NA, not available.

**Analytes Sample Extraction** 

3 IFs Soy dry extract Sonication/

products

7 IFs, Cou\* 2 plant extracts Refluxing or

5 IFs, Cou\* 7 plant extracts Maceration or

12 IFs Soybeans, soy

**method**

144 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Steam bath

Maceration

percolation

**Table 4.** HPLC and UPLC methods applied for analysis of isoflavones in different samples.

17 IFs Soymilk Refluxing LC-ESI(+)-MS/

5 IFs Legumes SPE C18 UHLPC-

5 IFs Coffee SPE C18 HPLC-ESI-MS/

3 IFs, Cou\* 10 plant species UAE\*\* ULPC-PDA 4 1.97–4.08 ng/mL [19] 12 IFs Soybean seeds Maceration HPLC-UV 60 NA# [70]

3 IFs Coffee Refluxing HPLC-DAD 35 13.7–25.0 ng/mL [14]

MS

LC-ESI(−)-MS/ MS

> ULPC-ESI(+)-MS/ MS

> ESI(+)-MS/ MS

> > MS

**Detection Run time (min) LOQ References**

NA# NA# [74]

18 40 ng/mL [20]

5.5 5–10.78 ng/mL [28]

18 0.1–1 ng/mL [73]

18 0.05–1 ng/mL [75]

HPLC-DAD 20 40–100 ng/mL [71]

Maceration HPLC-DAD 30 <600 nmol/L [72]

For isoflavone identification, the following chromatographic methods are used: gas chromatography coupled with mass spectrometry (GC-MS) [5, 15], high-performance liquid chromatography (HPLC) with UV detector (photodiode array, PDA) [28, 70, 71], fluorescence detector (FLD), electrochemical detector (ECD) or mass spectrometer detector (MS) [20, 74, 75], and, less often, capillary electrophoresis (CE).

Quantification of isoflavones and their derivatives can be achieved in two ways: (a) by determining the free aglycons after a prior acid hydrolysis [19, 70, 72], alkaline hydrolysis [72], or enzymatic hydrolysis [72] of the glycosides in the sample and (b) by simultaneously analyzing the glycosides and aglycones present in the sample [20, 28]. GC-MS methods are used less lately, because they require an additional step of isoflavone derivatization to the volatile compounds [5, 15]. This additional step increases both the time and the cost of the analysis and represents a potential source of error [28].

Generally, HPLC-UV is not sensitive enough (**Table 4**) for the quantification of small levels of isoflavones from plant extracts [19] or human plasma [5]. This method often requires a hydrolysis step to transform glycosides into aglycones followed by the quantification of total aglycones from the sample [71].

In order to correctly identify new isoflavones or isoflavone derivatives present in the samples analyzed, liquid chromatography coupled with mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) are the preferred methods (**Table 4**), due to the advantages: speed, selectivity, sensitivity, and robustness. In addition, mass spectrometry detection allows sure determination of the compounds based on molecular weight and ion charge. For the quantification of isoflavones, the pseudo-molecular ions or the ionic fragments resulted after fragmentation are monitored. In LC-MS/MS analysis, compound identification can be achieved even if their separation is not complete, and it is an advantage [74]. A shorter analysis can be realized by ultra-performance liquid chromatography (UPLC) [19, 28]. This method uses columns with very small size of the packing particles (1.7 μm) and consequently performs separations with superior resolution in a shorter time and a lower consumption of the mobile phase.

The isoflavones have polyphenolic structure and can easily lose a proton to form negative pseudo-molecular ions [M-H]− [20]. However, they can also be detected after ionization in positive mode to [M + H]+ [74]. Isoflavones are polar compounds and they form ions in solution. For these type of compounds, electro-spray ionization (ESI) is the most commonly used source to obtain analytical ions. Atmospheric pressure chemical ionization (APCI) is the source preferred for non-polar analytes that ionize in the gas phase. The isoflavones often give poor response in this ionization source [28]. The fragmentation patterns of isoflavone glycosides (malonyl-glycosides, acetyl-glycosides, glycosides, aglycones) follow a similar trend. However each compound has a unique fragmentation pattern that allows their accurate identification (**Table 5**) [74].


**Table 5.** Ions (*m*/*z*) and transitions monitored for isoflavone quantification.

## **6. Conclusion**

determination of the compounds based on molecular weight and ion charge. For the quantification of isoflavones, the pseudo-molecular ions or the ionic fragments resulted after fragmentation are monitored. In LC-MS/MS analysis, compound identification can be achieved even if their separation is not complete, and it is an advantage [74]. A shorter analysis can be realized by ultra-performance liquid chromatography (UPLC) [19, 28]. This method uses columns with very small size of the packing particles (1.7 μm) and consequently performs separations with superior resolution in a shorter time and a lower consumption of the mobile

146 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

The isoflavones have polyphenolic structure and can easily lose a proton to form negative

tion. For these type of compounds, electro-spray ionization (ESI) is the most commonly used source to obtain analytical ions. Atmospheric pressure chemical ionization (APCI) is the source preferred for non-polar analytes that ionize in the gas phase. The isoflavones often give poor response in this ionization source [28]. The fragmentation patterns of isoflavone glycosides (malonyl-glycosides, acetyl-glycosides, glycosides, aglycones) follow a similar trend. However each compound has a unique fragmentation pattern that allows their accurate iden-

**Isoflavone [M + H]+ Transitions [M − H]− Tranzitions** Daidzein 255 [28, 74] →199 [28] 253 [20, 73, 75] →208, 132 [73, 75] Formononetin 269 [28] →197 [28] 267 [20, 75] →252, 223 [75] Genistein 271 [28, 73, 74] →153 [28] 269 [20, 73, 75] →159, 133 [73, 75] Biochanin A 283 [73, 75] →268, 239 [73, 75]

166 [74]

Ononin 429 [20] →267 [20]

[73, 75]

[73]

Glycitein 285 [74] →270, 257, 229, 196,

Daidzin 417 [28, 73, 74] →255 [28, 73, 74], 199

Genistin 433 [28, 74, 75] →271 [28, 73–75], 91

**Table 5.** Ions (*m*/*z*) and transitions monitored for isoflavone quantification.

Glycitin 447 [74] →428, 285 [74] Ac-daidzin 459 [74] →441, 255 [74] Ac-genistin 475 [74] →431, 271 [74] Ac-glycitin 489 [74] →471, 285 [74] Mal-daidzin 503 [74] →485, 255 [74] Mal-genistin 519 [74] →501, 271 [74]

[20]. However, they can also be detected after ionization in

[74]. Isoflavones are polar compounds and they form ions in solu-

283 [20]

415 [20] →253 [20]

431 [20] →268, 269 [20]

phase.

pseudo-molecular ions [M-H]−

positive mode to [M + H]+

tification (**Table 5**) [74].

Dietary intake of isoflavones is widespread, mainly due to the high consumption of soybean products. Health benefits of isoflavones justify the interest for this class of bioactive compounds, but the controversial outcomes of some clinical and epidemiological studies require further investigations. In the context of these researches, the analytical methods applied for assessment of isoflavones are very valuable. They allow for the evaluation of dietary intake of isoflavones, equating the health benefits and the circumstances in which they are exerted, and highlight the natural sources of isoflavones with phytotherapeutic potential.

## **Author details**

Daniela-Saveta Popa1 \* and Marius Emil Rusu<sup>2</sup>

\*Address all correspondence to: dpopa@umfcluj.ro

1 Department of Toxicology, Faculty of Pharmacy, "Iuliu Haţieganu" University of Medicine and Pharmacy Cluj-Napoca, Cluj-Napoca, Romania

2 Department of Pharmaceutical Technology and Biopharmacy, Faculty of Pharmacy, "Iuliu Haţieganu" University of Medicine and Pharmacy Cluj-Napoca, Cluj-Napoca, Romania

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## **Anthocyanins in Berries and Their Potential Use in Human Health Anthocyanins in Berries and Their Potential Use in Human Health**

Daniela Peña-Sanhueza, Daniela Peña-Sanhueza, Claudio Inostroza-Blancheteau,

Claudio Inostroza-Blancheteau, Alejandra Ribera-Fonseca and Marjorie Reyes-Díaz

Alejandra Ribera-Fonseca and Marjorie Reyes-Díaz

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67104

#### **Abstract**

Anthocyanin pigments are responsible for the red, purple, and blue colors of many fruits, vegetables, cereal grains, and flowers, increasing the interest due to their strong antioxi‐ dant capacity and their possible use to the benefit of human health. Abundant evidence is available about the preventive and therapeutic roles of anthocyanin in different kinds of chronic diseases. According to the structural differences and anthocyanin content of berries such as blackberry, blueberry, chokeberry, and others, there are different healthy properties in the treatments of circulatory disorders, cancer cell lines, and diabetes as well as antiviral and antimicrobial activities. On the other hand, molecular aspects play an important role in anthocyanin biosynthesis, making it possible to determine how biotic and abiotic factors impact its biosynthesis complex. Thus, the aim of this chapter was to describe the use of anthocyanins from berries for human health and their poten‐ tial use as a pharmacological bioresource in the prevention of chronic diseases. In addi‐ tion, an update of the molecular mechanisms involved in anthocyanin biosynthesis will be discussed.

**Keywords:** anthocyanins, berries, cancer, transcription factors

## **1. Introduction**

The scientific evidence regarding the positive relationship between diet and health has in creased consumer demand for more information related to healthy diets, including fruits and vegetables, with functional characteristics that help to delay the aging process and

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

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2017 The Author(s). Licensee InTech. This chapter is 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.

reduce the risk of several diseases, mainly cardiovascular diseases and cancer [1]. Berries are recognized as an important component of healthy diets due to their bioactive compounds. In this sense, commercial berry species such as blackberry (*Rubus* sp.), bilberry (*Vaccinium myrtillus* L*.*), blackcurrant (*Ribes rugrum* L*.*), chokeberry (*Aronia melanocarpa* (Michx.) Elliott.), cranberry (*V. macrocarpon* Ait.), bayberry (*Myrica* sp.), raspberry (*Rubus ideaus* L.), black raspberry (*Rubus occidentalis* L.), strawberry (*Fragaria ananassa* Duch.), highbush blueberry (*V. corymbosum* L.), maqui (*Aristotelia chilensis*), murtilla (*Ugni molinae* Turcz.), and calafate (*Berberis microphylla* G. Forst.) are particularly rich sources of antioxidants, which are usually consumed in fresh and processed products [2–5]. Higher plants, especially berry species, synthesize a diverse group of phenolic compounds such as flavonoids. These plant second‐ ary metabolites have many biological functions, including their key role in plantmicrobe interaction, plantpathogen interaction, pollentube growth, UV radiation protection, tissue pigmentation, and others [6, 7]. Flavonoid compounds, which include flavonols, flavones, flavanols, flavanones, isoflavonoids, and anthocyanins, are molecules widely accumulated in vascular plants and to a lesser extent in mosses, being accumulated in all organs and tis‐ sues at different stages of development and depending on the environmental conditions [6].

Anthocyanins are natural pigments responsible for the blue, purple, red, and orange col‐ ors of many fruits and vegetables [8, 9]. Anthocyanins are a glycoside form of anthocyani‐ dins [9], and the structural differences among them are related to the number of hydroxyl group, position, and kind and/or number of sugars linked to the molecule [10, 11]. These compounds appear to be an interesting natural resource of watersoluble dyes because they are easily incorporated in aqueous media [12]. Another important property of antho‐ cyanins is their remarkable antioxidant activity, playing a vital role in the prevention of neuronal and cardiovascular illnesses, diabetes, cancer, etc. [11, 13]. Many reports have focused on the effect of anthocyanins in cancer prevention [14], human nutrition [15], and their biological activity [10]. Nowadays, there is an increased interest in explaining the role of anthocyanins as a natural antioxidant and their mechanism of action on human health as well as the treatment of chronic diseases and their use as a natural dye, substituting the synthetic dyes, which can be toxic to humans. This review endeavors to describe the use of anthocyanins from berries for human health and their potential use as a pharmacological bioresource in the prevention of chronic diseases. In addition, an update of the molecular mechanisms involved in anthocyanin biosynthesis will be discussed. Finally, recent clini‐ cal and preclinical studies about anthocyanin use in the prevention of human diseases are reported.

## **2. Anthocyanin and phenolic compounds in berries**

Phenolic acid, organic acids, tannins, anthocyanins, and flavonoids are phenolic bioactive compounds with a high concentration in the berry fruits [16]. The chemical structure of phenolic compounds is characterized by one or more aromatic rings with hydroxyl groups. According to their structural characteristics, phenolic compounds are classified into five major groups: phenolic acids, stilbenes, flavonoids (flavonols or catechins, flavonols, fla‐ vones, flavonones, isoflavonoids, anthocyanins), tannins, and lignans [13]. The concentra‐ tion of phenolic compounds in berry fruits is altered by many factors, such as genotype, species, agronomic management, climatic factors, ripening stage, harvesting time, and postharvest management [17, 18]. Given the plant phenol attributes of berry species, atten‐ tion has largely focused on anthocyanin and flavonol antioxidant action on human health. In this way, substantial epidemiological and experimental research suggests that intakes of recognized nutritional antioxidants such as vitamin E and carotenoids can decrease the oxidative damage of proteins, lipids, and DNA *in vivo* and may reduce the incidence of developing many chronic diseases in humans [19]. The *in vitro* antioxidant effectiveness of anthocyanins and other polyphenols is due to its donation of free hydrogen atom from an aromatic hydroxyl group of the antioxidant molecules, acting as radical scavenger [20].

reduce the risk of several diseases, mainly cardiovascular diseases and cancer [1]. Berries are recognized as an important component of healthy diets due to their bioactive compounds. In this sense, commercial berry species such as blackberry (*Rubus* sp.), bilberry (*Vaccinium myrtillus* L*.*), blackcurrant (*Ribes rugrum* L*.*), chokeberry (*Aronia melanocarpa* (Michx.) Elliott.), cranberry (*V. macrocarpon* Ait.), bayberry (*Myrica* sp.), raspberry (*Rubus ideaus* L.), black raspberry (*Rubus occidentalis* L.), strawberry (*Fragaria ananassa* Duch.), highbush blueberry (*V. corymbosum* L.), maqui (*Aristotelia chilensis*), murtilla (*Ugni molinae* Turcz.), and calafate (*Berberis microphylla* G. Forst.) are particularly rich sources of antioxidants, which are usually consumed in fresh and processed products [2–5]. Higher plants, especially berry species, synthesize a diverse group of phenolic compounds such as flavonoids. These plant second‐ ary metabolites have many biological functions, including their key role in plantmicrobe interaction, plantpathogen interaction, pollentube growth, UV radiation protection, tissue pigmentation, and others [6, 7]. Flavonoid compounds, which include flavonols, flavones, flavanols, flavanones, isoflavonoids, and anthocyanins, are molecules widely accumulated in vascular plants and to a lesser extent in mosses, being accumulated in all organs and tis‐ sues at different stages of development and depending on the environmental conditions [6].

156 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Anthocyanins are natural pigments responsible for the blue, purple, red, and orange col‐ ors of many fruits and vegetables [8, 9]. Anthocyanins are a glycoside form of anthocyani‐ dins [9], and the structural differences among them are related to the number of hydroxyl group, position, and kind and/or number of sugars linked to the molecule [10, 11]. These compounds appear to be an interesting natural resource of watersoluble dyes because they are easily incorporated in aqueous media [12]. Another important property of antho‐ cyanins is their remarkable antioxidant activity, playing a vital role in the prevention of neuronal and cardiovascular illnesses, diabetes, cancer, etc. [11, 13]. Many reports have focused on the effect of anthocyanins in cancer prevention [14], human nutrition [15], and their biological activity [10]. Nowadays, there is an increased interest in explaining the role of anthocyanins as a natural antioxidant and their mechanism of action on human health as well as the treatment of chronic diseases and their use as a natural dye, substituting the synthetic dyes, which can be toxic to humans. This review endeavors to describe the use of anthocyanins from berries for human health and their potential use as a pharmacological bioresource in the prevention of chronic diseases. In addition, an update of the molecular mechanisms involved in anthocyanin biosynthesis will be discussed. Finally, recent clini‐ cal and preclinical studies about anthocyanin use in the prevention of human diseases are

Phenolic acid, organic acids, tannins, anthocyanins, and flavonoids are phenolic bioactive compounds with a high concentration in the berry fruits [16]. The chemical structure of phenolic compounds is characterized by one or more aromatic rings with hydroxyl groups. According to their structural characteristics, phenolic compounds are classified into five

**2. Anthocyanin and phenolic compounds in berries**

reported.

It has been reported that the antioxidant capacity of flavonoids is stronger than vitamins C and E [21, 22], and under *in vitro* conditions, flavonoids can prevent injury in differ‐ ent ways, acting as a suppressor of reactive oxygen formation, scavenging free radicals by hydrogen atom donation [22, 23], activating antioxidant enzymes [23, 24], chelating metal, reducing αtocopheryl radicals, inhibiting oxidases, oxidative stress mitigation by nitric oxide, increasing uric acid levels, and increasing antioxidant properties of lowmolecular antioxidants [22]. Anthocyanin concentration in blackberry is much higher than in raspberry and strawberry and similar to red currant blueberry, depending on the cultivar (see **Table 1**).

Anthocyanin concentration widely differs significantly among plant species, even among species of the same genus. In **Table 1**, anthocyanin and total phenolic compounds of differ‐ ent species and cultivars and their analysis are detailed. In blackberry, anthocyanin content is generally similar in all species, but phenolic content shows strong differences (**Table 1**). Anthocyanin content in *Rubus insularis* F. Aresch. represents 36% of the phenolic com‐ pounds, whereas in *R. fructicosus* cultivar Hull Thornless, it only represents 6.4% of the total phenolic compounds (**Table 1**). Raspberry (*R. innominatus* S. Moore) showed higher anthocy‐ anin level, representing 41.2% of the total phenolic compounds. *R. ideaus* show high phenolic compounds; however, their high content does not necessarily represent a high anthocyanin content. *R. ideaus* Heritage cultivar has showed the highest anthocyanin percent with respect to the total phenolic compounds, representing 3.8% (**Table 1**). Additionally, blueberry cul‐ tivars showed low differences between anthocyanin and phenolic compounds, but they showed greater health benefits than other berries due to their particularly high proportion of anthocyanins. In some cases, high anthocyanin content in blueberries is related to high antioxidant capacity, but the anthocyanin contents and composition are different in each species and cultivar (**Table 1**). More specifically, the *V. corymbosum* cultivar (Duke) contains 63% anthocyanin with respect to the total phenolic compounds, followed by the cultivars CVAC5.001 and Brigitta, with 46 and 41%, respectively, and finally by Bluecrop with 27%. It is therefore necessary to evaluate the correlation between anthocyanin content and total phenolic compounds, because the ratio can exist between the two parameters, but it is not necessary to estimate in all species or among cultivars of the same genus (**Table 1**) [25–29].


**Table 1.** Total anthocyanin and phenolic content of berry fruits.

Berry species with higher anthocyanin content are interesting for use in breeding programs for increasing their content in fruits, enhancing their antioxidant capacity, and obtaining fruit products with health properties. In addition, the understanding of the molecular net‐ work of genes involved in anthocyanin biosynthesis and how biotic and abiotic factors could affect their concentration and gene regulation are a key to use it in genetic engineering and agronomic management.

## **3. Molecular regulation of anthocyanin biosynthesis**

Six structural genes are common in the anthocyanin pathway in all angiosperms, which are divided into two main groups. The first group is the upstream genes or early biosynthesis genes, for example, chalcone synthase (CHS), chalcone flavanone isomerase (CHI), and fla‐ vanone 3hydroxylase (F3H), coding for enzymes that produce precursors for one or more important nonanthocyanin flavonoids. The second group is the downstream genes or late biosynthesis genes, for example, anthocyanidin synthase (ANS), dihydroflavonol4reductase (DFR), and UDPglucose flavonoid 3oxyglucosyltransferase (UF3GT), coding for enzymes specific to anthocyanin synthesis [30–32]. In the anthocyanin pathway, l‐phenylalanine is converted to naringenin by phenylalanine ammonia lyase (PAL), cinnamate 4hydroxy‐ lase (C4H), 4coumarate CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomer‐ ase (CHI). Then, the next pathway is catalyzed by the formation of complex aglycone and anthocyanin composition by flavanone 3hydroxylase (F3H), flavonoid 3'hydroxylase (F3'H), dihydroflavonol 4reductase (DFR), anthocyanidin synthase (ANS), UDPglucoside flavonoid glucosyltransferase (UFGT), and methyl transferase (MT) [33]. It has been described that the transcription of early and late biosynthesis genes to produce anthocyanins appears to be regu‐ lated by R2R3MYB and basic helixloophelix (bHLH, also known as MYC) called transcrip‐ tion factors in collaboration with tryptophanaspartic acid repeat (WDR) or WD40 proteins [32, 34–37].

## **3.1. MYB transcription factor**

**Scientific name Common name Cultivar Anthocyanins\* Phenolics\*\* References** *Rubus cyri* Juz. Blackberry Native 143 545 [25] *Rubus georgicus* Focke Blackberry Native 89 561 [25] *Rubus insularis* F. Aresch. Blackberry Native 170 472 [25]

158 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

*Rubus fructicosus* L. Blackberry Chactaw 125 1703 [26] *Rubus fructicosus* Blackberry T. evergreen 146 2061 [26] *Rubus fructicosus* Blackberry Hull Thornless 152 2349 [26] *Rubus idaeus* L. Raspberry Native 65 517 [27] *Rubus innominatus* S. Moore Raspberry Native 52 126 [25] *Rubus niveus* Thunb. Raspberry Native 230 402 [25] *Rubus ideaus* Raspberry Heritage 49 1280 [26] *Rubus ideaus* Raspberry Autumm Bliss 39 2494 [26] *Rubus ideaus* Raspberry Fallgold 3 1459 [26] *Rubus ideaus* Raspberry Meeker 42 2116 [26] *Ribes sativum* Red currants London Market 7.8 1115 [26]

*Ribes sativum* Red currants White Versailes 1.4 657 [26] *Ribes nigrum* L. Red currants Alagan 169 694 [25] *Ribes nigrum* Red currants Ben Lomond 261 933 [25] *Ribes nigrum* Red currants Ojebyn 165 830 [25] *Ribes nigrum* Red currants Consort 411 1342 [25] *Vaccinium corymbosum* L. Blueberry Bluecrop 84 304 [25] *Vaccinium corymbosum* Blueberry Briggita 103 246 [25] *Vaccinium corymbosum* Blueberry Duke 173 274 [25] *Vaccinium corymbosum* Blueberry CVAC5.001 430 868 [25] *Vaccinium corymbosum* Blueberry Native 62‐235 181–473 [28] *Vaccinium corymbosum* Blueberry Bluegold 206 432 [29] *Vaccinium corymbosum* Blueberry Briggita 190 468 [29] *Vaccinium corymbosum* Blueberry Legacy 226 570 [29] *Vaccinium angustifolium* Ait. Blueberry Native 208 692 [25] *Vaccinium myrtillus* L. Bilberry Native 300 525 [28]

Blackberry Native 211 629 [25]

Red currants Rovada 7.5 1193 [26]

*Rubus ursinus* (Douglas ex

*Ribes sativum* (Lam.) Mert.

\*mg cyanidin 3glucoside eq./100 g fresh weight. \*\*mg gallic acid eq./100 g fresh weight.

**Table 1.** Total anthocyanin and phenolic content of berry fruits.

& Kock

Hook.)

The MYB transcription factors involved in the flavonoid pathway have been identified and described for several kinds of model plants, crops, and ornamental plants. The first identi‐ fied and reported MYB transcription factor in plants was in *Zea mays*, which included C1 (Colorless 1) and PL1 (Purple Leaf 1) [38]. The MYB transcription factors are composed of the socalled Nterminal MYB domain, consisting of one to three imperfect repeats of almost 52 amino acids (R1, R2, and R3), beginning with R2R3, the most abundant subfamily in plants [39]. The MYB domain is involved in DNA binding and dimerization. The Ctermi‐ nal region is responsible for establishing protein‐protein and regulates activation or repres‐ sion of gene expression [34, 40, 41]. The MYB genes are exclusive to eukaryotic organisms [42]. In animals, these genes are associated with cell proliferation and differentiation [43, 44], whereas in plants, MYB is associated with responses to different biotic and abiotic stressors (drought, cold, pathogen disease resistance), plant development (trichome formation, seed development), stomatal movement, and many other functions [34, 40, 45, 46]. Anthocyanin biosynthesis mediated by MYB transcription factors has been reported in *Arabidopsis thaliana*

(L.) Heynh. [41, 47–49], strawberry (*F. ananassa*) [50], Chilean strawberry (*Fragaria chiloen‐ sis* (L.) Duch.) [51], apple (*Malus domestica* Borkh.) [52–54], and tomato (*Solanum lycopersi‐ cum* L.) [55]. Grape (*Vitis vinifera* L.) is the main plant species studied in this way due to its agricultural and commercial importance worldwide. Thus, many MYB transcription factors have been reported in this species by different researchers. *VvMYBPA1* and *VvMYBPA2* are involved in proanthocyanidin synthesis [46, 56], while *VvMYBF1* regulates flavonol synthesis [57]. In addition, *MYBA1* and *MYBA2* genes control the last biosynthetic step of anthocyanin synthesis [58, 59]. It is reported that a glycosylation reaction mediated by the UDPglucose flavonoid3Oglucosyltransferase (UFGT) enzyme produces anthocyanins in grapes [31, 39]. It is important to highlight that MYB transcription factor is conserved between different spe‐ cies and is one of the most important primary proteins involved in structural and biological functions. Furthermore, MYB transcription factor regulates the flavonoid pathway appar‐ ently in two ways: (a) due to variations in the Cterminal region of the protein or (b) modulat‐ ing the interaction with DNA, bHLH, and WD40 protein [34, 60].

