277]; hence, it is relevant to assess the gastrointestinal bioavailability of any potentially interesting peptides. Several studies have accordingly provided evidence for this realization – as happened with Manchego cheese, as well as with other fermented solutions and infant formulae [100, 261, 278-281]; for instance, a potent antihypertensive peptide was released via gastrointestinal digestion from a precursor with poor ACE-inhibitory activity *in vitro* [282] – and some peptides possess a remarkable intrinsic stability, whereas others are susceptible to unwanted degradation [136, 261, 281]; however, whether of any of those options will apply cannot be known in advance.

Animal and human trials are therefore nuclear when assessing bioactivity of peptides; peptides that do not show *in vitro* activity may exhibit *in vivo* antihypertensive activity, and vice versa. For instance, YKVPQL identified in a casein hydrolyzate and released by a proteinase from *L. helveticus* CP790, had a high *in vivo* ACE-inhibitory activity (IC50 22 M) but did not show any antihypertensive one [282] – probably as a consequence of degradation during the digestion process [137]. When the hydrolyzate was purified, another peptide sequence (KVLPVPQ) was found. Unlike the previous case – with a low *in vitro* ACE-inhibitory activity (IC50 > 1000 M), the latter showed a potent *in vivo* antihypertensive activity. It was claimed that this was due to pancreatic digestion that releases Gln, thus forming KVLPVP; furthermore, this fragment showed ACE-inhibitory activity *in vitro*, characterized by an IC50 of only 5 M. Finally, there are reports on peptides with a low ACEinhibitory activity *in vitro* that possess antihypertensive activity *in vivo* – owing to a hypotensive mechanism of action distinct from that of ACE inhibition. One example is YP, the IC50 of which is 720 M; however, it significantly decreases blood pressure between 2 and 8 h after oral administration to SHR [283]. It should be emphasized that *in vivo* tests of (putatively) promising bioactive peptides should not come into play before careful *in vitro* models have been checked – as they can provide useful preliminary information on the stability of such peptides upon exposure to the various peptidases and proteinases that they will likely find in the gastrointestinal tract, prior to eventual transport across the intestinal

Simulated (physiological) digestion is a useful tool to assess the stability of peptides with ACE-inhibitory activity against digestive enzymes. However, the degree of hydrolysis of a given peptide depends not only on its size and nature, but also on the presence of other peptides in its vicinity [272] – which would make it difficult to test the required number of possibilities in a rather limited experimental program. Several *in vitro* studies were carried out that show the importance of digestion upon formation and degradation of ACEinhibitor peptides [107, 272, 278-280, 284]. In these studies, peptides were subjected to two stages of hydrolysis that mimic digestion in the body. First, hydrolysis with pepsin, at acidic pH, intended to simulate the digestion process prevailing in the stomach; and second, digestion with a pancreatic extract, at basic pH as prevailing during intestinal digestion.

lumen – e.g. via interaction with receptors on the intestinal wall [97, 265, 276].

barrier [278-279].

#### *5.1.2. Bioavailability*

Among the several bioactive peptides studied to date, ACE-inhibitory peptides have received particular attention because of their beneficial effects upon hypertension [226, 233, 267]. Note that such effects depend on their ability to reach the target organs without having undergone decay or transformation. Tests encompassing hypertensive animals and human clinical trials have shown that certain sequences can lower blood pressure; however, it is difficult to establish a direct link between the ability to inhibit ACE *in vitro* and the actual antihypertensive activity *in vivo*. Knowledge of the mechanism of action of such bioactive peptides is obviously crucial before manufacture of functional foods with physiological properties is in order [268].

Some peptides with ACE-inhibitory and antihypertensive activities can be transported through the intestinal mucosa via the PepT1 transporter [269]; likewise, there is evidence that other peptides may exert a direct role upon the intestinal lumen [151, 270-271]. Digestive enzymes, absorption through the intestinal tract and blood proteases can bring about hydrolysis of ACE-inhibitor peptides, thus producing fragments with lower or greater activity than their precursor sequences [216]. Hence, for ACE-inhibitor peptides exert an *in vivo* effect, they should not act as substrates of the enzyme. Peptides may accordingly be classified into three groups based on their behavior regarding ACE: (1) true inhibitors, for which IC50 is not modified when incubated with the enzyme; (2) ACE-substrates, which are hydrolyzed during incubation, thus giving rise to fragments with a lower ACE-inhibition activity; and (3) peptides that are converted to real inhibitors by ACE and gastrointestinal protease action. Note that only sequences belonging to groups 1 and 3 may show an antihypertensive effect [245].

Effective inclusion of ACE-inhibitory peptides in the diet consequently requires them to somehow resist the strong stomach hydrolysis that may cause loss of bioactivity [104], and afterwards be able to pass into the blood stream – where they should be resistant to peptidases therein, so as to eventually reach the target sites where they are supposed to exert their physiological effects *in vivo*. The structure and bioactivity of short-chain peptides are more easily preserved through gastrointestinal passage than those of their long-chain counterparts [272] – whereas sequences containing Pro residue(s) are generally more resistant to degradation by digestive enzymes [273]. Furthermore, peptides absorbed following digestion may accumulate in specific organs, and then exert their action in a systematic and gradual manner [274, 275]. However, antihypertensive peptides that cannot be absorbed from the digestive tract may still exert their function directly in the intestinal lumen – e.g. via interaction with receptors on the intestinal wall [97, 265, 276].

92 Bioactive Food Peptides in Health and Disease

*5.1.2. Bioavailability* 

properties is in order [268].

antihypertensive effect [245].

Manchego cheese; despite having the same C-terminal tripeptide sequence, they exhibit IC50 values of 24.1 M and 249.5 M, respectively – i.e. the latter is 10-fold less active than the former. If Val were replaced by a dicarboxylic amino acid at the fourth position of the Cterminus, e.g. via synthesis of ERVL, the IC50 measured would be 200.3 M, which corresponds to an ACE-inhibitory activity 8-fold lower than VRYL – hence demonstrating

Among the several bioactive peptides studied to date, ACE-inhibitory peptides have received particular attention because of their beneficial effects upon hypertension [226, 233, 267]. Note that such effects depend on their ability to reach the target organs without having undergone decay or transformation. Tests encompassing hypertensive animals and human clinical trials have shown that certain sequences can lower blood pressure; however, it is difficult to establish a direct link between the ability to inhibit ACE *in vitro* and the actual antihypertensive activity *in vivo*. Knowledge of the mechanism of action of such bioactive peptides is obviously crucial before manufacture of functional foods with physiological

Some peptides with ACE-inhibitory and antihypertensive activities can be transported through the intestinal mucosa via the PepT1 transporter [269]; likewise, there is evidence that other peptides may exert a direct role upon the intestinal lumen [151, 270-271]. Digestive enzymes, absorption through the intestinal tract and blood proteases can bring about hydrolysis of ACE-inhibitor peptides, thus producing fragments with lower or greater activity than their precursor sequences [216]. Hence, for ACE-inhibitor peptides exert an *in vivo* effect, they should not act as substrates of the enzyme. Peptides may accordingly be classified into three groups based on their behavior regarding ACE: (1) true inhibitors, for which IC50 is not modified when incubated with the enzyme; (2) ACE-substrates, which are hydrolyzed during incubation, thus giving rise to fragments with a lower ACE-inhibition activity; and (3) peptides that are converted to real inhibitors by ACE and gastrointestinal protease action. Note that only sequences belonging to groups 1 and 3 may show an

Effective inclusion of ACE-inhibitory peptides in the diet consequently requires them to somehow resist the strong stomach hydrolysis that may cause loss of bioactivity [104], and afterwards be able to pass into the blood stream – where they should be resistant to peptidases therein, so as to eventually reach the target sites where they are supposed to exert their physiological effects *in vivo*. The structure and bioactivity of short-chain peptides are more easily preserved through gastrointestinal passage than those of their long-chain counterparts [272] – whereas sequences containing Pro residue(s) are generally more resistant to degradation by digestive enzymes [273]. Furthermore, peptides absorbed following digestion may accumulate in specific organs, and then exert their action in a systematic and gradual manner [274, 275]. However, antihypertensive peptides that cannot

the crucial role of Val in that position for the intended bioactivity [261].

Besides carrying out protein degradation to varying extents, gastrointestinal digestion plays a key role in formation of ACE-inhibitory peptides [216, 277]; hence, it is relevant to assess the gastrointestinal bioavailability of any potentially interesting peptides. Several studies have accordingly provided evidence for this realization – as happened with Manchego cheese, as well as with other fermented solutions and infant formulae [100, 261, 278-281]; for instance, a potent antihypertensive peptide was released via gastrointestinal digestion from a precursor with poor ACE-inhibitory activity *in vitro* [282] – and some peptides possess a remarkable intrinsic stability, whereas others are susceptible to unwanted degradation [136, 261, 281]; however, whether of any of those options will apply cannot be known in advance.

Animal and human trials are therefore nuclear when assessing bioactivity of peptides; peptides that do not show *in vitro* activity may exhibit *in vivo* antihypertensive activity, and vice versa. For instance, YKVPQL identified in a casein hydrolyzate and released by a proteinase from *L. helveticus* CP790, had a high *in vivo* ACE-inhibitory activity (IC50 22 M) but did not show any antihypertensive one [282] – probably as a consequence of degradation during the digestion process [137]. When the hydrolyzate was purified, another peptide sequence (KVLPVPQ) was found. Unlike the previous case – with a low *in vitro* ACE-inhibitory activity (IC50 > 1000 M), the latter showed a potent *in vivo* antihypertensive activity. It was claimed that this was due to pancreatic digestion that releases Gln, thus forming KVLPVP; furthermore, this fragment showed ACE-inhibitory activity *in vitro*, characterized by an IC50 of only 5 M. Finally, there are reports on peptides with a low ACEinhibitory activity *in vitro* that possess antihypertensive activity *in vivo* – owing to a hypotensive mechanism of action distinct from that of ACE inhibition. One example is YP, the IC50 of which is 720 M; however, it significantly decreases blood pressure between 2 and 8 h after oral administration to SHR [283]. It should be emphasized that *in vivo* tests of (putatively) promising bioactive peptides should not come into play before careful *in vitro* models have been checked – as they can provide useful preliminary information on the stability of such peptides upon exposure to the various peptidases and proteinases that they will likely find in the gastrointestinal tract, prior to eventual transport across the intestinal barrier [278-279].

