**Probiotics and Oxidative Stress**

Tiiu Kullisaar, Epp Songisepp and Mihkel Zilmer *University of Tartu, Bio-competence Centre of Healthy Dairy products LCC, Estonia*

#### **1. Introduction**

A large number of reports about the health benefits of probiotics could be found in the PubMed database. Very little information is available about probiotics possessing physiologically relevant antioxidative properties. Quite scarce is information on the influence of probiotics on human body oxidative stress status and a limited number of clinical trials have been conducted on the effect of antioxidative lactic acid bacteria on human oxidative stressdriven cardiovascular disease-related aspects. In this chapter possibilities of antioxidative probiotics to influence on oxidative stress status in human body are discussed.

#### **2. Short survey of probiotics**

The potential life-lengthening properties of lactic acid bacteria (LAB) were proposed by Metchnikoff already at the beginning of the 20th century. The term "probiotic" is an etymological hybrid derived from Greek and Latin meaning "for life" (Hamilton-Miller et al., 2003). Today probiotics are defined as live microorganisms which, when consumed in appropriate amounts, confer a health benefit on the host (FAO/WHO, 2002). Genera most commonly used as probiotics are *Lactobacillus* and *Bifidobacterium*, but other LAB such as lactococci, streptococci, enterococci as well as propionibacteria, bacilli (e.g. *Bacillus subtilis*) and yeasts (e.g. *Saccharomyces boulardii*) are applied. However, probiotics are usually LAB. Introducing a new probiotic into the market involves a step-wise process in order to obtain a functional and safe product (Saarela et al., 2000; Vankerckhoven et al., 2008). Exact requirements are set for probiotic bacteria. Centuries-long use of LAB in the food industry has proven their safety. Nevertheless, it is compulsory to test the safety of each new potential probiotic. The recommendations include an absence of hemolytic activity and the transferable antibiotic resistance of the selected strain, while safety should be proven in various animal models (FAO/WHO, 2002; Vesterlund et al., 2007; Kõll et al., 2010). There is a necessity for pilot clinical trials on healthy volunteers to exclude the adverse effects of probiotic administration on gut health, biochemical and cellular indices of blood reflecting the proper functions of human organs (Reid, 2005; Rijkers et al., 2010). Probiotics must be able to resist stomach acid, bile and the effects of digestive enzymes. Thus, potential probiotic candidates will be selected mostly from human normal microflora. The ability to survive in the GI tract, adhere to intestinal epithelium and maintain its metabolic activity is directly related to the manifestation of probiotic properties in the human body. Probiotic properties are strictly strain-specific. Even the related microbial species may have very

Probiotics and Oxidative Stress 205

below). To maintain the physiological grade of OxS needed for a number's biofunctions like intracellular messaging, growth, cellular differention, phagocytosis, immune response, etc the human body has an integrated antioxidative defence system (IADS, Table. 1). Several antioxidative components for this human IADS are derived from foodstuffs and provided by GI microbiota. Interestingly, it became more and more apparent that the IADS of the host and GI microbiota are tightly linked and some specific strains with physiologically effective antioxidative activity may have a great impact on the management of the OxS level in the gut lumen, inside mucosa cells and even in the host blood, to support the functionality of the IADS of the human body. Thus, experiments to find out strains with physiologically relevant antioxidative properties/effects as well as trials (including special clinical trials) using capsules of such strains or foodstuffs enriched with antioxidative strains are needed. Unfortunately, scientific data on probiotic LAB with physiologically relevant antioxidative properties is very limited and the data of experiments/trials about both intestinal antioxidative protection/influence and systemic antioxidative protection/influence (effects

Oxidative stressors (pro-oxidants) Integrated antioxidative defence system

Table 1. A net effect of oxidative stressors and the potency of the integrated antioxidant defence system (IADS) of the body are normally balanced. An imbalance leads to potentially harmful oxidative stress. PUFA, polyunsaturated fatty acids; SOD, superoxide dismutase; GPx, glutathione peroxidase; CAT, catalase; HO1, haem oxygenase1; GSH, reduced

However, as a certain progress has been made during recent years and we will give a

**metabolic syndrome, allergy, atopic dermatitis, radiation induced problems** 

A large body of evidence exists that high-grade OxS has one of the crucial roles in the pathogenesis of disorders/diseases of the vascular system (atherosclerosis, myocardial infarction, stroke, peripheral artery disease), the nervous system (Alzheimer's disease, Parkinson's disease), the liver (cirrhosis, ethanol damage), the skin (dermatoses), the pancreas (diabetes mellitus), metabolic syndrome, obesity, premature ageing, the eyes (age-related macular degeneration, retinopathy), development of some tumors and the GI (inflammatory bowel disease, coeliac disease, etc), etc (Halliwell & Gutterridge, 1999; Stocker & Keaney, 2004; Kals et al., 2006; Stojiljkovic et al., 2007; Krzystek-Korpacka et al., 2008; Tsukahara, 2007; Suzuki et al., 2007; Vincent et al., 2007; Castellani et al., 2008)*.* It has recently reviewed that harmful GI consequences of radiation therapy have OxS-related background (Spyropoulos

**4. Short survey of oxidative stress-related pathological states (CVD,** 

Vitamin E, C, Q, A Enzymes as antioxidants (SOD, GPx, CAT, HO1) Other antioxidants

(GSH, plasma albumin, uric acid, Bilirubin, carotenoids, etc)

of OxS-related indices) are scarce.

Smoking, Inflammation, xenobiotics

summarized overview about probiotics and OxS.

Radiation, Exhaustive exercises Prolonged severe emotional stress

Ischemia/reperfusion

**in the intestinal tract)** 

PUFA megadoses Iron or copper excess

glutathione.

different clinical effects. Thus, one cannot arbitrarily attribute the properties of one probiotic strain to another, even within the same species (Vaughan et al., 2005).

Probiotic effects have a dosage threshold. The minimum effective dose, which affects the intestinal environment and provides beneficial effects on human health, is considered to be 106-109 live microbial cells per day. The minimum dose depends on the particular strain and the type of foodstuffs (Reid, 2005, Williams, 2010, Champagne et al., 2011). Probiotics have been demonstrated to be effective in a variety of conditions including the relaxation of intestinal discomfort (bloating and pain), the alleviation of chronic intestinal inflammatory diseases, the prevention and treatment of pathogen-induced diarrhea, lowering lactose intolerance and food allergies, the lowering of cholesterol levels, the prevention of urogenital infections and the reduction of atopic diseases (Andersson et al. 2001; Chapman et al., 2011). The important area of human physiology that is relevant to functional food science according to the ILSI and FUFOSE (the European Commission Concerted Action on Functional Food Science in Europe) is, among others, the modulation of the defence against high-grade oxidative stress. The latter is one of the principal players in the pathogenesis of CVD and other diseases. Thousands of reports reflecting the abovementioned different health benefits of probiotics could be found in the databases. However, scarce information is available regarding probiotics possessing physiologically relevant antioxidative properties and a limited number of clinical trials on the effect of antioxidative LAB on human CVDrelated aspects have been conducted*.*

#### **3. Short survey of oxidative stress**

A net of pro-oxidants and the potency of an antioxidant defence system normally balanced in the body. Principal pro-oxidants are reactive species (including free radicals) divided into reactive oxygen species (ROS) and reactive nitrogen species (RNS) and they mediate the main effects of other pro-oxidative factors (Sies, 1991; Halliwell & Gutterridge, 1999). In the organisms the crucial ROS are superoxide radical, hydroxyl radical, lipid peroxyl radical and non-radical hydrogen peroxide (the latter is produced from superoxide by superoxide dismutase) and the principal RNS are nitric oxide and non-radical peroxynitrite. The pathological efficiency of the hydroxyl radical is the most potent and it is rapidly generated via the Fenton cycle where free iron (a very potent pro-oxidant) reacts with hydrogen peroxide (Halliwell & Gutterridge, 1999). Most of the mentioned reactive species (RS) come from endogenous sources as by-products of normal essential metabolic processes, while exogenous sources involve exposure to cigarette smoke, environmental pollutants, radiation, drugs, bacterial infections, excess of food iron, dysbalanced intestinal microflora, etc. Several diseases are associated with the toxic effect of the transition metals (iron, copper, cadmium). Thus, abnormal formation of the RS can occur *in vivo* and that leads to the damage of lipids, proteins, nucleic acids and carbohydrates of cells and tissues. An excessive production of RS causes an imbalance in the pro-oxidants/antioxidants system. Any imbalance in favour of the pro-oxidants potentially leading to damage was termed 'oxidative stress' (Sies, 1991). Recently an additional adapted conception of oxidative stress (OxS) was advanced as "a disruption of redox signalling and control"(Jones, 2006), emphasizing an impact of the redox ratio as good tools for the quantification of OxS. It is remarkable that the glutathione redox ratio has a crucial impact concerning this conception. A large body of evidence confirms that high-grade OxS is one of the crucial players in the pathogenesis of disorders/diseases (cf

different clinical effects. Thus, one cannot arbitrarily attribute the properties of one probiotic

Probiotic effects have a dosage threshold. The minimum effective dose, which affects the intestinal environment and provides beneficial effects on human health, is considered to be 106-109 live microbial cells per day. The minimum dose depends on the particular strain and the type of foodstuffs (Reid, 2005, Williams, 2010, Champagne et al., 2011). Probiotics have been demonstrated to be effective in a variety of conditions including the relaxation of intestinal discomfort (bloating and pain), the alleviation of chronic intestinal inflammatory diseases, the prevention and treatment of pathogen-induced diarrhea, lowering lactose intolerance and food allergies, the lowering of cholesterol levels, the prevention of urogenital infections and the reduction of atopic diseases (Andersson et al. 2001; Chapman et al., 2011). The important area of human physiology that is relevant to functional food science according to the ILSI and FUFOSE (the European Commission Concerted Action on Functional Food Science in Europe) is, among others, the modulation of the defence against high-grade oxidative stress. The latter is one of the principal players in the pathogenesis of CVD and other diseases. Thousands of reports reflecting the abovementioned different health benefits of probiotics could be found in the databases. However, scarce information is available regarding probiotics possessing physiologically relevant antioxidative properties and a limited number of clinical trials on the effect of antioxidative LAB on human CVD-

A net of pro-oxidants and the potency of an antioxidant defence system normally balanced in the body. Principal pro-oxidants are reactive species (including free radicals) divided into reactive oxygen species (ROS) and reactive nitrogen species (RNS) and they mediate the main effects of other pro-oxidative factors (Sies, 1991; Halliwell & Gutterridge, 1999). In the organisms the crucial ROS are superoxide radical, hydroxyl radical, lipid peroxyl radical and non-radical hydrogen peroxide (the latter is produced from superoxide by superoxide dismutase) and the principal RNS are nitric oxide and non-radical peroxynitrite. The pathological efficiency of the hydroxyl radical is the most potent and it is rapidly generated via the Fenton cycle where free iron (a very potent pro-oxidant) reacts with hydrogen peroxide (Halliwell & Gutterridge, 1999). Most of the mentioned reactive species (RS) come from endogenous sources as by-products of normal essential metabolic processes, while exogenous sources involve exposure to cigarette smoke, environmental pollutants, radiation, drugs, bacterial infections, excess of food iron, dysbalanced intestinal microflora, etc. Several diseases are associated with the toxic effect of the transition metals (iron, copper, cadmium). Thus, abnormal formation of the RS can occur *in vivo* and that leads to the damage of lipids, proteins, nucleic acids and carbohydrates of cells and tissues. An excessive production of RS causes an imbalance in the pro-oxidants/antioxidants system. Any imbalance in favour of the pro-oxidants potentially leading to damage was termed 'oxidative stress' (Sies, 1991). Recently an additional adapted conception of oxidative stress (OxS) was advanced as "a disruption of redox signalling and control"(Jones, 2006), emphasizing an impact of the redox ratio as good tools for the quantification of OxS. It is remarkable that the glutathione redox ratio has a crucial impact concerning this conception. A large body of evidence confirms that high-grade OxS is one of the crucial players in the pathogenesis of disorders/diseases (cf

strain to another, even within the same species (Vaughan et al., 2005).

related aspects have been conducted*.*

**3. Short survey of oxidative stress** 

below). To maintain the physiological grade of OxS needed for a number's biofunctions like intracellular messaging, growth, cellular differention, phagocytosis, immune response, etc the human body has an integrated antioxidative defence system (IADS, Table. 1). Several antioxidative components for this human IADS are derived from foodstuffs and provided by GI microbiota. Interestingly, it became more and more apparent that the IADS of the host and GI microbiota are tightly linked and some specific strains with physiologically effective antioxidative activity may have a great impact on the management of the OxS level in the gut lumen, inside mucosa cells and even in the host blood, to support the functionality of the IADS of the human body. Thus, experiments to find out strains with physiologically relevant antioxidative properties/effects as well as trials (including special clinical trials) using capsules of such strains or foodstuffs enriched with antioxidative strains are needed. Unfortunately, scientific data on probiotic LAB with physiologically relevant antioxidative properties is very limited and the data of experiments/trials about both intestinal antioxidative protection/influence and systemic antioxidative protection/influence (effects of OxS-related indices) are scarce.


Table 1. A net effect of oxidative stressors and the potency of the integrated antioxidant defence system (IADS) of the body are normally balanced. An imbalance leads to potentially harmful oxidative stress. PUFA, polyunsaturated fatty acids; SOD, superoxide dismutase; GPx, glutathione peroxidase; CAT, catalase; HO1, haem oxygenase1; GSH, reduced glutathione.

However, as a certain progress has been made during recent years and we will give a summarized overview about probiotics and OxS.