## **3.2. Basic helix‐loop‐helix (bHLH)**

After MYB, bHLH proteins, also known as MYC, are the second most important family of transcription factors involved in anthocyanin biosynthesis [34, 61]. The bHLH protein domain is constituted of about 60 amino acids and is characterized by the presence of 19 conserved amino acids, five in the basic region, five in the first helix, one in the loop, and eight in the final second helix [61]. The basic region of bHLH has basic residues (5.8 on aver‐ age) essential for DNA binding. In Arabidopsis, 20% of bHLH transcription factors do not have this domain and can act as a repressor because forming heterodimers are unable to bind to DNA [61]. Two ciselement boxes have been reported to bind with bHLH proteins, the Ebox (5'CANNTG3'), and Gbox (5'CACGTG3') elements. The Gbox is the most commonly recognized sequence representing 81% of the proteins predicted to bind DNA [61, 62]. In the basic region, two amino acids conferred the property on binding DNA in Arabidopsis plants. The Glu13 and Arg16 are the Ebox recognition motif [63]. Glu13 has contact with CA bases of Ebox and Arg16, apparently helping Glu13 to bind and stabi‐ lize. In Gbox, specific stabilization is mediated by His/Lys9, Glu13, and Arg17. The Arg17 interacts with inner G base, and His/Lys9 interacts with the last G of the Gbox [61, 62]. The alphahelix function is involved in homo and heterodimerization and is formed by hydrophobic residues of isoleucine, leucine, and valine [34, 61]. Arabidopsis has been dem‐ onstrated that this residue is conserved in all bHLH proteins, indicating the importance of the basic region of the bHLH transcription factor in DNA binding [61, 63]. The second helix is involved in DNA binding through direct contact with the Ebox. Finally, the loop is responsible for the three‐dimensional arrangement of alpha‐helices, and residues from the first helix loop junction are involved in association with bHLH proteins [34, 61, 63, 64]. Basic helixloophelix transcription factors in plants are involved in processes such as flower development [65, 66], hormonal response [67, 68], metal homeostasis [69], and oth‐ ers. Regarding bHLH and their relation to flavonoid synthesis, the first bHLH involved in this pathway was detected in maize in 1989 [70]. In this context, in *Z. mays* (ZmB, ZmR, and ZmLc), bHLH is involved in the regulation of the anthocyanin pathway [70–72], and ZmIn1 is involved in the repression of flavonoid gene expression in maize aleurone [73]. In *A. thaliana*, it has been reported that *AtTT8* gene encodes a bHLH transcription factor involved in the control of proanthocyanidins and anthocyanins in seeds and seedlings [74]. Quatroccio et al. [75, 76]. reported PhAN1, PhJAF13 hBLH transcription factor from *Petunia hibrida* as being involved in the control of the anthocyanin pathway in flowers. For *Vitis vinifera,* VvMYCA1 (also known as bHLH) was reported as involved in promotion of antho‐ cyanin accumulation in grape cells [37].

## **3.3. WDR proteins**

(L.) Heynh. [41, 47–49], strawberry (*F. ananassa*) [50], Chilean strawberry (*Fragaria chiloen‐ sis* (L.) Duch.) [51], apple (*Malus domestica* Borkh.) [52–54], and tomato (*Solanum lycopersi‐ cum* L.) [55]. Grape (*Vitis vinifera* L.) is the main plant species studied in this way due to its agricultural and commercial importance worldwide. Thus, many MYB transcription factors have been reported in this species by different researchers. *VvMYBPA1* and *VvMYBPA2* are involved in proanthocyanidin synthesis [46, 56], while *VvMYBF1* regulates flavonol synthesis [57]. In addition, *MYBA1* and *MYBA2* genes control the last biosynthetic step of anthocyanin synthesis [58, 59]. It is reported that a glycosylation reaction mediated by the UDPglucose flavonoid3Oglucosyltransferase (UFGT) enzyme produces anthocyanins in grapes [31, 39]. It is important to highlight that MYB transcription factor is conserved between different spe‐ cies and is one of the most important primary proteins involved in structural and biological functions. Furthermore, MYB transcription factor regulates the flavonoid pathway appar‐ ently in two ways: (a) due to variations in the Cterminal region of the protein or (b) modulat‐

After MYB, bHLH proteins, also known as MYC, are the second most important family of transcription factors involved in anthocyanin biosynthesis [34, 61]. The bHLH protein domain is constituted of about 60 amino acids and is characterized by the presence of 19 conserved amino acids, five in the basic region, five in the first helix, one in the loop, and eight in the final second helix [61]. The basic region of bHLH has basic residues (5.8 on aver‐ age) essential for DNA binding. In Arabidopsis, 20% of bHLH transcription factors do not have this domain and can act as a repressor because forming heterodimers are unable to bind to DNA [61]. Two ciselement boxes have been reported to bind with bHLH proteins, the Ebox (5'CANNTG3'), and Gbox (5'CACGTG3') elements. The Gbox is the most commonly recognized sequence representing 81% of the proteins predicted to bind DNA [61, 62]. In the basic region, two amino acids conferred the property on binding DNA in Arabidopsis plants. The Glu13 and Arg16 are the Ebox recognition motif [63]. Glu13 has contact with CA bases of Ebox and Arg16, apparently helping Glu13 to bind and stabi‐ lize. In Gbox, specific stabilization is mediated by His/Lys9, Glu13, and Arg17. The Arg17 interacts with inner G base, and His/Lys9 interacts with the last G of the Gbox [61, 62]. The alphahelix function is involved in homo and heterodimerization and is formed by hydrophobic residues of isoleucine, leucine, and valine [34, 61]. Arabidopsis has been dem‐ onstrated that this residue is conserved in all bHLH proteins, indicating the importance of the basic region of the bHLH transcription factor in DNA binding [61, 63]. The second helix is involved in DNA binding through direct contact with the Ebox. Finally, the loop is responsible for the three‐dimensional arrangement of alpha‐helices, and residues from the first helix loop junction are involved in association with bHLH proteins [34, 61, 63, 64]. Basic helixloophelix transcription factors in plants are involved in processes such as flower development [65, 66], hormonal response [67, 68], metal homeostasis [69], and oth‐ ers. Regarding bHLH and their relation to flavonoid synthesis, the first bHLH involved in this pathway was detected in maize in 1989 [70]. In this context, in *Z. mays* (ZmB, ZmR, and ZmLc), bHLH is involved in the regulation of the anthocyanin pathway [70–72], and

ing the interaction with DNA, bHLH, and WD40 protein [34, 60].

160 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**3.2. Basic helix‐loop‐helix (bHLH)**

Tryptophanaspartic acid repeat protein (WDR) or WD40 proteins are characterized by around 44–66 amino acids, delimited by the GH dipeptide on the Nterminal size (11–24 resi‐ dues from the Nterminus) and the WD dipeptide on the Cterminus [34, 77]. In Arabidopsis, WDR protein contains four (or more) tandem repeats composed of around 40 amino acids [78]. In contrast to the majority of proteins, WDR is not involved in catalytic activities such as DNA binding or gene expression regulation, mostly acting as a platform due to its capacity to interact with more than one protein at the same time [34, 78]. The work of WDR involves eukaryotic cellular process such as cell division, vesicle formation, signal transduction, RNA processing, and transcription regulation [78]. On the other hand, MYB and bHLH tran‐ scription factors have few WDR proteins involved in the flavonoid pathway, as shown in *Z. mays* (ZmPAC1), where it regulates the anthocyanin pathway in seed aleurone [79]. In Arabidopsis (AtTTG1), WDR proteins control trichomes, root hair, and seed mucilage pro‐ duction [80]. In petunia, AN11 regulates anthocyanin production as well as the pH of the flower vacuole [81], whereas in grape, *V. vinifera* WDR1 contributes to anthocyanin accu‐ mulation [37]. Although WDR proteins are not directly involved in the flavonoid pathway, particularly in anthocyanin synthesis, it is important to note that these proteins are highly conserved among species [34]. Nevertheless, few WDR proteins have been reported in plants, and it must be highlighted that WDR is involved in several metabolic and physiological pro‐ cesses [79, 80, 82]. To clarify the characteristics of WDR proteins and the complex formed with MYB and bHLH, which is involved in anthocyanin biosynthesis, species such as petunia and Arabidopsis have been used [34, 35].

## **3.4. MYB‐bHLH‐WDR (MBW complex)**

MBW complex has been reported in Arabidopsis, petunia, and some varieties of grape [35, 82]. The most important function of these transcription factors is involved in the process related to DNA binding, activation of gene expression involved in the flavonoid pathway, and stabilization of the threedimensional configuration of the complex [34]. Basic helixloophelixWDR interaction is needed to WDR protein translocation into the nucleus, and this was demonstrated in onion cells using green protein fluorescent (GPF), which when expressed alone is localized in the cytosol, whereas its coexpression with PFWD and MYCRP enables the transport and localization in the nucleus [35]. The AN11 from petunia showed the same results, being detected in the cytosol [81]. *V. vinifera* subjected to high salt concentrations showed a cultivardependent response for anthocyanin accumulation, which was correlated with the expression of MYBA12, MYCA1 and WDR1 genes [37].

## **4. Antioxidant capacity of anthocyanins in berries and their use in human health**

The radical scavenging activity (RSA) of anthocyanins is largely due to the presence of hydroxyl groups in position 3 of ring C and also in the 3', 4', and 5' positions in ring B of the molecule. In general, RSA of anthocyanidins (aglycons) is superior to their respective antho‐ cyanins (glycosides), and this decreases when the number of sugar increases [16]. Hanachi et al. [83] showed that fruits of *Berberis vulgaris* L. (barberry) have a high antioxidant activ‐ ity, reducing the viability of cell cultures associated with liver cancer (HepG2). Furthermore, extracts of leaves and twigs of *B. vulgaris* have more antioxidants than fruits. Končić et al. [84] studied the antioxidant activity of extracts of leaves, branches, and roots of two species of *B. vulgaris* and *Berberis croatica* and demonstrated that all these organs exhibited antioxidant activity. In all cases, the activity was positively correlated with the content of phenolic acids and flavonols, and the flavonols played the main role in the total antioxidant activity of the studied species [84]. They also concluded that the antioxidant activities were significantly dif‐ ferent (being higher in *B. croatica* than *B. vulgaris*) and among organs (being higher in leaves followed by branches and roots). The result of the anthocyanin concentration in different organs besides the fruits is interesting, because acquisition of anthocyanin in every season of the year has advantages for making new products with health properties. Thus, interesting results such as a new natural resource for promoting these compounds for human health have been reported. Končić et al. [84] suggested that studies into different species are needed to analyze all the organs of the plant, not just the fruits. Shin et al. [85] reported that in human liver cancer HepG2, cell proliferation was inhibited by strawberry extracts. Moreover, Chang et al. [86] reported that *Hibiscus sabdariffa* Linne (roselle) anthocyanin extracts mediated the apoptosis of human promyelocytic leukemia cells via the p38/Fas and Bid pathways. Research examining the use of black currant extract (BCE) with high concentrations of phenolic com‐ pounds on antiproliferative activity against gastric cancer SGC7901 cells showed a positive antiradical activity and anticarcinogenic effects [87]. Moreover, extracts of mulberry showed an inhibition on the growth of human gastric carcinoma cells [88]. In this study, anthocyanins extracted from mulberry had notable promotive effects on the p38/jun/Fas/FasL and p38/p53/ Bax signaling pathways, which accounted for its *in vitro* and *in vivo* growthinhibitory and apoptotic responses in AGS (gastric cancer) cells. The effects of berries on diseases are shown in **Table 2**.

With respect to *in vivo* studies, Wang and Stoner [89] reported the effect of an anthocy‐ aninrich extract from black raspberries on the development of tumors in rat esophagus by Nnitrosomethylbenzylamine (NMBA), the most potent inducer of tumors in rat esopha‐ gus. This extract inhibited cell proliferation, inflammation, and induced apoptosis in the esophageal tissues (**Table 2**). Stoner et al. [90] compared the effect of black raspberry, red raspberry, strawberry, and blueberry anthocyanin and ellagitannins in fruit extract on the prevention of esophageal cancer induced by Nnitrosomethylbenzylamine (NMBA) in rats. Inhibition of NMBAinduced tumorigenesis in the rat esophagus was observed. The authors detected a reduction in cytokine levels in serum, interleukin 5 (IL5), and GRO/KC, which is the rat homolog for human interleukin8 (IL8), and these cytokines


**4. Antioxidant capacity of anthocyanins in berries and their use in** 

162 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

The radical scavenging activity (RSA) of anthocyanins is largely due to the presence of hydroxyl groups in position 3 of ring C and also in the 3', 4', and 5' positions in ring B of the molecule. In general, RSA of anthocyanidins (aglycons) is superior to their respective antho‐ cyanins (glycosides), and this decreases when the number of sugar increases [16]. Hanachi et al. [83] showed that fruits of *Berberis vulgaris* L. (barberry) have a high antioxidant activ‐ ity, reducing the viability of cell cultures associated with liver cancer (HepG2). Furthermore, extracts of leaves and twigs of *B. vulgaris* have more antioxidants than fruits. Končić et al. [84] studied the antioxidant activity of extracts of leaves, branches, and roots of two species of *B. vulgaris* and *Berberis croatica* and demonstrated that all these organs exhibited antioxidant activity. In all cases, the activity was positively correlated with the content of phenolic acids and flavonols, and the flavonols played the main role in the total antioxidant activity of the studied species [84]. They also concluded that the antioxidant activities were significantly dif‐ ferent (being higher in *B. croatica* than *B. vulgaris*) and among organs (being higher in leaves followed by branches and roots). The result of the anthocyanin concentration in different organs besides the fruits is interesting, because acquisition of anthocyanin in every season of the year has advantages for making new products with health properties. Thus, interesting results such as a new natural resource for promoting these compounds for human health have been reported. Končić et al. [84] suggested that studies into different species are needed to analyze all the organs of the plant, not just the fruits. Shin et al. [85] reported that in human liver cancer HepG2, cell proliferation was inhibited by strawberry extracts. Moreover, Chang et al. [86] reported that *Hibiscus sabdariffa* Linne (roselle) anthocyanin extracts mediated the apoptosis of human promyelocytic leukemia cells via the p38/Fas and Bid pathways. Research examining the use of black currant extract (BCE) with high concentrations of phenolic com‐ pounds on antiproliferative activity against gastric cancer SGC7901 cells showed a positive antiradical activity and anticarcinogenic effects [87]. Moreover, extracts of mulberry showed an inhibition on the growth of human gastric carcinoma cells [88]. In this study, anthocyanins extracted from mulberry had notable promotive effects on the p38/jun/Fas/FasL and p38/p53/ Bax signaling pathways, which accounted for its *in vitro* and *in vivo* growthinhibitory and apoptotic responses in AGS (gastric cancer) cells. The effects of berries on diseases are shown

With respect to *in vivo* studies, Wang and Stoner [89] reported the effect of an anthocy‐ aninrich extract from black raspberries on the development of tumors in rat esophagus by Nnitrosomethylbenzylamine (NMBA), the most potent inducer of tumors in rat esopha‐ gus. This extract inhibited cell proliferation, inflammation, and induced apoptosis in the esophageal tissues (**Table 2**). Stoner et al. [90] compared the effect of black raspberry, red raspberry, strawberry, and blueberry anthocyanin and ellagitannins in fruit extract on the prevention of esophageal cancer induced by Nnitrosomethylbenzylamine (NMBA) in rats. Inhibition of NMBAinduced tumorigenesis in the rat esophagus was observed. The authors detected a reduction in cytokine levels in serum, interleukin 5 (IL5), and GRO/KC, which is the rat homolog for human interleukin8 (IL8), and these cytokines

**human health**

in **Table 2**.

**Table 2.** Anticarcinogenic effects of anthocyanin/anthocyaninrich extract from different berry species under *in vivo* and *in vitro* conditions in different chronic diseases.

were associated with an increase in serum antioxidant capacity. At molecular level, Stoner et al. [90] also reported that the use of extracts showed a differential expression in 626 and 625 genes per 4807 and 17846 of preneoplastic esophagus and esophageal papilloma genes, respectively. These genes are involved in carbohydrate and lipid metabolism, cell death and proliferation, and inflammation. These results are an important approach to estimate the relation of anthocyanin gene expression and its influence on proteins associ‐ ated with cell proliferation, apoptosis, angiogenesis, and esophageal carcinogenesis. Lala et al. [91] observed an anticarcinogenic effect of anthocyanins on colon cancer induced by azoxymethane in a rat model. In that study, anthocyaninrich extract from bilberry, choke‐ berry, and grapes significantly reduced azoxymethaneinduced aberrant crypt foci and decreased cell proliferation and *COX‐2* gene expression [91]. Delphinidin and pomegran‐ ate extracts enriched with anthocyanins, and tannins showed an inhibition in skin cancer induced by UVB or TPA (12O tetradecanoylphorbol13acetate) when applied to mouse skin [92, 93]. Here, delphinidin inhibited DNA damage mediated by UVB radiation, and pomegranate modulated the mitogenactivated protein kinase (MAPK) and nuclear fac‐ torkappa B (NFkB) pathways. Similarly, studies on the protective effect and antioxidant mechanism of anthocyanin extract from blueberries were conducted using a liver injury induced by CC4 in mice—the effect of which increased lipid peroxidation and reduced liver cell viability [94]. The results indicate that anthocyanin extract effectively protected mice from CC4induced liver injury by attenuation of lipid peroxidation. In mammary adenocarcinoma induced by dimethylbenzaanthracene (DMBA) in rats, the antitumoral effect of grape juice was evaluated by Singletary et al. [95]. They demonstrated that the tumor mass was ultimately reduced by suppressing cell proliferation (**Table 2**). In general, the strong antioxidant capacity of berry species is attributed to their anthocyanin content, suggesting that it might offer potential chemopreventive properties, including the inhibi‐ tion of gastric, leukemia, liver, and breast cancer cell proliferation, among others; how‐ ever, the mechanism of action must be evaluated for each disease because apparently their mechanism of effects varies (inhibiting cell proliferation, activating different enzymatic activity, inducing or repressing gene expression, etc.) depending on the extract from each plant species.

#### **5. Conclusions and future challenges**

The potential use of anthocyanins from different plant species as natural compounds with a health benefit for humans opens a new trend for the prevention and alternative treatments of chronic diseases. Several reports have demonstrated that anthocyanins from berries could inhibit or decrease the growth of carcinogenic tumors by affecting cell proliferation, increas‐ ing or inhibiting enzymatic systems, and increasing expression of genes involved in cell protection. On the other hand, it is important to highlight that synthesis of anthocyanins in different tissues of plants species should be considered. In addition, the discovery and char‐ acterization of new regulatory elements of anthocyanin biosynthesis are crucial to understand and manipulate this pathway in breeding programs. Improving knowledge about increas‐ ing anthocyanin synthesis in crops of research and commercial interest, together with more animal and human model studies under *in vivo* conditions, is essential to generate better human anticarcinogenic or antichronic disease supplement products with chemopreventive effects from berries.

## **Acknowledgements**

We are very grateful to PIAUFRO 160006 and DI 162013 projects from the Dirección de Investigación at the Universidad de La Frontera, Temuco, Chile.

## **Author details**

azoxymethane in a rat model. In that study, anthocyaninrich extract from bilberry, choke‐ berry, and grapes significantly reduced azoxymethaneinduced aberrant crypt foci and decreased cell proliferation and *COX‐2* gene expression [91]. Delphinidin and pomegran‐ ate extracts enriched with anthocyanins, and tannins showed an inhibition in skin cancer induced by UVB or TPA (12O tetradecanoylphorbol13acetate) when applied to mouse skin [92, 93]. Here, delphinidin inhibited DNA damage mediated by UVB radiation, and pomegranate modulated the mitogenactivated protein kinase (MAPK) and nuclear fac‐ torkappa B (NFkB) pathways. Similarly, studies on the protective effect and antioxidant mechanism of anthocyanin extract from blueberries were conducted using a liver injury induced by CC4 in mice—the effect of which increased lipid peroxidation and reduced liver cell viability [94]. The results indicate that anthocyanin extract effectively protected mice from CC4induced liver injury by attenuation of lipid peroxidation. In mammary adenocarcinoma induced by dimethylbenzaanthracene (DMBA) in rats, the antitumoral effect of grape juice was evaluated by Singletary et al. [95]. They demonstrated that the tumor mass was ultimately reduced by suppressing cell proliferation (**Table 2**). In general, the strong antioxidant capacity of berry species is attributed to their anthocyanin content, suggesting that it might offer potential chemopreventive properties, including the inhibi‐ tion of gastric, leukemia, liver, and breast cancer cell proliferation, among others; how‐ ever, the mechanism of action must be evaluated for each disease because apparently their mechanism of effects varies (inhibiting cell proliferation, activating different enzymatic activity, inducing or repressing gene expression, etc.) depending on the extract from each

164 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

The potential use of anthocyanins from different plant species as natural compounds with a health benefit for humans opens a new trend for the prevention and alternative treatments of chronic diseases. Several reports have demonstrated that anthocyanins from berries could inhibit or decrease the growth of carcinogenic tumors by affecting cell proliferation, increas‐ ing or inhibiting enzymatic systems, and increasing expression of genes involved in cell protection. On the other hand, it is important to highlight that synthesis of anthocyanins in different tissues of plants species should be considered. In addition, the discovery and char‐ acterization of new regulatory elements of anthocyanin biosynthesis are crucial to understand and manipulate this pathway in breeding programs. Improving knowledge about increas‐ ing anthocyanin synthesis in crops of research and commercial interest, together with more animal and human model studies under *in vivo* conditions, is essential to generate better human anticarcinogenic or antichronic disease supplement products with chemopreventive

We are very grateful to PIAUFRO 160006 and DI 162013 projects from the Dirección de

Investigación at the Universidad de La Frontera, Temuco, Chile.

plant species.

effects from berries.

**Acknowledgements**

**5. Conclusions and future challenges**

Daniela Peña‐Sanhueza1 , Claudio InostrozaBlancheteau1, 2, Alejandra RiberaFonseca1, 3 and Marjorie ReyesDíaz1, 4\*

\*Address all correspondence to: marjorie.reyes@ufrontera.cl

1 Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco, Chile

2 Núcleo de Investigación en Producción Alimentaria (NIPAUCT), Escuela de Agronomía, Facultad de Recursos Naturales, Universidad Católica de Temuco, Temuco, Chile

3 Departamento de Producción Agropecuaria, Facultad de Ciencias Agropecurias y Forestales, Universidad de La Frontera, Temuco, Chile

4 Departamento de Ciencias Químicas y Recursos Naturales, Facultad de Ingeniería y Ciencias, Universidad de La Frontera, Temuco, Chile

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#### **Protective Effects of** *Curcumin* **on Gastric Inflammation and Liver Disease Protective Effects of** *Curcumin* **on Gastric Inflammation and Liver Disease**

Duangporn Werawatganon Duangporn Werawatganon

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66119

#### **Abstract**

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Curcumin (diferuloylmethane), an anti-inflammatory and antioxidant compound, is isolated from the rhizomes of the plant *Curcuma longa Linn*. Most of the anti-inflammatory effects can be explained by the efficient inhibition of nuclear factor-κB–mediated and activation of PPARγ expression. These studies have been investigating the effects of curcumin on the gastric microcirculation, cytokine production after *Helicobacter pylori*–induced gastric inflammation, gastric cancer, drug-induced liver injury, and alcoholic liver disease (ALD). The results show that curcumin prevents indomethacininduced gastropathy via decreased leukocyte-endothelium interaction at postcapillary venule, decreased ICAM-1 and TNF-α level, and improved gastric microcirculation. Curcumin attenuated gastric inflammation and gastric cancer via reduced NF-κB p65 expression, decreased vascular endothelial growth factor (VEGF) level, and macromolecular leakage in the gastric mucosa. Curcumin prevented liver injury through decreased oxidative stress, reduced liver inflammation, and restored GSH. Moreover, curcumin could decrease hepatocyte apoptosis and improved PPARγ protein expression in alcohol-induced liver injury.