Simulated (physiological) digestion is a useful tool to assess the stability of peptides with ACE-inhibitory activity against digestive enzymes. However, the degree of hydrolysis of a given peptide depends not only on its size and nature, but also on the presence of other peptides in its vicinity [272] – which would make it difficult to test the required number of possibilities in a rather limited experimental program. Several *in vitro* studies were carried out that show the importance of digestion upon formation and degradation of ACEinhibitor peptides [107, 272, 278-280, 284]. In these studies, peptides were subjected to two stages of hydrolysis that mimic digestion in the body. First, hydrolysis with pepsin, at acidic pH, intended to simulate the digestion process prevailing in the stomach; and second, digestion with a pancreatic extract, at basic pH as prevailing during intestinal digestion. Results encompassing prior or subsequent hydrolysis of peptides showed that *in vitro* digestion controls bioavailability of ACE-inhibitor peptides [162, 278]

Whey Proteins as Source of Bioactive Peptides Against Hypertension 95

encompassing those molecules and their hydrolysates. The technology developed is not excessively expensive, and can easily be implemented in dairy plants – of either small or large dimension. Most whey peptides bearing biological activity are released by enzymatic hydrolysis, so new alternatives to enzymes of animal origin have been under

This chapter focused on studies of whey peptides with antihypertensive activity – including their mechanisms of action (especially ACE inhibition), as well as the bioavailability of these peptides, and highlighting the main *in vitro* and *in vivo* results, as well as clinical trials in

Although a good deal of data have been generated encompassing food bioactive peptides, much is still left to do with whey peptides. Hence, several opportunities for further research exist, on incorporation of said ingredients in food products for human consumption. However, several scientific, technological and regulatory issues should be addressed before such peptide concentrates (and pure peptides) will have a chance to be marketed at large,

More detailed studies are indeed welcome for a better understanding of antihypertensive mechanisms. In particular, the antihypertensive activity should be checked with extra detail – including deep studies on the blood pressure-reducing mechanisms, such as the effects of peptides on neutral endopeptidases and their putative beneficial activity upon cardiovascular diseases. The pharmacological effect of said peptides should be determined both on post- and prejunctional receptors. More extensive clinical trials should also be performed – after thorough bioavailability studies *in vitro*, such as stability to gastrointestinal digestion and passage through the blood barrier, have taken

*Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal* 

*Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal* 

This work received partial finantial support from project NEW PROTECTION – NativE, Wild PRObiotic sTrain EffecCT In Olives in briNe (ref. PTDC/AGR-ALI/117658/2010), from

*Department of Chemical Engineering, University of Porto, Porto, Portugal* 

scrutiny.

humans.

place.

 \*

**Author details** 

Tânia G. Tavares

F. Xavier Malcata\*

**Acknowledgement** 

Coresponding Author

FCT, Portugal, coordinated by author F. X. M.

aiming at both human nutrition and health.

Some authors used whey proteins, fermented (or not at all) with *L. helveticus* and *Saccharomyces cerevisiae*, and then subjected them to gastrointestinal digestion; they reached a maximum ACE-inhibitory activity, and unfermented samples were the most active. However, some peptides with *in vivo* antihypertensive activity – as is the sequence KVLPVPQ, and which did not show *in vitro* ACE-inhibitory activity, could be transformed to active forms via gastrointestinal digestion [282]. Simulation of digestion is also useful in studies of the mechanism of action of antihypertensive peptides with demonstrated *in vivo* activity. For example, Miguel [277] found that YAEERYPIL derived from ovalbumin – which is a powerful ACE-inhibitor (IC50 = 4.7 M) and exhibits antihypertensive activity, was susceptible to degradation by digestive enzymes; that peptide was indeed fully hydrolyzed during simulated gastrointestinal digestion, thus giving rise to fragments YAEER and YPI. Tests on mice showed that YAEER could not significantly lower blood pressure, but the peptide YPI exhibited a significant antihypertensive effect. This fragment may possibly be the active form hidden in the sequence YAEERYPIL, and may exert its action via a different mechanism of ACE-inhibition [285].

*In vitro* models provide useful information to assess the stability of bioactive peptides to different peptidases and proteinases of the body, yet transport across the intestinal barrier raises an extra resistance – so they have limitations. *In vitro* simulated digestion is in fact not entirely reliable; the degree of hydrolysis depends on the size, nature and neighborhood of the peptide [272], so *in vivo* studies (with laboratory animals and human volunteers) are eventually necessary to ascertain in full the behavior of the peptide. Another example is the release of *potent* ACE-inhibitory peptides from WPC brought about by aqueous extracts from the plant *C. cardunculus.* A peptide mixture – in which 3 peptides were pinpointed: α-La f(16-26), with the sequence KGYGGVSLPEW; α-La f(97-104), with the sequence DKVGINYW; and β-Lg f(33-42), with the sequence DAQSAPLRVY, produced ACEinhibition (see Table 4). Such peptides were then exposed to simulated gastrointestinal digestion: no peptide was able to keep its integrity, but even total hydrolysis to smaller peptides did not significantly compromise the overall ACE-inhibitory activity observed. In view of their ACE-inhibitory activities, both in the absence or following gastrointestinal digestion, peptides KGYGGVSLPEW and DAQSAPLRVY are expected to eventually exhibit notable antihypertensive activities *in vivo* [107].

## **6. Concluding remarks**

Processing of whey proteins yields several bioactive peptides able to trigger physiological effects in the human body. Such peptides, in concentrated form, can be commercially appealing because their claimed health-promoting features are nowadays an important driver for consumers' food choices. Hence, they may constitute an excellent alternative for whey upgrade. Use of selective membranes to isolate, and eventually purify whey proteins and peptides has substantially increased the number and depth of studies encompassing those molecules and their hydrolysates. The technology developed is not excessively expensive, and can easily be implemented in dairy plants – of either small or large dimension. Most whey peptides bearing biological activity are released by enzymatic hydrolysis, so new alternatives to enzymes of animal origin have been under scrutiny.

This chapter focused on studies of whey peptides with antihypertensive activity – including their mechanisms of action (especially ACE inhibition), as well as the bioavailability of these peptides, and highlighting the main *in vitro* and *in vivo* results, as well as clinical trials in humans.

Although a good deal of data have been generated encompassing food bioactive peptides, much is still left to do with whey peptides. Hence, several opportunities for further research exist, on incorporation of said ingredients in food products for human consumption. However, several scientific, technological and regulatory issues should be addressed before such peptide concentrates (and pure peptides) will have a chance to be marketed at large, aiming at both human nutrition and health.

More detailed studies are indeed welcome for a better understanding of antihypertensive mechanisms. In particular, the antihypertensive activity should be checked with extra detail – including deep studies on the blood pressure-reducing mechanisms, such as the effects of peptides on neutral endopeptidases and their putative beneficial activity upon cardiovascular diseases. The pharmacological effect of said peptides should be determined both on post- and prejunctional receptors. More extensive clinical trials should also be performed – after thorough bioavailability studies *in vitro*, such as stability to gastrointestinal digestion and passage through the blood barrier, have taken place.

## **Author details**

94 Bioactive Food Peptides in Health and Disease

Results encompassing prior or subsequent hydrolysis of peptides showed that *in vitro*

Some authors used whey proteins, fermented (or not at all) with *L. helveticus* and *Saccharomyces cerevisiae*, and then subjected them to gastrointestinal digestion; they reached a maximum ACE-inhibitory activity, and unfermented samples were the most active. However, some peptides with *in vivo* antihypertensive activity – as is the sequence KVLPVPQ, and which did not show *in vitro* ACE-inhibitory activity, could be transformed to active forms via gastrointestinal digestion [282]. Simulation of digestion is also useful in studies of the mechanism of action of antihypertensive peptides with demonstrated *in vivo* activity. For example, Miguel [277] found that YAEERYPIL derived from ovalbumin – which is a powerful ACE-inhibitor (IC50 = 4.7 M) and exhibits antihypertensive activity, was susceptible to degradation by digestive enzymes; that peptide was indeed fully hydrolyzed during simulated gastrointestinal digestion, thus giving rise to fragments YAEER and YPI. Tests on mice showed that YAEER could not significantly lower blood pressure, but the peptide YPI exhibited a significant antihypertensive effect. This fragment may possibly be the active form hidden in the sequence YAEERYPIL, and may exert its

*In vitro* models provide useful information to assess the stability of bioactive peptides to different peptidases and proteinases of the body, yet transport across the intestinal barrier raises an extra resistance – so they have limitations. *In vitro* simulated digestion is in fact not entirely reliable; the degree of hydrolysis depends on the size, nature and neighborhood of the peptide [272], so *in vivo* studies (with laboratory animals and human volunteers) are eventually necessary to ascertain in full the behavior of the peptide. Another example is the release of *potent* ACE-inhibitory peptides from WPC brought about by aqueous extracts from the plant *C. cardunculus.* A peptide mixture – in which 3 peptides were pinpointed: α-La f(16-26), with the sequence KGYGGVSLPEW; α-La f(97-104), with the sequence DKVGINYW; and β-Lg f(33-42), with the sequence DAQSAPLRVY, produced ACEinhibition (see Table 4). Such peptides were then exposed to simulated gastrointestinal digestion: no peptide was able to keep its integrity, but even total hydrolysis to smaller peptides did not significantly compromise the overall ACE-inhibitory activity observed. In view of their ACE-inhibitory activities, both in the absence or following gastrointestinal digestion, peptides KGYGGVSLPEW and DAQSAPLRVY are expected to eventually exhibit

Processing of whey proteins yields several bioactive peptides able to trigger physiological effects in the human body. Such peptides, in concentrated form, can be commercially appealing because their claimed health-promoting features are nowadays an important driver for consumers' food choices. Hence, they may constitute an excellent alternative for whey upgrade. Use of selective membranes to isolate, and eventually purify whey proteins and peptides has substantially increased the number and depth of studies

digestion controls bioavailability of ACE-inhibitor peptides [162, 278]

action via a different mechanism of ACE-inhibition [285].

notable antihypertensive activities *in vivo* [107].

**6. Concluding remarks** 

Tânia G. Tavares *Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal* 

F. Xavier Malcata\*

*Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Department of Chemical Engineering, University of Porto, Porto, Portugal* 

## **Acknowledgement**

This work received partial finantial support from project NEW PROTECTION – NativE, Wild PRObiotic sTrain EffecCT In Olives in briNe (ref. PTDC/AGR-ALI/117658/2010), from FCT, Portugal, coordinated by author F. X. M.

<sup>\*</sup> Coresponding Author

#### **7. References**

[1] Diplock AT, Aggett PJ, Ashwell M, Bornet F, Fern EB, Roberfroid MB. Scientific concepts of functional foods in Europe consensus document. British Journal of Nutrition 1999; 81, 1-27.

Whey Proteins as Source of Bioactive Peptides Against Hypertension 97

[19] Whitney RM. Milk Proteins In: Fundamentals of dairy chemistry. van Nostrand

[20] Law AJR, Leaver J, Banks JM, Horne DS. Quantitative fractionation of whey proteins by

[21] Creamer L. K., Sawyer L. -Lactoglobulin. In: Roginski H, Fuquay JW, Fox PF (ed)

[22] Sawyer L, Kontopidis G. The core lipocalin, bovine -lactoglobulin. Biochimica et

[23] Imafidon IG, Farkye YN, Spanier MA. Isolation, purification, and alteration of some functional groups of major milk proteins: a review. Food Science and Nutrition 1997; 37,

[24] Gough P, Jenness R. Heat denaturation of β-lactoglobulins A and B. Journal of Dairy

[25] Walstra P, Geurts TJ, Noomen A, Tellema A, van Bockel. MAJS. Principles of milk properties and processes – Dairy Technology. New York, USA: Marcel Dekker; 1999. [26] Chatterton DEW, Smithers G, Roupas P, Brodkorb A. Bioactivity of -lactoglobulin and -lactalbumin – technological implications for processing. International Dairy Journal

[27] Papiz MZ, Sawyer L, Eliopoulos EE, North AC, Findlay JB, Sivaprasadarao R, Jones TA, Newcomer ME, Kraulis PJ. The structure of -lactoglobulin and its similarity to plasma

[28] Mansouri A, Haertle T, Gerard A, Gerard H, Gueant JL. Retinol free and retinol complexed -lactoglobulin binding sites in bovine germ cells. Biochimica et Biophysica

[29] Mansouri A, Gueant JL, Capiaumont J, Pelosi P, Nabet P, Haertle T. Plasma membrane receptor for -lactoglobulin and retinol-binding protein in murine hybridomas.