#### **4. Short survey of oxidative stress-related pathological states (CVD, metabolic syndrome, allergy, atopic dermatitis, radiation induced problems in the intestinal tract)**

A large body of evidence exists that high-grade OxS has one of the crucial roles in the pathogenesis of disorders/diseases of the vascular system (atherosclerosis, myocardial infarction, stroke, peripheral artery disease), the nervous system (Alzheimer's disease, Parkinson's disease), the liver (cirrhosis, ethanol damage), the skin (dermatoses), the pancreas (diabetes mellitus), metabolic syndrome, obesity, premature ageing, the eyes (age-related macular degeneration, retinopathy), development of some tumors and the GI (inflammatory bowel disease, coeliac disease, etc), etc (Halliwell & Gutterridge, 1999; Stocker & Keaney, 2004; Kals et al., 2006; Stojiljkovic et al., 2007; Krzystek-Korpacka et al., 2008; Tsukahara, 2007; Suzuki et al., 2007; Vincent et al., 2007; Castellani et al., 2008)*.* It has recently reviewed that harmful GI consequences of radiation therapy have OxS-related background (Spyropoulos

Probiotics and Oxidative Stress 207

antinutritional factors, and/or modulation of GI physiology and reduction of pain perception. Special probiotic strains may induce the expression of receptors on epithelial cells that locally control the transmission of nociceptive information to the GI nervous system (Rousseaux et al., 2007). Beneficial bacteria have enzymatic equipment which enables them to break down a wide variety of food constituents that cannot be metabolized by the host such as galactooligosaccharides, inulin, resistant starches, and antinutritional factors such as tannins or phytates responsible for the chelation of minerals including iron, zinc, magnesium and calcium (Gilman & Cashman, 2006; Songre-Quattara et al., 2008; Cecconi et al., 2009). They can also modify the host gut physiology by increasing the production of growth factors (Alberto et al., 2007). LAB may thus be of benefit to health and help protect against diseases, like CVD, diabetes, metabolic syndrome, etc. As far as OxS is at least one of the components of initiation and/or the development of the mentioned diseases thus any kind of agent which can prevent the development of harmful OxS has a principal impact. Probiotics involve LAB or bifidobacteria of human origin. They can during the consumption period adhere to the epithelial cells of GI modulating the human physiological status via the gut associated immune system and/or directly due to the expression of receptors of GI and/or systematically. LAB beneficial effects are strain-specific. *In vitro* and cellular models, the probiotic properties of lactobacilli have been limited to few parameters such as the ability to survive low (pH 2-3) and bile salts, to produce pathogen inhibitory compounds (including hydrogen peroxide), to compete with energy availability or adhesion sites, and to enhance immune response (Ryan et., 2008; Todorov et al., 2008; Pfeiler & Klaenhammer, 2009). Along with the probiotics themselves, there are metabiotics i.e, the metabolic byproducts of probiotics. Metabiotics are beneficial in promoting a healthy GI by creating an environment most favorable to probiotics, by nourishing the enterocytes, reinforcing mucosal barrier function, by maintaining or supporting epithelial integrity or signaling the immune system to limit inflammatory responses both in the gut and through influencing Tcells throughout the body. The principal metabiotics are short-chain fatty acids but also other substances like polyamines (putrescine, spermidine, spermine) have an impact (Larqué et al., 2007). It has been demonstrated, that NO produced by LAB protects mucosa

for damages and excessive permeability (Payne et al., 1993; Korhonen et al., 2001).

Since 1993 when Kaizu and co-workers discovered antioxidative activity of LAB, a few of them have had effects in clinical human trials (Kaizu et al., 1993). One of them is antioxidative-antiatherogenic and antimicrobial probiotic *Lactobacillus fermentum* ME-3 (LfME-3). Tartu University has patented this strain in Estonia, Russia, USA and Europe. LfME-3 (DSM 14241) is of human origin (Sepp et al., 1997) and has proven its safety as a probiotic exhibiting both antimicrobial and antioxidative benefits under different *in vitro* and *in vivo* conditions (Kullisaar et al., 2002, 2003; Truusalu et al., 2004; Songisepp et al., 2005; Järvenpää et al., 2007). What makes this strain such a powerful multivalent antioxidant? It is confirmed that *in vitro* the superoxide anion scavenging efficiency of LfME-3 was more than 80-100 times stronger as compared with trolox or ascorbic acid (Ahotupa, personal communication). LfME-3 expresses Mn-superoxide dismutase (MnSOD) activity, can effectively eliminate hydroxyl and peroxyl radicals, and has the complete glutathione system (reduced glutathione, glutathione peroxidase, GPx, glutathione reductase, GRed) necessary for glutathione recycling, transporting and synthesis (Kullisaar et al., 2002, 2010). Mn-SOD is very important in the control of LP. Manganese and Mn-SOD

et al., 2011). Firstly, radiolysis of water molecules causes rapid production of ROS, secondly, an increase in oxygen radical production in the vascular wall has shown already 2h after irradiation with a more intense OxS observed at 6h, this second burst being produced mainly by infiltrating inflammatory cells (Molla & Panes, 2007).

Prolonged excessive ROS/RNS production can trigger chemical chain reactions with all major biomolecules such as DNA, proteins, and membrane lipids. DNA is affected with a variety of lesions like oxidized bases, stand brakes, as well as DNA–DNA and DNA–protein cross-links (Barker et al., 2005). Oxidatively damaged proteins are characterized by formation of carbonyl groups (Stadtman, 1992). Hydroxyl radicals depolymerize hyaluronic acid, degrade collagen, inactivate enzymes and transport proteins via sulfhydryl oxidation. RNS may induce nitration of protein tyrosine residues. Lipid peroxidation is the oxidative degradation of membrane lipids and oxidation that can cause severe impairment of membrane function through changes in membrane permeability and fluidity, its protein oxidation, ultimately leading to cell lysis (Halliwell & Gutterridge, 1999). Lipid peroxidation also damages blood lipoproteins. Therefore, prolonged high-grade OxS causes damages in biomolecules, cells, tissue and organ functionality. Reactive species-damage can be evaluated via markers for oxidized proteins (i.e. nitrated tyrosine, protein carbonyls); oxidized nucleic acid bases (8-oxo-2-deoxyguanosine), oxidized carbohydrates (glycated products) and oxidized lipids (F2-isoprostanes, oxidized low-density lipoproteins (oxLDL), etc). Additional approach for investigations of OxS is an assay of the capacity of IADS (i.e. assay of total antioxidative status or response (TAS, TAR), etc). All these markers are informative but they are not still ultimately accepted as new risk markers yet. However, recently pathogenetic relevance of isoprostanes and oxLDL has been accepted (Statements of European Food Safety Authority). A large number of articles shows that oxLDL level is associated with development of cardiovascular diseases (CVD). Thus, to describe both process and status of atherosclerosis common risk markers like low-density lipoprotein or LDL-cholesterol, HDL-cholesterol, fasting triglycerides (TG), plasma homocysteine as well as by new additional OxS- and inflammation-related indices (oxLDL, 8-isoprostanes, highly sensitive C-reactive protein) should be used. All these markers are also diet-related markers (Mensink et al., 2003). It is reviewed that OxS indices (oxLDL, urine 8-isoprostanes, etc) together with the increased inflammatory markers (white blood cells (WBCs), highly sensitive C-reactive protein have been shown to be characteristic of patients with atherosclerotic lesions of the vascular system (Stocker, Keaney, 2004). Consequently, probiotics with physiologically relevant multivalent antioxidative properties/effects expressed via a positive influence both on a GI and systemic OxS level may have impact concerning the pathogenesis of different disorders/diseases, particularly CVD.

#### **5. Properties of probiotics necessary to have an influence on oxidative stress status**

#### **5.1 Role of probiotics in intestinal antioxidative protection (possible action mechanisms)**

The most documented effects of LAB in humans are the stimulation of the immune system, the prevention and the reduction of the intensity and duration of diarrhea, and reduction of lactose intolerance (Wolvers et al., 2010). LAB also have some other beneficial effects such as vitamin synthesis, improvement of mineral and nutrient absorption, degradation of

et al., 2011). Firstly, radiolysis of water molecules causes rapid production of ROS, secondly, an increase in oxygen radical production in the vascular wall has shown already 2h after irradiation with a more intense OxS observed at 6h, this second burst being produced

Prolonged excessive ROS/RNS production can trigger chemical chain reactions with all major biomolecules such as DNA, proteins, and membrane lipids. DNA is affected with a variety of lesions like oxidized bases, stand brakes, as well as DNA–DNA and DNA–protein cross-links (Barker et al., 2005). Oxidatively damaged proteins are characterized by formation of carbonyl groups (Stadtman, 1992). Hydroxyl radicals depolymerize hyaluronic acid, degrade collagen, inactivate enzymes and transport proteins via sulfhydryl oxidation. RNS may induce nitration of protein tyrosine residues. Lipid peroxidation is the oxidative degradation of membrane lipids and oxidation that can cause severe impairment of membrane function through changes in membrane permeability and fluidity, its protein oxidation, ultimately leading to cell lysis (Halliwell & Gutterridge, 1999). Lipid peroxidation also damages blood lipoproteins. Therefore, prolonged high-grade OxS causes damages in biomolecules, cells, tissue and organ functionality. Reactive species-damage can be evaluated via markers for oxidized proteins (i.e. nitrated tyrosine, protein carbonyls); oxidized nucleic acid bases (8-oxo-2-deoxyguanosine), oxidized carbohydrates (glycated products) and oxidized lipids (F2-isoprostanes, oxidized low-density lipoproteins (oxLDL), etc). Additional approach for investigations of OxS is an assay of the capacity of IADS (i.e. assay of total antioxidative status or response (TAS, TAR), etc). All these markers are informative but they are not still ultimately accepted as new risk markers yet. However, recently pathogenetic relevance of isoprostanes and oxLDL has been accepted (Statements of European Food Safety Authority). A large number of articles shows that oxLDL level is associated with development of cardiovascular diseases (CVD). Thus, to describe both process and status of atherosclerosis common risk markers like low-density lipoprotein or LDL-cholesterol, HDL-cholesterol, fasting triglycerides (TG), plasma homocysteine as well as by new additional OxS- and inflammation-related indices (oxLDL, 8-isoprostanes, highly sensitive C-reactive protein) should be used. All these markers are also diet-related markers (Mensink et al., 2003). It is reviewed that OxS indices (oxLDL, urine 8-isoprostanes, etc) together with the increased inflammatory markers (white blood cells (WBCs), highly sensitive C-reactive protein have been shown to be characteristic of patients with atherosclerotic lesions of the vascular system (Stocker, Keaney, 2004). Consequently, probiotics with physiologically relevant multivalent antioxidative properties/effects expressed via a positive influence both on a GI and systemic OxS level may have impact concerning the

mainly by infiltrating inflammatory cells (Molla & Panes, 2007).

pathogenesis of different disorders/diseases, particularly CVD.

**status** 

**mechanisms)** 

**5. Properties of probiotics necessary to have an influence on oxidative stress** 

The most documented effects of LAB in humans are the stimulation of the immune system, the prevention and the reduction of the intensity and duration of diarrhea, and reduction of lactose intolerance (Wolvers et al., 2010). LAB also have some other beneficial effects such as vitamin synthesis, improvement of mineral and nutrient absorption, degradation of

**5.1 Role of probiotics in intestinal antioxidative protection (possible action** 

antinutritional factors, and/or modulation of GI physiology and reduction of pain perception. Special probiotic strains may induce the expression of receptors on epithelial cells that locally control the transmission of nociceptive information to the GI nervous system (Rousseaux et al., 2007). Beneficial bacteria have enzymatic equipment which enables them to break down a wide variety of food constituents that cannot be metabolized by the host such as galactooligosaccharides, inulin, resistant starches, and antinutritional factors such as tannins or phytates responsible for the chelation of minerals including iron, zinc, magnesium and calcium (Gilman & Cashman, 2006; Songre-Quattara et al., 2008; Cecconi et al., 2009). They can also modify the host gut physiology by increasing the production of growth factors (Alberto et al., 2007). LAB may thus be of benefit to health and help protect against diseases, like CVD, diabetes, metabolic syndrome, etc. As far as OxS is at least one of the components of initiation and/or the development of the mentioned diseases thus any kind of agent which can prevent the development of harmful OxS has a principal impact. Probiotics involve LAB or bifidobacteria of human origin. They can during the consumption period adhere to the epithelial cells of GI modulating the human physiological status via the gut associated immune system and/or directly due to the expression of receptors of GI and/or systematically. LAB beneficial effects are strain-specific. *In vitro* and cellular models, the probiotic properties of lactobacilli have been limited to few parameters such as the ability to survive low (pH 2-3) and bile salts, to produce pathogen inhibitory compounds (including hydrogen peroxide), to compete with energy availability or adhesion sites, and to enhance immune response (Ryan et., 2008; Todorov et al., 2008; Pfeiler & Klaenhammer, 2009). Along with the probiotics themselves, there are metabiotics i.e, the metabolic byproducts of probiotics. Metabiotics are beneficial in promoting a healthy GI by creating an environment most favorable to probiotics, by nourishing the enterocytes, reinforcing mucosal barrier function, by maintaining or supporting epithelial integrity or signaling the immune system to limit inflammatory responses both in the gut and through influencing Tcells throughout the body. The principal metabiotics are short-chain fatty acids but also other substances like polyamines (putrescine, spermidine, spermine) have an impact (Larqué et al., 2007). It has been demonstrated, that NO produced by LAB protects mucosa for damages and excessive permeability (Payne et al., 1993; Korhonen et al., 2001).

Since 1993 when Kaizu and co-workers discovered antioxidative activity of LAB, a few of them have had effects in clinical human trials (Kaizu et al., 1993). One of them is antioxidative-antiatherogenic and antimicrobial probiotic *Lactobacillus fermentum* ME-3 (LfME-3). Tartu University has patented this strain in Estonia, Russia, USA and Europe. LfME-3 (DSM 14241) is of human origin (Sepp et al., 1997) and has proven its safety as a probiotic exhibiting both antimicrobial and antioxidative benefits under different *in vitro* and *in vivo* conditions (Kullisaar et al., 2002, 2003; Truusalu et al., 2004; Songisepp et al., 2005; Järvenpää et al., 2007). What makes this strain such a powerful multivalent antioxidant? It is confirmed that *in vitro* the superoxide anion scavenging efficiency of LfME-3 was more than 80-100 times stronger as compared with trolox or ascorbic acid (Ahotupa, personal communication). LfME-3 expresses Mn-superoxide dismutase (MnSOD) activity, can effectively eliminate hydroxyl and peroxyl radicals, and has the complete glutathione system (reduced glutathione, glutathione peroxidase, GPx, glutathione reductase, GRed) necessary for glutathione recycling, transporting and synthesis (Kullisaar et al., 2002, 2010). Mn-SOD is very important in the control of LP. Manganese and Mn-SOD

Probiotics and Oxidative Stress 209

On the basis of simplified general understandings it can be speculated that there are several factors that may have an impact on OxS. This is only one of the examples. It can be speculated that the suppression of *Helicobacter pylori* infection by some LAB (Wang et al., 2004; Cruchet et al., 2003; Linsalata et al., 2004) may have a certain effect on the host OxSrelated indices in blood. However, such approaches are actually only speculations. Why? An analysis of scientific literature allows one to conclude that for a real effect on the systemic OxS-related indices of a host, a specific probiotic strain should have multifunctional bioquality: a) to have positive effects on GI total lactobacilli counts; b) to be able to suppress putative contaminants of food; c) to have biovaluable different antioxidative properties; d) to have a positive effect on OxS-related CVD markers, like TG, oxLDL, etc. In section 5.1. it was explained that the probiotic LfME-3 carries first three types (a,b,c) of properties. Thus, these multifunctional properties of LfME-3 may protect the host against both food-derived infections and help in the prevention of the oxidative damage of food. For example, the antioxidative protection provided by the LfME-3 strain for the prevention of the oxidative spoilage of semi-soft cheeses was found out (Järvenpaa et al., 2007). Thus, points a, b and c have an impact on the role of probiotics for systemic antioxidant defence. However, it is crucial also to have data (according to point d) about the specific influence of probiotics on OxS-related CVD markers. Since LfME-3 has been carefully investigated, concerning the latter we will use gathered information as a model to discuss possible mechanisms on how

probiotics may have an influence on the OxS-driven CVD risk markers of a host.