**Keywords:** curcumin, gastric inflammation, gastric cancer, liver disease

## **1. Introduction**

Curcumin (diferuloylmethane), the natural yellow pigment in tumeric, is isolated from the rhizomes of the plant *Curcuma longa* Linn. (*C. longa* L.). *C. longa* belongs to the Zingiberaceae family. It is a perennial herb that is distributed throughout tropical and subtropical regions of the world and is widely cultivated in Asian countries, such as India, Thailand, and China. The rhizomes are used as a traditional remedy in Nepal [1]. The powder form, called turmeric, is

© 2017 The Author(s). Licensee InTech. This chapter is 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. © 2017 The Author(s). Licensee InTech. This chapter is 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.

bright yellow and has been used as a food-coloring agent in the United States. In India, it has been used as a spice, as a food preservative, and as therapeutic agent. The current Indian medicine claims the usage of tumeric is effective against biliary disorders, anorexia, coryza, cough, diabetic wounds, hepatic disorder, rheumatism, and sinusitis [2].

In the nineteenth century, there has been considerable interest in the active compounds in tumeric called curcuminoids. Curcumin is the major curcuminoid compound that makes up approximately 90% of the curcuminoid content in tumeric, followed by demethoxycurcumin and bisdemethoxycurcumin [3]. The chemical structure of curcumin was determined by Roughley and Whiting (**Figure 1**) [4].

**Figure 1.** Chemical structure of curcumin (diferuloylmethane) [4].

Curcumin can be dissolved in organic solvents such as dimethylsulfoxide (DMSO), oil, alcohol, and petroleum agents. Interestingly, curcumin has been demonstrated to be safe for human and animals use. Human appeared to be able to tolerate high doses of curcumin without significant side-effects. A phase 1 study by Cheng et al. [5] found no adverse effects of curcumin ingestion for 3 months of dosage up to 8000 mg/day. Other human studies of curcumin included the following: a double-blinded, crossover trial in 18 patients with rheumatoid arthritis [6], a randomized, placebo-controlled trial with 45 postsurgical patients [7]. The doses of curcumin in these studies ranged from 1125 to 2500 mg/day. Only one postsurgical patient reported mild transient giddiness. No other serious adverse reactions were reported, including any changes in blood chemistry reports. Thus, curcumin appears to be safe in human even with ingestion at a high dosage.

In animals, the previous study demonstrated that curcumin is rapidly metabolized and poorly absorbed in Sprague-Dawley rats. Administrating curcumin orally was carried out by Wahlström and Blennow [8]. They demonstrated that this compound with a dose of 1–5 g/kg BW given to rats apparently did not cause any adverse effects and it was excreted about 75% in the feces, while traces found in the urine. In addition, measurements of blood plasma levels and biliary excretion showed that curcumin was poorly absorbed by the gastrointestinal (GI) tract. Curcumin could not be detected after 30 minutes when added to microsomes suspensions or hepatocyte suspensions. Furthermore, it was capable of disappearing from the blood after intravenous injection or after addition to the liver perfusion system. Moreover, oral LD50 was found to be 12.2 g/kg BW in rats [9]. In addition, a study in which rats were fed with curcumin 1.8 g/kg BW per day for 90 days and monkeys were fed with curcumin 0.8 mg/kg BW per day for 90 days showed no adverse effects [10].

bright yellow and has been used as a food-coloring agent in the United States. In India, it has been used as a spice, as a food preservative, and as therapeutic agent. The current Indian medicine claims the usage of tumeric is effective against biliary disorders, anorexia, coryza, cough, diabetic

In the nineteenth century, there has been considerable interest in the active compounds in tumeric called curcuminoids. Curcumin is the major curcuminoid compound that makes up approximately 90% of the curcuminoid content in tumeric, followed by demethoxycurcumin and bisdemethoxycurcumin [3]. The chemical structure of curcumin was determined by

Curcumin can be dissolved in organic solvents such as dimethylsulfoxide (DMSO), oil, alcohol, and petroleum agents. Interestingly, curcumin has been demonstrated to be safe for human and animals use. Human appeared to be able to tolerate high doses of curcumin without significant side-effects. A phase 1 study by Cheng et al. [5] found no adverse effects of curcumin ingestion for 3 months of dosage up to 8000 mg/day. Other human studies of curcumin included the following: a double-blinded, crossover trial in 18 patients with rheumatoid arthritis [6], a randomized, placebo-controlled trial with 45 postsurgical patients [7]. The doses of curcumin in these studies ranged from 1125 to 2500 mg/day. Only one postsurgical patient reported mild transient giddiness. No other serious adverse reactions were reported, including any changes in blood chemistry reports. Thus, curcumin appears to be safe in human even

In animals, the previous study demonstrated that curcumin is rapidly metabolized and poorly absorbed in Sprague-Dawley rats. Administrating curcumin orally was carried out by Wahlström and Blennow [8]. They demonstrated that this compound with a dose of 1–5 g/kg BW given to rats apparently did not cause any adverse effects and it was excreted about 75% in the feces, while traces found in the urine. In addition, measurements of blood plasma levels and biliary excretion showed that curcumin was poorly absorbed by the gastrointestinal (GI) tract. Curcumin could not be detected after 30 minutes when added to microsomes suspensions or hepatocyte suspensions. Furthermore, it was capable of disappearing from the blood after intravenous injection or after addition to the liver perfusion system. Moreover, oral LD50 was found to be 12.2 g/kg BW in rats [9]. In addition, a study in which rats were fed with curcumin

wounds, hepatic disorder, rheumatism, and sinusitis [2].

174 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**Figure 1.** Chemical structure of curcumin (diferuloylmethane) [4].

Roughley and Whiting (**Figure 1**) [4].

with ingestion at a high dosage.

Curcumin has been tested to demonstrate pharmacoprotective effects in various gastrointestinal (GI) and liver diseases. We highlight studies on its potential mechanism of action classified into four categories: (i) Curcumin protects against *Helicobacter pylori* infection and gastric cancer, (ii) curcumin protects against nonsteroidal anti-inflammatory drug (NSAID)-induced ulcer; (iii) curcumin protects against drug-induced liver injury; and (iv) curcumin protects against alcoholic liver disease (ALD).

## **2. Curcumin protects against** *H. pylori* **infection and gastric cancer**

The discovery of *H. pylori* was first reported in 1984 by two Australian investigators, Barry Marshall and Robin Warren [11], who isolated the bacteria from mucosal biopsies of patients with chronic active gastritis. Its name was changed from *Campylobacter pyloridis, Campylobacter pylori*, and *Campylobacter*-like organism when the biochemical and genetic characterization has shown that it is in the genus *Helicobacter* [12].

*H. pylori* is a noninvasive, nonspore-forming, and spiral shaped gram-negative bacterium measuring approximately 3.5 × 0.5 μm. It has four to six sheathed flagella at one pole. These flagella and spiral shape of *H. pylori* help the bacterial movement into the mucus of stomach. It slowly grows in microaerophilic condition, 5% oxygen, 50% carbon dioxide at 37°C [13]. *H. pylori* is an unusual organism with a remarkably high level of genetic diversity [14], which means, it can survive in the human stomach and also multiply in high-acid environment of the stomach. When *H. pylori* infected human, it adheres on the gastric epithelial cells and induces chronic active gastritis, peptic ulcer, mucosal-associated lymphoid tissue (MALT) lymphoma, and gastric cancer.

*H. pylori* is highly adapted to the stomach environment. To avoid the acidic environment of the stomach lumen, *H. pylori* uses its flagella to permit entry into the mucus. It adheres to the epithelial cells by producing adhesins for attachment to epithelial cells. *H. pylori* produces a potent urease enzyme. Urease generates carbon dioxide and ammonia, which potentially buffer the surrounding microenvironment and the bacterial cytosol [15]. In addition, urea is an important source of nitrogen for the bacteria.

Powerful flagella help the bacteria to swim through the viscous mucous layer covering the gastric epithelium, where bacterial adhesion proteins mediate a close interaction with the host cells [16]. *H. pylori* can bind tightly to epithelial cells by multiple bacterial surface components. The outer-membrane protein (Hop), such as BabA, binds to the fucosylated Lewis B bloodgroup antigen on the gastric epithelial cells [17]. Several Hop protein families also mediate adhesion to epithelial cells. When *H. pylori* adheres on gastric epithelial cells, it releases virulent factors to immune subversion. The host response to *H. pylori* participates in the induction of gastric epithelial damage and therefore has an integral role in *H. pylori* pathogenesis. *H. pylori* adheres on the gastric epithelial cells by bacterial adhesion proteins. Then, virulent factors are delivered into host cells. Especially, cytotoxin-associated gene A (CagA) induces many pathological conditions. For example, activation of NF-κB that causes production of many inflammatory mediators inducing gastric inflammation [18].

**Figure 2.** Protective mechanism of curcumin on *H. pylori* infection [19].

Effect of curcumin was examined by using rats [19]. The scheme of the effects was shown in **Figure 2**. Host inflammatory responses were measured by the following parameters: leakage

of macromolecules from gastric postcapillary venules (PCVs), serum level of vascular endothelial growth factor (VEGF), and the expression of NF-κB subunit p65.

delivered into host cells. Especially, cytotoxin-associated gene A (CagA) induces many pathological conditions. For example, activation of NF-κB that causes production of many

inflammatory mediators inducing gastric inflammation [18].

176 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**Figure 2.** Protective mechanism of curcumin on *H. pylori* infection [19].

Effect of curcumin was examined by using rats [19]. The scheme of the effects was shown in **Figure 2**. Host inflammatory responses were measured by the following parameters: leakage The successful inoculation of *H. pylori* was 85%. *H. pylori* infection led to the macromolecular leakage, the NF-κB-p65 expression, and increase of VEGF level compared with control group. Curcumin alone did not significantly change baseline of these parameters. There were

**Figure 3.** Protective mechanism of curcumin on N-methyl-N-nitrosourea (MNU) and saturated sodium chloride (s-NaCl)-induced gastric cancer [20].

significant decrease of macromolecular leakage and NF-κB-p65 expression (*p* ≤ 0.05) in the curcumin-treated groups (curcumin 200 mg/kg and 600 mg/kg BW) compared with *H. pylori*infected group, respectively. These results could be concluded that *H. pylori* infection increased macromolecular leakage, NF-κB-p65 expression, and serum VEGF level. Curcumin can reduce macromolecular leakage, decrease serum VEGF level, and NF-κB-p65 expression. It is implied that curcumin may have an anti-inflammatory effect on *H. pylori* infection [19].

In addition, the present study to examine the protective effect of curcumin on gastric cancer induced by N-methyl-N-nitrosourea (MNU) and saturated sodium chloride (s-NaCl) administration [20]. The scheme of the results was shown in **Figure 3**. Gastric cancer can generate in any part of the stomach. Cancers were found in the forestomach of all rats induced by MNU and s-NaCl. Curcumin supplementations showed 40–50% reduction of cancer incidence. Expressions of 8-OHdG, cyclin D1, and Bcl-2 significantly increased in rat with MNU and s-NaCl administration compared with control group. The phospho-IκBα expression had a tendency to increase in MNU and s-NaCl group compared with control group. Immunoreactive cells of 8-OHdG in curcumin supplementation significantly decreased when compared with MNU and s-NaCl group. The relative intensity of phospho-IκBα in curcumin group tended to reduce when compared with MNU and s-NaCl group. Curcumin can attenuate cancer via a reduction of phospho-IκBα and 8-OHdG expressions, which may play a promising role in gastric carcinogenesis [20].

## **3. Curcumin protects against NSAID-induced ulcer**

Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly prescribed drugs worldwide. It is well-known that NSAIDs cause gastric mucosal damage ranging from nonspecific dyspepsia to ulceration, upper gastrointestinal (GI) bleeding, and death. These can summarize by the term "NSAIDs gastropathy." NSAIDs caused topical damage from "ion trapping" effect [21], the reduction of the hydrophobicity of the gastric mucosal surface, and uncoupling of oxidative phosphorylation [22, 23]. The systemic effect caused by inhibiting cyclo-oxygenases (COX). NSAIDs block the formation not only of proinflammatory cytokines but also of gastroprotective prostaglandins those maintain gastric mucosal blood flow and bicarbonate production [24]. The enhanced synthesis of leukotrienes may occur by shunting the arachidonic acid metabolism towards the 5-lipoxygenase pathway [25–27]. COX inhibition, enhanced synthesis of leukotrienes, contributing to gastric mucosal injury by promoting tissue ischemia and inflammation [28–31].

Mechanism of NSAID-induced gastric ulceration is a neutrophil-dependent process. NSAIDs induced neutrophil adherence to vascular endothelium [32]. Neutrophils play an important role by releasing a variety of inflammatory mediators, including neutrophil elastase and ROS caused gastric mucosal injury. Furthermore, adhesion molecules expressed on activated neutrophils, such as CD11b and CD18, play an important role in neutrophil-induced tissue injury [33–35].

Protective effects of curcumin on NSAIDs were examined [36]. The scheme of the effects was shown in **Figure 4**. The study demonstrates effects of curcumin on gastric microcirculation, tumor necrosis factor (TNF)-α, and intercellular adhesion molecule (ICAM)-1 levels on rat with NSAID-induced gastric injury. The stomach histopathology in NSAIDs group showed multiple erosions with mild to moderate inflammation. Serum of ICAM-1, TNF-α levels, and leukocyte-endothelium interaction increased significantly when compared with control group. Pretreatment with curcumin group resulted in decreasing the elevation serum of ICAM-1, TNF-α levels, and leukocyte-endothelium interaction. The stomach histopathology was improved in curcumin administration group. Therefore, curcumin accomplishes the protective effect on NSAID-induced gastric mucosal injury on improving gastric microcirculation and reducing inflammatory cytokines [36].

significant decrease of macromolecular leakage and NF-κB-p65 expression (*p* ≤ 0.05) in the curcumin-treated groups (curcumin 200 mg/kg and 600 mg/kg BW) compared with *H. pylori*infected group, respectively. These results could be concluded that *H. pylori* infection increased macromolecular leakage, NF-κB-p65 expression, and serum VEGF level. Curcumin can reduce macromolecular leakage, decrease serum VEGF level, and NF-κB-p65 expression. It is implied

In addition, the present study to examine the protective effect of curcumin on gastric cancer induced by N-methyl-N-nitrosourea (MNU) and saturated sodium chloride (s-NaCl) administration [20]. The scheme of the results was shown in **Figure 3**. Gastric cancer can generate in any part of the stomach. Cancers were found in the forestomach of all rats induced by MNU and s-NaCl. Curcumin supplementations showed 40–50% reduction of cancer incidence. Expressions of 8-OHdG, cyclin D1, and Bcl-2 significantly increased in rat with MNU and s-NaCl administration compared with control group. The phospho-IκBα expression had a tendency to increase in MNU and s-NaCl group compared with control group. Immunoreactive cells of 8-OHdG in curcumin supplementation significantly decreased when compared with MNU and s-NaCl group. The relative intensity of phospho-IκBα in curcumin group tended to reduce when compared with MNU and s-NaCl group. Curcumin can attenuate cancer via a reduction of phospho-IκBα and 8-OHdG expressions, which may play a promising

Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly prescribed drugs worldwide. It is well-known that NSAIDs cause gastric mucosal damage ranging from nonspecific dyspepsia to ulceration, upper gastrointestinal (GI) bleeding, and death. These can summarize by the term "NSAIDs gastropathy." NSAIDs caused topical damage from "ion trapping" effect [21], the reduction of the hydrophobicity of the gastric mucosal surface, and uncoupling of oxidative phosphorylation [22, 23]. The systemic effect caused by inhibiting cyclo-oxygenases (COX). NSAIDs block the formation not only of proinflammatory cytokines but also of gastroprotective prostaglandins those maintain gastric mucosal blood flow and bicarbonate production [24]. The enhanced synthesis of leukotrienes may occur by shunting the arachidonic acid metabolism towards the 5-lipoxygenase pathway [25–27]. COX inhibition, enhanced synthesis of leukotrienes, contributing to gastric mucosal injury by promoting tissue

Mechanism of NSAID-induced gastric ulceration is a neutrophil-dependent process. NSAIDs induced neutrophil adherence to vascular endothelium [32]. Neutrophils play an important role by releasing a variety of inflammatory mediators, including neutrophil elastase and ROS caused gastric mucosal injury. Furthermore, adhesion molecules expressed on activated neutrophils, such as CD11b and CD18, play an important role in neutrophil-induced tissue

that curcumin may have an anti-inflammatory effect on *H. pylori* infection [19].

178 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

role in gastric carcinogenesis [20].

ischemia and inflammation [28–31].

injury [33–35].

**3. Curcumin protects against NSAID-induced ulcer**

**Figure 4.** Protective mechanism of curcumin on NSAID-induced gastric mucosal injury [36].

## **4. Curcumin protects against drug-induced liver injury**

N-acetyl-P-aminophenol (APAP) or paracetamol is a widely used analgesic and antipyretic drugs [37, 38]. APAP toxicity is one of the most common drug-induced liver damages worldwide, where major liver major complication is caused due to APAP overdose. APAP metabolites produced in the liver and other organs are the main contributors for the mechanism of its toxicity [39, 40].

The scheme of liver injury by drug was shown in **Figure 5**. In therapeutic doses, APAP is mainly metabolized via glucuronidation and sulfation and in conjugated forms are excreted from the body. Besides, APAP partly is metabolized by cytochrome P450 (CYP 450), to some metabolites, mainly N-acetyl-*p*-benzoquinone imine (NAPQI), which are dramatically increased in high APAP concentrations. These metabolites of APAP are detoxified by glutathione (GSH) and removed from the body. Then, in APAP overdose causes increasing of toxic metabolites. These metabolites interact with a range of cellular proteins via covalent binding, which disrupting hepatocyte function causing necrosis, apoptosis, and liver injury occurs [41, 42].

**Figure 5.** Protective mechanism of curcumin on paracetamol overdose-induced hepatitis [43].

The protective effects of curcumin on paracetamol overdose-induced hepatitis in mice were studied. The effect was shown in **Figure 5**. The results showed that serum transaminases, Hepatic malondialdehyde (MDA), and inflammatory cytokines (TNF-α and IL18) were increased significantly in the 400 mg/kg of APAP group compared with the control group. Curcumin treatment groups (curcumin 200 mg/kg and 600 mg/kg) were significantly decreased these parameters compared with the APAP group. The level of GSH decreased significantly in the APAP compared with the control group. Curcumin treatment groups (curcumin 200 mg/kg and 600 mg/kg) were significantly increased GSH level compared with the APAP group. The histological appearance of the liver in the control group showed normal. In the APAP group, the liver showed damage with extensive hemorrhagic hepatic necrosis at all zones. Curcumin treatment groups (curcumin 200 mg/kg and 600 mg/kg) improved the liver histopathology. In curcumin 200 mg/kg group, the liver showed mild focal necrosis and the normal architecture was well preserved in curcumin 600 mg/kg group. The results indicated that curcumin prevented APAP-induced hepatitis through decreased oxidative stress, reduced liver inflammation, and restored GSH, which caused the improvement of liver histopathology [43]

## **5. Curcumin protects against alcoholic liver disease**

**4. Curcumin protects against drug-induced liver injury**

180 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

toxicity [39, 40].

N-acetyl-P-aminophenol (APAP) or paracetamol is a widely used analgesic and antipyretic drugs [37, 38]. APAP toxicity is one of the most common drug-induced liver damages worldwide, where major liver major complication is caused due to APAP overdose. APAP metabolites produced in the liver and other organs are the main contributors for the mechanism of its

The scheme of liver injury by drug was shown in **Figure 5**. In therapeutic doses, APAP is mainly metabolized via glucuronidation and sulfation and in conjugated forms are excreted from the body. Besides, APAP partly is metabolized by cytochrome P450 (CYP 450), to some metabolites, mainly N-acetyl-*p*-benzoquinone imine (NAPQI), which are dramatically increased in high APAP concentrations. These metabolites of APAP are detoxified by glutathione (GSH) and removed from the body. Then, in APAP overdose causes increasing of toxic metabolites. These metabolites interact with a range of cellular proteins via covalent binding, which disrupting

hepatocyte function causing necrosis, apoptosis, and liver injury occurs [41, 42].

**Figure 5.** Protective mechanism of curcumin on paracetamol overdose-induced hepatitis [43].

Alcoholic liver disease (ALD) represents a spectrum of clinical illness and morphological changes that range from fatty liver, hepatic inflammation, and necrosis (alcoholic hepatitis) to progressive fibrosis (alcoholic cirrhosis) [44]. Ethanol oxidation generates toxic products such as acetaldehyde, and reactive oxygen species resulted in oxidative stress that initiates apoptosis and cell injury [45–48]. More than 80–90% of heavy drinkers develop fatty liver, but only up to 20–40% of this population develops more severe forms of alcoholic liver disease (ALD), including fibrosis, alcoholic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) [49].

Pathogenic mechanisms of alcoholic liver disease were proposed. Ethanol promotes the translocation of lipopolysaccharide from the gastrointestinal lumen to the portal vein. In Kupffer cells, lipopolysaccharide binds to CD14, which combines with Toll-like receptor 4 (TLR4) which is responsible for activating the innate immune system. The increase on inflammatory cytokine production in conjunction with a decrease in signal transducer and activator of transcription (STAT) factors' expression reduces liver regeneration. Long-term alcohol consumption alters the intracellular balance of antioxidants with subsequent decrease in the release of mitochondrial cytochrome c and expression of Fas ligand, leading to hepatic apoptosis. Activated Kupffer cells and hepatocytes are suggested to be sources of free radicals (especially ROS), which are responsible for lipid peroxidation and further apoptotic damage. Activation of hepatic stellate cells also contributes to the production of cytokines, ROS and TGF-β exacerbating liver fibrosis [49].

This study demonstrated effects of curcumin attenuated inflammation and liver pathology in rats with alcoholic liver disease. The effect was shown in **Figure 6**. The results showed that the liver histopathology in ethanol group revealed moderate steatosis and necroinflammation. In ethanol group, hepatic MDA, hepatocyte apoptosis, and NF-κB activation have increased significantly when compared with control. The 400 mg/kg BW of curcumin treatment revealed the decreased of hepatocyte apoptosis, hepatic MDA, NF-κB activation. The peroxisome proliferator-activated receptor gamma (PPARγ) protein expression increased in the curcumin groups. Therefore, curcumin improved liver damage in ethanol-induced hepatitis by reduction of oxidative stress, inhibition of NF-κB activation, and restoration of PPARγ [50].

**Figure 6.** Protective mechanism of curcumin on changes of PPARγ expression, NF-κB activation and oxidative stress in rats with alcoholic hepatitis [50].

## **6. Conclusion**

Curcumin prevents indomethacin-induced gastropathy by decreasing ICAM-1, TNF-α levels, and leukocyte-endothelium interaction. Curcumin reduces *H. pylori*-induced gastric inflammation and gastric cancer by reducing macromolecular leakage, decreasing serum VEGF level, and NF-κB-p65 expression. Curcumin improves liver damage caused by APAP overdose by decreasing hepatic MDA, TNF-α and IL18, and restoring GSH. Moreover, curcumin attenuates alcohol-induced liver injury by decreasing the elevation of hepatic MDA, inhibition of NF-κB activation and improving of liver pathology. Overall, the major mechanisms of curcumin are associated with reduction of oxidative stress, restoration of glutathione and PPARγ expression, inhibition the activation of NF-κB, attenuation of inflammation, and the improvement of histopathology. Therefore, these features make curcumin a very promising new therapeutic option for the treatment of gastrointestinal and liver diseases.

## **Author details**

ethanol group, hepatic MDA, hepatocyte apoptosis, and NF-κB activation have increased significantly when compared with control. The 400 mg/kg BW of curcumin treatment revealed the decreased of hepatocyte apoptosis, hepatic MDA, NF-κB activation. The peroxisome proliferator-activated receptor gamma (PPARγ) protein expression increased in the curcumin groups. Therefore, curcumin improved liver damage in ethanol-induced hepatitis by reduction

**Figure 6.** Protective mechanism of curcumin on changes of PPARγ expression, NF-κB activation and oxidative stress in

Curcumin prevents indomethacin-induced gastropathy by decreasing ICAM-1, TNF-α levels, and leukocyte-endothelium interaction. Curcumin reduces *H. pylori*-induced gastric inflammation and gastric cancer by reducing macromolecular leakage, decreasing serum VEGF level, and NF-κB-p65 expression. Curcumin improves liver damage caused by APAP overdose by

rats with alcoholic hepatitis [50].

**6. Conclusion**

of oxidative stress, inhibition of NF-κB activation, and restoration of PPARγ [50].

182 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Duangporn Werawatganon

Address all correspondence to: dr.duangporn@gmail.com

Department of Physiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

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#### **Glutamine: A Conditionally Essential Amino Acid with Multiple Biological Functions Glutamine: A Conditionally Essential Amino Acid with Multiple Biological Functions**

Alberto Leguina-Ruzzi and Marcial Cariqueo Alberto Leguina-Ruzzi and Marcial Cariqueo

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66488

#### **Abstract**

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[42] Ishibe T, Kimura A, Ishida Y, Takayasu T, Hayashi T, Tsuneyama K, Matsushima K, Sakata I, Mukaida N, Kondo T. Reduced acetaminophen-induced liver injury in mice

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186 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

2001; 31: 55–138

187.

number 981963.