[30] Barros RM, Ferreira CA, Silva SV, Malcata FX. Quantitative studies on the enzymatic hydrolysis of milk proteins brought about by cardosins precipitated by ammonium

[31] Anderson ME. Glutathione: an overiew of biosynthesys and modulation. Chemico-

[32] Armstrong JM, McKenzie HA, Sawyer WH. On the fractionation of -lactoglobulin and

[33] Monaco HL, Zanotti G, Spadon P, Bolognesi M, Sawyer L, Eliopoulos EE. Crystal structure of the trigonal form of bovine -lactoglobulin and of its complex with retinol

[34] Felipe X, Law AJ. Preparative-scale fractionation of bovine, caprine and ovine whey proteins by gel permeation chromatography. Journal of Dairy Research 1997; 64, 459-

[35] de Jongh HH, Groneveld T, de Groot J. Mild isolation procedure discloses new protein structural properties of -lactoglobulin. Journal of Dairy Science 2001; 84, 562-571.

Reinhold. New York, USA; 1988.

Biophysica Acta 2000; 1482, 136-148.

Science 1961; 44, 1163-1168.

2006; 16, 1229-1240.

Acta 1997; 1357, 107-114.

Biofactors 1998; 7, 287-298.

464.

663-689.

gel permeation FPL. Milchwissenschaft 1993; 48, 663-666.

retinol-binding protein. Nature 1986; 324, 383-385.

sulfate. Enzyme and Microbial Technology 2001; 29, 541-547.


at 2.5 Å resolution. Journal of Molecular Biology 1987; 197, 695-706.

Biological Interaction – Limerick 1998; 111, 1-14.

Encyclopedia of Dairy Sciences. New York: Academic Press; 2003.


[19] Whitney RM. Milk Proteins In: Fundamentals of dairy chemistry. van Nostrand Reinhold. New York, USA; 1988.

96 Bioactive Food Peptides in Health and Disease

Nutrition 1999; 81, 1-27.

and Biochemistry 1996; 60, 9-15.

Dairy Journal 2006; 16, 945-960.

Ciencia y la Tecnología; 2005.

Wiley – VCH Publishers; 1996.

UK: Blackie Academic and Professional; 1998.

Elsevier; 1992. p369-404.

Food 1997; 41, 2-12.

Journal 1998; 8, 425-434.

USA: CRC, 2000.

Dairy Science 2003; 86, 1662-1672.

modificações. Varela, São Paulo 1996; 139-157.

and Nutrition Research 2003; 47, 175-276.

[1] Diplock AT, Aggett PJ, Ashwell M, Bornet F, Fern EB, Roberfroid MB. Scientific concepts of functional foods in Europe consensus document. British Journal of

[2] Arai S. Studies on functional foods in Japan–state of the art. Bioscience, Biotechnology

[3] Korhonen H, Pihlanto A. Food-derived bioactive peptides – opportunities for designing

[4] Korhonen H, Pihlanto A. Bioactive peptides: production and functionality. International

[5] Pintado ME, Pintado AE, Malcata FX. Controlled whey protein hydrolysis using two

[6] Recio I, López-Fandiño R. Ingredientes y productos lácteos funcionales, bases científicas de sus efectos en la salud. In: Alimentos funcionales (ed.) Fundación Espanõla para la

[7] Mulvihill DM. Production, functional properties and utilization of milk protein products. In: Fox PF. (ed.) Advanced dairy chemistry – proteins, vol 1. London, UK:

[8] Nakai S, Modler HW. Food proteins, properties and characterization. New York, USA:

[9] Fox PF, McSweeney PLH. Milk Proteins. In: Dairy chemistry and biochemistry. London,

[10] Pintado ME, Macedo AC, Malcata FX. Review: technology, chemistry and microbiology of whey cheeses. Food Science and Technology International 2001; 7, 105-116. [11] Blenford DE. Whey: from waste to gold. International Food Ingredients 1996; 1, 27-29. [12] Barth CA, Behnke U. Nutritional significance of whey and whey components. Nahrung-

[13] Walzem RL, Dilliard CJ, German JB. Whey components: millenia of evolution create functionalities for mammalian nutrition: what we know and what we may be

[15] Balagtas JV, Hutchinson FM, Krochta JM, Sumner DA. Anticipating market effects of new uses for whey and evaluating returns to research and development. Journal of

[16] Miller GD, Jarvis JK, Mcbeon LD. Handbook of Dairy Foods and Nutrition. Boca Raton,

[17] Sgarbieri VC. Proteinas em alimentos protéicos: propriedades, degradações,

[18] Pihlanto-Leppälä A, Korhonen H. Bioactive peptides and proteins. Advances in Food

overlooking. Critical Reviews in Food Science and Nutrition 2002; 42, 353-375. [14] McIntosh GH, Royle PJ, le Leu RK, Regester GO, Johnson MA, Grinsted RL, Kenward RS, Smithers GW. Whey proteins as functional food ingredients? International Dairy

future foods. Current Pharmaceutical Design 2003; 9, 1297-1308.

alternative proteases. Journal of Food Engineering 1999; 42, 1-13.

**7. References** 


[36] Outinen M, Tossavainen O, Tupasela T, Koskela P, Koskinen H, Rantamaki P, Syvaoja EL, Antila P, Kankare V. Fractionation of proteins from whey with different pilot scale processes. Food Science and Technology 1996; 29, 411-417.

Whey Proteins as Source of Bioactive Peptides Against Hypertension 99

[52] Delfour A, Jollès J, Alais C, Jollès P. Caseino-glycopeptides: characterization of a methionine residue and of the N-terminal sequence. Biochemical and Biophysical

[53] Manso MA, López-Fandiño R. -Casein macropeptides from cheese whey, physicochemical, biological, nutritional, and technological features for possible uses.

[54] Chobert JM, Touati A, Bertrand-Harb C, Dalgalarrondo M, Nicolas MG. Solubility and emulsifying properties of -casein and its caseinomacropeptide. Journal of Food

[55] Moreno FJ, López-Fandiño R, Olano A. Characterization and functional properties of lactosyl caseinomacropeptide conjugates. Journal of Agricultural and Food Chemistry,

[56] Dziuba J, Minkiewicz P. Influence of glycosylation on micelle-stabilizing ability and biological properties of C-terminal fragments of cow's -casein. International Dairy

[57] Kinsella E, Whitehead DM. Proteins in whey: chemical, physical and functional

[58] de Wit JN. The use of whey protein products. In: Fox PF. (ed.) Developments in dairy chemistry 4. Functional milk proteins. Barking, UK: Elsevier Science Publishers; 1989.

[59] Korhonen H, Marnila P, Gill H. Milk immunoglobulins and complement factors, a

[60] Steijns JM. Milk ingredients as nutraceuticals. International Journal of Dairy Technology

[61] Fonseca ML, Fonseca CSP, Brandão SCC. Propriedades anticarcinogênicas de

[64] Parodi PW. A role for milk protein in cancer prevention. Australian Journal of Dairy

[65] Regester GO, Mcintosh GH, Lee VWK, Smithers GW. Whey proteins as nutritional and

[66] Ha E, Zemel MB. Functional properties of whey, whey components, and essential amino acids: mechanisms underlying health benefits for active people (review). Journal

[67] Smithers GW. Whey and whey proteins – 'from gutter-to-gold'. International Dairy

[68] Bouchard D, Morisset D, Bourbonnais Y, Tremblay GM. Proteins with whey acidic-

[69] Gauthier SF, Pouliot Y, Saint-Sauveur D. Immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins. International Dairy Journal 2006; 16, 1315-1323.

[62] Alais C (ed.). Science du lait: principes des techniques laitières 4. Paris: SEPAIC; 1984. [63] Innocente N, Corradini C, Blecker C, Paquot M. Emulsifying properties of the total fraction and the hydrophobic fraction of bovine milk proteose-peptones. International

properties. Advances in Food and Nutrition Research 1989: 33, 343-438.

Research Communications 1965; 19, 452-455.

Food Reviews International 2004; 20, 329-355.

review. British Journal of Nutrition 2000; 84, 75-80.

componentes do leite. Indústria de Laticínios 1999; 4, 5-55.

functional food ingredients. Food Australia 1996; 48, 123-127.

protein motifs and cancer. Lancet Oncology 2006; 7, 167-174.

of Nutritional Biochemistry 2003; 14, 251-258.

Biochemistry 1989; 13, 457-473.

2002; 50, 5179-5184.

p323-345.

2001; 54, 81-88.

Journal 1996; 6, 1017-1044.

Dairy Journal 1998; 8, 981-985.

Technology 1998; 53, 37-47.

Journal 2008; 18, 695-704.


[52] Delfour A, Jollès J, Alais C, Jollès P. Caseino-glycopeptides: characterization of a methionine residue and of the N-terminal sequence. Biochemical and Biophysical Research Communications 1965; 19, 452-455.

98 Bioactive Food Peptides in Health and Disease

1104.

1599-1631.

298-300.

109-121.

1989; 6, 87-103.

102-109.

47, 83-229.

8, 901-911.

[36] Outinen M, Tossavainen O, Tupasela T, Koskela P, Koskinen H, Rantamaki P, Syvaoja EL, Antila P, Kankare V. Fractionation of proteins from whey with different pilot scale

[37] Hiraoka Y, Segawa T, Kuwajima K, Sugai S, Murai N. -Lactalbumin: a calcium metalloprotein. Biochemical and Biophysical Research Communication 1980; 95, 1098-

[38] Eigel WN, Butler JE, Ernstrom CA, Farrell H, Harwalkar VR, Jenness R, Whitney RM. Nomenclature of proteins of cows milk – 5th revision. Journal of Dairy Science, 1984; 67,

[39] Law AJR, Horne DS, Banks JM, Leaver J. Heat-induced changes in the whey proteins

[40] Heine WE, Klein PD, Reeds PJ. The importance of -lactalbumin in infantil nutrition.