We repeatedly showed that administering a food products to humans comprising strain LfME-3 enhances the systemic antioxidative activity of sera (increases total antioxidative activity, TAA, or total antioxidative status, TAS), enhances the lag phase of LDL (increases oxi-resistance of LDL particles, i.e. suppresses production of atherogenic oxLDL) and decreases level of oxidized glutathione (pro-oxidant), oxLDL and BCD-LDL of sera (Kullisaar et al., 2003, 2011; Mikelsaar et al., 2007). Clinical trials showed that the strain LfME-3 alleviates inflammation and OxS-associated shifts in gut, skin and blood (Kullisaar et al., 2003, 2008; Kaur et al.2008). This realizes via complicated cross-talk between probiotic and host body cells via the integrated influence of several factors of strain LfME-3 like having complete glutathione system, the expression of antioxidative enzymes, the production of CLA and NO by strain LfME-3, etc (Mikelsaar & Zilmer, 2009; Kullisaar et al., 2010, 2011). This strain survives in different fermentation processes of milk due to its good tolerance to low temperature, acid and salt (Songisepp et al., 2004; Songisepp, 2005) and is able for temporal colonization of the GI tract of the consumer. All this is very important as the GI surface is a crucial host organism-environment boundary and the interactions of GI microbes inside the intestinal lumen and mucosal cells have impact for the metabolic activity both microbes and host cells. An impaired environment (the imbalance of GI microbiota, the increase of LP, decrease of the GSH) both at the intestinal surface and in the intestinal cells, are substantial modulators causing unhealthy outcomes in the host. In addition, data that these cellular modulators of the intestinal mucosal status can be repaired by applying of strain LfME-3 was confirmed by using a mouse model of experimental *S. Typhimurium* infection (Truusalu et al., 2004, 2008). Concerning this process the involvement of the glutathione system is crucial as GSH, in addition its role as a crucial cellular antioxidant, is the principal redox controller for a number of cellular processes. Glutathione-related information has impact for LfM-3 regarding next information: a) a recent adapted conceptions of OxS is advanced as "a disruption of redox signalling and control" (Jones, 2006) or "steady-state ROS" (Lushchak,

activity of LAB (not possessing catalase) is important for their survival in the oxidative milieu (milk, host) created by the production of hydrogen peroxide (Sanders et al., 1995). It has been shown that some LAB (*L.gasseri*) engineered to produce SOD reduce the inflammation in the case of colitis in interleukin-10-deficient mice (Carroll et al., 2007).

Glutathione (*L-*gamma-Glu-*L*-Cys-Gly or GSH) is a major cellular non-enzymatic antioxidant. It eliminates lipid- and hydroperoxides, hydroxyl radical and peroxynitrite mainly via cooperation with Se-dependent glutathione peroxidase (Zilmer et al., 2005). The GI surface is an important host organism-environment boundary and the interactions of gut microbes inside the intestinal lumen and mucosal cells are important for the host. An impaired environment such as the imbalance of GI microbiota, but also the increase of LP and decrease of the reduced GSH both at the GI surface and in the GI cells, are the mighty modulators causing different unhealthy outcomes in the host. In this process the involvement of the glutathione system is crucial as GSH, besides its role as a crucial antioxidant, is the principal redox controller for a number of processes in cells. Glutathione-related data has impact for LfME-3 regarding at several aspects (cf. 5.2). Thus, confirmation of the presence of all glutathione system components in a specific concrete LAB gives very valuable information as it shows that a specific LAB strain has especially high oxygen and ROS tolerance under different stress conditions. An essential physiological trait for probiotics is tolerance to stress in the GI as well as during the production of functional foods (Ross et al., 2005). Beside that GSH has essential role in maintaining mucosal integrity. Studies have shown diminished GSH levels in inflammatory diseases of intestine and GSH supplementation has beneficial effect (Coskun et al., 2010).

Evidently some probiotics are able to promote an elevation of the level of beneficial bacteria in the GI. In experiments and clinical trials, the administration of the LfME-3 strain has led to the improvement of the GI microbial ecology. More than a 10-fold increase of total lactobacilli counts in comparison with the individually different initial count was registered in the collected faecal samples (Mikelsaar & Zilmer, 2009). The metabolites secreted by LfME-3 into the GI tract could be used as a substrate by other lactobacilli. Adding LfM-3 as a probiotic into a dairy product (yoghurt, cheese, milk) also suppressed the putative contaminants of food (*Salmonella spp., Shigella spp*.,). The secreted substantial amount of hydrogen peroxide and the production of NO by LfME-3 are the main antimicrobial agents (Mikelsaar & Zilmer, 2009*.* Animal studies have confirmed that the increase in total LAB counts as much as the specific LfME-3 strain antioxidative action in the GI eradicated live salmonellas and prevented the formation of typhoid nodules in experimental *Salmonella Typhimurium* infections, resembling typhoid fever in humans (Truusalu et al., 2004, 2008). It was the first time that the antibiotic therapy of an invasive infection like enteric fever was shown to be more effective if administered together with a probiotic.

#### **5.2 Role of probiotics for systemic antioxidative defence (possible action mechanisms)**

Such information is limited. However, some specific multifunctional probiotics may have an influence on systemic (blood) antioxidative defence and the OxS status of host. Thus, to characterize the role of high-grade OxS in the pathogenesis of CVD, we will give an overview about the possible action mechanisms of probiotics on OxS-related indices of CVD.

activity of LAB (not possessing catalase) is important for their survival in the oxidative milieu (milk, host) created by the production of hydrogen peroxide (Sanders et al., 1995). It has been shown that some LAB (*L.gasseri*) engineered to produce SOD reduce the inflammation in the case of colitis in interleukin-10-deficient mice (Carroll et al., 2007).

Glutathione (*L-*gamma-Glu-*L*-Cys-Gly or GSH) is a major cellular non-enzymatic antioxidant. It eliminates lipid- and hydroperoxides, hydroxyl radical and peroxynitrite mainly via cooperation with Se-dependent glutathione peroxidase (Zilmer et al., 2005). The GI surface is an important host organism-environment boundary and the interactions of gut microbes inside the intestinal lumen and mucosal cells are important for the host. An impaired environment such as the imbalance of GI microbiota, but also the increase of LP and decrease of the reduced GSH both at the GI surface and in the GI cells, are the mighty modulators causing different unhealthy outcomes in the host. In this process the involvement of the glutathione system is crucial as GSH, besides its role as a crucial antioxidant, is the principal redox controller for a number of processes in cells. Glutathione-related data has impact for LfME-3 regarding at several aspects (cf. 5.2). Thus, confirmation of the presence of all glutathione system components in a specific concrete LAB gives very valuable information as it shows that a specific LAB strain has especially high oxygen and ROS tolerance under different stress conditions. An essential physiological trait for probiotics is tolerance to stress in the GI as well as during the production of functional foods (Ross et al., 2005). Beside that GSH has essential role in maintaining mucosal integrity. Studies have shown diminished GSH levels in inflammatory diseases of intestine and GSH

Evidently some probiotics are able to promote an elevation of the level of beneficial bacteria in the GI. In experiments and clinical trials, the administration of the LfME-3 strain has led to the improvement of the GI microbial ecology. More than a 10-fold increase of total lactobacilli counts in comparison with the individually different initial count was registered in the collected faecal samples (Mikelsaar & Zilmer, 2009). The metabolites secreted by LfME-3 into the GI tract could be used as a substrate by other lactobacilli. Adding LfM-3 as a probiotic into a dairy product (yoghurt, cheese, milk) also suppressed the putative contaminants of food (*Salmonella spp., Shigella spp*.,). The secreted substantial amount of hydrogen peroxide and the production of NO by LfME-3 are the main antimicrobial agents (Mikelsaar & Zilmer, 2009*.* Animal studies have confirmed that the increase in total LAB counts as much as the specific LfME-3 strain antioxidative action in the GI eradicated live salmonellas and prevented the formation of typhoid nodules in experimental *Salmonella Typhimurium* infections, resembling typhoid fever in humans (Truusalu et al., 2004, 2008). It was the first time that the antibiotic therapy of an invasive infection like enteric fever was

supplementation has beneficial effect (Coskun et al., 2010).

shown to be more effective if administered together with a probiotic.

**mechanisms)** 

**5.2 Role of probiotics for systemic antioxidative defence (possible action** 

Such information is limited. However, some specific multifunctional probiotics may have an influence on systemic (blood) antioxidative defence and the OxS status of host. Thus, to characterize the role of high-grade OxS in the pathogenesis of CVD, we will give an overview about the possible action mechanisms of probiotics on OxS-related indices of CVD.

On the basis of simplified general understandings it can be speculated that there are several factors that may have an impact on OxS. This is only one of the examples. It can be speculated that the suppression of *Helicobacter pylori* infection by some LAB (Wang et al., 2004; Cruchet et al., 2003; Linsalata et al., 2004) may have a certain effect on the host OxSrelated indices in blood. However, such approaches are actually only speculations. Why? An analysis of scientific literature allows one to conclude that for a real effect on the systemic OxS-related indices of a host, a specific probiotic strain should have multifunctional bioquality: a) to have positive effects on GI total lactobacilli counts; b) to be able to suppress putative contaminants of food; c) to have biovaluable different antioxidative properties; d) to have a positive effect on OxS-related CVD markers, like TG, oxLDL, etc. In section 5.1. it was explained that the probiotic LfME-3 carries first three types (a,b,c) of properties. Thus, these multifunctional properties of LfME-3 may protect the host against both food-derived infections and help in the prevention of the oxidative damage of food. For example, the antioxidative protection provided by the LfME-3 strain for the prevention of the oxidative spoilage of semi-soft cheeses was found out (Järvenpaa et al., 2007). Thus, points a, b and c have an impact on the role of probiotics for systemic antioxidant defence. However, it is crucial also to have data (according to point d) about the specific influence of probiotics on OxS-related CVD markers. Since LfME-3 has been carefully investigated, concerning the latter we will use gathered information as a model to discuss possible mechanisms on how probiotics may have an influence on the OxS-driven CVD risk markers of a host.

We repeatedly showed that administering a food products to humans comprising strain LfME-3 enhances the systemic antioxidative activity of sera (increases total antioxidative activity, TAA, or total antioxidative status, TAS), enhances the lag phase of LDL (increases oxi-resistance of LDL particles, i.e. suppresses production of atherogenic oxLDL) and decreases level of oxidized glutathione (pro-oxidant), oxLDL and BCD-LDL of sera (Kullisaar et al., 2003, 2011; Mikelsaar et al., 2007). Clinical trials showed that the strain LfME-3 alleviates inflammation and OxS-associated shifts in gut, skin and blood (Kullisaar et al., 2003, 2008; Kaur et al.2008). This realizes via complicated cross-talk between probiotic and host body cells via the integrated influence of several factors of strain LfME-3 like having complete glutathione system, the expression of antioxidative enzymes, the production of CLA and NO by strain LfME-3, etc (Mikelsaar & Zilmer, 2009; Kullisaar et al., 2010, 2011). This strain survives in different fermentation processes of milk due to its good tolerance to low temperature, acid and salt (Songisepp et al., 2004; Songisepp, 2005) and is able for temporal colonization of the GI tract of the consumer. All this is very important as the GI surface is a crucial host organism-environment boundary and the interactions of GI microbes inside the intestinal lumen and mucosal cells have impact for the metabolic activity both microbes and host cells. An impaired environment (the imbalance of GI microbiota, the increase of LP, decrease of the GSH) both at the intestinal surface and in the intestinal cells, are substantial modulators causing unhealthy outcomes in the host. In addition, data that these cellular modulators of the intestinal mucosal status can be repaired by applying of strain LfME-3 was confirmed by using a mouse model of experimental *S. Typhimurium* infection (Truusalu et al., 2004, 2008). Concerning this process the involvement of the glutathione system is crucial as GSH, in addition its role as a crucial cellular antioxidant, is the principal redox controller for a number of cellular processes. Glutathione-related information has impact for LfM-3 regarding next information: a) a recent adapted conceptions of OxS is advanced as "a disruption of redox signalling and control" (Jones, 2006) or "steady-state ROS" (Lushchak,

Probiotics and Oxidative Stress 211

bioquality (with lower levels of harmful oxidation products) and higher concentrations of antioxidant factors/enzymes. The higher bioquality of assembled lipoprotein particles leads to improvement of their metabolism/circulation in the host body. This is one of the explanations why strain LfME-3 exerted the prolonged resistance of the blood lipoprotein fraction to oxidation, lowered the level of oxLDL and enhanced the TAC of sera in both healthy and problematic consumers (Kullisaar et al., 2003, 2006, 2008, 2011; Songisepp et al., 2005). Recently it was showed that administration of strain LfME-3 alleviated the postprandial elevation of TG levels in the blood, and improves HDL bioquality (elevates of paraoxonase level in HDL particles) (Kullisaar et al., 2006; 2008; 2011). The antioxidant activity of HDL can be expressed via several mechanisms (Bruckert & Hansel B, 2009). Paraoxonase (PON), an antioxidant enzyme associated with HDL, hydrolyzes oxidized phospholipids and inhibits the LDL oxidation that is an important step in atherogenesis. In animals, the addition of oxidized lipids to the circulation reduces PON activity, and diets rich in oxidized fat accelerate the development of aterogenesis (Sutherland et al., 1999). Removal and inactivation of lipid peroxides accumulating during LDL oxidation may be the central mechanism accounting for HDL antioxidative properties and when HDL particles have poor bioquality (low antioxidant properties and anti-atherosclerotic potency), they may have even inflammatory effect (Navab et al., 2006). The increase in PON activity after LfME-3 consumption shows that protection of LDL particles against oxidative modification by ROS is improved. PON inhibits atherogenesis by hydrolyzing lipid hydroperoxides and cholesterol ester hydroperoxides, reducing peroxides to the hydroxides, and hydrolyzing homocycteine thiolactone which prevents protein homocycteinylation (Beltowski et al., 2003; Durrington et al., 2005). Therefore, an elevation of PON activity should decrease the level of oxLDL. Antioxidant action of HDL is noted as one of the principal mechanisms mediating its cardioprotective effect (Hansel et al., 2006). It should be noted that HDL-associated antioxidant activity information is also supported both by data of anti-inflammatory effects of strain LfM-3 on the liver (Truusalu, et al., 2008) and by a hepato-protective role for PON against inflammation and liver disease mediated by OxS (Marsillach et al., 2009). Next, it is accepted that postprandial abnormal events are crucial concerning the development of CVD (Lopez-Miranda et al., 2006). Recently a postprandial decrease of three different OxS-related parameters (oxLDL, BCD-LDL, Beta2- GPI-OxLDL) was established (Kullisaar et al., 2011)*.* Thus, the foodstuffs enriched with LfME-3 substantially improves postprandial indices both of lipid/lipoproteins and OxS (Kullisaar et al., 2006; 2008; 2011). The beneficial influence of such enriched food on the postprandial lipid metabolism and OxS is important as many links between OxS and metabolic syndrome occur during the postprandial period. These include an excessive and prolonged elevation of blood TG levels, impairment of the endothelial function, an intestinal overproduction of chylomicrons, a redundant load for insulin production, the elevation of levels of atherogenic oxLDL and possible disturbances in the antioxidative activity of HDL (Bae et al., 2001; Jackson et al., 2007; Perez-Martinez et al., 2009; Hopps et al.,2010). To summarize, a positive modulation of the postprandial situation, including postprandial OxS, is an important target

for dietary preventive actions concerning cardiovascular diseases.