Glutamine (Gln) is the most abundant free amino acid (AA) in the body with concen‐ trations fluctuating around 500–900 μmol/L. The biological functions of Gln have been widely studied, and they have opened new targets because Gln could modulate physiological functions such as immune enhancer, muscular maintainer, nitrogen transporter, neuronal mediator, pH homeostasis, gluconeogenesis, amino sugar synthesis, and insulin release modulation. In 1990, it was identified that Gln is a conditionally essential AA, meaning that in hypercatabolic or stress conditions, the body suffers depletion in its circulating levels. Moreover, this condition is an independ‐ ent risk factor of mortality, has been correlated with increase in infection rates, and length of hospital stay in intensive care units (ICU) patients. This characteristic confers the option of Gln use, meaning that through its targets, it could improve the outcome of patients who are suffering a hypercatabolic or hypermetabolic condition.

**Keywords:** glutamine, parenteral nutrition, enteral nutrition, amino acid

## **1. Introduction**

l‐Glutamine (abbreviated as Gln or Q; encoded by the codons CAA and CAG) is a charge neutral, polar (at physiological pH) α‐amino acid. Its free form has limited solubility and is unstable in aqueous solution. Gln solved in aqueous solution constructs the cyclized com‐ pound pyroglutamic acid (**Figure 1**), which is an uncommon amino acid associated with metabolism problems [1].

© 2017 The Author(s). Licensee InTech. This chapter is 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. © 2017 The Author(s). Licensee InTech. This chapter is 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.

**Figure 1.** Suggested mechanism for the formation of pyroglutamic acid from glutamine.

Gln is commonly used as a dipetide through a conjugation with l‐alanine (Ala‐Gln) in the use of nutritional supplement. The dipeptide showed not only dramatically high solubility and stability (**Table 1**) but also can liberate and excrete after absorption into body (**Figure 2**) and ensure the proper excretion [2].


**Table 1.** Solubility and stability profile of amino acids and conjugations.

**Figure 2.** Plasma concentrations of alanine, glutamine, and Ala‐Gln during and after continuous intravenous infusion of Ala‐Gln (mean ± SD) [2].

Gln is the most abundant amino acid in the body, representing around 30–35% of the amino acid nitrogen in the plasma. It contains two ammonia groups: one from its precursor glutamate and the other from free ammonia in the bloodstream. Because of it, one of its classic and first described functions was as a "nitrogen shuttle," which helps to protect the body from high concentrations of ammonia: Gln behaves as a buffer, receiving excess ammonia, and then releasing it when needed to form other amino acids, amino sugars, nucleotides, and urea.

Gln is mainly distributed in the skeletal muscle (60% of the total pool), short intestine, brain, kidney, and liver. This amino acid (AA) is supplied by specific organs for its metabolic use and adequate renal excretion (**Figure 3**) [3].

**Figure 3.** Glutamine distribution in the body.

**Figure 1.** Suggested mechanism for the formation of pyroglutamic acid from glutamine.

188 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Alanine 167.2 Yes Cystine 0.1 Yes Cystine‐HCl 252.0 No Bis‐L‐analyl‐L‐cystine >500.0 Yes Bis‐glycyl‐L‐cystine 541.0 Yes Tyrosine 0.4 Yes L‐alanyl‐L‐tyrosine 14.0 Yes Glycyl‐L‐tyrosine 30.0 Yes Glutamine 36.0 No L‐alanyl‐L‐glutamine 568.0 Yes Glycyl‐L‐glutamine 154.0 Yes

**Table 1.** Solubility and stability profile of amino acids and conjugations.

ensure the proper excretion [2].

of Ala‐Gln (mean ± SD) [2].

Gln is commonly used as a dipetide through a conjugation with l‐alanine (Ala‐Gln) in the use of nutritional supplement. The dipeptide showed not only dramatically high solubility and stability (**Table 1**) but also can liberate and excrete after absorption into body (**Figure 2**) and

**Figure 2.** Plasma concentrations of alanine, glutamine, and Ala‐Gln during and after continuous intravenous infusion

**Solubility (g/L H2O at 20°C) Stable in solution**

The role of nutrition in the Gln metabolism has been widely studied. The initial studies on its physiological role suggested that Gln is not always necessary to be ingested from diet because it can accumulate to a high amount; however, this concept has changed in the recent years. In fact, when 5–10 g\day of Gln is consumed in the diet, the de novo synthesis of Gln is regulated by a demand to maintain a balance [4].

An extensive study that evaluated the glutamine content of a wide range of food has been performed. The results showed that the content varied from 0.01 to 9.49 g/100 g of food and a ratio of Gln contained in total protein reached around 1–33% of the intake. The most Gln‐ enriched foods were the one directed from beef meat, milk, tofu, white rice, corn protein, among others [5].

As mentioned above, the food containing Gln at high concentrations may be used as superfood. In this chapter, we precisely introduce the role of AA in the body, its possibility to be considered as a super nutrient, and we discuss its effectiveness in clinical practice.

## **2. Glutamine metabolism**

Gln is considered to be a nonessential amino acid that was coined by Lacey JM and Wilmore in 1990, as human cells can readily synthesize by glutamine synthetase present in the skeletal muscles, liver, brain, and stomach tissues (**Figure 4**) [6].

**Figure 4.** Glutamine biosynthesis process.

Gln is a highly versatile AA as shown in **Figure 5**: it can be converted to other amino acids, to glucose in the liver, and contributes amino groups to nucleotide, amino sugar, and protein biosynthesis. Moreover, it is related to multiple functions and molecular targets in physiolog‐ ical pathways [7].

The uptake into cellular compartments is mediated by several membrane transporters that regulate the homeostasis by coordinating the absorption, reabsorption, and delivery to tissues. These redundant and ubiquitous located transporters belong to different protein families. The complex interplay between the cell polarity and types of Gln transporters have been sophis‐ tically reviewed by Pochini et al. They described the role of the glutamine transporters linked to their different transport modes and coupling with Na+ and H+ . Most transporters share the specific transport capacity with other neutral or cationic amino acids. Na+ ‐dependent co‐ transporters efficiently accumulate glutamine, while antiporters regulate the pools of gluta‐ mine and other amino acids.

**Figure 5.** Glutamine intracellular metabolic pathway.

fact, when 5–10 g\day of Gln is consumed in the diet, the de novo synthesis of Gln is regulated

An extensive study that evaluated the glutamine content of a wide range of food has been performed. The results showed that the content varied from 0.01 to 9.49 g/100 g of food and a ratio of Gln contained in total protein reached around 1–33% of the intake. The most Gln‐ enriched foods were the one directed from beef meat, milk, tofu, white rice, corn protein,

As mentioned above, the food containing Gln at high concentrations may be used as superfood. In this chapter, we precisely introduce the role of AA in the body, its possibility to be considered

Gln is considered to be a nonessential amino acid that was coined by Lacey JM and Wilmore in 1990, as human cells can readily synthesize by glutamine synthetase present in the skeletal

Gln is a highly versatile AA as shown in **Figure 5**: it can be converted to other amino acids, to glucose in the liver, and contributes amino groups to nucleotide, amino sugar, and protein biosynthesis. Moreover, it is related to multiple functions and molecular targets in physiolog‐

The uptake into cellular compartments is mediated by several membrane transporters that regulate the homeostasis by coordinating the absorption, reabsorption, and delivery to tissues.

as a super nutrient, and we discuss its effectiveness in clinical practice.

190 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

muscles, liver, brain, and stomach tissues (**Figure 4**) [6].

by a demand to maintain a balance [4].

**2. Glutamine metabolism**

**Figure 4.** Glutamine biosynthesis process.

ical pathways [7].

among others [5].

The most studied subfamilies of Gln transporters are the SLC1, 6, 7, and 38. The members involved in the homeostasis are the co‐transporters B0AT1 and the SNAT members 1, 2, 3, 5, and 7; the antiporters ASCT2, LAT1 and 2 (**Figure 6**) [8].

Additionally, limited information on glycosylation and/or phosphorylation regulatory sites of the Gln transporters has been described. More studies in the field are needed to fully under‐ stand their associated mechanisms.

As shown in **Figure 7**, the metabolic pathway of Gln is a complex network of transport, and the hyperglutaminemia is a highly cytotoxic state classically reported in kidney and liver failure [9]. The proper function of these organs is to ensure the safe excretion. Gln is trans‐ formed to urea through the metabolic hepatic pathway and is excreted by the kidney. Addi‐ tionally, in the intestine, muscle, and liver the Gln is converted to other compounds by chemical

reactions. At those organs, Gln is degraded and converted to glutamate, aspartate, CO2, pyruvate, lactate, alanine, and citrate, among other metabolites [10].

**Figure 6.** Glutamine transporters, modified from Pochini et al. [8].

**Figure 7.** Glutamine transport and excretion system (Ala: alanine, BCAA: Branched Chain Amino Acids, αKG: alpha ketoglutarate).

## **3. Glutamine depletion in hypercatabolism**

reactions. At those organs, Gln is degraded and converted to glutamate, aspartate, CO2,

**Figure 7.** Glutamine transport and excretion system (Ala: alanine, BCAA: Branched Chain Amino Acids, αKG: alpha

pyruvate, lactate, alanine, and citrate, among other metabolites [10].

192 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**Figure 6.** Glutamine transporters, modified from Pochini et al. [8].

ketoglutarate).

The depletion of glutamine is a generally accepted phenomenon particularly observed in the intensive care units (ICU) patients [11]. However, no clear mechanism of the onset of this alteration have been investigated. During hypercatabolic stress, proliferating lymphocytes and immune‐stimulated macrophages are major glutamine consumers [12]. The increased de‐ mands of Gln in hypercatabolic stress cause high secretion of Gln from skeletal muscle leading to muscle mass loss, a prevalent feature of ICU patients [13].

Of all AA, muscular Gln levels is the most important indicator to determine the restoring forth or surviving rate in ICU patients with prolonged sepsis [14], even more it has been reported that low Gln levels are an independent mortality predictor [11]. The decreased Gln plasma levels in ICU patients are around the 50% of normal levels and negatively correlated with the severity of the pathology.

Based on the clinical observations, the reduction in Gln is associated with higher mortality, length of hospital stay (LOS), and infection [15]. The clinical experts have suggested a series of metabolic alternations that in concentration would explain the poor outcome that an ICU patient with this depletion present (**Figure 8**).

**Figure 8.** Consequences of glutamine depletion for the organism, modified Stehle et al. [15].

## **4. Molecular targets of glutamine**

Several molecular actions of the Gln [16] have beneficial effects as a supplement with pharmaco logical actions. A supplement of Gln is effective for normalizing the metabolic processes that are altered in hypercatabolic patients. The last decades of biomedical research have identified the specific molecular targets in which this AA exerts its functions**<sup>1</sup>** .

## **4.1. Glutathione biosynthesis**

Glutathione (GSHis) is a reduced nonprotein thiol, which is present in all mammalian tissues and has important antioxidant and detoxification functions. Gln is a precursor of GSHis when combined with glycine and cysteine in the cytoplasmic compartment of the cell. This reduced metabolite has strong affinity to free radicals and toxins, via reaction to oxidized glutathione disulphide (GSSG). GSSG can be converted again to GSHis or be translocated to the vacuole for degradation. This conversion capacity confers its antioxidant and detoxicant effects to the cells (**Figure 9**).

**Figure 9.** Antioxidant and detoxicant mechanism of glutathione (Cys: cysteine, ATP: adenosine triphosphate, ADP: ad‐ enosine diphosphate, y‐ESC: gamma glutaminecystein dipeptide, Glu: glutamine, Gly: glycine, SG: glutathione disul‐ fide).

**<sup>1</sup>** From Leguina‐Ruzzi [16]. For the full compilation of references please check the cited article.

#### **4.2. Heat Shock Proteins genetic regulation**

**4. Molecular targets of glutamine**

**4.1. Glutathione biosynthesis**

cells (**Figure 9**).

fide).

**1**

Several molecular actions of the Gln [16] have beneficial effects as a supplement with pharmaco logical actions. A supplement of Gln is effective for normalizing the metabolic processes that are altered in hypercatabolic patients. The last decades of biomedical research have identified

Glutathione (GSHis) is a reduced nonprotein thiol, which is present in all mammalian tissues and has important antioxidant and detoxification functions. Gln is a precursor of GSHis when combined with glycine and cysteine in the cytoplasmic compartment of the cell. This reduced metabolite has strong affinity to free radicals and toxins, via reaction to oxidized glutathione disulphide (GSSG). GSSG can be converted again to GSHis or be translocated to the vacuole for degradation. This conversion capacity confers its antioxidant and detoxicant effects to the

**Figure 9.** Antioxidant and detoxicant mechanism of glutathione (Cys: cysteine, ATP: adenosine triphosphate, ADP: ad‐ enosine diphosphate, y‐ESC: gamma glutaminecystein dipeptide, Glu: glutamine, Gly: glycine, SG: glutathione disul‐

From Leguina‐Ruzzi [16]. For the full compilation of references please check the cited article.

.

the specific molecular targets in which this AA exerts its functions**<sup>1</sup>**

194 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

The Heat Shock Proteins (HSP) (also known as stress response proteins) are chaperones that are highly conserved and present in all cells. The important role of HSP is to participate in protein folding, assembly, and correct transport, enabling them to act normally. It has been shown that in sepsis or in inflammatory response syndrome, there is a significant reduction in the intracellular levels of HSP70, which correlates with severity of illness and mortality. Interestingly, administration of Gln enhanced the HSP70 expression through the crosstalk with the hexosamine pathway (**Figure 10**). An *in vitro* study demonstrated that the promotion in HSP70 synthesis by Gln is accompanied by a favorable inflammatory response mediated by IL‐8 and IL‐10 [17].

**Figure 10.** Enhancement of HSP synthesis by glutamine and hexosamine cross‐talk (GFAT: glutamine fructose‐6‐phos‐ pate aminotransferase, GlcN‐6‐P: glucosamine‐6‐phospato, UDP‐GlcNAc: uridine diphosphate N‐acetylglucosamine, UDP: uridine di phosphate, SP1: specific protein 1, HSF‐1: heat shock transcription factor 1, O‐GlcNAc: O‐linked N‐ acetylglucosamine, PRE: promotor regulatory elements).

#### **4.3. Enterocyte integrity**

The bacterial translocation (BT) is mainly occurring under pathological conditions because the passage of viable bacteria from the gastrointestinal tract to extraintestinal sites is opened in such conditions.

ICU patients are at higher risk of bacterial translocation: 15% of these patients is affected. BT is one of the main causes of sepsis and multiorgan failure.

It has been reported that the supplementation of Gln to the total parenteral nutrition (TPN) reduces the prevalence of BT and prevents inflammatory intestinal complications. *In vivo* and *in vitro* studies have reported that the enterocyte uses Gln as its principal energy source and enhances its growth and proliferation. Moreover, the enterocyte is capable of transporting Gln to and from the intestinal circulation, creating a bidirectional supply of this AA. This process uses a series of antiporters coupled with Na+ and H+ from the family of ASCT2. On the other hand, a deprivation of Gln facilitates BT, which suggests the importance of Gln in the intestinal barrier integrity (**Figure 11**.)

**Figure 11.** Glutamine transport and metabolism in the enterocyte.

## **4.4. Lymphocyte function**

The activation of naive T cells is a pivotal process for the immune response and is a highly energetic event in which T cells require an increase in nutrient metabolism. For the series of processes required, Gln uptake is a fundamental step highly regulated by the extracellular signal‐regulated kinases (ERK)/MAPK pathway (**Figure 12**).

*In vitro* studies have demonstrated that the Gln supplementation modulates the proliferation of the naive T cells, and the extracellular Gln is essential as a respiratory fuel. The supplemen‐ tation of Gln has a prominent effect on both activations of lymphocyte and modulation of secretory functions as well as killing bacteria by neutrophils and macrophage phagocytosis.

**Figure 12.** Metabolic processes required for lymphocyte activation (GLUT1: glucose transporter 1, ERK: extracellular signal‐regulated kinases).

## **4.5. Nitrogen balance**

ICU patients are at higher risk of bacterial translocation: 15% of these patients is affected. BT

It has been reported that the supplementation of Gln to the total parenteral nutrition (TPN) reduces the prevalence of BT and prevents inflammatory intestinal complications. *In vivo* and *in vitro* studies have reported that the enterocyte uses Gln as its principal energy source and enhances its growth and proliferation. Moreover, the enterocyte is capable of transporting Gln to and from the intestinal circulation, creating a bidirectional supply of this AA. This process

and H+

hand, a deprivation of Gln facilitates BT, which suggests the importance of Gln in the intestinal

The activation of naive T cells is a pivotal process for the immune response and is a highly energetic event in which T cells require an increase in nutrient metabolism. For the series of processes required, Gln uptake is a fundamental step highly regulated by the extracellular

*In vitro* studies have demonstrated that the Gln supplementation modulates the proliferation of the naive T cells, and the extracellular Gln is essential as a respiratory fuel. The supplemen‐ tation of Gln has a prominent effect on both activations of lymphocyte and modulation of secretory functions as well as killing bacteria by neutrophils and macrophage phagocytosis.

from the family of ASCT2. On the other

is one of the main causes of sepsis and multiorgan failure.

196 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

uses a series of antiporters coupled with Na+

**Figure 11.** Glutamine transport and metabolism in the enterocyte.

signal‐regulated kinases (ERK)/MAPK pathway (**Figure 12**).

**4.4. Lymphocyte function**

barrier integrity (**Figure 11**.)

The regulation of protein concentration in the body at a relatively normal level is a physio‐ logically favorable process to maintain the cellular integrity. It is widely accepted that ICU patients with TPN present a negative balance that correlates with the clinical outcome. The capacity of Gln pool and nitrogen balance was improved by its supplementation has shown the capacity to recover the nitrogen balance in three days after supplementation. Furthermore, the Gln‐supplemented diet did not affect portal ammonia concentration, showing that it does not affect the excretion pathway and did not cause anabolic effects that are associated with cardiovascular alterations [18].

Consequently, these clinical observations could be explained in part by the capacity of the Gln to act as a substrate for other AA or to construct more protein at the muscle.

#### **4.6. Insulin release**

The hyperglycemic condition is a metabolic emergency commonly associated with uncontrol‐ led diabetes mellitus, which may result in significant morbidity and death. The prevalence in UCI patients is approximately 40% and classically was presumed to be an adaptive response to the hypercatabolism. However, more recent reports have shown that hyperglycemia is associated with unfavorable clinical outcomes.

The role of Gln in insulin sensitivity and hyperglycemia is a hot topic that has been actively studied. A recent randomized clinical trial demonstrated that the supplementation of Gln to the TPN for more than 7 days reduces significantly the hyperglycemic episodes and the insulin requirement in ICU polytrauma patients. *In vivo* and *in vitro* studies have demonstrated the Gln stimulates calcium‐dependent insulin secretion and beta‐cell depolarization and enhances the insulin glucose response. The improved process involves the metabolism of the gamma‐ glutamyl cycle, the glutathione synthesis, and the mitochondrial function (**Figure 13**).

**Figure 13.** Glutamine‐dependent insulin release (TCA: tricarboxylic acid cycle, ATP: adenosine triphosphate, KATP‐ channel: ATP‐sensitive potassium channel).

#### **4.7. Others beneficial effects of glutamine**

The scientific community has been eager to understand the beneficial effects of Gln. Recent clinical, *in vivo* and *in vitro* studies have suggested the cardio protective role of Gln [19, 20] in ischemic heart disease and diabetic cardiomyopathy [21]. Additionally, studies suggest that the supplementation of Gln enhances the healing of the lung parenchymal injuries, reducing the air leakage [22], also regulates the pulmonary infiltration in sepsis, modulating the immunological function [23], and endogenous levels of this AA modulates the vasoactive response of the nitric oxide (NO) in pulmonary hypertension [24].

## **5. Enteral and parenteral glutamine supplementation**

The role of Gln in insulin sensitivity and hyperglycemia is a hot topic that has been actively studied. A recent randomized clinical trial demonstrated that the supplementation of Gln to the TPN for more than 7 days reduces significantly the hyperglycemic episodes and the insulin requirement in ICU polytrauma patients. *In vivo* and *in vitro* studies have demonstrated the Gln stimulates calcium‐dependent insulin secretion and beta‐cell depolarization and enhances the insulin glucose response. The improved process involves the metabolism of the gamma‐

198 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

glutamyl cycle, the glutathione synthesis, and the mitochondrial function (**Figure 13**).

**Figure 13.** Glutamine‐dependent insulin release (TCA: tricarboxylic acid cycle, ATP: adenosine triphosphate, KATP‐

The scientific community has been eager to understand the beneficial effects of Gln. Recent clinical, *in vivo* and *in vitro* studies have suggested the cardio protective role of Gln [19, 20] in ischemic heart disease and diabetic cardiomyopathy [21]. Additionally, studies suggest that the supplementation of Gln enhances the healing of the lung parenchymal injuries, reducing the air leakage [22], also regulates the pulmonary infiltration in sepsis, modulating the immunological function [23], and endogenous levels of this AA modulates the vasoactive

channel: ATP‐sensitive potassium channel).

**4.7. Others beneficial effects of glutamine**

response of the nitric oxide (NO) in pulmonary hypertension [24].

The benefits of parenteral and enteral Gln supplementation in critically ill patients have been shown in numerous clinical trials. Many authors reported in systematic reviews and meta‐ analyses that Gln supplementation, combined with enteral nutrition (EN) and parenteral nutrition (PN), is associated with reduced infectious morbidity and improved recovery from critical illness compared with standard [25]. The most relevant results have been observed with parenteral supplementation, a phenomenon that might be explained by highly regulated metabolism of the AA and the enterocyte participation (**Figure 11**).

Standard PN and EN formulations do not contain Gln as monopeptide due to the poor solubility and the instability in heat sterilization as described in Section 2. The solubility is limited to 35 g/l (3.5%) at 20°C, and the recommendation is not to use solutions of 2.5%, to avoid precipitation that could affect the proper nutritional administration. To solve these problems, the clinical use by parenteral formulation has been supported in the administration of the Gln dipeptides with other AA, which is more stable and allow prolonged conservation. The dipeptides are rapidly hydrolyzed by serum peptidases, allowing the utilization of 100% of Gln (**Figure 2**). The dipeptides are more soluble than Gln alone; the solubility of Gly‐L‐Gln is 154 g/l (15.4%) and the solubility of l‐Ala‐l‐Gln is 568 g/l (56.8%). Importantly, parenteral formulations contain 200 g/l (20%) of dipeptide l‐Ala‐l‐Gln which is equivalent to 134 g/l of Gln and enteral formulations contain 2–4 g/l (0.02–0.04%) [26].

Currently, the most commercially used Gln products are Dipeptiven® for PN and Reconvan® for EN produced by Fresenius Kabi Co.; however, it is important to mention that the basic enteral formulations contain Gln as a part of the protein composition and under low concen‐ trations; for example, the Fresubin® line by Fresenius Kabi (Bad Homburg vor der Höhe, Germany) fluctuates around 3.5—9.4 g per presentation bag.

The early enteral feeding has been associated with a substantial reduction in length of hospital stay (LOS) with a significant reduction in the frequency of acquired infections. The enteral Gln supplementation has been shown to be safe and well tolerated and may help to reduce infectious complications, oxidative stress, intestinal permeability, mortality, and LOS [27]. Houdijk [28]. However, in 2015, Van Zanten et al. published a systematic review and meta‐ analysis with a total of 11 studies involving 1079 adult critical ill patients and enteral Gln supplementation was not associated with a reduction of hospital mortality, infectious compli‐ cations, or stay in the intensive care unit. In the subset of patients with burns, there may be a significant benefit in hospital mortality [29].

The effective concentration of Gln in TPN has been suggested by different multidisciplinary medical groups. According to the ESPEN guideline, "when PN is indicated in ICU patients the amino acid solution should contain 0.2–0.4 g/kg/day of l‐glutamine (e.g. 0.3–0.6 g/kg/day alanyl‐glutamine dipeptide)" [30] and "glutamine should be added to a standard enteral formulation in burned patients and trauma patients" [31]. It is important to consider that the main contraindications are renal failure (Creatinine clearance <20 mL/min), metabolic acidosis, and liver failure (Liver function tests International Normalized Ratio >1.5) [32]. Interestingly, Helling et al. evidenced the association between liver failure and high plasma Gln levels [33], a medical situation that needs to be taken into consideration at the moment to prescribe Gln supplementation.

The use of EN with Gln may not be enough to full up plasma concentration up to normal level; new remarkable data showed that enteral supplementation only is not enough to revert the Gln depletion [34]. The values and data accumulated in PN cannot be directly extrapolated to EN supplementation and, therefore, cannot be used as the base for recommendation. Accord‐ ing to the trials, high protein enteral nutrition enriched with immuno modulator nutrients, such as Gln, did not improve infectious complications or other clinical end points compared to standard EN [35].