[41] Tolkach A, Kulozik, U. Fractionation of whey proteins and caseinomacropeptide by means of enzymatic crosslinking and membrane separation techniques. Journal of Food

[42] Chang J, Bulychev A, Li L. A stabilized molten globule protein. FEBS Letters 2000; 487,

[43] Dolgikh DA, Abaturov IA, Bolotina, Brazhnikov EV, Bychkova VE, Gilmanshin RI, Lebedev YO, Semisotnov GV, Tiktopulo EI, Ptitsyn OB. Compact state of a protein molecule with pronounced small-scale mobility. European Biophysics Journal 1985; 13,

[44] Vanderheeren G, Hanssens I. Thermal unfolding of bovine α-lactalbumin. Comparison of circular dichroism with hydrophobicity measurements. Journal of Biological

[45] Kuwajima K. The molten globule state as a clue for understanding the folding and cooperativity of globular-protein struture. Protein: Structure, Function and Genetics

[46] Kuwajima K, Hiraoka Y, Ikeguchi M, Sugai S. Comparison of the transient folding intermediates in lysozyme and -lactalbumin. Biochemistry 1985; 24, 874-881. [47] Kuwajima K. The molten globule state of -lactalbumin. The FASEB Journal 1996; 1,

[48] Dolgikh DA, Gilmanshin RI, Brazhnikov EV, Bychkova VE, Semisotnov GV, Venyaminov S, Ptitsyn OB. Alpha-lactalbumin: compact state with fluctuating terciary

[49] Ptitsyn OB. Molten globule and protein folding. Advances in Protein Chemistry 1995;

[50] Arai M, Kuwajima K. Role of the molten globule state in protein folding. Advances in

[51] Leandro P, Gomes CM. Protein misfolding in conformational disorders: rescue of folding defects and chemical chaperoning. Mini-Reviews in Medicinal Chemistry 2008;

processes. Food Science and Technology 1996; 29, 411-417.

and caseins. Milchwissenschaft 1994; 49, 125-129.

Journal of Nutrition 1991; 121, 277-283.

Engineering 2005; 67, 13-20.

Chemistry 1994; 269, 7090-7094.

structure? FEBS Letters 1981; 136, 311-315.

Protein Chemistry 2000; 53, 209-282.


[70] Xu RJ. Bioactive peptides in milk and their biological and health implications. Food Reviews International 1998; 14, 1-16.

Whey Proteins as Source of Bioactive Peptides Against Hypertension 101

[84] Pacheco MTB, Bighetti EA, Antônio M, Carvalho JE, Possenti A, Sgarbieri VC. Antiulcerogenic activity of fraction and hydrolyzate obtained from whey protein

[85] Mezzaroba LFH, Carvalho JE, Ponezi AN, Antônio MA, Monteiro KM, Possenti A, Sgarbieri, VC. Antiulcerative properties of bovine -lactalbumin. International Dairy

[86] Tavares TG, Monteiro KM, Possenti A, Pintado ME, Carvalho JE, Malcata FX. Antiulcerogenic activity of peptide concentrates obtained from hydrolysis of whey protein brought about by proteases from *Cynara cardunculus*. International Dairy

[87] Madureira AR, Pereira CI, Gomes AMP, Pintado ME, Malcata FX. Bovine whey proteins – overview on their main biological properties. Food Research International 2007; 40,

[88] Puyol P, Dolores-Perez M, Sanchez L, Ena JM, Calvo M. Uptake and passage of lactoglobulin, palmitic acid and retinol across the Caco-2 monolayer. Biochimica et

[89] Wu SY, Pérez MD, Puyol P, Sawyer L. -Lactoglobulin binds palmitate within its

[90] Puyol P, Pérez MD, Ena JM, Calvo M. Interaction of -lactoglobulin and other bovine and human whey proteins with retinol and fatty acids. Agricultural and Biological

[91] Bounous G, Batist G, Gold P. Whey proteins in cancer prevention. Cancer Letters 1991;

[92] Baruchel S, Wang T, Farah R, Jamali M, Batist G. *In vivo* modulation of tissue glutathione in rat mammary carcinoma model. Biochemical Pharmacology 1995; 50,

[93] Bounous G. Whey protein concentrate (WPC) and glutathione modulation in cancer

[94] Perez MD, Sanchez L, Aranda P, Ena JM, Oria R, Calvo M. Effect of -lactoglobulin on the activity of pregastric lipase. A possible role for this protein in ruminant milk.

[95] Warme PKF, Momany A, Rumball SV, Tuttle RW, Scherag HA. Computation of structures of homologous proteins. -Lactalbumin from lysozyme. Biochemistry 1974;

[96] Farrel HM, Bede MJ, Enyeart JA. Binding of *p*-nitrophenyl phosphate and other

[97] Meisel H, Schlimme E. Bioactive peptides derived from milk proteins: ingredients for functional foods? Kieler Milchwirtschaftliche und Forschungsberichte 1996; 48, 343-357. [98] Mullally MM, Meisel H, Fitzgerald RJ. Synthetic peptides corresponding to lactalbumin and -lactoglobulin sequences with angiotensin-I-converting enzyme

aromatic compounds by -Lg. Journal of Dairy Science 1987; 70, 252-258.

inhibitory activity. Biological Chemistry Hoppe-Seyler 1996; 377, 259-260.

Biophysica Acta – Biomembranes 1995; 1236, 149-154.

treatment. Anticancer Research 2000; 20, 4785-4792.

Biochimica et Biophysica Acta 1992; 1123, 151-155.

central cavity. Journal of Biological Chemistry 1999; 274, 170-177.

concentrate. Brazilian Journal of Food Technology, III JIPCA 2006; 15-22.

Journal 2006; 16, 1005-1012.

Journal 2011; 21, 934-939.

Chemistry 1991; 10, 2515-2520.

1197-1211.

57, 91-94.

1505-1508.

13, 768-782.


[84] Pacheco MTB, Bighetti EA, Antônio M, Carvalho JE, Possenti A, Sgarbieri VC. Antiulcerogenic activity of fraction and hydrolyzate obtained from whey protein concentrate. Brazilian Journal of Food Technology, III JIPCA 2006; 15-22.

100 Bioactive Food Peptides in Health and Disease

Campinas-Unicamp, 2003.

Toxicology 2001; 20, 165-174.

Prevention 2000; 9, 113-117.

Investigation, 2001; 31, 171-178.

Journal of Nutrition 2003; 89, 339-348.

18.

Journal of Nutrition 2001; 131, 3281-3287.

Reviews International 1998; 14, 1-16.

[70] Xu RJ. Bioactive peptides in milk and their biological and health implications. Food

[71] Prates JAM, Mateus CMRP. Functional foods from animal sources and their physiologically active components. Revue de Médicine Vétérinaire 2002; 153, 155-160. [72] Amaya-Farfán J. Avanços no conhecimento sobre a função das proteínas nas dietas para desempenho físico. 5º Simpósio Latino Americano de Ciência de Alimentos – Desenvolvimento Científico e Tecnológico e Inovação na Indústria de Alimentos.

[73] Hartmann R, Meisel H. Food-derived peptides with biological activity: from research to

[74] Gill HS, Rutherford KJ, Cross ML. Bovine milk: a unique source of immunomodulatory ingredients for functional foods. In: Buttriss J, Saltmarsh M. (eds.) Functional foods II – claims and evidence. Cambridge, UK: Royal Society of Chemistry Press; 2000. p82-90. [75] Badger TM, Ronis MJJ, Hakkak R. Developmental effects and health aspects of soy protein isolate, casein and whey in male and female rats. International Journal of

[76] Hakkak R, Korourian S, Shelnutt SR, Lensing S, Ronis MJJ, Badger TM. Diets containing whey proteins or soy protein isolate protect against 2,12-dimethylbenzanthraceneinduced mammary tumours in female rats. Cancer Epidemiology, Biomarkers and

[77] Rowlands JC, He L, Hakkak R, Ronis MJJ, Badger TM. Soy and whey proteins down regulate DMBA-induced liver and mammary gland CYP1 expression in female rats.

[78] Micke P, Beeh KM, Schlaak JF, Buhl R. Oral supplementation with whey proteins increases plasma GSH levels of HIV-infected patients. European Journal of Clinical

[79] Micke P, Beeh KM, Buhl R. Effects of long-term supplementation with whey proteins on plasma GSH levels of HIV-infected patients. European Journal of Nutrition 2002; 41, 12-

[80] Clare DA, Catignani GL, Swaisgood HE. Biodefense properties of milk, the role of antimicrobial proteins and peptides. Current Pharmaceutical Design 2003; 9, 1239-1255. [81] Hall WL, Millward DJ, Long SJ, Morgan LM. Casein and whey exert different effects on plasma amino acid profiles, gastrointestinal hormone secretion and appetite. British

[82] Tavares TG, Contreras MM, Amorim M, Martín-Álvarez PJ, Pintado ME, Recio I, Malcata FX. Optimization, by response surface methodology, of degree of hydrolysis, antioxidant and ACE-inhibitory activities of whey protein hydrolyzates obtained with

[83] Rosaneli CF, Bighetti AE, Antônio MA, Carvalho JE, Sgarbieri VC. Efficacy of a whey protein concentrate on the inhibition of stomach ulcerative lesions caused by ethanol

cardoon extract. International Dairy Journal 2011; 21, 926-933.

ingestion. Journal of Medicinal Food 2002; 5, 221-228.

food applications. Current Opinion in Biotechnology 2007; 18, 163-169.


[99] Pihlanto-Leppälä A, Paakkari I, Rinta-Koski M, Antila P. Bioactive peptide derived from *in vitro* proteolysis of bovine β-lactoglobulin and its effect on smooth muscle. Journal of Dairy Research 1997; 64, 149-155.

Whey Proteins as Source of Bioactive Peptides Against Hypertension 103

identification of aggregating peptides. Journal of Agricultural and Food Chemistry

[114] Nagaoka S, Futamura Y, Miwa K, Awano T, Yamauchi K, Kanamaru Y, Kojima T, Kuwata T. Identification of novel hypocholesterolemic peptides derived from bovine milk -lactoglobulin. Biochemical and Biophysical Research Communications 2001; 281,

[115] Antila P, Paakkari I, Järvinen A, Mattila MJ, Laukkanen M, Pihlanto-Leppälä A, Mäntsälä P, Hellman J. Opioid peptides derived from *in vitro* proteolysis of bovine

[116] Murakami M, Tonouchi H, Takahashi R, Kitazawa H, Kawai Y, Negishi H, Saito T. Structural analysis of a new anti-hypertensive peptide (β-lactosin B) isolated from a

[117] Tavares TG, Sevilla MA, Montero MJ, Carrón R, Malcata FX. Acute effects of whey peptides upon blood pressure of hypertensive rats, and relationship with their angiotensin-converting enzyme inhibitory activity. Molecular Nutrition and Food

[118] Yamauchi R, Sonoda S, Jinsmaa Y, Yoshikawa M. Antinociception induced by βlactotensin, a neurotensin agonist peptide derived from β-lactoglobulin, is mediated by

[119] de Wit JN. Nutritional and functional characteristics of whey proteins in food

[120] A, Zhivotovsky B, Orrenius S, Sabharwal H, Svanborg C. Apoptosis induced by a human milk protein. Proceedings of the National Academy of Sciences of USA 1995; 92,

[121] Svensson M, Sabharwal H, Hakansson A, Mossberg AK, Lipniunas P, Leffler H, Svanborg, C, Linse S. Molecular characterization of -lactalbumin folding variants that induce apoptosis in tumor cells. Journal of Biological Chemistry 1999; 274, 6388-6396. [122] Svensson M, Hakansson A, Mossberg AK, Linse S, Svanborg C. Conversion of lactalbumin to a protein inducing apoptosis. Proceedings of the National Academy of

[123] Markus CR, Olivier B, Haan E. Whey protein rich in -lactalbumin increases the ratio of plasma tryptophan to the sum of the other large neutral amino acids and improves cognitive performance in stress-vulnerable subjects. American Journal of Clinical

[124] Ganjam LS, Thornton WH, Marshall RT, MacDonald RS. Antiproliferative effects of yoghurt fractions obtained by membrane dialysis on cultured mammalian intestinal

[125] Hakansson A, Svensson M, Mossberg AK, Sabharwal H, Linse S, Lazou I, Lönnerdal B, Svanborg C. A folding variant of -lactalbumin with bactericidal activity against

[126] Markus CR, Olivier B, Panhuysen GEM, Gugten J, van Der, Alles MS, Tuiten A, Westenberg HGM, Fekkes D, Kopperschaar HF, Haan E. The bovine α-lactalbumin increases the plasma ratio of tryptophan to the other large neutral amino acids, and in

*Streptococcus pneumoniae*. Molecular Microbiology 2000; 35, 589-600.

commercial whey product. Journal of Dairy Science 2004; 87, 1967-1974.

whey proteins. International Dairy Journal 1991; 1, 215-229.