**6.1 Functional food and capsules** 

**6. Possibilities of the oxidative stress-targeted administration of probiotics** 

Functional foods are foods or dietary components (incl. probiotics) that may provide a health benefit beyond basic nutrition. Probiotic products may be conventional foods

2011) that emphasize an impact of GSH and its redox ratio for the quantification of OxS and the signalling role of GSH, described previously (Karelson et al., 2002; Zilmer et al., 2005); and b) there exists the possibility for the effective participation of LfM-3 in both enzymatic and non-enzymatic glutathione-driven protection as this strain carries all components needed for functionality of complete glutathione system (Kullisaar et al, 2010). It is interesting to add that recently it has been shown that just *L. fermentum* as a species significantly counteracted the depletion of colonic GSH content induced by some inflammatory processes (Peran et al., 2007) that also supported our understandings. There exists also a correlation between the glutathione redox ratio and DNA oxidative damages (de la Asuncion et al., 1996). Thus, consumption of multivalent probiotic LfME-3, which produces glutathione and has complete glutathione redox cycle enzymes (GPx and GRed), may contribute to the reduction of lipid hydroperoxides in the GI tract and in hepatocytes and prevent them from entering the circulation (Kullisaar et al., 2010). This may lead to an improvement of systemic picture in the host organism.

Data showed that the improvement of the intestinal extra- and intracellular environment yielded beneficial changes of some general (systemic) biochemical indices of the host organism*.* The administration of LfME-3 to healthy volunteers and atopic adults results in a reduction of LP and a counterbalance of the glutathione system both in blood and in skin. In addition, in several trials LfME-3 has beneficial effect on the blood LDL fraction: the prolongation of its resistance to oxidation, the lowering of the content of oxLDL (a potent inflammatory and atherogenic factor) and BDC-LDL and the enhancement of the TAS of sera (Kullisaar et al., 2003, 2011; Songisepp et al., 2005; Mikelsaar et al., 2008). In trial on elderly persons the lower content of oxLDL was significantly predicted by the higher count of live lactobacilli in the GI tract*.* Evidently, both the number special antioxidative characteristics of strain LfME-3 and the increase in lactobacilli counts induced by administration of LfME-3 are responsible for such effect on host lipoprotein circulation/metabolism. As we mentioned before, the status of OxS and blood lipoproteins are both related to the pathogenesis of different diseases, including inflammation-related diseases and CVD. Dzau et al (2006) presented in *Circulation* the pathophysiological continuum showing that traditional CVD risk factors all promote OxS and endothelial dysfunction as the first steps in a cascade of pathological events. Elevated OxS leads to the overproduction of oxLDL and the latter has accepted as one of the new systemic markers of the development of CVD (Bonaterra et al., 2010). The higher levels of circulating oxLDL are strongly (much more than LDL-cholesterol) associated with an increased incidence of metabolic syndrome already in people who are currently young and healthy according to a large population-based study (Holvoet et al., 2008). Next, oxLDL is an important determinant of structural changes of the arteries already in asymptomatic persons (Kals et al., 2006; Kampus et al., 2007). An increased production of atherogenic and inflammatory oxLDL within the vessel wall suppresses immunity-related cells, including regulatory T cells (George, 2008) exerting antiatherogenic and antiallergic effects.

The influence of strain LfM-3 on host systemic OxS markers has been showed also via the decline of the values of isoprostanes and 8-OHdG in urine (Kullisaar et al., 2003, 2008; Songisepp et al., 2005). These indices are very informative for systemic OxS burden (Halliwell & Gutteridge, 1999). Evidently the systemic antioxidative effect of strain LfME-3 begins from the alleviation of the OxS- and inflammation-related abnormalities in the GI cells that lead to the assembling of particles of chylomicrons, LDL and HDL with a higher

2011) that emphasize an impact of GSH and its redox ratio for the quantification of OxS and the signalling role of GSH, described previously (Karelson et al., 2002; Zilmer et al., 2005); and b) there exists the possibility for the effective participation of LfM-3 in both enzymatic and non-enzymatic glutathione-driven protection as this strain carries all components needed for functionality of complete glutathione system (Kullisaar et al, 2010). It is interesting to add that recently it has been shown that just *L. fermentum* as a species significantly counteracted the depletion of colonic GSH content induced by some inflammatory processes (Peran et al., 2007) that also supported our understandings. There exists also a correlation between the glutathione redox ratio and DNA oxidative damages (de la Asuncion et al., 1996). Thus, consumption of multivalent probiotic LfME-3, which produces glutathione and has complete glutathione redox cycle enzymes (GPx and GRed), may contribute to the reduction of lipid hydroperoxides in the GI tract and in hepatocytes and prevent them from entering the circulation (Kullisaar et al., 2010). This may lead to an

Data showed that the improvement of the intestinal extra- and intracellular environment yielded beneficial changes of some general (systemic) biochemical indices of the host organism*.* The administration of LfME-3 to healthy volunteers and atopic adults results in a reduction of LP and a counterbalance of the glutathione system both in blood and in skin. In addition, in several trials LfME-3 has beneficial effect on the blood LDL fraction: the prolongation of its resistance to oxidation, the lowering of the content of oxLDL (a potent inflammatory and atherogenic factor) and BDC-LDL and the enhancement of the TAS of sera (Kullisaar et al., 2003, 2011; Songisepp et al., 2005; Mikelsaar et al., 2008). In trial on elderly persons the lower content of oxLDL was significantly predicted by the higher count of live lactobacilli in the GI tract*.* Evidently, both the number special antioxidative characteristics of strain LfME-3 and the increase in lactobacilli counts induced by administration of LfME-3 are responsible for such effect on host lipoprotein circulation/metabolism. As we mentioned before, the status of OxS and blood lipoproteins are both related to the pathogenesis of different diseases, including inflammation-related diseases and CVD. Dzau et al (2006) presented in *Circulation* the pathophysiological continuum showing that traditional CVD risk factors all promote OxS and endothelial dysfunction as the first steps in a cascade of pathological events. Elevated OxS leads to the overproduction of oxLDL and the latter has accepted as one of the new systemic markers of the development of CVD (Bonaterra et al., 2010). The higher levels of circulating oxLDL are strongly (much more than LDL-cholesterol) associated with an increased incidence of metabolic syndrome already in people who are currently young and healthy according to a large population-based study (Holvoet et al., 2008). Next, oxLDL is an important determinant of structural changes of the arteries already in asymptomatic persons (Kals et al., 2006; Kampus et al., 2007). An increased production of atherogenic and inflammatory oxLDL within the vessel wall suppresses immunity-related cells, including regulatory T cells (George, 2008) exerting

The influence of strain LfM-3 on host systemic OxS markers has been showed also via the decline of the values of isoprostanes and 8-OHdG in urine (Kullisaar et al., 2003, 2008; Songisepp et al., 2005). These indices are very informative for systemic OxS burden (Halliwell & Gutteridge, 1999). Evidently the systemic antioxidative effect of strain LfME-3 begins from the alleviation of the OxS- and inflammation-related abnormalities in the GI cells that lead to the assembling of particles of chylomicrons, LDL and HDL with a higher

improvement of systemic picture in the host organism.

antiatherogenic and antiallergic effects.

bioquality (with lower levels of harmful oxidation products) and higher concentrations of antioxidant factors/enzymes. The higher bioquality of assembled lipoprotein particles leads to improvement of their metabolism/circulation in the host body. This is one of the explanations why strain LfME-3 exerted the prolonged resistance of the blood lipoprotein fraction to oxidation, lowered the level of oxLDL and enhanced the TAC of sera in both healthy and problematic consumers (Kullisaar et al., 2003, 2006, 2008, 2011; Songisepp et al., 2005). Recently it was showed that administration of strain LfME-3 alleviated the postprandial elevation of TG levels in the blood, and improves HDL bioquality (elevates of paraoxonase level in HDL particles) (Kullisaar et al., 2006; 2008; 2011). The antioxidant activity of HDL can be expressed via several mechanisms (Bruckert & Hansel B, 2009). Paraoxonase (PON), an antioxidant enzyme associated with HDL, hydrolyzes oxidized phospholipids and inhibits the LDL oxidation that is an important step in atherogenesis. In animals, the addition of oxidized lipids to the circulation reduces PON activity, and diets rich in oxidized fat accelerate the development of aterogenesis (Sutherland et al., 1999). Removal and inactivation of lipid peroxides accumulating during LDL oxidation may be the central mechanism accounting for HDL antioxidative properties and when HDL particles have poor bioquality (low antioxidant properties and anti-atherosclerotic potency), they may have even inflammatory effect (Navab et al., 2006). The increase in PON activity after LfME-3 consumption shows that protection of LDL particles against oxidative modification by ROS is improved. PON inhibits atherogenesis by hydrolyzing lipid hydroperoxides and cholesterol ester hydroperoxides, reducing peroxides to the hydroxides, and hydrolyzing homocycteine thiolactone which prevents protein homocycteinylation (Beltowski et al., 2003; Durrington et al., 2005). Therefore, an elevation of PON activity should decrease the level of oxLDL. Antioxidant action of HDL is noted as one of the principal mechanisms mediating its cardioprotective effect (Hansel et al., 2006). It should be noted that HDL-associated antioxidant activity information is also supported both by data of anti-inflammatory effects of strain LfM-3 on the liver (Truusalu, et al., 2008) and by a hepato-protective role for PON against inflammation and liver disease mediated by OxS (Marsillach et al., 2009). Next, it is accepted that postprandial abnormal events are crucial concerning the development of CVD (Lopez-Miranda et al., 2006). Recently a postprandial decrease of three different OxS-related parameters (oxLDL, BCD-LDL, Beta2- GPI-OxLDL) was established (Kullisaar et al., 2011)*.* Thus, the foodstuffs enriched with LfME-3 substantially improves postprandial indices both of lipid/lipoproteins and OxS (Kullisaar et al., 2006; 2008; 2011). The beneficial influence of such enriched food on the postprandial lipid metabolism and OxS is important as many links between OxS and metabolic syndrome occur during the postprandial period. These include an excessive and prolonged elevation of blood TG levels, impairment of the endothelial function, an intestinal overproduction of chylomicrons, a redundant load for insulin production, the elevation of levels of atherogenic oxLDL and possible disturbances in the antioxidative activity of HDL (Bae et al., 2001; Jackson et al., 2007; Perez-Martinez et al., 2009; Hopps et al.,2010). To summarize, a positive modulation of the postprandial situation, including postprandial OxS, is an important target for dietary preventive actions concerning cardiovascular diseases.

#### **6. Possibilities of the oxidative stress-targeted administration of probiotics**

#### **6.1 Functional food and capsules**

Functional foods are foods or dietary components (incl. probiotics) that may provide a health benefit beyond basic nutrition. Probiotic products may be conventional foods

Probiotics and Oxidative Stress 213

to the protective effect, which affects the survival of the ingested probiotic, milk contains lactose, minerals, vitamins and bioactive peptides, which enhance the metabolic activity of

Probiotics have been advocated for the prevention and treatment of a wide range of diseases, and there is a growing evidence for their efficacy in some clinical scenarios. Probiotics are now widely used in many countries by consumers and in clinical practice. Given the increasingly widespread use of probiotics, a thorough understanding of their benefits is imperative. The properties of different probiotic species vary and can be strainspecific. Therefore, the effects of one probiotic strain should not be generalized to others without confirmation in separate studies. The proposed health benefits of probiotics have undergone increasingly rigorous scientific evaluation in recent years, and there is now

A meta-analysis of randomized controlled trials (RCTs) has shown that many probiotics are effective in preventing antibiotic-associated diarrhoea (McFarland, 2006; Ruszczynski et al., 2008). A separate meta-analysis of RCTs has shown a variety of probiotics to be effective in the treatment of infective diarrhea in both adults and children (Allen et al., 2011) acute waterly diarrhoea (Dutta et al., 2011), *C. difficile* diarrhoea (Plummer et al., 2004), ulcerative colitis and necrotizing enterocolitis (Sari et al., 2011). There is also support from RCTs for the efficacy of a probiotic mix in patients with inflammatory bowel disease (Kajander et al., 2007; Hovyeda et al., 2009). Nevertheless the evidence to date suggests that the major clinical effects of probiotics are seen in prevention GI disorders, probiotic therapy has also been explored in non-GI diseases, including the treatment of atopic eczema in children and adults (Kalliomaki et al., 2001, 2007; Kaur et al., 2008). A specialprobiotic, LfME-3, offers a good potential also in cardiovascular health management. LfME-3 an antioxidativeantiatherogenic and antimicrobial probiotic decreases OxS level in human body. The foodstuffs enriched with this probiotic decrease the level of oxidized LDL, increases the level of HDL, modulates postprandial lipid profile and OxS, and decreases the level of 8 isoprostanes in urine (the markers of systemic OxS) and body overall OxS-load, indicating an atherogenic potential (Kullisaar et al., 2002, 2003, 2011; Songisepp et al., 2005; Mikelsaar,

Intense physical activity increases oxygen consumption and inflammation induced by tissue damage and the probiotic consumption decreased the OxS level (Martarelli et al., 2011). The emerging evidence of a role for GI microbiota on central nervous system functions suggests that the oral intake of probiotics may have benecial consequences on mood and psychological distress by the competitive exclusion of deleterious GI pathogens, decreases in proinflammatory cytokines and communication with the CNS, leading to changes in neurotransmitter level or function (Logan; Katzman, 2005; Messaoudi et al., 2011). Probiotics are widely used to promote host health. Despite the huge amount of *in vitro* and *in vivo* studies (including cell culture, animal and human studies) we still lack data on the exact mechanisms involved. Our recent results by using MALDI-TOF spectrometry proteomic analysis confirmed that the concentration of glutathione in the blood of the probiotic LfME-3 users increases substantially; that is in good correlation with earlier results. Thus, new proteomic and metabolomic data about LAB and the relation between the colonic microbiota

**6.2 Special clinical trial with lactobacillus strains concerning oxidative stress** 

strong evidence for their use in treating and preventing some human diseases.

the ingested probiotic strain in the GI tract.