On the other hand, administration of TPN with Gln is effective. In 2002, Goether showed improvement in a 6‐month survival in a patient with at least 9 days of parenteral Gln supple‐ mentation [28]. Until 2014, several trials showed that PN with Gln supplementation given in conjunction with nutrition support continues to be associated with a significant reduction in hospital mortality and hospital LOS. A systematic review published by Wischmeyer et al. in 2014 summarized all randomized controlled trials conducted from 1997 to 2013, showing the benefits of parenteral glutamine [36]. However, in 2013, the Reducing Deaths Due to Oxidative Stress (REDOX) study, the largest trial to date showed that the supplementation of Gln was associated with higher mortality and no beneficial effects were seen [37]. The results of this trial awaken a question to the safety and efficacy of the use of glutamine in critically ill patients, in high doses (much higher than recommended) may produce adverse effects. In this study, they used a combined enteral and IV Gln supplementation in higher doses than the recom‐ mended ones: the intervention setting was the Gln enteral 30 g/day plus parenteral 15 g/day giving around 1 g/kg/day, doses higher than the classically recommended by the clinical guidelines of the date. Furthermore, the heterogeneous enrolment included patients that fulfilled contraindication criteria for its supplementation.

A year later, the authors published a new analysis of the data (post hoc study) concluding that high doses of Gln may be associated with higher mortality in patients with multiorgan failure and particularly renal dysfunction [38]. These conclusions had a high impact not only on the pharmaceutical industry but also on the clinical practice: two of the most important guidelines changed dramatically the recommendation of the use of Gln. The Canadian Clinical Practice Guidelines (2016) [39] and ASPEN (2016) [40] downgraded the Gln supplementation based of the REDOX results and a series of studies that do not justify this.

It is important to highlight that even after these dramatic results, the research has given important steps in the years follow the REDOX. It gave not only answers to the basic questions such as the glutamine plasmatic levels in ICU patients and its safe use as supplement to TPN but also opened a whole perspective for further research and use. Pérez‐Bárcena et al. [41], in 2014, showed that low doses of IV Gln for 5 days did not show beneficial effects in the ICU patients, without causing any derogative effect. Additionally, plasmatic Gln measurement in the patients showed that those with lower levels presented a worse outcome (mortality, LOS, and infections) in which a supplementation with higher doses might be necessary.

In the same year, Grintescu et al. [42] showed that Gln supplementation in trauma patients reduces hyperglycemic episodes and improves insulin response. The conclusions suggest a role for Gln as an insulin sensitizer.

Despite the controversy, when parenteral nutrition is prescribed, the Gln supplementation is still recommended in critically ill patients. There are insufficient data to generate recommen‐ dations for IV glutamine in critically ill patients who are receiving EN. New large, multicenter, prospective randomized clinical trials are needed to confirm the beneficial effects of Gln in the mortality, LOS, and infections rates as main clinical outcomes are highly relevant in the critical care unit.

## **6. Conclusion**

Helling et al. evidenced the association between liver failure and high plasma Gln levels [33], a medical situation that needs to be taken into consideration at the moment to prescribe Gln

200 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

The use of EN with Gln may not be enough to full up plasma concentration up to normal level; new remarkable data showed that enteral supplementation only is not enough to revert the Gln depletion [34]. The values and data accumulated in PN cannot be directly extrapolated to EN supplementation and, therefore, cannot be used as the base for recommendation. Accord‐ ing to the trials, high protein enteral nutrition enriched with immuno modulator nutrients, such as Gln, did not improve infectious complications or other clinical end points compared

On the other hand, administration of TPN with Gln is effective. In 2002, Goether showed improvement in a 6‐month survival in a patient with at least 9 days of parenteral Gln supple‐ mentation [28]. Until 2014, several trials showed that PN with Gln supplementation given in conjunction with nutrition support continues to be associated with a significant reduction in hospital mortality and hospital LOS. A systematic review published by Wischmeyer et al. in 2014 summarized all randomized controlled trials conducted from 1997 to 2013, showing the benefits of parenteral glutamine [36]. However, in 2013, the Reducing Deaths Due to Oxidative Stress (REDOX) study, the largest trial to date showed that the supplementation of Gln was associated with higher mortality and no beneficial effects were seen [37]. The results of this trial awaken a question to the safety and efficacy of the use of glutamine in critically ill patients, in high doses (much higher than recommended) may produce adverse effects. In this study, they used a combined enteral and IV Gln supplementation in higher doses than the recom‐ mended ones: the intervention setting was the Gln enteral 30 g/day plus parenteral 15 g/day giving around 1 g/kg/day, doses higher than the classically recommended by the clinical guidelines of the date. Furthermore, the heterogeneous enrolment included patients that

A year later, the authors published a new analysis of the data (post hoc study) concluding that high doses of Gln may be associated with higher mortality in patients with multiorgan failure and particularly renal dysfunction [38]. These conclusions had a high impact not only on the pharmaceutical industry but also on the clinical practice: two of the most important guidelines changed dramatically the recommendation of the use of Gln. The Canadian Clinical Practice Guidelines (2016) [39] and ASPEN (2016) [40] downgraded the Gln supplementation based of

It is important to highlight that even after these dramatic results, the research has given important steps in the years follow the REDOX. It gave not only answers to the basic questions such as the glutamine plasmatic levels in ICU patients and its safe use as supplement to TPN but also opened a whole perspective for further research and use. Pérez‐Bárcena et al. [41], in 2014, showed that low doses of IV Gln for 5 days did not show beneficial effects in the ICU patients, without causing any derogative effect. Additionally, plasmatic Gln measurement in the patients showed that those with lower levels presented a worse outcome (mortality, LOS,

and infections) in which a supplementation with higher doses might be necessary.

fulfilled contraindication criteria for its supplementation.

the REDOX results and a series of studies that do not justify this.

supplementation.

to standard EN [35].

The multiple functions of Gln and the possibility of its use as a pharmaco‐nutrient under the hypercatabolic condition were introduced in this chapter. The studies on basic and clinical science showed the beneficial effects of Gln in the metabolism of subjects suffering from a catabolic stress condition. *In vitro* data performed under concentration ranges from 500 to 2000 μmol/L of Gln, and it could represent supplementation and supports the clinical findings. Gln pleiotropic functions make it a great candidate for its use in pathological conditions; however, important lessons have to be learned from the controversial evidence that impacts negatively on its use. Still nonconclusive data have weakened it role in oral and enteral supplementation making mandatory new research in this field.

## **Acknowledgements**

All figures were illustrated by CatarsisCreativa.com in Santiago, Chile.

*Authors contributions*: A. Leguina‐Ruzzi conceptualized and prepared the chapter. M. Cariqueo.

## **Author details**

Alberto Leguina‐Ruzzi1\* and Marcial Cariqueo2

\*Address all correspondence to: alberto@juntendo.ac.jp

1 Biochemistry Department, Research Institute for Disease of Old Age, Juntendo University, Tokyo, Japan

2 Intensive Care Unit, Clinical Hospital, University of Chile, Santiago, Chile

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204 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

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**Chapter 11 Provisional chapter**

## **The Effect of Dietary Intake of Omega-3 Polyunsaturated Fatty Acids on Cardiovascular Health: Revealing Potentials of Functional Food Polyunsaturated Fatty Acids on Cardiovascular Health: Revealing Potentials of Functional Food**

Ines Drenjančević, Gordana Kralik, Zlata Kralik, Martina Mihalj, Ana Stupin, Sanja Novak and Manuela Grčević Zlata Kralik, Martina Mihalj, Ana Stupin, Sanja Novak and Manuela Grčević Additional information is available at the end of the chapter

Ines Drenjančević, Gordana Kralik,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67033

#### **Abstract**

Functional food is a food containing components that show beneficial effects on one or more body functions and improve general condition and health or significantly affect lowering of disease risks. This chapter is aimed to examine the effect of dietary intake of omega‐3 polyunsaturated fatty acids (n3‐PUFA) on cardiovascular health. This chapter presents current knowledge on functional poultry products and the reasons to consume them, omega‐3 enrichment of eggs and poultry meat, and the differences in profile of fatty acids in conventional and omega‐3–enriched eggs. The second part of the chapter focuses on the metabolism of fatty acids and effectiveness of n‐3 PUFA in the improvement of endothelial function, improvement of elasticity of the vascular wall and the anti‐inflammatory effects in patients with chronic diseases, such as metabolic syndrome, diabetes mellitus and hypercholesterolemia, and overall effect on cardiovascular health and protection. To achieve long‐term protective effects, the functional food should be consumed on daily basis. There are no specific constrains in taking functional food; even more, it can be recommended to athletes and cardiovascular patients. General population can also benefit from eating functional food enriched with n‐3 PUFA due to their anti‐inflammatory and vascular‐protective effects.

**Keywords:** omega‐3 fatty acids, enriched eggs and poultry, cardiovascular risk

## **1. Introduction**

Definitions of functional food differ in different parts of the world; however, they all have in common the reference toward food of natural origin that contains ingredients with beneficial

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

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2017 The Author(s). Licensee InTech. This chapter is 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.

effect on human health. In the United States, the definition of functional food says that functional food was "natural or processed food that contains known or unknown biologically active ingredients, which in certain, effective and non‐toxic concentrations provide clinically proven and documented health benefits for prevention, treatment or healing of chronic diseases" [1]. This way of defining functional food is different from the definition in Europe, which does not mention effects of functional food in the treatment of diseases, but mainly refer to benefits in maintaining good health or reducing the risk of developing diseases. The European Commission document "Scientific Concepts of Functional Foods in Europe" states a working definition saying that food can be considered functional if it is satisfactorily shown that, in addition to appropriate nutritional effects, it has beneficial effects on one or more target functions of an organism, in a way that it is important for improving health condition and general well‐being or reducing disease risks. Functional food has to be food (not in the form of pills or capsules) and it has to show its effects when consumed in normal daily amounts [2]. Regardless of different definitions [3], concluded that the main purpose of functional food had to be clear—it improves human health and well‐being or general condition of the body.

Functional poultry products refer to meat and poultry eggs enriched with ingredients that have positive influence on human health. Poultry is particularly suitable for functional food products because of their ability to use the physiological and metabolic processes of their body to deposit beneficial ingredients from feed into products, that is, into meat and eggs. Meat and poultry eggs are enriched with functional ingredients (fatty acids, vitamins, and antioxidants) by feeding poultry feed supplemented with increased concentrations of those ingredients. The most common functional poultry products are meat and eggs with increased content of desirable omega‐3 fatty acids, vitamin E, selenium, and carotenoids. Poultry meat is rich in protein and low in fat. As of its nutritional composition, it can be considered a dietary foodstuff. It is easily digested and especially recommended for consumption of the elderly and children. If considering all stated nutritional benefits, poultry meat enriched with functional ingredients can be considered as functional product. Chicken meat is available to wide population of consumers because of its price, which is more affordable if compared to red meat. High‐quality nutritional composition of chicken meat is also one of the reasons for its frequent consumption, which is especially emphasized in recent years when consumers became more aware about the composition of foods and their effects on health.

Egg is a foodstuff that contains high‐quality and easily digestible proteins, where amino acid composition is the most similar to proteins of the human body. Egg proteins are fully exploited in the human body and have greater biological value than meat proteins. Egg yolk contains essential fatty acids, vitamins, and minerals needed for proper functioning of human organism. Egg is considered a natural functional foodstuff because of its nutritional value. When compared to poultry meat, enrichment of eggs with functional ingredients is easier because of the high content of fat in egg yolk [4].

If consuming meat and eggs enriched with functional ingredients, consumers can affect the increase of the content of such functional ingredients in blood and tissue in a natural way, thus avoiding taking in some dietary supplements. The importance of functional ingredients for human health is elaborated further in the text.

## **2. Functional poultry products production**

effect on human health. In the United States, the definition of functional food says that functional food was "natural or processed food that contains known or unknown biologically active ingredients, which in certain, effective and non‐toxic concentrations provide clinically proven and documented health benefits for prevention, treatment or healing of chronic diseases" [1]. This way of defining functional food is different from the definition in Europe, which does not mention effects of functional food in the treatment of diseases, but mainly refer to benefits in maintaining good health or reducing the risk of developing diseases. The European Commission document "Scientific Concepts of Functional Foods in Europe" states a working definition saying that food can be considered functional if it is satisfactorily shown that, in addition to appropriate nutritional effects, it has beneficial effects on one or more target functions of an organism, in a way that it is important for improving health condition and general well‐being or reducing disease risks. Functional food has to be food (not in the form of pills or capsules) and it has to show its effects when consumed in normal daily amounts [2]. Regardless of different definitions [3], concluded that the main purpose of functional food had to be clear—it improves human health

208 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Functional poultry products refer to meat and poultry eggs enriched with ingredients that have positive influence on human health. Poultry is particularly suitable for functional food products because of their ability to use the physiological and metabolic processes of their body to deposit beneficial ingredients from feed into products, that is, into meat and eggs. Meat and poultry eggs are enriched with functional ingredients (fatty acids, vitamins, and antioxidants) by feeding poultry feed supplemented with increased concentrations of those ingredients. The most common functional poultry products are meat and eggs with increased content of desirable omega‐3 fatty acids, vitamin E, selenium, and carotenoids. Poultry meat is rich in protein and low in fat. As of its nutritional composition, it can be considered a dietary foodstuff. It is easily digested and especially recommended for consumption of the elderly and children. If considering all stated nutritional benefits, poultry meat enriched with functional ingredients can be considered as functional product. Chicken meat is available to wide population of consumers because of its price, which is more affordable if compared to red meat. High‐quality nutritional composition of chicken meat is also one of the reasons for its frequent consumption, which is especially emphasized in recent years when consumers

became more aware about the composition of foods and their effects on health.

Egg is a foodstuff that contains high‐quality and easily digestible proteins, where amino acid composition is the most similar to proteins of the human body. Egg proteins are fully exploited in the human body and have greater biological value than meat proteins. Egg yolk contains essential fatty acids, vitamins, and minerals needed for proper functioning of human organism. Egg is considered a natural functional foodstuff because of its nutritional value. When compared to poultry meat, enrichment of eggs with functional ingredients is easier

If consuming meat and eggs enriched with functional ingredients, consumers can affect the increase of the content of such functional ingredients in blood and tissue in a natural way, thus avoiding taking in some dietary supplements. The importance of functional ingredients

and well‐being or general condition of the body.

because of the high content of fat in egg yolk [4].

for human health is elaborated further in the text.

## **2.1. Metabolism of n‐3 and n‐6 polyunsaturated fatty acids**

Fatty acids are constituent parts of fat and oil molecules. Polyunsaturated fatty acids (PUFAs) are divided into two groups of n‐3 and n‐6, depending on where the first double bond is found in the carbon chain, that is, where hydrogen atoms are missing. Linoleic fatty acid (LA) and arachidonic fatty acid (AA) are typical representatives of the n‐6 group, and α‐linolenic fatty acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) represent the n‐3 group. The metabolism and the role of PUFA n‐6 and n‐3 may differ in living organism. Fatty acids of the n‐6 and n‐3 groups (LA and ALA) cannot be synthesized in an organism and are therefore called essential fatty acids (EFAs).

Importance of designed products (enriched with n‐3 PUFA) is found in the fact that LA and ALA (precursors with 18 carbon atoms) may extend in human organism and desaturate into arachidonic acid and DHA. Processes are catalyzed by elongase, Δ6 ‐ and Δ<sup>5</sup> ‐desaturase [5]. The limiting factor of metabolizing n‐6 PUFA and n‐3 PUFA is the enzyme Δ6 ‐desaturase. Unfortunately, final conversion into docosapentaenoic acid (DPA) and docosahexaenoic acid is still not clear; however, the important role is attributed toΔ<sup>4</sup> ‐desaturase [6]. Infante and Huszagh stated that the biosynthesis of DHA took place in the mitochondria membranes, and biosynthesis of AA, EPA, and DPA occurred in the endoplasmic reticulum [7]. Supported by enzymes of cyclooxygenase (COX) and lipoxygenase (LX) within certain processes, EFA is converted into hormone‐like substances called eicosanoids. Numerous studies confirmed that linoleic, linolenic, and oleic acids during biosynthesis compete for the same Δ6 ‐desaturase. It was also found that linolenic acid acted as the inhibitor of n‐6 PUFA metabolism. At the same time, 10 times more linoleic acids are required to inhibit metabolism of n‐3 PUFA at the same level [6]. LA, ALA, and AA are essential fatty acids for poultry. The greatest importance in the composition of poultry feed should be given to those mentioned fatty acids because they are precursors to eicosapentaenoic acid and docosahexaenoic acid, both of which are also considered as essential for humans. Human organism requires daily intake of 290–390 mg of ALA and 100–200 mg of EPA and DHA. **Figure 1** depicts the metabolic pathways of fatty n‐3 PUFA and n‐6 PUFA.

## **2.2. Poultry meat and eggs enriched with n‐3 PUFA**

The intake of plant sources, especially linseed oil, significantly increases the content of omega‐3 fatty acids in the form of ALA; however, they fail to increase the content of long‐ chain omega‐3 fatty acids in meat and eggs. The best sources of long‐chain omega‐3 PUFA, EPA, and DHA are oils of sea organisms and of fish. The use of these oils is limited because of poorer organoleptic properties of final products [8]. In order to avoid unpleasant odor or taste in meat and eggs, portions of fish oil, as well as of linseed oil in feeding mixtures, must be taken into account.

In their research into effects of linseed contained in laying hens' feeding mixtures in different portions (0, 5, 10, and 15%) on the content of ALA in egg yolks, the increase of the content of

**Figure 1.** Fatty acids (FAs) source and metabolism.

ALA from 1.80% in the group without linseed to 7.07, 8.35, and 12.20% in the group with the highest content of linseed in feeding mixture [9] was determined. Valavan et al. recorded the increase of ALA content in egg yolks from 0.62% in the control group to 0.83, 0.93, and 1.00% in the experimental groups fed diets supplemented with linseed oil in the amounts of 1, 2, and 3% [10]. They also determined the increase in the content of EPA and DHA. Supplementation of linseed oil in the portion of 5 and 10% to laying hens' feed affected the increase of ALA portion from 0.37% to high 10.3% and 14.9% [11]. These results referring to the increase of ALA content in egg yolk correspond to the fact that linseed and linseed oil are rich in ALA.

Meluzzi et al. stated that the supplementation of 3% fish oil to laying hens' diet influenced the increase of EPA and DHA content in the egg of 19.53 and 143.70 mg/egg [12]. Gonzalez‐ Esquerra and Leeson pointed out that the supplementation of 6% fish oil to laying hens' diet affected the increase of EPA and DHA contents, as well as the content of total n‐3 PUFA, which amounted to 246 mg on average [13]. In their paper about the production of Bio‐omega‐3 eggs, Imran et al. fed laying hens with mixtures supplemented with extruded linseed and determined that an increased content of extruded linseed in feeding mixtures affected the increase of DHA portion in eggs and reduced AA portion, as well as the ratio of total n‐n‐3 PUFA [14]. Apart from oils that can be purchased on the market, there are different commercial preparations, which can be used in poultry feeding in order to achieve the increased content of fatty acids in their products. Kralik et al. investigated the influence of Pronova Biocare EPAX 3000 (PBE), which is rich in fish oil, on the profile of fatty acids in chicken eggs [15]. The authors determined that the replacement of 3.33% of corn in chicken diet with the PBE oil resulted in the reduction of arachidonic fatty acid portion in egg yolks (*C* = 1.66%, *E* = 0.58%), and in the increase of EPA (*C* = 0.01%, *E* = 0.24%) and of DHA (*C* = 0.72%, and *E* = 1.76%). Moreover, these authors reported that the n‐6/n‐3 PUFA ratio was lowered from 14.88 in the control to 7.25 in the experimental group.

The possibility of altering the fatty acid composition of chicken meat is an objective in many studies. Kralik et al. emphasized more favorable ratio of total n‐6/n‐3 PUFA in thigh muscle lipids of chickens fed diets supplemented with linseed oil, in comparison to the control group that consumed diets with sunflower oil (2.75 and 12.23, respectively) [16]. Since poultry diet is based on corn rich in saturated fatty acids (SFAs), which are then through feed deposited into muscle tissue, feeding mixtures for chickens should be supplemented with linseed or rapeseed or their oils or with fish oil if wanting to enrich their meat with desirable n‐3 fatty acids [16–18]. These authors agreed that dietary supplementation of plant oils (linseed and rapeseed oils) instead of sunflower oil affected the increase of n‐3 PUFA, and the reduction of n‐6 PUFA in poultry meat.

Fish oil or seafood oil are an excellent source of n‐3 PUFA fatty acid, such as EPA and DHA. Mirghelenj et al. reported that the increase in the portion of fish oil contained in broiler feed influenced the increase of EPA and DHA fatty acids content in thigh and breast muscles [19]. The content of EPA in breast muscle increased from 0.014 mg/g in the group K to 0.090 mg/g in the group P4, and the content of DHA was raised from 0.046 mg/g in the group K to 0.338 mg/g in the group P4. The authors also reported the increase of EPA in thigh meat, from 0.028 mg/g in the control to 0.232 mg/g in the group P4, while the content of DHA was the lowest in the control (0.0085 mg/g), and the highest in the group P5 (0.578 mg/g). Unlike plant oils used in feeding mixtures, fish oil can negatively affect the organoleptic properties of meat [19, 20].

## **2.3. Specifics of fatty acid profile in conventional and n‐3 PUFA‐enriched products**

ALA from 1.80% in the group without linseed to 7.07, 8.35, and 12.20% in the group with the highest content of linseed in feeding mixture [9] was determined. Valavan et al. recorded the increase of ALA content in egg yolks from 0.62% in the control group to 0.83, 0.93, and 1.00% in the experimental groups fed diets supplemented with linseed oil in the amounts of 1, 2, and 3% [10]. They also determined the increase in the content of EPA and DHA. Supplementation of linseed oil in the portion of 5 and 10% to laying hens' feed affected the increase of ALA portion from 0.37% to high 10.3% and 14.9% [11]. These results referring to the increase of ALA content in egg yolk correspond to the fact that linseed and linseed oil are rich in ALA.

210 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**Figure 1.** Fatty acids (FAs) source and metabolism.

Composition of fatty acids in eggs is influenced by many factors, such as genetic background and age of laying hens, housing system, and composition of feed [11, 21–23]. Simopoulos reported that eggs produced outdoors in the Peloponnese contained as much as 20 times more n‐3 fatty acids than conventional eggs [24]. Huyghebaert et al. confirmed that feed composition significantly affected the profile of fatty acids in egg yolk [25]. The content of DHA in egg yolk is in positive correlation with the content of ALA, EPA, and DHA contained in feed, but in negative correlation with LA. Bavelaar and Beynen pointed out that EPA contained in egg yolk could be modified through laying hens' feed containing EPA, while DHA in yolk might be increased if the feed was rich in ALA or DHA [26]. **Table 1** overviews the results of the authors' own research referring to enrichment of egg yolk with n‐3 PUFA.


2 ΣMUFA: C14:1 + C16:1 + C18:1n9t + C18:1n9c + C20:1n9 + C22:1n9.

3 Σn‐6 PUFA: C18:2n6 + C18:3n6 + C20:2n6 + C20:3n6 + C20:4n6 + C22:2n6.

**Table 1.** Profile of fatty acids in yolk lipids of conventional and n‐3 PUFA eggs (% of total fatty acids).

Eggs enriched with n‐3 PUFA contain 5.3 times more ALA, 20 times more EPA, and 3.5 times more DHA compared to conventional eggs. The sum of n‐3 PUFA in enriched eggs is 4.4 times higher than in conventional eggs. Samman et al. analyzed the profile of fatty acids in conventional table eggs bought in a store and omega‐3 eggs [27]. They determined that the ratio of n‐6/n‐3 PUFA in conventional eggs was 11.03, and in eggs enriched with n‐3 PUFA only 2.17. Our researches proved that lipids of omega‐3 eggs contain less percentage of SFA and n‐6 PUFA, and a higher percentage of ALA, EPA, and DHA than conventional eggs. The n‐6/n‐3 PUFA ratio in conventional eggs was 12.94 and 8.02, respectively, and in omega‐3 eggs it was only 2.67. Many health organizations recommend that the n‐6/n‐3 PUFA ratio shall range between 3:1 and 10:1. In the USA, it is determined as 15:1, and in Japan that ratio is only 1:1 to 3:1. In Croatia, such ratio is quite wide, from 11:1 to 35:1.


reported that eggs produced outdoors in the Peloponnese contained as much as 20 times more n‐3 fatty acids than conventional eggs [24]. Huyghebaert et al. confirmed that feed composition significantly affected the profile of fatty acids in egg yolk [25]. The content of DHA in egg yolk is in positive correlation with the content of ALA, EPA, and DHA contained in feed, but in negative correlation with LA. Bavelaar and Beynen pointed out that EPA contained in egg yolk could be modified through laying hens' feed containing EPA, while DHA in yolk might be increased if the feed was rich in ALA or DHA [26]. **Table 1** overviews the results of the authors' own research referring to enrichment of egg yolk with

**SFO\* SO\*\* MO\*\*\***

ΣSFA<sup>1</sup> 35.34 ± 1.77 34.72 ± 1.13 31.33 ± 0.64

ΣMUFA<sup>2</sup> 41.21 ± 2.03 41.82 ± 2.04 41.86 ± 1.08 Σn‐6 PUFA<sup>3</sup> 21.74 ± 1.44 20.85 ± 1.91 19.59 ± 0.83

ALA (C18:3n‐3) 0.89 ± 0.18 1.17 ± 0.15 4.73 ± 0.21 ETA (C20:3n3) 0.01 ± 0.005 0.02 ± 0.01 0.03 ± 0.21 EPA (C20:5n‐3) 0.01 ± 0.004 0.06 ± 0.02 0.20 ± 0.03

DHA (C22:6n‐3) 0.68 ± 0.22 1.35 ± 0.22 2.37 ± 0.18 Σn‐3 PUFA 1.68 ± 0.39 2.60 ± 0.18 7.32 ± 0.23 Σn‐6 PUFA/Σn‐3 PUFA 12.94 8.02 2.67

**Table 1.** Profile of fatty acids in yolk lipids of conventional and n‐3 PUFA eggs (% of total fatty acids).

Eggs enriched with n‐3 PUFA contain 5.3 times more ALA, 20 times more EPA, and 3.5 times more DHA compared to conventional eggs. The sum of n‐3 PUFA in enriched eggs is 4.4 times higher than in conventional eggs. Samman et al. analyzed the profile of fatty acids in conventional table eggs bought in a store and omega‐3 eggs [27]. They determined that the ratio of n‐6/n‐3 PUFA in conventional eggs was 11.03, and in eggs enriched with n‐3 PUFA only 2.17. Our researches proved that lipids of omega‐3 eggs contain less percentage of SFA and n‐6 PUFA, and a higher percentage of ALA, EPA, and DHA than conventional eggs. The n‐6/n‐3 PUFA ratio in conventional eggs was 12.94 and 8.02, respectively, and in omega‐3 eggs it was only 2.67. Many health organizations recommend that the n‐6/n‐3 PUFA ratio shall range between 3:1 and 10:1. In the USA, it is determined as 15:1, and in Japan that ratio is only 1:1 to

3:1. In Croatia, such ratio is quite wide, from 11:1 to 35:1.