Research 2011; doi: 10.1002/mnfr.201100381.

Sciences of USA 2002; 97, 4221-4226.

cells. Journal of Dairy Science 1997; 80, 2325-2329.

Nutrition 2002; 75, 1051-1056.

NT2 and D1 receptors. Life Sciences 2003; 73, 1917-1923.

products. Journal of Dairy Science 1998; 81, 597-608.

2003; 51, 4370-4375.

11-17.

8064-8068.


identification of aggregating peptides. Journal of Agricultural and Food Chemistry 2003; 51, 4370-4375.

[114] Nagaoka S, Futamura Y, Miwa K, Awano T, Yamauchi K, Kanamaru Y, Kojima T, Kuwata T. Identification of novel hypocholesterolemic peptides derived from bovine milk -lactoglobulin. Biochemical and Biophysical Research Communications 2001; 281, 11-17.

102 Bioactive Food Peptides in Health and Disease

1998; 8, 325-331.

Trivandrum; 2006. p37-60.

2007; 17, 471-480.

1526, 131-140.

Journal of Dairy Research 1997; 64, 149-155.

Journal of Dairy Technology 2006; 59, 118-125.

fermentation. International Dairy Journal 2007; 17, 641-647.

proteins. International Dairy Journal 2006; 16, 1294-1305.

Biochimica et Biophysica Acta 1999; 1426, 439-448.

[99] Pihlanto-Leppälä A, Paakkari I, Rinta-Koski M, Antila P. Bioactive peptide derived from *in vitro* proteolysis of bovine β-lactoglobulin and its effect on smooth muscle.

[100] Pihlanto-Leppälä A, Rokka T, Korhonen H. Angiotensin I converting enzyme inhibitory peptides derived from bovine milk proteins. International Dairy Journal

[101] Pihlanto L. Bioactive peptides derived from bovine whey proteins: opioid and ACEinhibitory peptides. Trends in Food Science and Technology 2000; 11, 347-356. [102] Ijäs H, Collin M, Finckenberg P, Pihlanto-Leppälä A, Korhonen H, Korpela R, Vapaatalo H, Nurminen ML. Antihypertensive opioid-like milk peptide -lactorphin: lacks effect on behavioural tests in mice. International Dairy Journal 2004; 14, 201-205. [103] Hernández-Ledesma B, López-Expósito I, Ramos M, Recio I. Bioactive peptides from milk proteins. In: Pizzano R (ed.) Immunochemistry in dairy research. Kerala, India:

[104] FitzGerald RJ, Murray BA. Bioactive peptides and lactic fermentations. International

[105] Chen G-W, Tsai J-S, Pan BS. Purification of angiotensin I-converting enzyme inhibitory peptides and antihypertensive effect of milk produced by protease-facilitated lactic

[106] Roufik S, Gauthier S, Turgeon S. Physicochemical characterization and *in vitro* digestibility of -lactoglobulin/-Lg f142-148 complexes. International Dairy Journal

[107] Tavares TG, Contreras MM, Amorim M, Pintado ME, Recio I, Malcata FX. Novel whey-derived peptides with inhibitory activity against angiotensin-converting enzyme: *in vitro* activity and stability to gastrointestinal enzymes. Peptides 2011; 32, 1013-1019. [108] Expósito IL, Récio I. Antibacterial activity of peptides and holding variants from milk

[109] Pihlanto-Leppälä A, Marnila P, Hubert L, Rokka T, Korhonen HJT, Karp M. The effect of α-lactalbumin and β-lactoglobulin hydrolysates on the metabolic activity of

[110] Pellegrini A, Thomas U, Bramaz N, Hunziker P, Fellenberg R. Isolation and identification of three bactericidal domains in the bovine α-lactalbumin molecule.

[111] Bruck WM, Graverholt G, Gibson GR. A two-stage continuous culture system to study the effect of supplemental -lactalbumin and glycomacropeptide on mixed populations of human gut bacteria challenged with enteropathogenic *Escherichia coli* and *Salmonella* 

[112] Pellegrini A, Dettling C, Thomas U, Hunziker P. Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin. Biochimica et Biophysica Acta 2001;

[113] Groleau PE, Morin P, Gauthier SF, Pouliot Y. Effect of physicochemical conditions on peptide-peptide interactions in a tryptic hydrolysate of β-lactoglobulin and

*Escherichia coli* JM103. Journal of Applied Microbiology 1999; 87, 540-545.

serotype *Typhimurium*. Journal of Applied Microbiology 2003; 95, 44-53.


vulnerable subjects raises brain serotonin activity, reduces cortisol concentration, and improves mood under stress. American Journal of Clinical Nutrition 2000; 71, 1536- 1544.

Whey Proteins as Source of Bioactive Peptides Against Hypertension 105

[141] Mitra AK, Mahalanabis D, Unicomb L, Eechels R, Tzipori S. Hyperimmune cow colostra reduces diarrhoea due to rotavirus: a double-blind, controlled clinical trial.

[142] Tomita M, Todhunter DA, Hogan JS, Smith KL. Immunisation of dairy cows with an *Escherichia coli* J5 lipopolysaccharide vaccine. Journal of Dairy Science 1995; 78, 2178-

[143] Oona M, Rägö T, Maaroos HI, Mikelsaar M, Loivukene K, Salminen S, Korhonen H. *Helicobacter pylori* in children with abdominal complaints: has immune bovine colostrum some influence on gastritis? Alpe Adria Microbiology Journal 1997; 6, 49-57. [144] Freedman DJ, Tacket CO, Delehanty A, Maneval DR, Nataro J, Crabb JH. Milk immunoglobulin with specific activity against purified colonization factor antigens can protect against oral challenge with enterotoxigenic *Escherichia coli*. Journal of Infectious

[145] Loimaranta V, Laine M, Söderling E, Vasara E, Rokka S, Marnila P, Korhonen H, Tossavainen O, Tenovuo J. Effects of bovine immune- and non-immune whey preparations on the composition and pH response of human dental plaque. European

[146] Okhuysen PC, Chappell CL, Crabb J, Valdez LM, Douglass E, DuPont HL. Prophylactic effect of bovine anti-*Cryptosporidium hyperimmune* colostra immunoglobulin in healthy volunteers challenged with *Cryptosporidium parvum*.

[147] Sharpe SJ, Gamble GD, Sharpe DN. Cholesterol-lowering and blood pressure effects of

[148] Jollès P, Fiat AM. The carbohydrate portions of milk glycoproteins. Journal of Dairy

[149] Jollès P, Levy-Toledano S, Fiat AM, Soria C, Cillessen D, Thomaidis A, Dunn FW, Caen JP. Analogy between fibrinogen and casein. Effect of an undecapeptide isolated from -casein on platelet function. European Journal of Biochemistry 1986; 158, 379-382. [150] Shebuski RJ, Berry DE, Bennett DB, Romoff T, Storer BL, Ali F, Samanen J. Demonstration of Ac-Arg-Gly-Asp-Ser-NH2 as an antiaggregatory agent in the dog by intracoronary administration. Journal of Thrombosis and Haemostasis 1989; 61, 183-188. [151] Chabance B, Marteau P, Rambaud JC, Migliore-Samour D, Boynard M, Perrotin P, Guillet R, Jollès P, Fiat AM. Casein peptide release and passage to the blood in humans

[152] Rutherford KJ, Gill H. Peptides affecting coagulation. British Journal of Nutrition 2000;

[153] Manso MA, Escudero C, Alijo M, López-Fandiño R. Platelet aggregation inhibitory activity of bovine, ovine and caprine -casein macropeptides and their tryptic

[154] Nakamura Y, Yamamoto N, Sakai K, Okubo A, Yamazaki S, Takano T. Purification and characterization of angiotensin I-converting enzyme inhibitors from sour milk.

immune milk. American Journal of Clinical Nutrition 1994; 59, 929-934.

during digestion of milk or yogurt. Biochimie 1998; 80, 155-165.

hydrolyzates. Journal of Food Protection 2002; 65, 1992-1996.

Journal of Dairy Science 1995; 78, 777-783.

Acta Paediatrica 1995; 84, 996-1001.

Diseases 1998; 177, 662-667.

Research 1979; 46, 187-191.

84, 99-102.

Journal of Oral Science 1999; 107, 244-250.

Clinical Infectious Diseases 1998; 26, 1324-1329.

2185.


[141] Mitra AK, Mahalanabis D, Unicomb L, Eechels R, Tzipori S. Hyperimmune cow colostra reduces diarrhoea due to rotavirus: a double-blind, controlled clinical trial. Acta Paediatrica 1995; 84, 996-1001.

104 Bioactive Food Peptides in Health and Disease

Nutrition 2000; 84, 27-31.

Science 2000; 66, 1535-1543.

Chemistry 2000; 48, 1473-1478.

Journal of Dairy Science 1998; 81, 3131-3138.

Journal of Nutrition 2004; 134, 980-988.

proteins. IDF Bulletin 1992; 272, 51-57.

1544.

1104-1111.

489.

32, 160-166.

vulnerable subjects raises brain serotonin activity, reduces cortisol concentration, and improves mood under stress. American Journal of Clinical Nutrition 2000; 71, 1536-

[127] Montagne PM, Cuiliere ML, Mole CM, Bene MC, Faure GC. Dynamics of the main immunologically and nutritionally available proteins of human milk during lactation.