Zilmer, 2009, Table 2).

consumed for nutritional purposes, but also for the probiotic effect or "medical foods" - the primary purpose is that food formulation is a delivery vehicle for probiotics or metabiotics (beneficial by-products of probiotics). Probiotics are also available as dietary supplements in capsule, powder or liquid extract form. In functional food products no more than two probiotic strains are used in combination as a rule. Probiotic dietary supplements can consist of one single strain or mixed cultures of two or even more strains. There is some evidence that multi-strain probiotic mixtures could be more effective than single strains, including strains that are components of the mixtures themselves (Chapman et al., 2011).

Many functional foods can be found in a form of synbiotics. Synbiotics have been dened as mixtures of probiotics and prebiotics (dietary fiber) (Schrezenmeir & de Vrese, 2001; Saulner et al., 2007). One of the main benefits of synbiotics is the increased persistence of probiotics in the GI tract. Probiotic dietary supplements (capsules, powders and chewing tablets) often additionally contain amino acids, vitamins and/or prebiotics. Probiotic functional foods could be fermented or non-fermented foods. Traditionally dairy products are the carriers of probiotics. A large variety of probiotic dairy products with particular functional properties are available on the market worldwide. Fermented dairy products, especially yoghurts and yoghurt-like products are most widely used. There is a technological reason for using dairy products as probiotic carriers: dairy products have been optimized for the survival of starter cultures (mostly LAB) and are relatively easily adapted to grant the survival of probiotic strains as well. Besides, dairy products have other advantages over other formulations. Dairy foods are refrigerated. Probiotic bacteria in cultured dairy products benefit, as they remain the most stable in a refrigerated storage condition.

Cheese is used as a probiotic vehicle to a lesser extent than fermented milk products (Songisepp et al., 2004; Ross et al., 2005, Ibrahim et al*.,* 2010). Cheese (especially cheddar) may offer certain advantages over other probiotic products such as yogurt or milk. The cheese is a protective environment for the microbes, as the anaerobic conditions, relatively high fat content and buffering capacity of the cheese matrix helps to protect the probiotic strain in the product. The longer cheese is aged for, the higher density of probiotic microbes and metabiotics it will contain. Although the sensitivity of probiotics to physical and chemical stress, heat and acidity makes the product development challenging for other type of food products, probiotics in addition to dairy have been applied in nontraditional foods such as chocolate, energy bars, juices, smoothies, vegetables, breakfast cereals and even meat products like salami etc (Saarela et al., 2000, Siro et al., 2008).

The physiological state of the bacteria in a functional product is an important factor for the survival of the probiotic strain in the product, but most important is the manifestation of functional/health promoting properties in the human body after ingestion. There is a crucial difference between functional food and dietary supplements concerning the physiological state of the probiotic culture. Microbes are often freeze dried by the process of lyophilization before being manufactured as a dietary supplement (free-flowing powders, capsules, tablets). The dryness keeps the probiotic in a quiescent state during storage, while in food products the bacteria are in a vegetative state with a potentially active metabolism. Besides, dried probiotic cultures may have undergone several stressful processes during their production that damage the cells and may affect their viability (Champagne et al., 2011). Milk as a delivery vehicle has a dual effect on the probiotic additive: the buffering capacity of milk protects the viability of the strain against the stomach's acidic conditions. In addition

consumed for nutritional purposes, but also for the probiotic effect or "medical foods" - the primary purpose is that food formulation is a delivery vehicle for probiotics or metabiotics (beneficial by-products of probiotics). Probiotics are also available as dietary supplements in capsule, powder or liquid extract form. In functional food products no more than two probiotic strains are used in combination as a rule. Probiotic dietary supplements can consist of one single strain or mixed cultures of two or even more strains. There is some evidence that multi-strain probiotic mixtures could be more effective than single strains, including

Many functional foods can be found in a form of synbiotics. Synbiotics have been dened as mixtures of probiotics and prebiotics (dietary fiber) (Schrezenmeir & de Vrese, 2001; Saulner et al., 2007). One of the main benefits of synbiotics is the increased persistence of probiotics in the GI tract. Probiotic dietary supplements (capsules, powders and chewing tablets) often additionally contain amino acids, vitamins and/or prebiotics. Probiotic functional foods could be fermented or non-fermented foods. Traditionally dairy products are the carriers of probiotics. A large variety of probiotic dairy products with particular functional properties are available on the market worldwide. Fermented dairy products, especially yoghurts and yoghurt-like products are most widely used. There is a technological reason for using dairy products as probiotic carriers: dairy products have been optimized for the survival of starter cultures (mostly LAB) and are relatively easily adapted to grant the survival of probiotic strains as well. Besides, dairy products have other advantages over other formulations. Dairy foods are refrigerated. Probiotic bacteria in cultured dairy products benefit, as they

Cheese is used as a probiotic vehicle to a lesser extent than fermented milk products (Songisepp et al., 2004; Ross et al., 2005, Ibrahim et al*.,* 2010). Cheese (especially cheddar) may offer certain advantages over other probiotic products such as yogurt or milk. The cheese is a protective environment for the microbes, as the anaerobic conditions, relatively high fat content and buffering capacity of the cheese matrix helps to protect the probiotic strain in the product. The longer cheese is aged for, the higher density of probiotic microbes and metabiotics it will contain. Although the sensitivity of probiotics to physical and chemical stress, heat and acidity makes the product development challenging for other type of food products, probiotics in addition to dairy have been applied in nontraditional foods such as chocolate, energy bars, juices, smoothies, vegetables, breakfast cereals and even meat

The physiological state of the bacteria in a functional product is an important factor for the survival of the probiotic strain in the product, but most important is the manifestation of functional/health promoting properties in the human body after ingestion. There is a crucial difference between functional food and dietary supplements concerning the physiological state of the probiotic culture. Microbes are often freeze dried by the process of lyophilization before being manufactured as a dietary supplement (free-flowing powders, capsules, tablets). The dryness keeps the probiotic in a quiescent state during storage, while in food products the bacteria are in a vegetative state with a potentially active metabolism. Besides, dried probiotic cultures may have undergone several stressful processes during their production that damage the cells and may affect their viability (Champagne et al., 2011). Milk as a delivery vehicle has a dual effect on the probiotic additive: the buffering capacity of milk protects the viability of the strain against the stomach's acidic conditions. In addition

strains that are components of the mixtures themselves (Chapman et al., 2011).

remain the most stable in a refrigerated storage condition.

products like salami etc (Saarela et al., 2000, Siro et al., 2008).

to the protective effect, which affects the survival of the ingested probiotic, milk contains lactose, minerals, vitamins and bioactive peptides, which enhance the metabolic activity of the ingested probiotic strain in the GI tract.

#### **6.2 Special clinical trial with lactobacillus strains concerning oxidative stress**

Probiotics have been advocated for the prevention and treatment of a wide range of diseases, and there is a growing evidence for their efficacy in some clinical scenarios. Probiotics are now widely used in many countries by consumers and in clinical practice. Given the increasingly widespread use of probiotics, a thorough understanding of their benefits is imperative. The properties of different probiotic species vary and can be strainspecific. Therefore, the effects of one probiotic strain should not be generalized to others without confirmation in separate studies. The proposed health benefits of probiotics have undergone increasingly rigorous scientific evaluation in recent years, and there is now strong evidence for their use in treating and preventing some human diseases.

A meta-analysis of randomized controlled trials (RCTs) has shown that many probiotics are effective in preventing antibiotic-associated diarrhoea (McFarland, 2006; Ruszczynski et al., 2008). A separate meta-analysis of RCTs has shown a variety of probiotics to be effective in the treatment of infective diarrhea in both adults and children (Allen et al., 2011) acute waterly diarrhoea (Dutta et al., 2011), *C. difficile* diarrhoea (Plummer et al., 2004), ulcerative colitis and necrotizing enterocolitis (Sari et al., 2011). There is also support from RCTs for the efficacy of a probiotic mix in patients with inflammatory bowel disease (Kajander et al., 2007; Hovyeda et al., 2009). Nevertheless the evidence to date suggests that the major clinical effects of probiotics are seen in prevention GI disorders, probiotic therapy has also been explored in non-GI diseases, including the treatment of atopic eczema in children and adults (Kalliomaki et al., 2001, 2007; Kaur et al., 2008). A specialprobiotic, LfME-3, offers a good potential also in cardiovascular health management. LfME-3 an antioxidativeantiatherogenic and antimicrobial probiotic decreases OxS level in human body. The foodstuffs enriched with this probiotic decrease the level of oxidized LDL, increases the level of HDL, modulates postprandial lipid profile and OxS, and decreases the level of 8 isoprostanes in urine (the markers of systemic OxS) and body overall OxS-load, indicating an atherogenic potential (Kullisaar et al., 2002, 2003, 2011; Songisepp et al., 2005; Mikelsaar, Zilmer, 2009, Table 2).

Intense physical activity increases oxygen consumption and inflammation induced by tissue damage and the probiotic consumption decreased the OxS level (Martarelli et al., 2011). The emerging evidence of a role for GI microbiota on central nervous system functions suggests that the oral intake of probiotics may have benecial consequences on mood and psychological distress by the competitive exclusion of deleterious GI pathogens, decreases in proinflammatory cytokines and communication with the CNS, leading to changes in neurotransmitter level or function (Logan; Katzman, 2005; Messaoudi et al., 2011). Probiotics are widely used to promote host health. Despite the huge amount of *in vitro* and *in vivo* studies (including cell culture, animal and human studies) we still lack data on the exact mechanisms involved. Our recent results by using MALDI-TOF spectrometry proteomic analysis confirmed that the concentration of glutathione in the blood of the probiotic LfME-3 users increases substantially; that is in good correlation with earlier results. Thus, new proteomic and metabolomic data about LAB and the relation between the colonic microbiota

Probiotics and Oxidative Stress 215

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and host status could give new information regarding the mechanism of probiotic beneficial effects, including the effects on the OxS status of a host organism.

It has been demonstrated that functional food products with special *Lactobacillus sp.* strains have the potential to lower blood pressure (Naruszewicz et al., 2002). We demonstrated that semi-hard Edam-type cheese comprising the strain *L. plantarum* TENSIA (DSM 21380, property of Bio-Competence Centre of Healthy Dairy Products LLC) helps to maintain normal systolic and diastolic blood pressure in healthy adults and elderly subjects, thus supporting the functions of the cardiovascular system (Songisepp et al. 2009). Lately we have found that a 3-week consumption of 50g of probiotic cheese comprising *L. plantarum*  TENSIA (daily dose 1010 of probiotic viable cells per serving) decreased both diastolic (diapason of change: -3.67.1 (median -2.3; p=0.01) and systolic (diapason of change: -4.48.2 (median -4.0, p=0.01) blood pressure in adult subjects with high normal blood pressure (130- 139.5 mmHg).


Table 2. Effects of foodstuffs enriched with probiotic LfME-3 on both oxidative stress-related indices and HDL-cholesterol level of human body. oxLDL, oxidized low-density lipoprotein; BDC-LDL, baseline conjugated diene in LDL; GSSG, oxidized glutathione; GSH, reduced glutathione; HDL-Chol, high-density lipoprotein cholesterol

In the elderly, the consumption of the same amount of probiotic cheese in a somewhat lower daily dose of probiotic viable cells per serving (108) decreased both diastolic (diapason of change -4.0 ± 5.2mmHg, median -5mm Hg; p= 0.004) and systolic (diapason of change: - 5.9±13.4, median -12 mmHg; p=0.038).

It is repeatedly declared that new approaches in global CVD risk reduction are needed (Elliott, 2008). It is stated that for the prevention of CVD risk the anti-inflammatory agents and antioxidants are considered as a possible "third great wave"' (Bhatt, 2008). Evidently, the prevention complexes of several diseases could become more successful by including probiotics with a special multivalent (including antioxidative properties) biopotency.

#### **7. References**

Alberto, MR., Arena, ME., & Manca de Nadra, M.C. (2007). Putrescine production from agmatine by Lactobacillus hilgardii: effect of phenolic compounds. *Food Control,* 18, 898–903.

and host status could give new information regarding the mechanism of probiotic beneficial

It has been demonstrated that functional food products with special *Lactobacillus sp.* strains have the potential to lower blood pressure (Naruszewicz et al., 2002). We demonstrated that semi-hard Edam-type cheese comprising the strain *L. plantarum* TENSIA (DSM 21380, property of Bio-Competence Centre of Healthy Dairy Products LLC) helps to maintain normal systolic and diastolic blood pressure in healthy adults and elderly subjects, thus supporting the functions of the cardiovascular system (Songisepp et al. 2009). Lately we have found that a 3-week consumption of 50g of probiotic cheese comprising *L. plantarum*  TENSIA (daily dose 1010 of probiotic viable cells per serving) decreased both diastolic (diapason of change: -3.67.1 (median -2.3; p=0.01) and systolic (diapason of change: -4.48.2 (median -4.0, p=0.01) blood pressure in adult subjects with high normal blood pressure (130-

> BCD-LDL

participants 169 63 106 54 130 63

\*17% p<0.02

Table 2. Effects of foodstuffs enriched with probiotic LfME-3 on both oxidative stress-related indices and HDL-cholesterol level of human body. oxLDL, oxidized low-density lipoprotein; BDC-LDL, baseline conjugated diene in LDL; GSSG, oxidized glutathione; GSH, reduced

In the elderly, the consumption of the same amount of probiotic cheese in a somewhat lower daily dose of probiotic viable cells per serving (108) decreased both diastolic (diapason of change -4.0 ± 5.2mmHg, median -5mm Hg; p= 0.004) and systolic (diapason of change: -

It is repeatedly declared that new approaches in global CVD risk reduction are needed (Elliott, 2008). It is stated that for the prevention of CVD risk the anti-inflammatory agents and antioxidants are considered as a possible "third great wave"' (Bhatt, 2008). Evidently, the prevention complexes of several diseases could become more successful by including

Alberto, MR., Arena, ME., & Manca de Nadra, M.C. (2007). Putrescine production from

agmatine by Lactobacillus hilgardii: effect of phenolic compounds. *Food Control,* 18,

probiotics with a special multivalent (including antioxidative properties) biopotency.