\*\*\*MO mixed oil (sunflower oil, soybean oil, linseed oil, fish oil).

ΣSFA: C14:0 + C15:0 + C16:0 + C17:0 + C18:0 + C20:0 + C21:0 + C23:0.

Σn‐6 PUFA: C18:2n6 + C18:3n6 + C20:2n6 + C20:3n6 + C20:4n6 + C22:2n6.

ΣMUFA: C14:1 + C16:1 + C18:1n9t + C18:1n9c + C20:1n9 + C22:1n9.

**Fatty acid Conventional eggs n‐3 PUFA eggs**

212 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

n‐3 PUFA.

\*

1

2

3

SFO sunflower oil. \*\*SO soybean oil, fish oil.

**Table 2.** Supplementation of oils to laying hens' diet and its effect on enrichment of eggs and breast meat with n‐3 PUFA.

The data presented in **Table 2** include the efficiency of enriching yolk lipids and broiler breast meat with n‐3 PUFA, as reported by various authors. Most authors used feeding treatment with sunflower or soybean oil in the control groups, and for the purpose of enriching eggs with n‐3 PUFA, those authors used rapeseed, linseed, and fish oils, as well as their combinations.

Results of their research showed that the most efficient enrichment of eggs with n‐3 PUFA was achieved by supplementing fish oil to laying hens' diet, as well as by combining fish, rapeseed, and soybean oils. Combination of sunflower oil and fish oil was less effective in the deposition of n‐3 PUFA in yolk lipids, if compared to treatments with a combination of fish oil and other plant oils. Enrichment of broiler breast meat with the n‐3 PUFA was also more successful when supplementing fish oil to diets. The best deposition of EPA and DHA in breast meat (1.32 and 8.95%, respectively) was achieved in feeding treatment with 2.5% fish oil and 2.5% rapeseed oil, thus achieving 1.67 times more EPA and 1.59 times more DHA than in feeding treatment with a combination of fish oil and sunflower oil [17].

When enriching products with EPA and DHA by using particular plant oils: sunflower, soybean, rapeseed, and linseed oils supplemented in the amount of 5% in feed, and the best efficiency was proven with linseed oil, which achieved 0.89% EPA and 1.85% DHA in muscle lipids, being 7.41 and 1.92 times more than in the control group with sunflower oil [33]. Selenium supplemented to broiler feed in the amount of 3 and 6% did not have influence on enriching breast meat with n‐3 PUFA.

Soybean and rapeseed oils are rich in monounsaturated fatty acids (MUFA >65%). Linseed oil is rich in n‐6 polyunsaturated fatty acids (n‐6 PUFA >37%) and n‐3 polyunsaturated fatty acids (n‐3 PUFA >28% α‐linolenic acid). Fish oil is rich mostly in saturated fatty acids (39.7%) and n‐3 PUFA (>31%). Our research confirmed that it was more efficient to use a combination of soybean, linseed, rapeseed, and fish oils as supplement to laying hens' diet than pure soybean oil. Results of our own research as well as of the abovementioned authors confirmed that the modification of poultry diets could influence the deposition of desirable n‐3 PUFA in lipids of egg yolks and broiler meat.

## **3. Health consequences of n3‐ and n6‐PUFA consumption**

#### **3.1. The fate of PUFA in human organism**

Linoleic acid and alpha linolenic acid belong to the n‐6 (omega‐6) and n‐3 (omega‐3) series of polyunsaturated fatty acids, respectively. They are defined "essential" fatty acids since they are not synthesized in the human body and are mostly obtained from the diet. The best food sources of ALA and LA are the most vegetable oils, cereals and walnuts, fish meat, and fish oil. The adequate intake (AI) determined by the Food and Drug Administration (FDA) is for α‐linolenic acid 1.6 g/day for men and 1.1 g/day for women, while the acceptable macronutrient distribution range (AMDR) is 0.6–1.2% of total energy [35]. The FDA has recommended that adults can safely consume a total of 3 g/day of combined DHA and EPA, with no more than 2 g/day coming from dietary supplements [36]. Linoleic acid (18:2, n‐6), the shortest‐ chained omega‐6 fatty acid, is one of many essential fatty acids. Mammalian cells lack the enzyme omega‐3 desaturase and therefore cannot convert omega‐6 fatty acids to omega‐3 fatty acids. This outlines the importance of the proportion of omega‐3 to omega‐6 fatty acids in a diet [35, 36]. Omega‐6 fatty acids are precursors to endocannabinoids, lipoxins, and specific eicosanoids [6, 7].

Arachidonic acid and EPA are precursors of different classes of pro‐inflammatory or anti‐ inflammatory eicosanoids, respectively (**Figure 2**). AA is found in small amounts in animal food sources (e.g., eggs and meats) and can also be formed by desaturation plus elongation reactions from its precursor, LA. The long‐chain n‐3 PUFA, DHA and EPA, can be formed in very limited amounts in the human body, or can be consumed preformed in the diet from sources rich in DHA/EPA such as fish meat or fish oils or enriched or fortified functional foods. Dietary n‐3 PUFA may counteract the inflammatory effects of AA's eicosanoids in three ways: by counteracting the effects of their AA‐derived counterparts via n‐3 PUFA derivatives; by displacement, since dietary n‐3 PUFA decreases tissue concentrations of AA, thus less of AA‐derived eicosanoids is synthesized; and by competitive inhibition with AA for the access to the cyclooxygenase and lipoxygenase enzymes [37]. AA replacement by EPA or DHA n‐3 PUFA results in reduced/inhibited production of pro‐inflammatory mediators such as prostaglandins, leukotrienes, and lipoxins. EPA and DHA compete with AA for the conversion by cytochrome P450 (CYP) enzymes, resulting in the formation of alternative, physiologically active, metabolites. Renal and hepatic microsomes, as well as various CYP isoforms, displayed equal or elevated activities when metabolizing EPA or DHA instead of AA. CYP2C/2J isoforms converting AA to epoxyeicosatrienoic acids (EETs) preferentially epoxidized the ‐3 double bond and thereby produced 17,18‐epoxyeicosatetraenoic (17,18‐EEQ) and 19,20‐epoxydocosapentaenoic acid (19,20‐EDP) from EPA and DHA. Those ‐3 epoxides are highly active as antiarrhythmic agents. Moreover, rats given dietary EPA/DHA supplementation exhibited substantial replacement of AA by EPA and DHA in membrane phospholipids in plasma, heart, kidney, liver, lung, and pancreas, with less pronounced changes in the brain [38]. The metabolic pathways competition of n3‐PUFA and n6‐PUFA is schematically represented in **Figure 2**.

**Figure 2.** n3‐PUFA and n6‐PUFA metabolic pathways competition.

Results of their research showed that the most efficient enrichment of eggs with n‐3 PUFA was achieved by supplementing fish oil to laying hens' diet, as well as by combining fish, rapeseed, and soybean oils. Combination of sunflower oil and fish oil was less effective in the deposition of n‐3 PUFA in yolk lipids, if compared to treatments with a combination of fish oil and other plant oils. Enrichment of broiler breast meat with the n‐3 PUFA was also more successful when supplementing fish oil to diets. The best deposition of EPA and DHA in breast meat (1.32 and 8.95%, respectively) was achieved in feeding treatment with 2.5% fish oil and 2.5% rapeseed oil, thus achieving 1.67 times more EPA and 1.59 times more DHA than

When enriching products with EPA and DHA by using particular plant oils: sunflower, soybean, rapeseed, and linseed oils supplemented in the amount of 5% in feed, and the best efficiency was proven with linseed oil, which achieved 0.89% EPA and 1.85% DHA in muscle lipids, being 7.41 and 1.92 times more than in the control group with sunflower oil [33]. Selenium supplemented to broiler feed in the amount of 3 and 6% did not have influence on

Soybean and rapeseed oils are rich in monounsaturated fatty acids (MUFA >65%). Linseed oil is rich in n‐6 polyunsaturated fatty acids (n‐6 PUFA >37%) and n‐3 polyunsaturated fatty acids (n‐3 PUFA >28% α‐linolenic acid). Fish oil is rich mostly in saturated fatty acids (39.7%) and n‐3 PUFA (>31%). Our research confirmed that it was more efficient to use a combination of soybean, linseed, rapeseed, and fish oils as supplement to laying hens' diet than pure soybean oil. Results of our own research as well as of the abovementioned authors confirmed that the modification of poultry diets could influence the deposition of desirable n‐3 PUFA in

Linoleic acid and alpha linolenic acid belong to the n‐6 (omega‐6) and n‐3 (omega‐3) series of polyunsaturated fatty acids, respectively. They are defined "essential" fatty acids since they are not synthesized in the human body and are mostly obtained from the diet. The best food sources of ALA and LA are the most vegetable oils, cereals and walnuts, fish meat, and fish oil. The adequate intake (AI) determined by the Food and Drug Administration (FDA) is for α‐linolenic acid 1.6 g/day for men and 1.1 g/day for women, while the acceptable macronutrient distribution range (AMDR) is 0.6–1.2% of total energy [35]. The FDA has recommended that adults can safely consume a total of 3 g/day of combined DHA and EPA, with no more than 2 g/day coming from dietary supplements [36]. Linoleic acid (18:2, n‐6), the shortest‐ chained omega‐6 fatty acid, is one of many essential fatty acids. Mammalian cells lack the enzyme omega‐3 desaturase and therefore cannot convert omega‐6 fatty acids to omega‐3 fatty acids. This outlines the importance of the proportion of omega‐3 to omega‐6 fatty acids in a diet [35, 36]. Omega‐6 fatty acids are precursors to endocannabinoids, lipoxins, and spe-

in feeding treatment with a combination of fish oil and sunflower oil [17].

214 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

**3. Health consequences of n3‐ and n6‐PUFA consumption**

enriching breast meat with n‐3 PUFA.

lipids of egg yolks and broiler meat.

cific eicosanoids [6, 7].

**3.1. The fate of PUFA in human organism**

n‐3 fatty acids, ALA, EPA, and DHA are especially important for good condition of heart and blood vessels, as well as for the prevention of diabetes and certain types of cancer [39]. Connor states that n‐3 PUFA prevents heart diseases by preventing the occurrence of arrhythmia [40]. They have anti‐inflammatory and hypolipidemic properties, they act antithrombotic, and slow down the development of atherosclerosis. Moreover, they also have a beneficial effect on digestion, improve the immune system, and reduce occurrence of allergic diseases [41]. DHA is an essential element in phospholipids of cell membranes, particularly in brain and eye retina. It is necessary for proper development and function of these organs, especially in fetuses and infants [42]. The anti‐inflammatory effects of n‐3 fatty acids through reduced production of pro‐inflammatory mediators include reduced/inhibited leukocyte chemotaxis, adhesion molecule expression, and leukocyte‐endothelial interactions. In addition, among the products of omega‐3 fatty acid metabolism are the resolvins, maresins, and protectins [43, 44] which have an indispensable role in the contraction of inflammation [45].

Both n‐3 and n‐6 PUFA are essential in the diet; however, their ratio affects the ratio of produced pro‐inflammatory and anti‐inflammatory metabolites. Healthy ratios of n‐6:n‐3, according to some authors, is the ratio of n6 to n3 of 1:1 to 1:4 (an individual needs more omega‐3 than omega‐6) [46]. Typical Western diets provide ratios of n6:n3 PUFA between 10:1 and 30:1 [47].

In human, after digestion in the small intestine and transport to the blood, the n‐6 and n‐3 PUFAs are assimilated within tissues themselves through the body. They can be used in energy metabolism by beta‐oxidation to form ATPEFAs and can also undergo esterification into cellular lipids including triglyceride, cholesterol ester, and phospholipid or can be stored in the form of triglycerides and released later by enzymatic/hydrolytic processes. EFAs can also be temporarily stored as cholesterol ester and also released to be utilized in energy metabolism. Both n‐6 and n‐3 PUFAs in the form of phospholipids are particularly important as they maintain both the structural integrity and the critical functioning of cellular membranes throughout the body. In addition, LA and ALA are activated to high‐energy forms known as fatty‐acyl CoA which provides the conversion of these dietary PUFAs into their longer‐chain and more polyunsaturated products as derived by a series of desaturation plus elongation reactions which are particularly active in the liver and to a lesser extent in other tissues [48].

## **3.2. Metabolic effects of n‐3 PUFA consumption on triacylglycerol and very low‐density lipoprotein**

It is known that n‐3 PUFA fatty acids reduce triacylglycerol (TG) synthesis in the liver and increase very low‐density lipoprotein (VLDL) clearance in the peripheral circulation. This occurs because n‐3 PUFA inhibit diacylglycerol acyl transferase (DGAT), and phosphatidic acid phosphohydrolase (PA), two crucial enzymes involved in hepatic TG biosynthesis which results in decreased hepatic VLDL secretion. Furthermore, the availability of FAs for TG synthesis is decreased because of increased peroxisomal beta‐oxidation of FA. Finally, due to the action of lipoprotein lipase, there is an increased plasma lipolytic activity in the peripheral circulation [49].

## **3.3. Anti‐inflammatory effects of n‐3 PUFA in chronic cardiometabolic diseases**

n‐3 fatty acids, ALA, EPA, and DHA are especially important for good condition of heart and blood vessels, as well as for the prevention of diabetes and certain types of cancer [39]. Connor states that n‐3 PUFA prevents heart diseases by preventing the occurrence of arrhythmia [40]. They have anti‐inflammatory and hypolipidemic properties, they act antithrombotic, and slow down the development of atherosclerosis. Moreover, they also have a beneficial effect on digestion, improve the immune system, and reduce occurrence of allergic diseases [41]. DHA is an essential element in phospholipids of cell membranes, particularly in brain and eye retina. It is necessary for proper development and function of these organs, especially in fetuses and infants [42]. The anti‐inflammatory effects of n‐3 fatty acids through reduced production of pro‐inflammatory mediators include reduced/inhibited leukocyte chemotaxis, adhesion molecule expression, and leukocyte‐endothelial interactions. In addition, among the products of omega‐3 fatty acid metabolism are the resolvins, maresins, and protectins [43, 44]

Both n‐3 and n‐6 PUFA are essential in the diet; however, their ratio affects the ratio of produced pro‐inflammatory and anti‐inflammatory metabolites. Healthy ratios of n‐6:n‐3, according to some authors, is the ratio of n6 to n3 of 1:1 to 1:4 (an individual needs more omega‐3 than omega‐6) [46]. Typical Western diets provide ratios of n6:n3 PUFA between 10:1 and 30:1 [47].

In human, after digestion in the small intestine and transport to the blood, the n‐6 and n‐3 PUFAs are assimilated within tissues themselves through the body. They can be used in energy metabolism by beta‐oxidation to form ATPEFAs and can also undergo esterification into cellular lipids including triglyceride, cholesterol ester, and phospholipid or can be stored in the form of triglycerides and released later by enzymatic/hydrolytic processes. EFAs can also be temporarily stored as cholesterol ester and also released to be utilized in energy metabolism. Both n‐6 and n‐3 PUFAs in the form of phospholipids are particularly important as they maintain both the structural integrity and the critical functioning of cellular membranes throughout the body. In addition, LA and ALA are activated to high‐energy forms known as fatty‐acyl CoA which provides the conversion of these dietary PUFAs into their longer‐chain and more polyunsaturated products as derived by a series of desaturation plus elongation reactions which are particularly active in the liver and to a lesser extent in

**3.2. Metabolic effects of n‐3 PUFA consumption on triacylglycerol and very low‐density** 

It is known that n‐3 PUFA fatty acids reduce triacylglycerol (TG) synthesis in the liver and increase very low‐density lipoprotein (VLDL) clearance in the peripheral circulation. This occurs because n‐3 PUFA inhibit diacylglycerol acyl transferase (DGAT), and phosphatidic acid phosphohydrolase (PA), two crucial enzymes involved in hepatic TG biosynthesis which results in decreased hepatic VLDL secretion. Furthermore, the availability of FAs for TG synthesis is decreased because of increased peroxisomal beta‐oxidation of FA. Finally, due to the action of lipoprotein lipase, there is an increased plasma lipolytic activity in the peripheral

which have an indispensable role in the contraction of inflammation [45].

216 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

other tissues [48].

**lipoprotein**

circulation [49].

n‐3 PUFAs have numerous positive effects on immune response [50]. They are components of the plasma membrane and as such are important for cell permeability, fluidity, and flexibility [51]. Increased intake of fish oil or n‐3 PUFA supplementations due to its anti‐inflammatory function has beneficial effects on cardiovascular disease (CVD), metabolic syndrome, diabetes mellitus, and other diseases [52, 53].

## **3.4. Anti‐inflammatory effect of n‐3 PUFA in adiposity and glucose metabolism**

Adipocytes play an important endocrine role regulating metabolism, and immune response by secreting adipokines [54]. Adipocyte in healthy subjects maintains the balance between pro‐ and anti‐inflammatory adipokines, but in obesity secretion of inflammatory adipokines is shifted to pro‐inflammatory cytokines [55], which may contribute to the pathogenesis of metabolic disorders. Accumulation of triacylglyceride in adipocytes results in adipocyte hypertrophy and dysregulation in secreting bioactive components. Obese patients are a high‐risk population for developing diabetes mellitus and cardiovascular complications because they become insulin insensitive, have higher blood pressure, and heart rate (HR). It has been shown that obesity‐related metabolic disorders originate from a low‐grade inflammation [56].

Macrophage, lymphocyte, adipose stem cells, and preadipocytes that residue in adipose tissue also contribute to increased secretion of pro‐inflammatory cytokines such as monocyte chemotactic protein (MCP)‐1, IL‐8, IL‐6, IL‐1, and tumor necrosis factor alpha (TNF‐α) [57].

Anti‐inflammatory effects of n‐3 PUFAs have a protective effect and decrease the pro‐inflammatory action of adiponectin [58, 59], as a result of the activation of AMP‐activated protein kinase [60], which further regulates carbohydrate metabolism [61], and reduce the risk of developing cardiovascular diseases [62]. Although mechanisms involved in anti‐inflammatory effect of n‐3 PUFA are poorly understood, G protein‐coupled receptor 120 (GPR120) is highly expressed on adipocytes and pro‐inflammatory macrophage serves as an n‐3 PUFA receptor. In mice fed with high‐fat diet, supplemented with n‐3 PUFA, inflammation was decreased (lower levels of TNF‐α and IL‐6) and systemic insulin sensitivity was enhanced, while in GPR120 knockout mice these effects were not observed [63]. They also showed that β‐arrestin2 and GPR120 signaling induce the inhibition of TAB‐mediated activation of transforming growth factor‐β activated kinase 1 (TAK1) and inhibit toll‐like receptor2/3/4 (TLR) and TNF‐α pro‐inflammatory‐signaling pathway.

Increased intake of n‐3 PUFA increases DHA and EPA in immune cells of experimental animals and human subjects [64, 65]. Since immune cells integrate more n‐3 PUFAs, there is a decrease in AA content, and therefore a drop of pro‐inflammatory eicosanoids secretion [50, 66]. Recent i*n vitro* and *in vivo* studies show that the anti‐inflammatory effects of n‐3 PUFA are mediated through the inhibition of NF‐κB‐signaling pathway and decreased macrophage TNF‐α transcription [67, 68]. Macrophages stimulated with LPS in n‐3 PUFA‐

enriched medium significantly decreased serine 32 phosphorylation [69]. Without proper phosphorylation, NF‐κB remains in the cytoplasm, inactively coupled with IκB, and in these conditions, the inflammatory response is downregulated or missing [70]. The fatty acid can also act as a ligand for peroxisome proliferator‐activated receptors—PPARα and PPARγ [71]. Specifically, 8(S)‐HETE and 15d‐J2‐PUFA metabolites are PPARs potent selective activators. PPAR receptors are a group of transcription factors regulating energy homeostasis [72] and inflammation and immunity directly inhibiting NF‐κB and its downstream effects [73]. All together, these studies prove n‐3 PUFA to be a potent anti‐inflammatory compounds.

Besides the anti‐inflammatory effect of the n‐3 PUFAs, study of Mori et al. showed that the incorporation of fish into a low‐fat, energy‐restricted diet has decreased triglyceride level, insulin‐glucose metabolism [74], and thereafter reducing the risk of developing metabolic disorders. In addition, n‐3 PUFA improves glucose tolerance and insulin sensitivity in mice models of type‐2 diabetes and metabolic syndrome [75]. Rats fed with high‐fat diet supplemented with n‐3 PUFAs showed increased insulin receptor (IR) density and increased IR and IRS1 phosphorylation, phosphatidylinositol (PI) 3′‐kinase activity, and GLUT‐4 content in muscles, but rats show no beneficial effect on hyperglycemia and hyperinsulinemia, indicating important role of liver in glucose metabolism [76]. Documented data seen in animal models were not always translated to human subjects [77]. Some studies show beneficial effects of n‐3 PUFA on glucose metabolism, and others not. Mostad et al. showed that n‐3 PUFA supplementation in type‐2 diabetic and obese patients did not improve insulin sensitivity, although those patients had improved lipid metabolism [78]. On the contrary, Albert et al. showed that higher n‐3 PUFA concentrations were associated with improved insulin sensitivity, lower free fatty acid, and C‐reactive protein (CRP) level in a group of middle‐ aged overweight men [79]. Another study in women patients with type‐2 diabetes showed that in 2 months of n‐3 PUFA supplementation, they had decreased adiposity, significantly lower plasma triacylglycerol, but without changes in insulin sensitivity [80]. These opposing results in human insulin sensitivity may be explained by different phenotypes, sex, age, adiposity, and environmental factors of patients, but also by different n‐3 PUFA dosage in studies [81].

n‐3 PUFA effects on human and animals are dose and tissue dependent [82]. n‐3 PUFA incorporates in the plasma membrane, and it binds to receptors as a ligand and modulates gene expression for immune and metabolic function, and by these decrease risk for cardiovascular disease. n‐3 PUFA‐enriched membranes have changed membrane fluidity and biophysics of lipid rafts affecting protein function and signaling events [53]. For example, n‐3 PUFAs modulate the function of Na+ and L‐type Ca2+ membrane ion channels, to prevent arrhythmias [83]. Altered channel function by n‐3 PUFA leads to reduced myocyte excitability and cytosolic calcium fluctuation of ischemic myocardium myocyte which becomes susceptible to partial depolarization (resting inactivation) and prevents arrhythmia, while membrane potential of myocytes in the nonischemic myocardium is not drastically affected [84]. Beneficial and protective effects of n‐3 PUFA are supported by many scientific studies, without notable side effect [53].