[128] Meisel H, FitzGerald RJ. Opioid peptides encrypted in milk proteins. Journal of

[129] Nurminen ML, Sipola M, Kaarto H, Pihlanto-Leppälä, Piilola K, Korpela R, Tossavainen O, Coronen H, Vapaatalo H. -Lactorphin lowers blood pressure measured by radiotelemetry in normotensive and spontaneously hypertensive rats. Life

[130] Matsumoto H, Shimokawa Y, Ushida Y, Toida T, Hayasawa H. New biological function of bovine -lactalbumin: protective effect against ethanol- and stress-induced gastric mucosal injury in rats. Bioscience, Biotechnology and Biochemistry 2001; 65,

[131] Uchida K, Tateda T, Takagi S. Hypothetical mechanism of prostaglandin E1-induced

[132] Rosaneli CF, Bighetti AE, Antônio MA, Carvalho JE, Sgarbieri VC. Protective effect of bovine milk whey protein concentrate on the ulcerative lesions caused by subcutaneous

[133] Tong LM, Sasaki S, McClements DJ, Decker EA. Mechanisms of the antioxidant activity of a high molecular weight fraction of whey. Journal of Agricultural and Food

[134] Smith C, Halliwell B, Aruoma O I. Protection of albumin against the pro-oxidant actions of phenolic dietary components. Food Chemistry and Toxicology 1992; 30, 483-

[135] Laursen I, Briand P, Lykkesfeldt AE. Serum albumin as a modulator of the human

[136] Abubakar A, Saito T, Kitazawa H, Kawai Y, Itoh T. Structural analysis of new antihypertensive peptides derived from cheese whey protein by proteinase K digestion.

[137] FitzGerald RJ, Murray BA, Walsh DJ. Hypotensive peptides from milk proteins.

[138] Yamauchi K. Biologically functional proteins of milk and peptides derived from milk

[139] Tani F, Shiota, Chiba H, Yosliikawa M. Serophin an opioid peptide derived from bovine serum albumin. In: Brantl V. (ed.) -Casomorphins and relate peptides: recent

[140] Ormrod DJ, Miller TE. The anti-inflammatory activity of a low molecular weight component derived from the milk of hyperimmunized cows. Agents and Actions 1991;

breast cancer cell line MCF-7. Anticancer Research 1990; 10, 343-351.

developments. Weinheim, Germany: VCH-Verlag; 1993. p49-53.

administration of indomethacin. Journal of Medicinal Food 2004; 7, 309-314.

Journal of Food Composition and Analysis 2000; 13, 127-137.

bronchoconstriction. Medical Hypotheses 2003; 61, 378-384.


[155] Manso MA, López-Fandiño R. Angiotensin I converting enzyme-inhibitory activity of bovine, ovine, and caprine -casein macropeptides and their tryptic hydrolysates. Journal of Food Protection 2003; 66, 1686-1692.

Whey Proteins as Source of Bioactive Peptides Against Hypertension 107

[171] Fox PF. Exogeneous enzymes in dairy technology. A review. Journal of Food

[172] Simões IIG. Caracterização molecular da acção das cardosinas A e B sobre caseínas - e

[173] López-Fandiño R, Otte J, van Camp J. Physiological, chemical and technological aspects of milk-protein-derived peptides with antihypertensive and ACE-inhibitory

[174] Schmidt DG, Meijer RJ, Slangen CJ, van Beresteijn EC. Raising the pH of the pepsincatalysed hydrolysis of bovine whey proteins increases the antigenicity of the

[175] Barros RM, Malcata FX. Molecular characterization of peptides released from lactoglobulin and -lactalbumin via cardosins A and B. Journal of Dairy Science 2006;

[176] Lamas EM, Barros RM, Balcão VM, Malcata FX. Hydrolysis of whey proteins by proteases extracted from *Cynara cardunculus* and immobilized onto highly activated

[177] Barros RM, Malcata FX. Modelling the kinetics of whey protein hydrolysis brought about by enzymes from *Cynara cardunculus*. Journal of Agricultural and Food Chemistry

[178] Barros RM, Malcata FX. A kinetic model for hydrolysis of whey proteins by cardosin A

[179] Saboya LV, Maubois JL. Current developments of microfiltration technology in the

[180] Muller A, Daufin G, Chaufer B. Ultrafiltration modes of operation for the separation of -lactalbumin from acid casein whey. Journal of Membrane Science 1999; 153, 9-21. [181] Cheang B, Zydney AL. Separation of -lactalbumin and -lactoglobulin using membrane ultrafiltration. Biotechnology and Bioengineering 2003; 83, 201-209. [182] Cheang B, Zydney AL. A two-stage ultrafiltration process for fractionation of whey

[183] Brans G, Schroën CGPH, van der Sman RGM, Boom RM. Membrane fractionation of milk: state of the art and challenges. Journal of Membrane Science 2004; 243, 263-272. [184] Díaz O, Pereira CD, Cobos A. Functional properties of ovine whey protein concentrates produced by membrane technology after clarification of cheese

[185] Atra R, Vatai G, Bekassy-Molnar E, Balint A. Investigation of ultra- and nanofiltration for utilization of whey protein and lactose. Journal of Food Engineering 2005; 67, 325-

[186] Zydney AL. Protein separations using membrane filtration: new opportunities for

[187] Rektor A, Vatai G. Membrane filtration of Mozzarella whey. Desalination 2004;162,

[188] Smithers GW, Ballard FJ, Copeland AD, Silva KJ, Dionysius DA, Francis GL, Goddard C, Grieve PA, Mcintosh GH, Mitchell IR, Pearce RJ, Regester GO. New opportunities


hydrolyzates. Clinical and Experimental Allergy 1995; 25, 1007-1017.

supports. Enzyme and Microbial Technology 2000; 28, 642-652.

extracted from *Cynara cardunculus*. Food Chemistry 2004; 88, 351-359.

protein isolate. Journal of Membrane Science 2004; 231, 159-167.

manufacture by-products. Food Hydrocolloids 2004; 18, 601-610.

whey fractionation. International Dairy Journal 1998; 8, 243-250.

activity. International Dairy Journal 2006; 16, 1277-1293.

Biochemistry 1993; 17, 173-199.

89, 483-494.

332.

279-286.

2002; 50, 4347-4356.

dairy industry. Le Lait 2000; 80, 541-553.


[171] Fox PF. Exogeneous enzymes in dairy technology. A review. Journal of Food Biochemistry 1993; 17, 173-199.

106 Bioactive Food Peptides in Health and Disease

3201-3208.

1788.

1994; 5, 578-584.

Development 1994; 34, 527-537.

methods. US Patent 0,059,495 A1.

Dairy Journal 2006; 16, 1306-1314.

Functional Foods 2009; 1, 177-187.

anesthetized rat. Peptides 2000; 21, 1527-1535.

Journal of Food Protection 2003; 66, 1686-1692.

Journal of Nutrition 2005; 94, 84-91.

[155] Manso MA, López-Fandiño R. Angiotensin I converting enzyme-inhibitory activity of bovine, ovine, and caprine -casein macropeptides and their tryptic hydrolysates.

[156] Mizuno S, Matsuura K, Gotou T, Nishimura S, Kajimoto O, Yabune M, Kajimoto Y, Yamamoto N. Antihypertensive effect of casein hydrolysate in a placebo-controlled study in subjects with high-normal blood pressure and mild hypertension. British

[157] Neeser JR, Chambaz A, del Vedovo S, Prigent MJ, Guggenheim B. Specific and nonspecific inhibition of adhesion of oral actinomyces and streptococci to erythrocytes and polystyrene by caseinoglycopeptide derivatives. Infection and Immunity 1988; 56,

[158] Kawasaki Y, Isoda H, Tanimoto M, Dosako S, Idota T, Ahiko K. Inhibition by lactoferrin and -casein glycomacropeptide of binding of cholera toxin to its receptor.

[159] Schupbach P, Neeser JR, Golliard M, Rouvet M, Guggenheim B. Incorporation of caseinoglycomacropeptide and caseinophosphopeptide into the salivary pellicle inhibits adherence of mutant streptococci. Journal of Dental Research 1996; 75, 1779-

[160] Oh S, Worobo RW, Kim B, Rheem S, Kim S. Detection of cholera toxin-binding activity of -casein macropeptide and optimization of its production by the response surface

[161] Bouhallab S, Favrot C, Maubois JL. Growth-promoting activity of tryptic digest of caseinomacropeptide of *Lactococcus lactis* subsp. *lactis*. Le Lait 1993; 73, 73-77. [162] Beucher S, Levenez F, Yvon M, Corring T. Effects of gastric digestive products from casein on CCK release by intestinal-cells in rat. Journal of Nutritional Biochemistry

[163] Yvon M, Beucher S, Guilloteau P, le Huerou-Luron I, Corring T. Effects of caseinomacropeptide (CMP) on digestion regulation. Reproduction, Nutrition and

[164] Pederson NLR, Nagain-Domaine C, Mahe S, Chariot J, Roze C, Tome D. Caseinomacropeptide specifically stimulates exocrine pancreatic secretation in the

[165] Dartey C, Leveille G, Sox TE. (2003). Compositions for appetite control and related

[168] Udenigwe CC, Aluko RE. Food protein-derived bioactive peptides: production, processing, and potential health benefits. Journal of Food Science 2012; 77, R11–R24. [169] Korhonen H. Milk-derived bioactive peptides: from science to applications. Journal of

[170] Ustunol Z, Zeckzer T. Relative proteolytic action of milk-cloting enzyme preparations

on bovine and casein. Journal of Food Science 1996; 61, 1136-1138.

[166] Hasler C. A new look at an ancient concept. Chemistry and Industry 1998; 2, 84-89. [167] Pihlanto-Leppälä A. Antioxidative peptides derived from milk proteins. International

methodology. Bioscience, Biotechnology and Biochemistry 2000; 64, 516-522.

Bioscience, Biotechnology and Biochemistry 1992; 56, 195-198.


from the isolation and utilization of whey proteins. Journal of Dairy Science 1996; 79,1454-1459.

Whey Proteins as Source of Bioactive Peptides Against Hypertension 109

[203] Fujita H, Usui H, Kurahashi K, Yoshikawa M. Isolation and characterization of ovokinin, a bradykinin b-1 agonist peptide derived from ovalbumin. Peptides 1995; 16,

[204] Matoba N, Usui H, Fujita H, Yoshikawa M. A novel anti-hypertensive peptide derived from ovalbumin induces nitric oxide-mediated vasorelaxation in an isolated SHR

[205] Erdmann K, Grosser N, Schipporeit K, Schröder H. The ACE inhibitory dipeptide Met-Tyr diminishes free radical formation in human endothelial cells via induction of heme

[206] Maes W, Van Camp J, Vermeirssen V, Hemeryck M, Ketelslegers JM, Schrezen-meir J, Van Oostveldt P, Huyghebaert. Influence of the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR) on the release of endothelin-1 by endothelial cells. Regulatory Peptides

[207] Hernández-Ledesma B, Miralles B, Amigo L, Ramos M, Recio I. Identification of antioxidant and ACE-inhibitory peptides in fermented milk. Journal of the Science of

[208] Martínez-Maqueda D, Miralles B, Recio I, Hernández-Ledesma B. Antihypertensive

[209] Sánchez D, Kassan M, Contreras MM, Carrón R, Recio I, Montero MJ, Sevilla MA. Long-term intake of a milk casein hydrolysate attenuates the development of hypertension and involves cardiovascular benefits. Pharmacological Research 2011; 63,

[210] Quirós A, Ramos M, Muguerza B, Delgado MA, Miguel M, Aleixandre A, Recio I. Identification of novel antihypertensive peptides in milk fermented with *Enterococcus* 

[211] Miguel M, Contreras MM, Recio I, Aleixandre A. ACE-inhibitory and antihypertensive properties of a bovine casein hydrolysate. Food Chemistry 2009; 112, 211-214. [212] Hernández-Ledesma B, Contreras MM, Recio I. Antihypertensive peptides: production, bioavailability and incorporation into foods. Advances in Colloid and

[213] Gobetti M, Minervini F, Grizzello C. Angiotensin-I-converting-enzyme-inhibitory and antimicrobial bioactive peptides. International Journal of Dairy Technology 2004; 57,

[214] Meisel H. Biochemical properties of peptides encrypted in bovine milk proteins.