Glutathione redox ratio (GSSG/GSH)

> \*33% p<0.03

Total antioxidative activity

**\*\***20% p<0.005

8-isoprostanes

> \*26% p<0.03

effects, including the effects on the OxS status of a host organism.

Chol

\*\*7% p<0.003

glutathione; HDL-Chol, high-density lipoprotein cholesterol

139.5 mmHg).

Number of

Decrease\* or increase\*\* of level compared to baseline

**7. References** 

898–903.

Marker oxLDL HDL-

5.9±13.4, median -12 mmHg; p=0.038).

\*16% p<0.03


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*1Department of Nutrition, Faculty of Health and Nutrition,* 

*3Department of Nutrition, Faculty of Health and Nutrition,* 

 *Tabriz University of Medical Sciences, Tabriz, 2Department of Food Science and Technology,* 

*Tabriz University of Medical Sciences, Tabriz,* 

*Tabriz University of Medical Sciences, Tabriz,* 

*Faculty of Health and Nutrition,* 

*Iran* 

B. Alipoor1\*, A. Homayouni Rad2 and E. Vaghef Mehrabany3

Diabetes mellitus is the most common serious metabolic disorder in the world. Diabetes is characterized by a hyperglycemia that results from an absolute or relative insulin deficiency and is associated with long term complications affecting the eyes, kidneys, heart and nerves (Baydas *et al*., 2003). Oxidative stress is defined as imbalance between the generation of reactive oxygen species and antioxidant defense capacity of the body that is closely associated with aging and a number of diseases including cancer, cardiovascular diseases, diabetes and diabetic complications (Atalay *et al*., 2002). Irregular cellular metabolism in diabetes leads to production of free oxygen radicals and imbalanced antioxidant capacity

Recent studies have shown that both types of diabetes can increase oxidative stress in blood and treatment with antioxidants such as vitamin E and flavonoids may be used for decreasing of oxidative stress and diabetic complications (Baydas *et al*., 2003; Vincent *et al*., 2004). There is good evidence that tea flavonoids intake have a role in protection against degenerative diseases and long-term intake of tea flavonoids can prevent obesity in high fat diet. Also it has positive effects against glucose metabolism disorders and diabetes-induced fat disorders that lead to lowering the risk of diabetes complications (Crespy *et al*., 2004). Flavonoids have antioxidant properties, and tea is one of the main sources of flavonoids. Tea (from the plant Camellia Sinensis) is the most popular beverage next to water, consumed by over two-thirds of the world's population. About three billion kilograms of tea are produced and consumed yearly (Yang, 2000; Gupta *et al*., 2002; Crespy *et al*., 2004). Regular intake of tea is associated with an improved antioxidant status in vivo conditions that may contribute to the lowering risk of certain types of cancer, coronary heart disease, atherosclerosis, stroke, reduced mutagenicity and inflammation, protection against neurodegenerative diseases and increasing insulin sensitivity (Luximon-Ramma *et al*., 2005;

**1. Introduction** 

Alipoor et al., 2011).

Corresponding Author

 \*

(oxidative stress) of the body (Vincent *et al*., 2004).

Zilmer, M., Soomets, U., Rehema, A., & Langel, Ü. (2005). The glutathione system as an attractice therapeutic target. *Drug Design Reviews-Online,* 2, 121-127. **11** 

B. Alipoor1\*, A. Homayouni Rad2 and E. Vaghef Mehrabany3

*1Department of Nutrition, Faculty of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz, 2Department of Food Science and Technology, Faculty of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz, 3Department of Nutrition, Faculty of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran* 

#### **1. Introduction**

222 Oxidative Stress – Environmental Induction and Dietary Antioxidants

Zilmer, M., Soomets, U., Rehema, A., & Langel, Ü. (2005). The glutathione system as an

Diabetes mellitus is the most common serious metabolic disorder in the world. Diabetes is characterized by a hyperglycemia that results from an absolute or relative insulin deficiency and is associated with long term complications affecting the eyes, kidneys, heart and nerves (Baydas *et al*., 2003). Oxidative stress is defined as imbalance between the generation of reactive oxygen species and antioxidant defense capacity of the body that is closely associated with aging and a number of diseases including cancer, cardiovascular diseases, diabetes and diabetic complications (Atalay *et al*., 2002). Irregular cellular metabolism in diabetes leads to production of free oxygen radicals and imbalanced antioxidant capacity (oxidative stress) of the body (Vincent *et al*., 2004).

Recent studies have shown that both types of diabetes can increase oxidative stress in blood and treatment with antioxidants such as vitamin E and flavonoids may be used for decreasing of oxidative stress and diabetic complications (Baydas *et al*., 2003; Vincent *et al*., 2004). There is good evidence that tea flavonoids intake have a role in protection against degenerative diseases and long-term intake of tea flavonoids can prevent obesity in high fat diet. Also it has positive effects against glucose metabolism disorders and diabetes-induced fat disorders that lead to lowering the risk of diabetes complications (Crespy *et al*., 2004). Flavonoids have antioxidant properties, and tea is one of the main sources of flavonoids. Tea (from the plant Camellia Sinensis) is the most popular beverage next to water, consumed by over two-thirds of the world's population. About three billion kilograms of tea are produced and consumed yearly (Yang, 2000; Gupta *et al*., 2002; Crespy *et al*., 2004). Regular intake of tea is associated with an improved antioxidant status in vivo conditions that may contribute to the lowering risk of certain types of cancer, coronary heart disease, atherosclerosis, stroke, reduced mutagenicity and inflammation, protection against neurodegenerative diseases and increasing insulin sensitivity (Luximon-Ramma *et al*., 2005; Alipoor et al., 2011).

<sup>\*</sup> Corresponding Author

tolerance (IGT). Although lifestyle modifications are difficult to maintain, there is evidence that intensive intervention results in continued preventive benefit after the stopping of

The prevalence of diabetes for all age-groups worldwide was estimated to be 2.8% in 2000 and 4.4% in 2030. The total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030. Quantifying the prevalence of diabetes and the number of people affected by diabetes, now and in the future, is important to allow rational

Diabetes is a heterogeneous disorder both genetically and clinically and is hyperglycemia, attributable to either insulin insufficiency or insulin resistance. The traditional classification separates out hyperglycemic conditions into these groups: insulin-dependant diabetes mellitus (IDDM or type 1), non-insulin-dependent diabetes mellitus (NIDDM or type 2), other specific types of diabetes and gestational diabetes mellitus (GDM). Type 1 diabetes accounts for approximately 5% of diabetes and is manifested by insulin deficiency caused by destruction of the pancreatic β cells. Type 2 diabetes accounts for about 90% of diabetes and is characterized by two primary defects: insulin resistance (diminished tissue sensitivity to insulin) and impaired β-cell function (delayed or inadequate insulin release). Other causes account for the remaining 5% of diabetes. Classic symptoms such as polydipsia, polyuria, and rapid weight loss associated with gross and unequivocal elevation of blood glucose (≥200 mg/dl) make the diagnosis of Diabetes mellitus. A fasting plasma glucose level greater than or equal to 126 mg/dl on two occasions is diagnostic of diabetes (Shils *et al*.,

Associated to insulin-dependent diabetes (type 1), makes the disease one of the worst by considering the human suffering and the socio-economic trouble. In developed countries the number of diabetic patients is increasing all the time and both inability and mortality values are staggering. There is a dedication of studies aiming first to block or slow down the onset of type 1 diabetes, secondly to identify the numerous environmental and genetic factors causing type 2 diabetes and thirdly to suggest possible ways for the prevention or the postponement of crippling complications. The initial problem of diabetes is the hyperglycemia due to the inability of several control systems to maintain a normal glycemic plasma level. A first question is: can diabetic complications be prevented or delayed by normalizing hyperglycemia? This can be achieved at least in part if a meticulous control of glycemia is kept with an appropriate diet, oral anti-diabetic drugs, or insulin administration associated with daily exercise and a correct lifestyle. However, owing to genetic factors and in spite of a serious control, complications are found even in patients with a transitory and slight hyperglycemia. Circulatory abnormalities are the common denominator and they are present under the form of micro- and macro-vascular disease. Throughout the years the following complications may develop with different

structured counseling (Bharati *et al*., 2011).

**2.3 Classification and diagnosis** 

2006).

**2.4 Diabetic complication** 

intensity and localization:

planning and allocation of resources (Wild *et al*., 2011).

### **2. Diabetes**

Diabetes resembles fasting, especially regarding the responses of the liver, muscle cells, and adipose tissues. With low serum ratios of insulin to glucagon and high levels of fatty acids, the liver produces glucose, whereas other tissues use fatty acids and ketones instead of glucose. Muscle cells and adipose tissue respond by using ketones and fatty acids. Although these resemblances between fasting and diabetes are striking, pathologically low serum insulin levels disrupt the efficiency seen during fasting. With low insulin levels, key glycolytic enzyme activities decrease. Glucose use decreases to levels far below those seen during fasting. Concurrently, hepatic gluconeogenic enzyme activities and gluconeogenic rates increase.Bombarded with free fatty acids, the liver increases gluconeogenesis, secreting large amounts of very low density lipoproteins (VLDLs) and accumulating fatty acids in droplet form. A long-term toxic effect of diabetes is the accumulation of 25% more lipid than normal. In the diabetic state, the liver oxidizes these fatty acids and produces acetone, acetoacetate, and β-hydroxybutyrate. Muscle cells and adipose tissue also show major metabolic changes in diabetes. Muscle glycogen almost disappears, and muscle protein is broken down to support gluconeogenesis. Cardiac and skeletal muscles meet their energy needs from ketones and fatty acids. Fat cells actively release fatty acids under the lipolytic stimuli of glucagon, catecholamines, and insulin deficiency (Shils *et al*., 2006).

#### **2.1 Historical overview**

Diabetes mellitus is a chronic disease that has affected mankind throughout the world. The records of the ancient civilizations of Egypt, India, Japan, Greece, and Rome describe the symptoms of the disease and usually include recommendations for treatment. The wasting away of flesh, copious urination, and the sweet taste of the urine were frequently noted by the ancient medical writers. Aretaeus of Cappadocia, who lived between A.D. 30 and 90, not only named the disease diabetes, which means "to run through or to siphon" but also recommended, "The food is to be milk and with in the cereals, starch, autumn fruits and sweet wines". The term mellitus, which means honey like, was added by a London physician, Willis, in 1675 (Robinson, 1972).

#### **2.2 Epidemiology and etiology**

Diabetes mellitus has reached epidemic proportions worldwide. There is an apparent epidemic of diabetes which is strongly related to lifestyle and economic change (WHO). Over the next decade the projected number will exceed 200 million. Most will have type2 diabetes, and all are at risk of the development of complications. Diabetes mellitus is a heterogeneous group of diseases that develops dangerously and characterized by a state of chronic hyperglycemia, resulting from a diversity of etiologies, environmental and genetic. Diabetes mellitus is increasing due to population growth, aging, consequences of industrialization and urbanization, preference of high fat containing fast foods, sedentary life and obesity. Given the enormous public health and economic burden posed by the global epidemic of type 2 diabetes mellitus (T2DM), intervention in the pre-diabetes stage of disease to prevent progression to T2DM and its vascular complications seems the most sensible approach. Prudent lifestyle changes have been shown to significantly reduce the risk of progression in individuals with impaired fasting glucose (IFG) and impaired glucose

Diabetes resembles fasting, especially regarding the responses of the liver, muscle cells, and adipose tissues. With low serum ratios of insulin to glucagon and high levels of fatty acids, the liver produces glucose, whereas other tissues use fatty acids and ketones instead of glucose. Muscle cells and adipose tissue respond by using ketones and fatty acids. Although these resemblances between fasting and diabetes are striking, pathologically low serum insulin levels disrupt the efficiency seen during fasting. With low insulin levels, key glycolytic enzyme activities decrease. Glucose use decreases to levels far below those seen during fasting. Concurrently, hepatic gluconeogenic enzyme activities and gluconeogenic rates increase.Bombarded with free fatty acids, the liver increases gluconeogenesis, secreting large amounts of very low density lipoproteins (VLDLs) and accumulating fatty acids in droplet form. A long-term toxic effect of diabetes is the accumulation of 25% more lipid than normal. In the diabetic state, the liver oxidizes these fatty acids and produces acetone, acetoacetate, and β-hydroxybutyrate. Muscle cells and adipose tissue also show major metabolic changes in diabetes. Muscle glycogen almost disappears, and muscle protein is broken down to support gluconeogenesis. Cardiac and skeletal muscles meet their energy needs from ketones and fatty acids. Fat cells actively release fatty acids under the lipolytic

stimuli of glucagon, catecholamines, and insulin deficiency (Shils *et al*., 2006).

Diabetes mellitus is a chronic disease that has affected mankind throughout the world. The records of the ancient civilizations of Egypt, India, Japan, Greece, and Rome describe the symptoms of the disease and usually include recommendations for treatment. The wasting away of flesh, copious urination, and the sweet taste of the urine were frequently noted by the ancient medical writers. Aretaeus of Cappadocia, who lived between A.D. 30 and 90, not only named the disease diabetes, which means "to run through or to siphon" but also recommended, "The food is to be milk and with in the cereals, starch, autumn fruits and sweet wines". The term mellitus, which means honey like, was added by a London

Diabetes mellitus has reached epidemic proportions worldwide. There is an apparent epidemic of diabetes which is strongly related to lifestyle and economic change (WHO). Over the next decade the projected number will exceed 200 million. Most will have type2 diabetes, and all are at risk of the development of complications. Diabetes mellitus is a heterogeneous group of diseases that develops dangerously and characterized by a state of chronic hyperglycemia, resulting from a diversity of etiologies, environmental and genetic. Diabetes mellitus is increasing due to population growth, aging, consequences of industrialization and urbanization, preference of high fat containing fast foods, sedentary life and obesity. Given the enormous public health and economic burden posed by the global epidemic of type 2 diabetes mellitus (T2DM), intervention in the pre-diabetes stage of disease to prevent progression to T2DM and its vascular complications seems the most sensible approach. Prudent lifestyle changes have been shown to significantly reduce the risk of progression in individuals with impaired fasting glucose (IFG) and impaired glucose

**2. Diabetes** 

**2.1 Historical overview** 

physician, Willis, in 1675 (Robinson, 1972).