## **3.5. Effectiveness of n‐3 polyunsaturated fatty acids in the improvement of endothelial function and improvement of elasticity of the vascular wall**

enriched medium significantly decreased serine 32 phosphorylation [69]. Without proper phosphorylation, NF‐κB remains in the cytoplasm, inactively coupled with IκB, and in these conditions, the inflammatory response is downregulated or missing [70]. The fatty acid can also act as a ligand for peroxisome proliferator‐activated receptors—PPARα and PPARγ [71]. Specifically, 8(S)‐HETE and 15d‐J2‐PUFA metabolites are PPARs potent selective activators. PPAR receptors are a group of transcription factors regulating energy homeostasis [72] and inflammation and immunity directly inhibiting NF‐κB and its downstream effects [73]. All together, these studies prove n‐3 PUFA to be a potent anti‐inflammatory

218 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Besides the anti‐inflammatory effect of the n‐3 PUFAs, study of Mori et al. showed that the incorporation of fish into a low‐fat, energy‐restricted diet has decreased triglyceride level, insulin‐glucose metabolism [74], and thereafter reducing the risk of developing metabolic disorders. In addition, n‐3 PUFA improves glucose tolerance and insulin sensitivity in mice models of type‐2 diabetes and metabolic syndrome [75]. Rats fed with high‐fat diet supplemented with n‐3 PUFAs showed increased insulin receptor (IR) density and increased IR and IRS1 phosphorylation, phosphatidylinositol (PI) 3′‐kinase activity, and GLUT‐4 content in muscles, but rats show no beneficial effect on hyperglycemia and hyperinsulinemia, indicating important role of liver in glucose metabolism [76]. Documented data seen in animal models were not always translated to human subjects [77]. Some studies show beneficial effects of n‐3 PUFA on glucose metabolism, and others not. Mostad et al. showed that n‐3 PUFA supplementation in type‐2 diabetic and obese patients did not improve insulin sensitivity, although those patients had improved lipid metabolism [78]. On the contrary, Albert et al. showed that higher n‐3 PUFA concentrations were associated with improved insulin sensitivity, lower free fatty acid, and C‐reactive protein (CRP) level in a group of middle‐ aged overweight men [79]. Another study in women patients with type‐2 diabetes showed that in 2 months of n‐3 PUFA supplementation, they had decreased adiposity, significantly lower plasma triacylglycerol, but without changes in insulin sensitivity [80]. These opposing results in human insulin sensitivity may be explained by different phenotypes, sex, age, adiposity, and environmental factors of patients, but also by different n‐3 PUFA dosage in

n‐3 PUFA effects on human and animals are dose and tissue dependent [82]. n‐3 PUFA incorporates in the plasma membrane, and it binds to receptors as a ligand and modulates gene expression for immune and metabolic function, and by these decrease risk for cardiovascular disease. n‐3 PUFA‐enriched membranes have changed membrane fluidity and biophysics of lipid rafts affecting protein function and signaling events [53]. For example, n‐3 PUFAs modu-

Altered channel function by n‐3 PUFA leads to reduced myocyte excitability and cytosolic calcium fluctuation of ischemic myocardium myocyte which becomes susceptible to partial depolarization (resting inactivation) and prevents arrhythmia, while membrane potential of myocytes in the nonischemic myocardium is not drastically affected [84]. Beneficial and protective effects of n‐3 PUFA are supported by many scientific studies, without notable side

and L‐type Ca2+ membrane ion channels, to prevent arrhythmias [83].

compounds.

studies [81].

effect [53].

late the function of Na+

A large empirical data indicate that the consumption of n‐3 PUFA has beneficial effect on the risk and progression of cardiovascular diseases acting via multiple pathways and molecular mechanisms [53, 85, 86]. Since atherosclerosis is one of the main features of CVDs characterized by morphological and functional changes in blood vessel wall and its endothelium, attention has been given to numerous studies to investigate whether n‐3 PUFA may prevent or delay atherosclerosis progression acting on the initial steps in its pathogenesis—endothelium and vascular wall function.

Endothelium plays a critical role in maintaining vascular tone and the term "endothelial function" is commonly used to describe its ability to release vasoactive substances, thereby regulating the blood flow [87]. Classically, endothelial dysfunction (ED) refers to reduced production and/or bioavailability of the main vasodilator nitric oxide (NO) and/or an imbalance in the relative contribution of other endothelium‐derived relaxing (e.g. cyclooxygenase‐1 and ‐2 (COX‐1,2) or CYP450‐epoxygenase‐derived metabolites) and contracting (e.g. COX‐1,‐2 or CYP450‐hydroxylase‐derived metabolites) metabolites of AA, resulting in impaired vascular relaxation mechanisms [88]. It is considered that increased oxidative stress level is one of the main causes for ED and the development in various pathological states associated with vascular diseases such as hypertension, diabetes mellitus, hypercholesterolemia, smoking, and aging. A number of studies have shown that there is a cross‐talk between the enzymes producing the vasoactive metabolites (NOS, COX‐1,‐2, CYP450) and reactive oxygen species (ROS), in which ROS may affect the bioavailability of NO and/or affecting other enzymes to shift their production from vasodilators to vasoconstrictors [89]. ED becomes an accepted prognostic value for future cardiovascular events in both populations at low and high cardiovascular risk and its noninvasive assessment by flow‐mediated dilation (FMD) of brachial artery (gold standard) is being routinely used not only in research but in clinical practice, as well [90].

As elaborated previously, the mechanism by which n‐3 PUFA may influence endothelial function is its ability to incorporate into membrane phospholipids in which signaling molecules and receptors for endothelial cell function are located [91]. Some of the possible pathways activated in this way result in increased NO production and reduced synthesis of pro‐inflammatory mediators [92]. Enhanced eNOS activity/expression by n‐3 PUFA administration was demonstrated in several endothelial cell cultures or experimental animal studies [93–95]. In addition, n‐3 PUFAs increase NO production by directly stimulating eNOS gene and protein expression, which was reported by several studies in healthy and disease animals including atherosclerosis, diabetes mellitus, and menopause [96–102].

Taken together, these results strongly suggested that n‐3 PUFA increases the bioavailability of NO acting via different molecular mechanisms. Despite that high doses of n‐3 PUFA have been considered as to have a pro‐oxidant effect, several studies on cell cultures and isolated blood vessels have shown that n‐3 PUFA may reduce the oxidative stress level by attenuating ROS production via its direct effect on ROS formation, or reducing peroxynitrite production [97, 102, 103]. Both in vitro and in vivo experiments have demonstrated that n‐3 PUFAs reduce the concentration of soluble cell adhesion molecules (sCAMs) VCAM‐1 and E‐selectin, as well as IL‐6 and C reactive protein level resulting in the attenuation of cellular and systemic inflammation [104, 105]. It is important to emphasize that relatively high dose of n‐3 PUFAs is needed to achieve this anti‐inflammatory effect. Interestingly, high doses of n‐3 PUFA significantly reduce triglycerides level, which indirectly also contributes to improved endothelial function in these conditions [86]. Taken together, these data suggest that n‐3 PUFA has the potential to improve endothelial function by acting on the bioavailability of NO by various mechanisms, reducing oxidative stress and inflammation and thereby reducing pathological activation of the endothelium. The results of a number of functional vascular studies have been summarized in several recent meta‐analyses; however, the conclusions of these meta‐ analyses have been a bit inconsistent. There are a few studies, both in animals and in humans which aimed to distinct the effect of n‐3 PUFA on endothelium‐independent vasodilation, as well, and whose results suggest that the effect of n‐3 PUFA on endothelium‐independent vasodilation (contribution of vascular smooth muscle cells) is negligible, as demonstrated in the meta‐analysis by Wang et al. [106]. One of the main shortcomings of these functional studies was the lack of basal measurement of n‐3 PUFA in the studied population. Another lack of mentioned studies is significant heterogeneity in the number of participants, inclusion criteria such as age of participants or whether participants were healthy or disease, markers of endothelial function that were measured, dose and duration of n‐3 PUFA supplementation, forms of n‐3 PUFA that were administered (EPA, DHA, or ALA) alone or in combination and concomitant therapy that was used. Because of the abovementioned structure heterogeneity of functional studies, conclusions that indicate that n‐3 PUFA improves endothelial function are still adopted with great caution. Meta‐analysis of Wang et al. from 2012 identified totally 16 eligible studies which investigated the effect of n‐3 PUFA supplementation on endothelial function measured by FMD and involving 901 participants, which reported that n‐3 PUFA supplementation significantly increased FMD by 2.30% at a dose range from 0.45 to 4.5 g/ day during a median of 56 days. Furthermore, results of this meta‐analysis suggested that the effect on n‐3 PUFA on endothelial function can be modified by the health status of the participants or by the dose of n‐3 PUFA supplementation [106]. A review on human intervention studies by Egert and Stehle reported that n‐3 PUFA supplementation improved endothelial function in overweight DM type 2 patients with dyslipidemia; however, conflicting results were observed in CVD patients. The authors concluded that reasons for these discrepancies between studies lie in the heterogeneity in the participants' health status and age, as well as in dose, duration, and the type of n‐3 PUFA supplementation [107]. A third large meta‐analysis of randomized controlled trials on the fish oil supplementation on endothelial vascular function published in 2012 included 16 studies with 1385 participants involved and reported that fish oil supplementation significantly improved FMD. Furthermore, endothelial function was significantly improved particularly in normoglycemic subjects and participants with lower diastolic blood pressure [108]. But contradictory, sensitivity analysis including only double‐ blind, placebo‐controlled studies indicated that fish oil supplementation has no significant effect on endothelial function. All together, these studies provide many indices that n‐3 PUFA supplementation has beneficial effect and improves endothelial function, but large‐scale and high‐quality clinical trials are needed to evaluate this effect to get a definite conclusion.

Beside impairment of endothelial function, CVDs and atherosclerosis are closely linked to increased arterial wall stiffness which represents progressive deterioration in vessel elasticity [109]. Arterial wall stiffness is characterized by morphological changes in blood vessel wall structure and in mechanical properties of vascular wall, which result in changed functional possibilities of such blood vessels. The arterial stiffness, in addition to the changes in the anatomical structure of the blood vessel wall, is closely related to impaired endothelial function in promoting atherosclerosis development [86]. Therefore, the assessment of arterial stiffness became an accepted predictive factor for future cardiovascular events and mortality in patients with CVDs.

tion [97, 102, 103]. Both in vitro and in vivo experiments have demonstrated that n‐3 PUFAs reduce the concentration of soluble cell adhesion molecules (sCAMs) VCAM‐1 and E‐selectin, as well as IL‐6 and C reactive protein level resulting in the attenuation of cellular and systemic inflammation [104, 105]. It is important to emphasize that relatively high dose of n‐3 PUFAs is needed to achieve this anti‐inflammatory effect. Interestingly, high doses of n‐3 PUFA significantly reduce triglycerides level, which indirectly also contributes to improved endothelial function in these conditions [86]. Taken together, these data suggest that n‐3 PUFA has the potential to improve endothelial function by acting on the bioavailability of NO by various mechanisms, reducing oxidative stress and inflammation and thereby reducing pathological activation of the endothelium. The results of a number of functional vascular studies have been summarized in several recent meta‐analyses; however, the conclusions of these meta‐ analyses have been a bit inconsistent. There are a few studies, both in animals and in humans which aimed to distinct the effect of n‐3 PUFA on endothelium‐independent vasodilation, as well, and whose results suggest that the effect of n‐3 PUFA on endothelium‐independent vasodilation (contribution of vascular smooth muscle cells) is negligible, as demonstrated in the meta‐analysis by Wang et al. [106]. One of the main shortcomings of these functional studies was the lack of basal measurement of n‐3 PUFA in the studied population. Another lack of mentioned studies is significant heterogeneity in the number of participants, inclusion criteria such as age of participants or whether participants were healthy or disease, markers of endothelial function that were measured, dose and duration of n‐3 PUFA supplementation, forms of n‐3 PUFA that were administered (EPA, DHA, or ALA) alone or in combination and concomitant therapy that was used. Because of the abovementioned structure heterogeneity of functional studies, conclusions that indicate that n‐3 PUFA improves endothelial function are still adopted with great caution. Meta‐analysis of Wang et al. from 2012 identified totally 16 eligible studies which investigated the effect of n‐3 PUFA supplementation on endothelial function measured by FMD and involving 901 participants, which reported that n‐3 PUFA supplementation significantly increased FMD by 2.30% at a dose range from 0.45 to 4.5 g/ day during a median of 56 days. Furthermore, results of this meta‐analysis suggested that the effect on n‐3 PUFA on endothelial function can be modified by the health status of the participants or by the dose of n‐3 PUFA supplementation [106]. A review on human intervention studies by Egert and Stehle reported that n‐3 PUFA supplementation improved endothelial function in overweight DM type 2 patients with dyslipidemia; however, conflicting results were observed in CVD patients. The authors concluded that reasons for these discrepancies between studies lie in the heterogeneity in the participants' health status and age, as well as in dose, duration, and the type of n‐3 PUFA supplementation [107]. A third large meta‐analysis of randomized controlled trials on the fish oil supplementation on endothelial vascular function published in 2012 included 16 studies with 1385 participants involved and reported that fish oil supplementation significantly improved FMD. Furthermore, endothelial function was significantly improved particularly in normoglycemic subjects and participants with lower diastolic blood pressure [108]. But contradictory, sensitivity analysis including only double‐ blind, placebo‐controlled studies indicated that fish oil supplementation has no significant effect on endothelial function. All together, these studies provide many indices that n‐3 PUFA supplementation has beneficial effect and improves endothelial function, but large‐scale and high‐quality clinical trials are needed to evaluate this effect to get a definite conclusion.

220 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

One of the most straightforward and reliable methods for large artery stiffness assessment is noninvasive measurement of pulse wave velocity (PWV), and the most commonly used methods are carotid‐femoral PWV and brachial‐ankle PWV. PWV presents the speed at what the so‐called pulse wave (arterial pulsation produced by the ejection of blood from the heart) propagates from heart to the periphery. Higher PWV is associated with the greater blood vessel wall rigidity that is interpreted as increased arterial wall stiffness [110]. n‐3 PUFA administration may influence arterial stiffness acting via passive mechanisms involving mechanical and elastic arterial wall properties, just as via active mechanisms involving cellular and molecular functions of endothelium, VMS, and extracellular matrix of blood vessel wall [86, 109]. It is well known that chronically increased blood pressure levels also increase arterial stiffness by remodeling of the artery wall itself. A large body of evidence indicates that n‐3 PUFAs are able to decrease blood pressure level, and therefore act to reduce arterial stiffness as well [111–113]. A second possible link between n‐3 PUFA and arterial stiffness is blood triglyceride levels, which are decreased by n‐3 PUFA supplementation. Since abnormalities in lipid metabolism are considered to be one of the fundamental determinants for the atherosclerosis developments, its effect on stiffening of the arteries should be taken into account as well [114, 115]. It is considered that n‐3 PUFA may act on arterial stiffness by reducing heart rate, since numerous studies in both animal model and humans reported that an increased heart rate is associated with an increased risk for CV events, and is independently associated with the progression of arterial stiffness. It has been speculated that n‐3 PUFA may lower HR acting directly on cardiac electrophysiology, or through a modulation of vagal and sympathetic balance [53, 116, 117]. Therefore, beneficial effect of n‐3 PUFA on arterial stiffness is multifactorial affecting both passive and active mechanisms relating to the structure and function of the arterial wall, which are very often changed and/or damaged by some major cardiovascular risk factors (such as hypertension, obesity, smoking, menopause, hyperlipidemia, etc.).

Recently, numerous studies tried to investigate the effect of n‐3 PUFA supplementation on arterial stiffness in a variety of conditions associated with increased cardiovascular risk in both experimental animals and humans. Regarding results of studies in experimental animals, they have described beneficial effect of n‐3 PUFA in animals with insulin resistance, hypertension, and in ovariectomized animals which presented an experimental mode for menopause [118–122]. In the meta‐analysis from 2011 on the n‐3 PUFA interventions to arterial stiffness which included nine studies (one on the acute effects of n‐3 PUFA in healthy volunteers and others on the chronic supplementation in patients with various CVDs), all but one study reported improvement in PWV or capacitive arterial compliance compared to the controls. Furthermore, combined supplementation of EPA and DHA had greater effect on arterial stiffness improvement than that of EPA alone, while one study reported that DHA supplementation alone had no significant effect on arterial stiffness. Just as studies on the effect on n‐3 PUFA on endothelial function, the disadvantage of the above mentioned experiments is heterogeneity in the sample population and supplementation dose sizes. Yet, the authors of this meta‐analysis pointed out that if the different doses of n‐3 PUFA supplementation acted to improve arterial stiffness in diverse populations these findings could be potentially translated to general population [109].

In conclusion, there is a growing evidence that n‐3 PUFA supplementation by targeting to endothelial function and vascular wall stiffness may have beneficial effect in preventing the development and progression of atherosclerosis and incidents related to CVDs. So far, we can concisely presume that this benevolent effect of n‐3 PUFA on vascular health is a sum of their actions on vasodilator mediators' bioavailability, antioxidant and anti‐inflammatory capacity, modulation of lipid profile, and structural arterial remodeling. Still, stronger evidence from large clinical trials with more homogeneous experimental populations and supplementation dose is needed before n‐3 PUFAs can find their place in the clinical prevention and treatment of CVDs.

## **4. Conclusions**

The production of functional food enriched with n‐3 PUFA (i.e. eggs and poultry meat) is a well‐established process and the food is available at the market. Despite inconsistency in designs of the analyzed studies, up to date numerous accumulated data demonstrate beneficial effects of n‐3 PUFA for human health, particularly in relation to cardiovascular and metabolic conditions. To achieve long‐term protective effects, the functional food enriched with omega‐3 fatty acids should be consumed on daily basis. There are no specific constrains in taking functional food; even more, it can be recommended to athletes and cardiovascular patients. General population can also benefit from eating functional food enriched with n‐3 PUFA due to their anti‐inflammatory and vascular‐protective effects.

## **Author details**

Ines Drenjančević1, 2\*, Gordana Kralik1, 2, Zlata Kralik1, 2, Martina Mihalj1, 2, Ana Stupin1, 2, Sanja Novak1, 2 and Manuela Grčević1, 2


## **References**

but one study reported improvement in PWV or capacitive arterial compliance compared to the controls. Furthermore, combined supplementation of EPA and DHA had greater effect on arterial stiffness improvement than that of EPA alone, while one study reported that DHA supplementation alone had no significant effect on arterial stiffness. Just as studies on the effect on n‐3 PUFA on endothelial function, the disadvantage of the above mentioned experiments is heterogeneity in the sample population and supplementation dose sizes. Yet, the authors of this meta‐analysis pointed out that if the different doses of n‐3 PUFA supplementation acted to improve arterial stiffness in diverse populations these findings could be

222 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

In conclusion, there is a growing evidence that n‐3 PUFA supplementation by targeting to endothelial function and vascular wall stiffness may have beneficial effect in preventing the development and progression of atherosclerosis and incidents related to CVDs. So far, we can concisely presume that this benevolent effect of n‐3 PUFA on vascular health is a sum of their actions on vasodilator mediators' bioavailability, antioxidant and anti‐inflammatory capacity, modulation of lipid profile, and structural arterial remodeling. Still, stronger evidence from large clinical trials with more homogeneous experimental populations and supplementation dose is needed before n‐3 PUFAs can find their place in the clinical prevention and

The production of functional food enriched with n‐3 PUFA (i.e. eggs and poultry meat) is a well‐established process and the food is available at the market. Despite inconsistency in designs of the analyzed studies, up to date numerous accumulated data demonstrate beneficial effects of n‐3 PUFA for human health, particularly in relation to cardiovascular and metabolic conditions. To achieve long‐term protective effects, the functional food enriched with omega‐3 fatty acids should be consumed on daily basis. There are no specific constrains in taking functional food; even more, it can be recommended to athletes and cardiovascular patients. General population can also benefit from eating functional food enriched with n‐3

Ines Drenjančević1, 2\*, Gordana Kralik1, 2, Zlata Kralik1, 2, Martina Mihalj1, 2, Ana Stupin1, 2,

1 Faculty of Medicine Osijek, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia

2 Scientific Center of Excellence for Personalized Health Care University of Osijek, Osijek,

PUFA due to their anti‐inflammatory and vascular‐protective effects.

\*Address all correspondence to: ines.drenjancevic@mefos.hr

potentially translated to general population [109].

treatment of CVDs.

**4. Conclusions**

**Author details**

Croatia

Sanja Novak1, 2 and Manuela Grčević1, 2


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**Provisional chapter**

## **Evolution and Therapy of Brain by Foods Containing Unsaturated Fatty Acids Evolution and Therapy of Brain by Foods Containing**

Roberto Carlos Burini,

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Caroline das Neves Mendes Nunes and Roberto Carlos Burini, Caroline das Neves Mendes Nunes and Franz Homero Paganini

Franz Homero Paganini Burini Burini

**Unsaturated Fatty Acids**

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67321

#### **Abstract**

About 6 million years ago, our ancestors had experienced a tremendous brain growth, widely viewed as a "major adaptive shift" in human evolution. Half of human brain composition is fat and 20% of its dry weight is long‐chain polyunsaturated fatty acids (LCPUFA). Consequently, improvements in consumption of dietary fat were necessary condition for promoting encephalization. Dietary fat quantity and quality have been sub‐ jected to tremendous change over the past 10,000 years with the introduction of industri‐ ally produced *trans* fatty acids and reduced intakes of ω‐3 fatty acids. The *absolute human* brain size reached its peak of approximately 90,000 years ago and has decreased by 11% since 35,000 years ago, most of it (8%) coming in the last 10,000 years. The shortfall in consumption of animal foods since the late Paleolithic and mainly consequent shortfall in consumption of preformed LCPUFA would be the plausible hypothesis for the brain size decreasing. Genetically, we are still adapted to the East African ecosystem on which our genome evolved, with some adaptations since the Out‐of‐Africa Diaspora. Dietary fat quantity and quality change has caused a conflict with our slowly adapting genome and this mismatch is likely to be at the basis of "typically Western" diseases. Many rec‐ ommendations for the intakes of EPA + DHA have been issued, notably for prevention. However, the ultimate goal might be to return to the fat quality of our ancient diet on which our genes have evolved during the past million years of evolution.

**Keywords:** human encephalization, LCPUFA sources, dietary transition, LPUFA in health, W‐3/W‐6 LCFA and modern diseases, therapeutical W‐3 LCPUFA

Commons

© 2017 The Author(s). Licensee InTech. This chapter is 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.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative

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.

## **1. Introduction**

The evolution of Homo erectus in Africa is widely viewed as a "major adaptive shift" in human evolution. Humans share a common ancestor with the chimpanzee and bonobo that probably lived in East Africa and, since some 6 million years ago our ancestors had experi‐ enced a tremendous brain growth and assumed an upright position [1].

Half of brain composition is fat, but the central nervous system contains almost a quarter of the unesterified cholesterol present in the whole human body and, long‐chain polyunsatu‐ rated fatty acids (LCPUFAs) make up to 20% of brain dry weight, including 6% for arachi‐ donic acid (AA) and 8% for docosahexaenoic acid (DHA) [2].

## **2. Polyunsaturated fatty acids**

LCPUFAs are building blocks of the membrane phospholipids of all cells. Nevertheless, LCPUFAs are not only important structural elements of membranes, together with their highly potent metabolites (prostaglandins, thromboxanes, leukotrienes, resolvins, and (neuro)protectins), LCPUFAs are involved in the functioning of membrane‐bound receptors, transporters, ion channels, and enzymes, and also in signal transduction and gene expression.

LCPUFAs are ligands of nuclear transcription factors (PPARS, SREBPs, NF‐kB, and others) [3–6] that coordinate expression and repression of key enzymes and proteins participating in interme‐ diary metabolism in glycolysis and "*de novo*" lipogenesis, thermoregulation, energy partitioning, growth and differentiation, hemostasis, and (W‐3/W‐6 ratio) inflammatory responses [7–11].

## **3. Effect of diet on brain development**

The anatomic trends of human evolution (large body sizes, bigger brains, craniofacial, and intestinal changes) clearly suggest major energetic and dietary shifts [12–14].

Improvements in dietary quality and the increased consumption of dietary fat appear to have been a necessary condition for promoting encephalization in the human lineage [15].

Primitive humans with enlarging brains developed more sophisticated tool technology (including the fire cooking) and became more efficient hunter/gatherers and so gained greater access to more nutritious and easily digestible foods (e.g., fruits, nuts, and meat) [11].

Consequently, reductions of posterior tooth size (and grinding teeth) and, also the size of the face and so, no longer needed the large gastrointestinal tract. Key genetic mutations during later hominid evolution were critical to promoting the enhanced lipid metabolism necessary for subsisting on diets with greater levels of animal material [16].

In fact, associated with the evolution of our high‐quality diet, humans developed distinct molecular pathways for detecting and metabolizing high‐fat foods [17].

The ability to effectively detect, metabolize, and store fats likely provided tremendous selec‐ tive advantages to our hominid ancestors, allowing them to expand into diverse ecosystems around the world [18].