[215] Silva SV, Malcata FX. Caseins as source of bioactive peptides. International Dairy

[216] Vermeirssen V, Verstraete W, van Camp J. Bioavailability of angiotensin I converting

[217] Pihlanto-Leppälä A. Bioactive peptides derived from bovine whey proteins: opioid and ACE-inhibitory peptides. Trends in Food Science and Technology 2001; 11, 347-356.

enzyme inhibitory peptides. British Journal of Nutrition 2004; 92, 357-366.

peptides from food proteins: a review. Food and Function 2012; 3, 350-361.

oxygenase-1 and ferritin. Journal of Nutrition 2006; 136, 2148-2152.

mesenteric artery. FEBS Letters 1999; 452, 181-184.

Food and Agriculture 2005; 85, 1041–1048.

*faecalis*. International Dairy Journal 2007; 17, 33-41.

Current Medicinal Chemistry 2005; 12, 1905-1919.

Interface Science 2011; 165, 23-35.

785-790.

2004; 118, 105-109.

398-404.

173-188.

Journal 2005; 15, 1-15.


[203] Fujita H, Usui H, Kurahashi K, Yoshikawa M. Isolation and characterization of ovokinin, a bradykinin b-1 agonist peptide derived from ovalbumin. Peptides 1995; 16, 785-790.

108 Bioactive Food Peptides in Health and Disease

Engineering 2012; 110, 547-552.

Pressure. JAMA 2003; 289, 2560-2571.

Harvard School of Public Health; 1996.

Journal of Nutrition 2000; 84, S147-S153.

Chemistry 2008; 106, 552-558.

Research 1991; 68, 450-456.

749-756.

137, 825S-829S.

Research 2002; 69, 103-111.

79,1454-1459.

from the isolation and utilization of whey proteins. Journal of Dairy Science 1996;

[189] Wong CW, Seow HF, Husband AJ, Regester GO, Watson DL. Effects of purified bovine whey factors on cellular immune functions in ruminants. Veterinary

[190] Tavares TG, Amorim M, Gomes D, Pintado ME, Pereira CD, Malcata FX. Bioactive peptide-rich concentrates from whey: pilot process characterization. Journal of Food

[191] Eastern Stroke and Coronary Heart Disease Collaborative Research Group. Blood

[192] Chalmers J. Blood pressure burden: vascular changes and cerebrovascular

[193] Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL, Jones DW, Materson BJ, Oparil S, Wright JT, Roccella EJ. The seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood

[194] Tsai JS, Chen TJ, Pan BS, Gong SD, Chung MY. Antihypertensive effect of bioactive peptides produced by protease-facilitated lactic acid fermentation of milk. Food

[195] Murray CJL, Lopez AD (eds.). The global burden of disease: a comprehensive assessment of mortality and disability from disease, injuries and risk factors in 1990 and projected to 2020. Global Burden of Disease and Injury Series, vol 1. Cambridge:

[196] Geisterfer AA, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circulation Research 1988; 62,

[197] Daemen MJ, Lombardi DM, Bosman FT, Schwartz SM. Angiotensin II induces smooth muscle cell proliferation in the normal and injured rat arterial wall. Circulation

[198] Geerlings A, Villar IC, Zarco FH, Sánchez M, Vera R, Gomez AZ, Boza J, Duarte J. Identification and characterization of novel angiotensin-converting enzyme inhibitors

[199] Hong F, Ming L, Yi S, Zhanxia L, Yongquan W, Chi L. The antihypertensive effect of

[200] Scholz-Ahrens K, Schrezenmeir J. Effects of bioactive substances in milk on mineral and trace element metabolism with special reference to casein phosphopeptides. British

[201] Jauhiainen T, Korpela R. Milk peptides and blood pressure. Journal of Nutrition 2007;

[202] Sipola M, Finckenberg P, Korpela R, Vapaatalo H, Nurminen ML. Effect of long-term intake of milk products on blood pressure in hypertensive rats. Journal of Dairy

obtained from goat milk. Journal of Dairy Science 2006; 89, 3326-3335.

peptides: a novel alternative to drugs? Peptides 2008; 29, 1062-1071.

pressure, cholesterol and stroke in Eastern Asia. Lancet 1998; 352, 1801-807.

Immunology and Immunopathology 1997; 56, 85-96.

complications. Journal of Hypertension 2000; 18, S1-S2.

	- [218] Lee SH, Song KB. Isolation of an angiotensin-converting enzyme inhibitory peptide from irradiated bovine blood plasma protein hydrolysates. Food Chemistry and Toxicology, 2003; 68, 2469-2472.

Whey Proteins as Source of Bioactive Peptides Against Hypertension 111

[233] Pihlanto-Leppälä A, Koskinen P, Piilola K, Tupasela T, Korhonen H. Angiotensin-I converting enzyme inhibitory properties of whey proteins digests: concentration and

[235] Ortiz-Chao P, Gómez-Ruiz JA, Rastall RA, Mills D, Cramer R, Pihlanto A, Korhonen H, Jauregi P. Production of novel ACE inhibitory peptides from -lactoglobulin using

[236] Hernández-Ledesma B, Miguel M, Amigo L, Aleixandre MA, Recio I. Effect of simulated gastrointestinal digestion on the antihypertensive properties of synthetic -

[238] Chiba H, Yoshikawa M. Bioactive peptides derived from food proteins. Kagaku to

[239] Miguel M, Manso MA, López-Fandiño R, Alonso MJ, Salaices M. Vascular effects and antihypertensive properties of κ-casein macropeptide. International Dairy Journal 2007;

[240] Ruiz-Giménez P, Ibáñez A, Salom JB, Marcos JF, López-Díez JJ, Vallés S, Torregrosa G, Alborch E, Manzanares P. Antihypertensive properties of lactoferricin B-derived

[241] Kang DG, Kim YC, Sohn EJ, Lee YM, Lee AS, Yin MH, Lee HS. Hypotensive effect of butein via the inhibition of angiotensin-converting enzyme. Biological and

[242] Miguel M, López F, Ramos M, Aleixandre A. Short-term effect of egg-white hydrolysate products on the arterial blood pressure of hypertensive rats. British Journal

[243] Okamoto K, Aoki K. Development of a strain of spontaneously hypertensive rats.

[244] Nakamura Y, Yamamoto N, Sakai K, Takano T. Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors to angiotensin I-converting enzyme.

[245] Fujita H, Yokoyama K, Yoshikawa M. Classification and antihypertensive activity of angiotensin I-converting enzyme inhibitory peptides derived from food proteins.

[246] Muguerza B, Ramos M, Sánchez E, Manso MA, Miguel M, Aleixandre A, Delgado MA, Recio I. Antihypertensive activity of milk fermented by *Enterococcus faecalis* strains

[247] Contreras MM, Carrón R, Montero MJ, Ramos M, Recio I. Novel casein-derived peptides with antihypertensive activity. International Dairy Journal 2009; 19, 566-573.

isolated from raw milk. International Dairy Journal 2006; 16, 61-69.

peptides. Journal of Agricultural and Food Chemistry 2010; 58, 6721-6727.

lactoglobulin peptide sequences. Journal of Dairy Research 2007; 74, 336-339. [237] Otte J, Shalaby S, Zakora M, Nielsen MS. Fractionation and identification of ACEinhibitory peptides from -lactalbumin and -casein produced by thermolysin-

catalysed hydrolysis. International Dairy Journal 2007; 17, 1460-1472.

Seibutsu (in Japanese) 1991; 29, 454-458.

Pharmaceutical Bulletin 2003; 26, 1345-1347.

Japanese Circulation Journal 1963; 27, 282-293.

Journal of Dairy Science 1995; 78, 1253-1257.

Journal of Food Science 2000; 65, 564-569.

of Nutrition 2005; 94, 731-737.

characterization of active peptides. Journal of Dairy Research 2000; 67, 53-64. [234] Schlothauer RC, Schollum LM, Reid JR, Harvey SA, Carr A, Fanshawe RL. Improved bioactive whey protein hydrolyzate. Patent PCT/NZ01/00188 (WO 02/19837 A1), New

Protease N Amano. International Dairy Journal 2009; 19, 69-76.

Zealand 2002.

17, 1473-1477.


[233] Pihlanto-Leppälä A, Koskinen P, Piilola K, Tupasela T, Korhonen H. Angiotensin-I converting enzyme inhibitory properties of whey proteins digests: concentration and characterization of active peptides. Journal of Dairy Research 2000; 67, 53-64.

110 Bioactive Food Peptides in Health and Disease

Toxicology, 2003; 68, 2469-2472.

[218] Lee SH, Song KB. Isolation of an angiotensin-converting enzyme inhibitory peptide from irradiated bovine blood plasma protein hydrolysates. Food Chemistry and

[219] Mine Y, Kovacs-Nolan J. New insights in biologically active proteins and peptides

[220] Matsumura N, Fujii M, Takeda Y, Shimizu T. Isolation and characterization of angiotensin I-converting enzyme-inhibitory peptides derived from bonito bowels.

[221] Wung J, Ding X. Hypotensive and physiological effects of angiotensin converting enzyme inhibitory peptides derived from soy protein on spontaneously hypertensive

[222] Takayanagi T, Yokotsuka K. Angiotensin I converting enzyme-inhibitory peptides

[223] Miyoshi S, Ishikawa H, Kaneko, Fukui F, Tanaka H, Maruyama S. Structures and activity of angiotensin-converting enzyme inhibitors in α-zein hydrolysate. Agricultural

[224] Maruyama S, Suzuki H. A peptide inhibitor of angiotensin I converting enzyme in the tryptic hydrolysate of casein. Agricultural and Biological Chemistry 1982; 46, 1393-1394. [225] Maruyama S, Mitachi H, Tanaka H, Tomizuka N, Suzuki H. Studies on the active site and antihypertensive activity of angiotensin I-converting enzyme inhibitors derived

[226] Mullally MM, FitzGerald RJ, Meisel H. Identification of a novel angiotensin-Iconverting enzyme inhibitory peptide corresponding to a tryptic fragment of bovine -

[227] FitzGerald RJ, Meisel H. Lactokinins, whey protein derived ACE inhibitory peptides.