**2.2 Epidemiology and etiology** 

tolerance (IGT). Although lifestyle modifications are difficult to maintain, there is evidence that intensive intervention results in continued preventive benefit after the stopping of structured counseling (Bharati *et al*., 2011).

The prevalence of diabetes for all age-groups worldwide was estimated to be 2.8% in 2000 and 4.4% in 2030. The total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030. Quantifying the prevalence of diabetes and the number of people affected by diabetes, now and in the future, is important to allow rational planning and allocation of resources (Wild *et al*., 2011).

#### **2.3 Classification and diagnosis**

Diabetes is a heterogeneous disorder both genetically and clinically and is hyperglycemia, attributable to either insulin insufficiency or insulin resistance. The traditional classification separates out hyperglycemic conditions into these groups: insulin-dependant diabetes mellitus (IDDM or type 1), non-insulin-dependent diabetes mellitus (NIDDM or type 2), other specific types of diabetes and gestational diabetes mellitus (GDM). Type 1 diabetes accounts for approximately 5% of diabetes and is manifested by insulin deficiency caused by destruction of the pancreatic β cells. Type 2 diabetes accounts for about 90% of diabetes and is characterized by two primary defects: insulin resistance (diminished tissue sensitivity to insulin) and impaired β-cell function (delayed or inadequate insulin release). Other causes account for the remaining 5% of diabetes. Classic symptoms such as polydipsia, polyuria, and rapid weight loss associated with gross and unequivocal elevation of blood glucose (≥200 mg/dl) make the diagnosis of Diabetes mellitus. A fasting plasma glucose level greater than or equal to 126 mg/dl on two occasions is diagnostic of diabetes (Shils *et al*., 2006).

#### **2.4 Diabetic complication**

Associated to insulin-dependent diabetes (type 1), makes the disease one of the worst by considering the human suffering and the socio-economic trouble. In developed countries the number of diabetic patients is increasing all the time and both inability and mortality values are staggering. There is a dedication of studies aiming first to block or slow down the onset of type 1 diabetes, secondly to identify the numerous environmental and genetic factors causing type 2 diabetes and thirdly to suggest possible ways for the prevention or the postponement of crippling complications. The initial problem of diabetes is the hyperglycemia due to the inability of several control systems to maintain a normal glycemic plasma level. A first question is: can diabetic complications be prevented or delayed by normalizing hyperglycemia? This can be achieved at least in part if a meticulous control of glycemia is kept with an appropriate diet, oral anti-diabetic drugs, or insulin administration associated with daily exercise and a correct lifestyle. However, owing to genetic factors and in spite of a serious control, complications are found even in patients with a transitory and slight hyperglycemia. Circulatory abnormalities are the common denominator and they are present under the form of micro- and macro-vascular disease. Throughout the years the following complications may develop with different intensity and localization:

through its derivative, the hydroxyl radical, stimulates the activation of guanylate cyclase and formation of the "second messenger" cGMP. Similar effects were reported for the superoxide derivative hydrogen peroxide. It was discovered that nitric oxide (NO) has independently role as a regulatory molecule in the control of smooth muscle relaxation and

Also it is found that in activated T-cells the superoxide anion or low micromolar concentrations of hydrogen peroxide increase the production of the T-cell growth factor, interleukin-2 which is an immunologically important T-cell protein. Studies have shown that hydrogen peroxide induces the expression of the heme oxygenase (HO-1) gene and hydrogen peroxide has induction effects on various genes in bacteria, as well as activation of the transcription factor nuclear factor κB (NF-κB) in mammalian cells. At the beginning of the 21st century, there is a large amounts of evidence showing that living organisms have not only adapted to an unfriendly coexistence with free radicals but have, in fact, developed mechanisms for the advantageous use of free radicals. Important physiological functions that involve free radicals or their derivatives include the following: regulation of vascular tone, sensing of oxygen tension and regulation of functions that are controlled by oxygen concentration, enhancement of signal transduction from various membrane receptors including the antigen receptor of lymphocytes, and oxidative stress responses that ensure

The field of redox regulation is also receiving growing attention from clinical colleagues in view of the role that oxidative stress has been found to play in numerous disease conditions. These pathological conditions demonstrate the biological relevance of redox regulation. The delicate balance between the advantageous and detrimental effects of free radicals is clearly an important aspect of life. The science of biological "redox regulation" is a rapidly growing field of research that has impact on diverse disciplines including physiology, cell biology,

Measuring biomarkers of oxidative stress is an essential step toward better understanding the pathogenesis and developing treatments for diabetic. There are several approaches that may be adopted, including measurements of the depletion of antioxidant reserves, changes in the activities of antioxidant enzymes, free radical production, and presence of protein, lipid, and DNA free radical adducts. For the purposes of clinical assessment, measurements of end products of free radical attack may be the most reliable determination of the occurrence of oxidative stress because enzyme activities and cellular antioxidants are likely to display transient changes. The enzymes responsible for detoxifying free radicals or regenerating antioxidant molecules can provide an indication of the stress level experienced in a cell or tissue. These enzymes are usually measured by *in vitro* activity assays, although changes in transcription can also provide evidence of cell stress. In long-term diabetes, catalase, GSH reductase, GSH peroxidase, and SOD decrease in complication-prone tissue

There is a growing awareness that oxidative stress plays a role in various clinical conditions. Malignant diseases, diabetes, atherosclerosis, chronic inflammation, human immunodeficiency

in the inhibition of platelet adhesion (Droge, 2002).

the maintenance of redox homeostasis (Droge, 2002).

and clinical medicine (Droge, 2002).

**3.2 Biomarkers of oxidative stress** 

(Vincent *et al*., 2004).

**3.3 Oxidative stress and disease** 


Early detection and appropriate management of diabetes is essential to reduce major morbidity and mortality, however these strategies are not implemented in many countries of the world. In the diabetes centre in Isfahan*,* I.R. Iran*,* the rate of complications among approximately 4000 type 2 diabetes patients have been recorded as: ischemic heart disease 34%, hypertension 50%, congestive heart failure 12%, retinopathy 44%, cataract 5%, bacteriuria 27%, nephropathy 19%, neuropathy 27%,depression 60%, diabetic foot 2.5%, hypercholesterolemia 37%, and hypertriglyceridemia 37%. Among 296 cases of nontraumatic amputations, 38% were diabetes-related; 27% of stroke cases (cerebrovascular accident), 15% of patients with acute myocardial infarction and 15% of dialysis patients were also diabetics (Azizi *et al*., 2003).

#### **3. Oxidative stress**

Oxidative stress happens in a cellular system when the production of free radical moieties exceeds the antioxidant capacity of that system. If cellular antioxidants do not remove free radicals, radicals attack and damage proteins, lipids and nucleic acids. The oxidized or nitrosylated products of free radical attack have decreased biological activity, leading to loss of energy metabolism, cell signaling, transport, and other major functions. These altered products also are objected for proteosome degradation, further decreasing cellular function. Accumulation of such injury ultimately leads a cell to die through necrotic or apoptotic mechanisms (Vincent *et al*., 2004).

#### **3.1 Historical overview**

The presence of free radicals in biological materials was discovered less than 50 years ago. Thereafter, Denham Harman hypothesized that oxygen radicals may be formed as byproducts of enzymatic reactions in vivo*.* In 1956, he described free radicals as a Pandora's Box of evils that may account for gross cellular damage, mutagenesis, cancer, and last but not least, the degenerative process of biological aging. The science of free radicals in living organisms entered a second time after McCord and Fridovich discovered the enzyme superoxide dismutase (SOD) and, finally convinced most colleagues that free radicals are important in biology. Numerous researchers were now inspired to investigate oxidative damage inflicted by radicals upon DNA, proteins, lipids, and other components of the cell. A third period began with the first reports describing advantageous biological effects of free radicals. Mittal and Murard provided suggestive evidence that the superoxide anion,

1. Diabetic retinopathy is a leading cause of blindness in about 85% of patients aged 20-75

2. Diabetic nephropathy occurs in 20-40% of patients and when the GFR is <15 ml/min, the end stage renal disease (ESRD) is a leading cause of disability and premature death. 3. Diabetic foot disease normally caused by several factors such as peripheral vascular disease (PVD), altered biomechanics, possibly polyneuropathy and infected foot ulcers. 4. Neuropathy involving both the somatic and autonomic nervous system with neuromuscular dysfunction and muscular wasting is another major cause of morbidity. 5. Accelerated atherosclerosis frequently manifests itself with myocardial infarction,

6. Lipodistrophy, seemingly due to ineffective leptin activity or/and fatty acids dysmetabolism, represents another aspect of the metabolic syndrome (Bocci *et al*., 2011).

Early detection and appropriate management of diabetes is essential to reduce major morbidity and mortality, however these strategies are not implemented in many countries of the world. In the diabetes centre in Isfahan*,* I.R. Iran*,* the rate of complications among approximately 4000 type 2 diabetes patients have been recorded as: ischemic heart disease 34%, hypertension 50%, congestive heart failure 12%, retinopathy 44%, cataract 5%, bacteriuria 27%, nephropathy 19%, neuropathy 27%,depression 60%, diabetic foot 2.5%, hypercholesterolemia 37%, and hypertriglyceridemia 37%. Among 296 cases of nontraumatic amputations, 38% were diabetes-related; 27% of stroke cases (cerebrovascular accident), 15% of patients with acute myocardial infarction and 15% of dialysis patients

Oxidative stress happens in a cellular system when the production of free radical moieties exceeds the antioxidant capacity of that system. If cellular antioxidants do not remove free radicals, radicals attack and damage proteins, lipids and nucleic acids. The oxidized or nitrosylated products of free radical attack have decreased biological activity, leading to loss of energy metabolism, cell signaling, transport, and other major functions. These altered products also are objected for proteosome degradation, further decreasing cellular function. Accumulation of such injury ultimately leads a cell to die through necrotic or apoptotic

The presence of free radicals in biological materials was discovered less than 50 years ago. Thereafter, Denham Harman hypothesized that oxygen radicals may be formed as byproducts of enzymatic reactions in vivo*.* In 1956, he described free radicals as a Pandora's Box of evils that may account for gross cellular damage, mutagenesis, cancer, and last but not least, the degenerative process of biological aging. The science of free radicals in living organisms entered a second time after McCord and Fridovich discovered the enzyme superoxide dismutase (SOD) and, finally convinced most colleagues that free radicals are important in biology. Numerous researchers were now inspired to investigate oxidative damage inflicted by radicals upon DNA, proteins, lipids, and other components of the cell. A third period began with the first reports describing advantageous biological effects of free radicals. Mittal and Murard provided suggestive evidence that the superoxide anion,

stroke and limb vascular occlusion complicated with necrotic ulcers.

years.

were also diabetics (Azizi *et al*., 2003).

mechanisms (Vincent *et al*., 2004).

**3.1 Historical overview** 

**3. Oxidative stress** 

through its derivative, the hydroxyl radical, stimulates the activation of guanylate cyclase and formation of the "second messenger" cGMP. Similar effects were reported for the superoxide derivative hydrogen peroxide. It was discovered that nitric oxide (NO) has independently role as a regulatory molecule in the control of smooth muscle relaxation and in the inhibition of platelet adhesion (Droge, 2002).

Also it is found that in activated T-cells the superoxide anion or low micromolar concentrations of hydrogen peroxide increase the production of the T-cell growth factor, interleukin-2 which is an immunologically important T-cell protein. Studies have shown that hydrogen peroxide induces the expression of the heme oxygenase (HO-1) gene and hydrogen peroxide has induction effects on various genes in bacteria, as well as activation of the transcription factor nuclear factor κB (NF-κB) in mammalian cells. At the beginning of the 21st century, there is a large amounts of evidence showing that living organisms have not only adapted to an unfriendly coexistence with free radicals but have, in fact, developed mechanisms for the advantageous use of free radicals. Important physiological functions that involve free radicals or their derivatives include the following: regulation of vascular tone, sensing of oxygen tension and regulation of functions that are controlled by oxygen concentration, enhancement of signal transduction from various membrane receptors including the antigen receptor of lymphocytes, and oxidative stress responses that ensure the maintenance of redox homeostasis (Droge, 2002).

The field of redox regulation is also receiving growing attention from clinical colleagues in view of the role that oxidative stress has been found to play in numerous disease conditions. These pathological conditions demonstrate the biological relevance of redox regulation. The delicate balance between the advantageous and detrimental effects of free radicals is clearly an important aspect of life. The science of biological "redox regulation" is a rapidly growing field of research that has impact on diverse disciplines including physiology, cell biology, and clinical medicine (Droge, 2002).

#### **3.2 Biomarkers of oxidative stress**

Measuring biomarkers of oxidative stress is an essential step toward better understanding the pathogenesis and developing treatments for diabetic. There are several approaches that may be adopted, including measurements of the depletion of antioxidant reserves, changes in the activities of antioxidant enzymes, free radical production, and presence of protein, lipid, and DNA free radical adducts. For the purposes of clinical assessment, measurements of end products of free radical attack may be the most reliable determination of the occurrence of oxidative stress because enzyme activities and cellular antioxidants are likely to display transient changes. The enzymes responsible for detoxifying free radicals or regenerating antioxidant molecules can provide an indication of the stress level experienced in a cell or tissue. These enzymes are usually measured by *in vitro* activity assays, although changes in transcription can also provide evidence of cell stress. In long-term diabetes, catalase, GSH reductase, GSH peroxidase, and SOD decrease in complication-prone tissue (Vincent *et al*., 2004).