**1. Introduction**

The evolution of Homo erectus in Africa is widely viewed as a "major adaptive shift" in human evolution. Humans share a common ancestor with the chimpanzee and bonobo that probably lived in East Africa and, since some 6 million years ago our ancestors had experi‐

Half of brain composition is fat, but the central nervous system contains almost a quarter of the unesterified cholesterol present in the whole human body and, long‐chain polyunsatu‐ rated fatty acids (LCPUFAs) make up to 20% of brain dry weight, including 6% for arachi‐

LCPUFAs are building blocks of the membrane phospholipids of all cells. Nevertheless, LCPUFAs are not only important structural elements of membranes, together with their highly potent metabolites (prostaglandins, thromboxanes, leukotrienes, resolvins, and (neuro)protectins), LCPUFAs are involved in the functioning of membrane‐bound receptors, transporters, ion channels, and enzymes, and also in signal transduction and gene expression. LCPUFAs are ligands of nuclear transcription factors (PPARS, SREBPs, NF‐kB, and others) [3–6] that coordinate expression and repression of key enzymes and proteins participating in interme‐ diary metabolism in glycolysis and "*de novo*" lipogenesis, thermoregulation, energy partitioning, growth and differentiation, hemostasis, and (W‐3/W‐6 ratio) inflammatory responses [7–11].

The anatomic trends of human evolution (large body sizes, bigger brains, craniofacial, and

Improvements in dietary quality and the increased consumption of dietary fat appear to have

Primitive humans with enlarging brains developed more sophisticated tool technology (including the fire cooking) and became more efficient hunter/gatherers and so gained greater

Consequently, reductions of posterior tooth size (and grinding teeth) and, also the size of the face and so, no longer needed the large gastrointestinal tract. Key genetic mutations during later hominid evolution were critical to promoting the enhanced lipid metabolism necessary

In fact, associated with the evolution of our high‐quality diet, humans developed distinct

been a necessary condition for promoting encephalization in the human lineage [15].

access to more nutritious and easily digestible foods (e.g., fruits, nuts, and meat) [11].

intestinal changes) clearly suggest major energetic and dietary shifts [12–14].

for subsisting on diets with greater levels of animal material [16].

molecular pathways for detecting and metabolizing high‐fat foods [17].

enced a tremendous brain growth and assumed an upright position [1].

234 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

donic acid (AA) and 8% for docosahexaenoic acid (DHA) [2].

**2. Polyunsaturated fatty acids**

**3. Effect of diet on brain development**

Mammalian brain growth is dependent upon sufficient amounts of two LCPUFAs: DHA and AA [19].

It appears that mammals have a limited capacity to synthesize these fatty acids from dietary precursors. Because the composition of all mammalian brain tissue is similar with respect to these two fatty acids, species with higher levels of encephalization have greater require‐ ments for DHA and AA. Consequently, dietary sources of DHA and AA were likely limiting nutrients that constrained the evolution of larger brain size in many mammalian lineages [18].

On average, we consume higher levels of dietary fat than other primates, and much higher levels of key LCPUFAs are critical to brain development [20, 21].

Hominins had experienced a tremendous brain growth which coincided with a change from a vegetarian to a hunting‐gathering omnivore‐carnivore [22–25].

Greater consumption of animal foods would have increased total dietary fat consumption in early Homo, and markedly increased the levels of key fatty acids (AA and DHA) necessary for brain development. The available evidence seems to best support a mixed dietary strategy in early Homo that involved the consumption of larger amounts of animal foods than with the australopithecines. Brain tissue is a rich source of both AA and DHA, whereas liver and muscle tissues are good sources of AA and moderate sources of DHA [18].

Dietary fat quantity and quality have been subjected to tremendous change over the past 10,000 years. Important changes are the introduction of manufactured linoleic acid (LA), trans‐fatty acids, and reduced intakes of vegetal‐derived alpha‐linolenic acid (ALA) and fish‐ derived eicosapentaenoic acid (EPA) and DHA, overall leading to a reduced supplying of omega‐3 fatty acids [11].

Analysis of changes in brain size in humans over the last 1.8 million years found that encepha‐ lization quotient (EQ) began reaching its peak with the first anatomically modern humans of approximately 90,000 years ago and has since remained fairly constant. Most surprisingly, however, absolute brain size has decreased by 11% since 35,000 years ago, with most of this decrease (8%) coming in just the last 10,000 years. Therefore, a genetic mutation is no more likely as an explanation for the decrease in absolute brain size. The most notorious dietary change in the last 10,000 years has been the decreased consumption of animal food (roughly from 50 to 10%) by the adventure of agriculture, followed by an increased consumption of grains. Hence, the most feasible biological hypothesis for the absolute decrease in brain size is the reduction of animal food intake with a consequent reduction of preformed long‐chain fatty acids. The brain is dependent on the DHA, docosatetraenoic acid (DTA), and AA to sup‐ port its growth during the formative years. These are far more plentiful in animal foods than plant. It is possible that the levels of essential fatty acids (EFAs) provided in the prehistoric diets were sufficient to support the brain expansion and evolution from prehistoric times to the present, and the current low levels of EFA intake (provided by agricultural diets) may explain the recent smaller human brain size [26].

## **4. Transition of diet and the development of brain disease**

Dietary fat quantity and quality change have, together with other man‐made changes in our environment, caused a conflict with our slowly adapting genome [11].

In fact, the EFA and other changes in our diet together with an energy intake that does not match with our current sedentary lifestyle have caused a conflict with our genome that is likely to be at the basis of typically "Western" diseases and their basis on the conflict (mis‐ match) caused by our current sedentary‐energy rich industrialized diet, way of life with our ancestrally molded genome. The dietary composition of our ancestors has also become clear from our current (patho)physiology: epidemiological data demonstrated a negative associa‐ tion of fish consumption with coronary artery disease (CAD) and (postpartum) depression, while landmark trials with ALA and fish oil in CAD, and with EPA in depression and schizo‐ phrenia supported the causality of these relations.

The similarity among diseases currently associated with dietary risk factors adds the notion that there is a common insult originated from our changed environment. These effects hit different organs and systems varying in genetic susceptibility and life stages, but extremely dependent on the doses and exposure time. Low‐grade inflammation might be a strong candidate for this common denominator. Low‐grade inflammation can be found in metabolic syndrome and its sequelae, some psychiatric and neurodegenerative diseases. The LCPUFA, AA, EPA, and DHA are intimately related to the initiation and resolution of inflammatory responses [11].

The current balance between AA and EPA + DHA is disturbed by the dominance of AA, which originates from the diet or synthesis from LA. Higher ratio of AA/EPA+DHA might led to a pro‐inflammatory condition that may precipitate a hyper‐inflammatory response ("sys‐ temic inflammatory response syndrome‐SIRS") with collateral damage, scarring and fibrosis and the subsequent development of an immune paralysis ("compensatory anti‐inflammatory response syndrome‐CARS") evidenced by debilitated host defense and secondary infection susceptibility [27, 28].

The chronic inflammation resulting from the unbalanced AA/EPA + DHA ratio might be cen‐ tral in the pathogenesis of the diseases of the metabolic syndrome and neurodegenerative disease, explain the relation between inflammation, depression, and dementia [29].

## **5. Therapy of modern diseases with polyunsaturated fatty acids**

Dietary supplementation of LCFA, especially EPA and DHA, has been used during pregnancy or early postnatal life for improvement of fetal and newborn brain development, at primary and secondary CAD preventions and psychiatric diseases. Consensus has been reached that those in CAD and depression are positive but not in all others. LCPω3 supplements might especially be effective in prevention, as suggested by the outcomes of epidemiological studies on CAD and prospective studies on Alzheimer's disease, and also from the favorable effects of LCPω3 in early disease stages [30].

It takes 20 years before the human brain obtains its complex adult configuration but the most dramatic neurodevelopmental changes occur prenatally and early post‐natal, including a major transformation in cortical organization 3–4 months after term and, considerable evi‐ dence indicates that prenatal and neonatal LCPUFA status is associated with neurodevel‐ opmental outcome. Therefore, maternal and neonatal concentrations of DHA and AA are associated with improved outcomes in early infancy, and concentrations of DHA are associ‐ ated with favorable neurodevelopmental outcome beyond early infancy [31].

**4. Transition of diet and the development of brain disease**

236 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

environment, caused a conflict with our slowly adapting genome [11].

phrenia supported the causality of these relations.

susceptibility [27, 28].

of LCPω3 in early disease stages [30].

Dietary fat quantity and quality change have, together with other man‐made changes in our

In fact, the EFA and other changes in our diet together with an energy intake that does not match with our current sedentary lifestyle have caused a conflict with our genome that is likely to be at the basis of typically "Western" diseases and their basis on the conflict (mis‐ match) caused by our current sedentary‐energy rich industrialized diet, way of life with our ancestrally molded genome. The dietary composition of our ancestors has also become clear from our current (patho)physiology: epidemiological data demonstrated a negative associa‐ tion of fish consumption with coronary artery disease (CAD) and (postpartum) depression, while landmark trials with ALA and fish oil in CAD, and with EPA in depression and schizo‐

The similarity among diseases currently associated with dietary risk factors adds the notion that there is a common insult originated from our changed environment. These effects hit different organs and systems varying in genetic susceptibility and life stages, but extremely dependent on the doses and exposure time. Low‐grade inflammation might be a strong candidate for this common denominator. Low‐grade inflammation can be found in metabolic syndrome and its sequelae, some psychiatric and neurodegenerative diseases. The LCPUFA, AA, EPA, and DHA

The current balance between AA and EPA + DHA is disturbed by the dominance of AA, which originates from the diet or synthesis from LA. Higher ratio of AA/EPA+DHA might led to a pro‐inflammatory condition that may precipitate a hyper‐inflammatory response ("sys‐ temic inflammatory response syndrome‐SIRS") with collateral damage, scarring and fibrosis and the subsequent development of an immune paralysis ("compensatory anti‐inflammatory response syndrome‐CARS") evidenced by debilitated host defense and secondary infection

The chronic inflammation resulting from the unbalanced AA/EPA + DHA ratio might be cen‐ tral in the pathogenesis of the diseases of the metabolic syndrome and neurodegenerative

Dietary supplementation of LCFA, especially EPA and DHA, has been used during pregnancy or early postnatal life for improvement of fetal and newborn brain development, at primary and secondary CAD preventions and psychiatric diseases. Consensus has been reached that those in CAD and depression are positive but not in all others. LCPω3 supplements might especially be effective in prevention, as suggested by the outcomes of epidemiological studies on CAD and prospective studies on Alzheimer's disease, and also from the favorable effects

disease, explain the relation between inflammation, depression, and dementia [29].

**5. Therapy of modern diseases with polyunsaturated fatty acids**

are intimately related to the initiation and resolution of inflammatory responses [11].

Given the fact that LCPUFA accretion is especially abundant during the third trimester of gestation, it suggests that preterm infants would particularly profit from LCPUFA supple‐ mentation. However, studies of LCPUFA supplementation in preterm infants have not shown evidence of a positive effect on neurodevelopmental outcome. On the other hand, studies in full‐term infants indicated that DHA supplementation promotes neurodevelopmental out‐ come in early infancy but no longer positive effects later on, being virtually absent at school age or later. Generally, the literature suggests that LCPUFA supplementation in term infants does not affect outcomes beyond the age of 4 months [31].

It is known that up to 45% of the fatty acids of synaptic membranes are EFAs [32].

There is a well‐established positive correlation between depression and coronary artery dis‐ ease. In fact, epidemiological studies in various countries suggest that decreased ω‐3 fatty acid consumption correlates with increasing rates of depression and, adequate long‐chain polyunsaturated fatty acids, particularly DHA, may reduce the development of depression just as ω‐3 polyunsaturated fatty acids may reduce coronary artery disease [33].

Eight database trials that randomly assigned participants to receive ω‐3 PUFAs/fish, with mea‐ sured depressed mood, using human participants, came to the conclusion that trial evidence of the effects of ω‐3 PUFAs on depressed mood has increased. However, the considerable het‐ erogeneity of the studies made them difficult to summarize the results. Overall, the available evidence supports the benefit of ω‐3 PUFAs in individuals with diagnosed depressive illness but no evidence of any benefit in individuals without a diagnosis of depressive illness [34].

The association between fish and meat consumption and risk of dementia in populations in developing countries was investigated in low‐ and middle‐ income countries of China, India, Cuba, the Dominican Republic, Venezuela, Mexico, and Peru. The found associations of fish and meat consumption with dementia risk to populations were consistent with mechanistic data on the neuroprotective actions of ω‐3 PUFAs commonly found in fish. However, the inverse association between fish and prevalent dementia is unlikely to result from poorer dietary habits among demented individuals (reverse causality) because meat consumption was higher in those with a diagnosis of dementia. But anyway, the found beneficial effects of fish consumption on dementia provide preliminary evidence of the etiological significance of diet in dementia [35].

Given the fact that PUFAs are naturally occurring endogenous substances, present in almost all tissues and are essential components of all mammalian cells and can be taken safely for long periods of time (from few months to few years) we can conclude that PUFAs, especially ω‐3 fatty acids, are useful in the prevention and treatment of Alzheimer' disease, schizophre‐ nia, and depression [36].

The pioneering studies in Greenland Eskimos almost 30 years ago suggested that ingestion of *n*‐3 fatty acids conveys protection from cardiovascular diseases [37].

Since then, many interventions have been conducted with LCPUFA, especially EPA and DHA, aiming at primary and secondary CAD preventions. From that, in most of the prospec‐ tive cohort studies, *n*‐3 fatty acids were found to be beneficial [38–43] but there were also exceptions with no effect [44, 45].

By comparing people who never or ate fish less than once per month, a meta‐analysis of 11 prospective studies (11.8 years follow‐up of more than 220 thousand subjects) showed the odds ratio for CHD mortality as 0.85 for fish consumption once per week, 0.77 for 2– 4 times/ week, and 0.62 for 5 times/week. The authors calculated that each 20 g/day increase in fish intake was associated with a 7% lower risk of coronary heart disease mortality [46].

Many international organizations have made recommendations to increase the intake of EPA plus DHA, and these are summarized by the International Society for the Study of Fatty Acids and Lipids [47]. In general, these recommendations are for 200 mg/day of EPA plus DHA for all adults. The United States has also issued a Dietary Reference Intake for *n*‐3 fatty acids [48].

The 2002 American Heart Association recommendations for dietary intake of *n*‐3 fatty acids recommended: (1) in the absent of documented CHD it is advised to eat fish twice per week plus oils and foods rich in ALA (flaxseed, canola, soy, walnuts), this accomplishes 500 mg/day of *n*‐3 fatty acids. (2) Individuals already with CHD are advised to eat 1 g/day of EPA plus DHA, preferably from oily fish, but could take EPA plus DHA supplements. (3) Individuals with hypertriglyceridemia could take 2–4 g/day of EPA plus DHA, under prescription care [49].

Several mechanisms have been proposed to explain how EPA plus DHA might beneficially influence cardiovascular disease. These include preventing arrhythmias, lowering plasma triacylglycerols, decreasing blood pressure, decreasing platelet aggregation, improving vas‐ cular reactivity, and decreasing inflammation. Overall, the therapeutic effect appears to be due to suppression of fatal arrhythmias rather than stabilization of atherosclerotic plaques [50–60].

Elevated plasma triacylglycerol concentrations have been associated with increased risk of coronary heart disease (CHD). Prospective evidence shows that nonfasting plasma triacylg‐ lycerol concentration is a strong and independent predictor of future myocardial infarction once elevated postprandial triacylglycerolemia leads to a series of metabolic reactions that reduce high‐density lipoprotein (HDL)‐cholesterol concentrations and promote the formation of small, dense low‐density lipoprotein (LDL) particles. Metabolism of plasma triacylglycer‐ ols also influences postprandial factor VII activation [61].

EPA and DHA are *n*‐3 PUFAs in fish oil which are effective hypotriacylglycerolemic agents, even when consumed at low doses (1 g *n*‐3 PUFA/d). Therefore, consumption of *n*‐3 PUFAs provides a realistic option for the optimization of plasma triacylglycerol metabolism [61].

Omega‐3 fatty acid supplementation provided additional benefits to Lifestyle Modification Program (LSMP) in the resolution of metabolic syndrome of free living adults. The fish oil group received 3 g of fish oil per day (360 mg of DHA and 540 mg of EPA) (G2, *n* = 23)) during 20 weeks. Compared to the control group (only LSMP) the intervened group showed a signifi‐ cant decrease in waist circumference (1.3%) followed by metabolic syndrome reduction (29%) mainly due to normalization of blood pressure (33.3%) and triacylglycerol (27.3%). Some the‐ ories have been proposed to explain how omega‐3 reduces triacylglycerol. The strongest evi‐ dence is the reduction in hepatic lipogenesis, reducing hepatic secretion of very low‐density lipoprotein (VLDL). Additionally, omega‐3 inhibits certain enzymes involved in the hepatic synthesis of triacylglycerol, reducing its plasma level [62].

The pioneering studies in Greenland Eskimos almost 30 years ago suggested that ingestion of

Since then, many interventions have been conducted with LCPUFA, especially EPA and DHA, aiming at primary and secondary CAD preventions. From that, in most of the prospec‐ tive cohort studies, *n*‐3 fatty acids were found to be beneficial [38–43] but there were also

By comparing people who never or ate fish less than once per month, a meta‐analysis of 11 prospective studies (11.8 years follow‐up of more than 220 thousand subjects) showed the odds ratio for CHD mortality as 0.85 for fish consumption once per week, 0.77 for 2– 4 times/ week, and 0.62 for 5 times/week. The authors calculated that each 20 g/day increase in fish

Many international organizations have made recommendations to increase the intake of EPA plus DHA, and these are summarized by the International Society for the Study of Fatty Acids and Lipids [47]. In general, these recommendations are for 200 mg/day of EPA plus DHA for all adults. The United States has also issued a Dietary Reference Intake for *n*‐3 fatty

The 2002 American Heart Association recommendations for dietary intake of *n*‐3 fatty acids recommended: (1) in the absent of documented CHD it is advised to eat fish twice per week plus oils and foods rich in ALA (flaxseed, canola, soy, walnuts), this accomplishes 500 mg/day of *n*‐3 fatty acids. (2) Individuals already with CHD are advised to eat 1 g/day of EPA plus DHA, preferably from oily fish, but could take EPA plus DHA supplements. (3) Individuals with hypertriglyceridemia could take 2–4 g/day of EPA plus DHA, under

Several mechanisms have been proposed to explain how EPA plus DHA might beneficially influence cardiovascular disease. These include preventing arrhythmias, lowering plasma triacylglycerols, decreasing blood pressure, decreasing platelet aggregation, improving vas‐ cular reactivity, and decreasing inflammation. Overall, the therapeutic effect appears to be due to suppression of fatal arrhythmias rather than stabilization of atherosclerotic plaques

Elevated plasma triacylglycerol concentrations have been associated with increased risk of coronary heart disease (CHD). Prospective evidence shows that nonfasting plasma triacylg‐ lycerol concentration is a strong and independent predictor of future myocardial infarction once elevated postprandial triacylglycerolemia leads to a series of metabolic reactions that reduce high‐density lipoprotein (HDL)‐cholesterol concentrations and promote the formation of small, dense low‐density lipoprotein (LDL) particles. Metabolism of plasma triacylglycer‐

EPA and DHA are *n*‐3 PUFAs in fish oil which are effective hypotriacylglycerolemic agents, even when consumed at low doses (1 g *n*‐3 PUFA/d). Therefore, consumption of *n*‐3 PUFAs provides a realistic option for the optimization of plasma triacylglycerol metabolism [61].

ols also influences postprandial factor VII activation [61].

intake was associated with a 7% lower risk of coronary heart disease mortality [46].

*n*‐3 fatty acids conveys protection from cardiovascular diseases [37].

238 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

exceptions with no effect [44, 45].

acids [48].

[50–60].

prescription care [49].

A significant decrease of plasma oxidative‐stress markers in patients with ulcerative colitis was shown when fish oil ω‐3 fatty acids were used in combination with sulfasalazine [63].

Regarding type 2 diabetes mellitus (T2DM), a prospective cohort analysis of men and women showed that long‐term dietary intake of long‐chain omega 3 fatty acids does not decrease the risk of T2DM. Instead, a modestly but significantly higher incidence of T2DM was associated with higher fish and long‐chain omega 3 fatty acid consumption [64].

Doses 3 g/day, EPA plus DHA can improve cardiovascular disease risk factors, including decreasing plasma triacylglycerols, blood pressure, platelet aggregation, and inflammation, while improving vascular reactivity [50].

By the fact that EFAs and their long‐chain metabolites and other products prevent plate‐ let aggregation, lower blood pressure, reduce LDL‐cholesterol, and ameliorate the adverse actions of homocysteine, the EFAs and their metabolites show all the actions expected of the "polypill" [65].

The concept of cardiovascular "polypill" was coined [66] by the fact that, when combined, the effects of statins, aspirin, and blood pressure lowering drugs reduced the all causes mortality in CHD patients.

In conclusion, it is evident that PUFAs, especially an optimal combination of EPA, DHA, and possibly, gamma‐linolenic acid (GLA), dihomo‐gamma‐linolenic acid (DGLA), and AA show all the qualities of the suggested "polypill", viz., aspirin‐like action, inhibition of HMG‐ CoA and angiotensin‐converting enzymes (ACEs), and possess diuretic, antihypertensive, and beta‐blocker‐like actions. Additionally, given the fact that PUFAs are naturally occur‐ ring endogenous substances, present in almost all tissues and are essential components of all mammalian cells and can be taken safely for long periods of time (from few months to few years), we can conclude that PUFAs, especially ω‐3 fatty acids, are useful in the preven‐ tion and treatment of Alzheimer' disease, schizophrenia, and depression [36], suggesting that PUFAs have a much wider benefit compared to the "polypill" [65].

Thus, LCPω3 supplements might especially be effective in prevention, as suggested by the outcomes of epidemiological studies on CAD and prospective studies on Alzheimer's disease, and also from the favorable effects of LCPω3 in early disease stages [30]. Consensus has been reached that those interventions in CAD and depression are positive but not in all others.

## **6. Future directions**

Genetically, we are for the greater part still adapted to the East African ecosystem on which our genome evolved, with some adaptations since the Out‐of‐Africa Diaspora. Dietary fat quantity and quality change have, together with other man‐made changes in our environ‐ ment, caused a conflict with our slowly adapting genome that is implicated in "typically Western" diseases. Fortunately, the majority of Western diseases occur typically after repro‐ ductive age. Rather than reducing our life expectancy, these diseases notably diminish our number of years in health.

Many recommendations for the intakes of saturated fat, *trans* fat, and EPA + DHA have been issued, notably for prevention. The ultimate goal might be, however, translate to the culture of the current society that our genes had evolved for million years in an entirely different dietary composition and lifestyle and therefore we must return to the fat quality of our ancient diet [11].

## **Author details**

Roberto Carlos Burini1 \*, Caroline das Neves Mendes Nunes<sup>2</sup> and Franz Homero Paganini Burini<sup>3</sup>

\*Address all correspondence to: burini@fmb.unesp.br

1 Public Health Department, UNESP Medical School, Botucatu, Brazil

2 Pathology Department, UNESP Medical School, Botucatu, Brazil

3 UNESP Medical School Clinical Hospital, Botucatu, Brazil

## **References**


[5] Jump D.B. Fatty acid regulation of gene transcription. Critical Reviews in Clinical Laboratory Sciences. 2004;41:41–78. DOI: 10.1080/10408360490278341

**6. Future directions**

number of years in health.

**Author details**

Burini<sup>3</sup>

**References**

nature01669

Roberto Carlos Burini1

\*Address all correspondence to: burini@fmb.unesp.br

10.1097/01.mol.0000113205.97033.22

1 Public Health Department, UNESP Medical School, Botucatu, Brazil

2 Pathology Department, UNESP Medical School, Botucatu, Brazil

3 UNESP Medical School Clinical Hospital, Botucatu, Brazil

Genetically, we are for the greater part still adapted to the East African ecosystem on which our genome evolved, with some adaptations since the Out‐of‐Africa Diaspora. Dietary fat quantity and quality change have, together with other man‐made changes in our environ‐ ment, caused a conflict with our slowly adapting genome that is implicated in "typically Western" diseases. Fortunately, the majority of Western diseases occur typically after repro‐ ductive age. Rather than reducing our life expectancy, these diseases notably diminish our

240 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Many recommendations for the intakes of saturated fat, *trans* fat, and EPA + DHA have been issued, notably for prevention. The ultimate goal might be, however, translate to the culture of the current society that our genes had evolved for million years in an entirely different dietary composition and lifestyle and therefore we must return to the fat quality of our ancient diet [11].

\*, Caroline das Neves Mendes Nunes<sup>2</sup>

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## *Edited by Naofumi Shiomi and Viduranga Waisundara*

Superfoods and functional foods are receiving increasing attention because of their important roles in health. This book focuses on the production of superfoods and functional foods and their role as medicine. In the early chapters, prominent researchers introduce the roles and production of microalgae and functional fruits through metabolic engineering, the use of food waste, and effective cooking procedures. In the latter chapters, other prominent researchers introduce the medical effects of polyphenols, glutamine, and unsaturated fatty acids, which are contained in superfoods and functional foods. They suggest the importance of superfoods and functional foods in the treatment and prevention of many diseases. It is also recommended for readers to take a look at a related book, Superfood and Functional Food: An Overview of Their Processing and Utilization.

Photo by Elenathewise / iStock

Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Superfood

and Functional Food

The Development of Superfoods

and Their Roles as Medicine

*Edited by Naofumi Shiomi* 

*and Viduranga Waisundara*