[228] Hernández-Ledesma B, Recio I, Ramos M, Amigo L. Preparation of ovine and caprine β-lactoglobulin hydrolysates with ACE-inhibitory activity. Identification of active peptides from caprine β-lactoglobulin hydrolysed with thermolysin. International Dairy

[229] Ondetti MA, Rubin B, Cushman DW. Design of specific inhibitors of angiotensinconverting enzyme: new class of orally active antihypertensive agents. Science 1977;

[230] Jeunemaitre X, Soubrier F, Kotelevtsev YV, Lifton RP, Williams CS, Charru A, Hunt SC, Hopkins PN, Williams RR, Lalouel JM, Corvol P. Molecular basis of human

[231] Timmermans PBMWM, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JAM, Smith RD. Angiotensin II receptors and angiotensin II receptor

[232] Nelson KM, Yeager BF. What is the role of angiotensin-converting enzyme inhibitors in congestive heart failure and after myocardial infarction? Annals of Pharmacotherapy

hypertension: role of angiotensinogen. Cell 1992; 71, 169-180.

antagonists. Pharmacological Reviews 1993; 45, 205-251.

derived from hen egg. World Poultry Science Journal 2006; 62, 87-96.

Bioscience, Biotechnology and Biochemistry 1993; 57, 1743-1744.

rats. Journal of Agricultural and Food Chemistry 2001; 49, 501- 506.

and Biological Chemistry 1991; 55, 1313-1318.

lactoglobulin. FEBS Letters 1997; 402, 99-101.

Nährung-Food 1999; 43, 165-167.

Journal 2002; 12, 805-812.

196, 441-444.

1996; 30, 986-993.

from wine. American Journal of Enology and Viticulture 1999; 50, 65-68.

from casein. Agricultural and Biological Chemistry 1987; 51, 1581-1586.

	- [248] Matsui T, Matsufugi HY, Osajima Y. Colorimetric measurement of angiotensin Iconverting enzyme inhibitory activity with trinitrobenzene sulfonate. Bioscience, Biotechnology and Biochemistry 1992; 56, 517-518.

Whey Proteins as Source of Bioactive Peptides Against Hypertension 113

[264] Gobbetti M, Stepaniak L, de Angelis M, Corsetti A, Cagno RD. Latent bioactive peptides in milk proteins, proteolytic activation and significance in dairy processing.

[265] Meisel H. Biochemical properties of regulatory peptides derived from milk proteins.

[266] Georgiadis D, Beau F, Czarny B, Cotton J, Yiotakis A, Dive V. Roles of the two active sites of somatic angiotensin-converting enzyme in the cleavage of angiotensin-I and bradykinin, insights from selective inhibitors. Circulation Research 2003; 93, 148-154. [267] Saito T. Antihypertensive peptides derived from bovine casein and whey proteins. In: Bösze, Z (ed.). Advances in experimental medicine and biology: bioactive components

[268] Shimizu M. Food-derived peptides and intestinal functions. Biofactors 2004; 21, 43-47. [269] Yang CY, Dantzig AH, Pidgeon C. Intestinal peptide transport systems and oral drug

[270] Gardner MLG. Intestinal assimilation of intact peptides and proteins from the diet – a neglected field. Biological Reviews of the Cambridge Philosophical Society 1984; 59,

[271] Roberts PR, Burney JD, Black KW, Zaloga GP. Effects of chain length on absortion of biologically active peptides from the gastrointestinal tract. Digestion 1999; 60, 332-337. [272] Roufik S, Gauthier SF, Turgeon SL. *In vitro* digestibility of bioactive peptides derived

[273] Kim YS, Bertwhistle W, Kim YW. Peptide hydrolyses in the brush borde rand soluble fractions of small intestinal mucosa of rat and man. Journal of Clinical Investigation

[274] Masuda O, Nakamura Y, Takano T. Antihypertensive peptides are present in aorta after oral administration of sour milk containing these peptides in spontaneously

[275] Matsui T, Yukiyoshi A, Doi S, Sugimoto H, Yamada H, Matsumoto K. Gastrointestinal enzyme production of bioactive peptides from royal jelly protein and their antihypertensive ability in SHR. Journal of Nutritional Biochemistry 2002; 13, 80-86. [276] Langley-Danysz P. Des hydrolysats protéiques pour développer des aliments santé.

[277] Miguel M, Recio I, Ramos I, Delgado MA, Aleixandre MA. Antihypertensive effect of peptides obtained from *Enterococcus faecalis*-fermented milk in rats. Journal of Dairy

[278] Vermeirssen V, van Camp J, Decroos K, van Wijmelbeke L, Verstraete W. The impact of fermentation and *in vitro* digestion on the formation of angiotensin-I-converting enzyme inhibitory activity from pea and whey protein. Journal of Dairy Science 2003;

[279] Hernández-Ledesma B, Amigo L, Ramos M, Recio I. Angiotensin converting enzyme inhibitory activity in commercial fermented products. Formation of peptides under simulated gastrointestinal digestion. Journal of Agricultural and Food Chemistry, 2004;

from bovine -lactoglobulin. International Dairy Journal 2006; 16, 294-302.

Critical Reviews in Food Science and Nutrition 2002; 42, 223-239.

of milk, vol. 606. New York, USA: Springer 2008; 295-317.

availability. Pharmaceutical Research 1999; 16, 1331-1343.

hypertensive rats. Journal of Nutrition 1996; 126, 3063-3068.

RIA Technology Veille 1998; 581, 38-40.

Science 2006; 89, 3352-3359.

86, 429-438.

52, 1504-1510.

Biopolymers 1997; 43, 119-128.

289-331.

1972; 51, 1419-1430.


[264] Gobbetti M, Stepaniak L, de Angelis M, Corsetti A, Cagno RD. Latent bioactive peptides in milk proteins, proteolytic activation and significance in dairy processing. Critical Reviews in Food Science and Nutrition 2002; 42, 223-239.

112 Bioactive Food Peptides in Health and Disease

1637-1648.

1390-1394.

Chemistry 1980; 25, 401-407.

Food Chemistry 2001; 49, 2992-2997.

Biotechnology and Biochemistry 1992; 56, 517-518.

Journal of Chromatography A 1999; 853, 185-188.

and Biological Chemistry 1989; 53, 2107-2114.

Journal of Clinical Nutrition 2003; 77, 326-330.

[248] Matsui T, Matsufugi HY, Osajima Y. Colorimetric measurement of angiotensin Iconverting enzyme inhibitory activity with trinitrobenzene sulfonate. Bioscience,

[249] Friedland J, Silverstein E. A sensitive fluorometric assay for serum angiotensin converting enzyme. American Journal of Clinical Pathology 1976; 66, 416-424. [250] Shihabi ZK. Analysis of angiotensin-converting enzyme by capillary electrophoresis.

[251] Cushman DW, Cheung HS. Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochemical Pharmacology 1971; 20,

[252] Vermeirssen V, van Camp J, Verstraete W. Optimisation and validation of an angiotensin-converting enzyme inhibition assay for the screening of bioactive peptides.

[253] Kohmura M, Nio N, Kubo K, Minoshima Y, Munekata E, Ariyoshi Y. Inhibition of angiotensin-converting enzyme by synthetic peptides of human -casein. Agricultural

[254] Seppo L, Jauhiainen T, Poussa T, Korpela R. A fermented milk high in bioactive peptides has a blood pressure-lowering effect in hypertensive subjects. American

[255] Lv GS, Huo GC, Fu XY. Expression of milk-derived antihypertensive peptide in

[256] Ondetti MA, Cushman DW. Enzymes of the renin-angiotensin system and their

[257] Cushman DW, Cheung HS, Sabo EF, Ondetti MA. Development and design of specific inhibitors of angiotensin-converting enzyme. American Journal of Cardiology 1982; 49,

[258] Cheung HS, Wang FL, Ondetti MA, Sabo EH, Cushman DW. Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Journal of Biological

[259] Stevens RL, Micalizzi ER, Fessler DC, Pals DT. Angiotensin I converting enzyme of calf lung. Method of assay and partial purification. Biochemistry 1972; 11, 2999-3007. [260] Kim SK, Byun HG, Park PY, Fereidoon S. Angiotensin I converting enzyme inhibitory peptides purified from bovine skin gelatin hydrolysate. Journal of Agricultural and

[261] Gómez-Ruiz JA, Ramos M, Recio I. Angiotensin-converting enzyme-inhibitory activity of peptides isolated from Manchego cheese. Stability under simulated gastrointestinal

[262] Pripp AH, Isaksson T, Stepaniak L, Sørhaug T. Quantitative structure-activity relationship modelling of ACE-inhibitory peptides derived from milk proteins.

[263] Wu JP, Aluko RE, Nakai S. Structural requirements of antiotensin I-converting enzyme inhibitory peptides. Quantitative structure-activity relationship study of di- and

tripeptides. Journal of Agricultural and Food Chemistry 2006; 54, 732-738.

Journal of Biochemical and Biophysical Methods 2002; 51, 75-87.

*Escherichia coli*. Journal of Dairy Science 2003; 86, 1927-1931.

inhibitors. Annual Reviews of Biochemistry 1982; 51, 283-308.

digestion. International Dairy Journal 2004; 14, 1075-1080.

European Food Research and Technology 2004; 219, 579-583.


[280] Hernández-Ledesma B, Amigo L, Ramos M, Recio I. Release of angiotensin converting enzyme-inhibitory peptides by simulated gastrointestinal digestion of infant formulas. International Dairy Journal 2004; 14, 889-898.

**Chapter 5** 

© 2013 Abdou et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Abdou et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Functional Proteins and Peptides** 

Hen's egg has long history as a food. It contains a great variety of nutrients to sustain both life and growth. Egg provides an excellent, inexpensive and low calorie source of highquality proteins. Moreover, Eggs are a good source of several important nutrients including protein, total fat, monounsaturated fatty acids, polyunsaturated fatty acids, cholesterol, choline, folate, iron, calcium, phosphorus, selenium, zinc and vitamins A, B2, B6, B12, D, E and K [1]. Eggs are also a good source of the antioxidant carotenoids, lutein and zeaxanthin [2]. The high nutritional properties of eggs make them ideal for many people with special

Egg proteins are nutritionally complete with a good balance of essential amino acids which are needed for building and repairing the cells in muscles and other body tissues [3]. Egg proteins are distributed in all parts of the egg, but most of them are present in the egg white and egg yolk amounting to 50% and 40%, respectively. The remaining amount of protein is

In addition to excellent nutritional value, egg proteins have unique biological activities. Hyperimmunized hens could provide a convenient and economic source of specific immunoglobulin in their yolks (IgY) that have been found to be effective in preventing many bacteria and viruses infections [4]. Proteins in the egg white as lysozyme, ovotransferrin, and avidin have proven to exert numerous biological activities. Moreover, a specific protein in eggshell matrix shows unique activity; enhancement of calcium

It is well-known that egg proteins are a source of biologically active peptides. Many researches are aiming to unlock the hidden biological functions of peptides hidden in egg proteins. These peptides are inactive within the sequence of parent proteins and can be released during gastrointestinal digestion or food processing and exerting biological

Adham M. Abdou, Mujo Kim and Kenji Sato

Additional information is available at the end of the chapter

**of Hen's Egg Origin** 

http://dx.doi.org/10.5772/54003

**1. Introduction** 

dietary requirements.

in the egg shell and egg shell membranes.

transportation in the human intestinal epithelial cells.