#### **3.3 Oxidative stress and disease**

There is a growing awareness that oxidative stress plays a role in various clinical conditions. Malignant diseases, diabetes, atherosclerosis, chronic inflammation, human immunodeficiency

To better understand the role of tea antioxidants in either preventing diabetes or reducing its complications, one must first know the mechanisms through which oxidative stress contributes to the development of this chronic disease and disorders following it. Some evidence on how tea antioxidants, in particular, can prevent diabetes development and its

As mentioned previously, type1 diabetes mellitus which is less prevalent than type 2 is a genetic autoimmune disorder affecting the islet cells leading to insulin deficiency and thus hyperglycemia. Type 2 diabetes however is a multi-factorial disease. Insulin resistance most often precedes the onset of this type by many years and can be caused by acquired factors. Elevations in glucose and free fatty acids have been shown to induce oxidative stress which in turn can play a key role in causing insulin resistance and β-cell dysfunction (Evans *et al*., 2002). One most favored hypothesis on how hyperglycemia and elevated free fatty acids (FFA) can lead to oxidative stress is that as energy intake exceeds energy expenditure, generation of excess mitochondrial NADH (mNADH) and the level of reactive oxygen species (ROS) is increased due to the greater activity of citric acid cycle, induced by the abundance of substrates. Reducing ROS formation or increasing its removal is the way through which cells can protect themselves. The mechanism of preventing excessive mNADH generation may be inhibiting insulin-stimulated nutrient uptake and preventing the entrance of pyruvate and fatty acids into the mitochondria. Either of glucose or FFA enters the citric acid cycle after being converted to acetyl-CoA which then combines with oxaloacetate to form citrate. Greater availability of substrates will result in greater production of mNADH which is beyond the capability of oxidative phosphorylation to dissipate it all, leading to increased mitochondrial proton gradient. Thus single electrons are transferred to molecular

One way the cells can reduce free radical generation is inhibition of FFA oxidation. This will increase intracellular FFA which in turn leads to reduced GLUT4 translocation to the plasma membrane resulting in resistance to insulin-stimulated glucose uptake in muscle and adipose tissue. *In vitro* studies have shown that antioxidants may have role in reducing

Chronic exposure to abnormally high levels of glucose and FFA leads to toxic effect on βcells of pancreas. Furthermore as aforementioned, hyperglycemia and high levels of FFA leads to increased oxidative stress. ß-cells are particularly susceptible to the damages inflicted by oxidative stress; since they are low in free radical quenching enzymes such as catalase, glutathione peroxidase and superoxide dismutase. ß-cells are responsible for the sensing glucose and secreting appropriate amount of insulin in response to glucose boots. This process is pretty complex, but the critical significance of mitochondrial glucose metabolism in linking stimulus to secretion is well established. This is one reason that oxidative stress can blunt insulin secretion due to its ability to damage mitochondria (Evans

Chronic oxidative stress can also affect insulin gene expression. At least two critical proteins that activate the insulin promoter are involved in defects in insulin gene expression. One is PDX-1 and the other is RIPE-3b1 activator recently identified as MafA. Glucose toxicity and lipotoxicity both of which lead to increased oxidative stress have been shown to leave

progression will be presented next.

oxygen forming free radicals, especially superoxide anion.

insulin resistance (Ceriello *et al*., 2004).

*et al.*, 2003; Robertson *et al.*, 2004).

deleterious effects on islet cells (Robertson, 2004).

virus (HIV) infection, ischemia reperfusion injury, and sleep apnea are important examples. These diseases fall into two major categories. In the first category, diabetes mellitus and cancer show commonly a pro-oxidative shift in the systemic thiol/disulfide redox state and impaired glucose clearance, suggesting that skeletal muscle mitochondria may be the major site of elevated reactive oxygen species (ROS) production. These conditions may be referred to as "mitochondrial oxidative stress." Without therapeutic intervention these conditions lead to massive skeletal muscle wasting, reminiscent of aging-related wasting. The second category may be referred to as "inflammatory oxidative conditions" because it is typically associated with an excessive stimulation of NAD(P)H oxidase activity by cytokines or other agents. In this case increased ROS levels or changes in intracellular glutathione levels are often associated with pathological changes indicative of a dysregulation of signal cascades and/or gene expression, exemplified by altered expression of cell adhesion molecules (Droge, 2002).

#### **3.4 Oxidative stress and diabetes**

Increased oxidative stress is widely accepted as a major role player in both development and progression of diabetes (Maritim *et al*, 2002). Figure 1 summarizes the relationship between oxidative stress, development of diabetes and resulting complications.

Fig. 1.Overnutrition leads to oxidative stress which in turn results in diabetes and its complications (FFA: Free Fatty Acids, IGT: Impaired Glucose Tolerance, CVD: Cardiovascular Disease)

virus (HIV) infection, ischemia reperfusion injury, and sleep apnea are important examples. These diseases fall into two major categories. In the first category, diabetes mellitus and cancer show commonly a pro-oxidative shift in the systemic thiol/disulfide redox state and impaired glucose clearance, suggesting that skeletal muscle mitochondria may be the major site of elevated reactive oxygen species (ROS) production. These conditions may be referred to as "mitochondrial oxidative stress." Without therapeutic intervention these conditions lead to massive skeletal muscle wasting, reminiscent of aging-related wasting. The second category may be referred to as "inflammatory oxidative conditions" because it is typically associated with an excessive stimulation of NAD(P)H oxidase activity by cytokines or other agents. In this case increased ROS levels or changes in intracellular glutathione levels are often associated with pathological changes indicative of a dysregulation of signal cascades and/or gene

expression, exemplified by altered expression of cell adhesion molecules (Droge, 2002).

oxidative stress, development of diabetes and resulting complications.

Increased oxidative stress is widely accepted as a major role player in both development and progression of diabetes (Maritim *et al*, 2002). Figure 1 summarizes the relationship between

> **Glucose FFA Cellular Overload**

> > **Muscle Adipose**

**Insulin resistance**

**Metabolic Syndrome**

Fig. 1.Overnutrition leads to oxidative stress which in turn results in diabetes and its complications (FFA: Free Fatty Acids, IGT: Impaired Glucose Tolerance, CVD:

**Oxidative Stress**

**βcells**

**Altered insulin secretion**

**IGT (postprandial hyperglycemia**

**Diabetes (chronic hyperglycemia)**

**3.4 Oxidative stress and diabetes** 

**Overnutrition Decreased physical activity**

**Genetic** 

**predisposition**

Cardiovascular Disease)

**Endothelial calls**

**Endothelial dysfunction**

**CVD**

To better understand the role of tea antioxidants in either preventing diabetes or reducing its complications, one must first know the mechanisms through which oxidative stress contributes to the development of this chronic disease and disorders following it. Some evidence on how tea antioxidants, in particular, can prevent diabetes development and its progression will be presented next.

As mentioned previously, type1 diabetes mellitus which is less prevalent than type 2 is a genetic autoimmune disorder affecting the islet cells leading to insulin deficiency and thus hyperglycemia. Type 2 diabetes however is a multi-factorial disease. Insulin resistance most often precedes the onset of this type by many years and can be caused by acquired factors. Elevations in glucose and free fatty acids have been shown to induce oxidative stress which in turn can play a key role in causing insulin resistance and β-cell dysfunction (Evans *et al*., 2002).

One most favored hypothesis on how hyperglycemia and elevated free fatty acids (FFA) can lead to oxidative stress is that as energy intake exceeds energy expenditure, generation of excess mitochondrial NADH (mNADH) and the level of reactive oxygen species (ROS) is increased due to the greater activity of citric acid cycle, induced by the abundance of substrates. Reducing ROS formation or increasing its removal is the way through which cells can protect themselves. The mechanism of preventing excessive mNADH generation may be inhibiting insulin-stimulated nutrient uptake and preventing the entrance of pyruvate and fatty acids into the mitochondria. Either of glucose or FFA enters the citric acid cycle after being converted to acetyl-CoA which then combines with oxaloacetate to form citrate. Greater availability of substrates will result in greater production of mNADH which is beyond the capability of oxidative phosphorylation to dissipate it all, leading to increased mitochondrial proton gradient. Thus single electrons are transferred to molecular oxygen forming free radicals, especially superoxide anion.

One way the cells can reduce free radical generation is inhibition of FFA oxidation. This will increase intracellular FFA which in turn leads to reduced GLUT4 translocation to the plasma membrane resulting in resistance to insulin-stimulated glucose uptake in muscle and adipose tissue. *In vitro* studies have shown that antioxidants may have role in reducing insulin resistance (Ceriello *et al*., 2004).

Chronic exposure to abnormally high levels of glucose and FFA leads to toxic effect on βcells of pancreas. Furthermore as aforementioned, hyperglycemia and high levels of FFA leads to increased oxidative stress. ß-cells are particularly susceptible to the damages inflicted by oxidative stress; since they are low in free radical quenching enzymes such as catalase, glutathione peroxidase and superoxide dismutase. ß-cells are responsible for the sensing glucose and secreting appropriate amount of insulin in response to glucose boots. This process is pretty complex, but the critical significance of mitochondrial glucose metabolism in linking stimulus to secretion is well established. This is one reason that oxidative stress can blunt insulin secretion due to its ability to damage mitochondria (Evans *et al.*, 2003; Robertson *et al.*, 2004).

Chronic oxidative stress can also affect insulin gene expression. At least two critical proteins that activate the insulin promoter are involved in defects in insulin gene expression. One is PDX-1 and the other is RIPE-3b1 activator recently identified as MafA. Glucose toxicity and lipotoxicity both of which lead to increased oxidative stress have been shown to leave deleterious effects on islet cells (Robertson, 2004).

Fig. 2. Hyperglycemia results in endothelial dysfunction and diabetic complications through

Several free radical species are normally produced in the body to perform specific functions. O2-., H2O2 and NO are three free radical reactive oxygen species (ROS) that are essential for

causing oxidative stress

**3.5 Oxidative stress and antioxidants** 

Apoptosis is one other way through which oxidative stress can cause beta-cell dysfunction. There is some evidence that NF-κB is in part responsible for the induction of apoptosis in β cells; NF-κB production is stimulated by oxidative stress (Evans *et al*., 2002; Robertson, 2004).

Uncoupling proteins (UCP) are carriers expressed in the mitochondrial inner membrane that uncouple oxygen consumption by the respiratory chain from ATP synthesis and can play a significant role in diabetes. These proteins can control ROS production in the mitochondria. UCP2 and UCP3 are expressed in adipose tissue and skeletal muscles, the tissues important for thermogenesis and substrate oxidation. Elevated expression of UCP2 has been shown to exert negative regulation of β-cell insulin secretion and contribute to the impairment of β-cell function. UCP3 level has been reported to be decreased in diabetic patients and is assumed to facilitate fatty acid oxidation and minimize ROS production (Maiese *et al.*, 2007).

As mentioned previously, hyperglycemia increases peroxide generation in mitochondria which then through many different routes results in endothelial dysfunction and other diabetic complications in the end. Figure 2 illustrates how oxidative stress induced by hyperglycemia leads to the downstream events.

Superoxide overproduction decreases eNOS activity, but increases iNOS expression through NF-κB and protein kinase C (PKC); the final effect is greater NO generation and strong oxidant peroxynitrite which in turn produces in iNOS and eNOS, an uncoupled state resulting in the production of superoxide rather than NO, and damages DNA. DNA damage is necessary for the activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). This reduces the intracellular concentration of NAD+ which it uses as a substrate. The rate of glycolysis, electron transport and ATP formation reduces as a result of decreased NAD+ and an ADP-ribosylation of the GAPDH (glyceraldehydes 3-phosphate dehydrogenase) occurs. This process results in acute endothelial dysfunction in diabetic blood vessels, which contributes to the development of diabetic complications. NF-κB activation also induces a proinflammatory conditions and overexpression of the adhesion molecules overexpression. All these alterations end in the diabetic complications, and cardiovascular disorders.

NF-κB, one major intracellular target of hyperglycemia and oxidative stress which can be activated by a number of stimuli including hyperglycemia, elevated FFA, ROS, TNF-α, IL-1β, and other proinflammatory cytokines, AGE (advanced glycation end product)-binding to RAGE (receptor for AGE), DNA damage, viral infection and UV irradiation, regulates the expression of a large number of genes, including growth factors (vascular endothelial growth factor (VEGF), proinflammatory cytokines like TNF-α and IL-1β, RAGE, adhesion molecules like vascular cell adhesion molecule-1, and many others).

VEGF has been identified as a primary initiator of proliferative diabetic retinopathy and as a potential mediator of nonproliferative retinopathy. It is also involved in the development of nephropathy and neuropathy. Thus VEGF seems to play an important role in the etiology of several complications of diabetes (Ishii *et al.*, 2001; Evans *et al.*, 2002; Esposito *et al.*, 2002; Ceriello, 2003; Ceriello, 2006; Negrean *et al.*, 2007)

Apoptosis is one other way through which oxidative stress can cause beta-cell dysfunction. There is some evidence that NF-κB is in part responsible for the induction of apoptosis in β cells; NF-κB production is stimulated by oxidative stress (Evans *et al*., 2002; Robertson,

Uncoupling proteins (UCP) are carriers expressed in the mitochondrial inner membrane that uncouple oxygen consumption by the respiratory chain from ATP synthesis and can play a significant role in diabetes. These proteins can control ROS production in the mitochondria. UCP2 and UCP3 are expressed in adipose tissue and skeletal muscles, the tissues important for thermogenesis and substrate oxidation. Elevated expression of UCP2 has been shown to exert negative regulation of β-cell insulin secretion and contribute to the impairment of β-cell function. UCP3 level has been reported to be decreased in diabetic patients and is assumed to facilitate fatty acid oxidation and minimize ROS

As mentioned previously, hyperglycemia increases peroxide generation in mitochondria which then through many different routes results in endothelial dysfunction and other diabetic complications in the end. Figure 2 illustrates how oxidative stress induced by

Superoxide overproduction decreases eNOS activity, but increases iNOS expression through NF-κB and protein kinase C (PKC); the final effect is greater NO generation and strong oxidant peroxynitrite which in turn produces in iNOS and eNOS, an uncoupled state resulting in the production of superoxide rather than NO, and damages DNA. DNA damage is necessary for the activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). This reduces the intracellular concentration of NAD+ which it uses as a substrate. The rate of glycolysis, electron transport and ATP formation reduces as a result of decreased NAD+ and an ADP-ribosylation of the GAPDH (glyceraldehydes 3-phosphate dehydrogenase) occurs. This process results in acute endothelial dysfunction in diabetic blood vessels, which contributes to the development of diabetic complications. NF-κB activation also induces a proinflammatory conditions and overexpression of the adhesion molecules overexpression. All these alterations end in the diabetic complications, and

NF-κB, one major intracellular target of hyperglycemia and oxidative stress which can be activated by a number of stimuli including hyperglycemia, elevated FFA, ROS, TNF-α, IL-1β, and other proinflammatory cytokines, AGE (advanced glycation end product)-binding to RAGE (receptor for AGE), DNA damage, viral infection and UV irradiation, regulates the expression of a large number of genes, including growth factors (vascular endothelial growth factor (VEGF), proinflammatory cytokines like TNF-α and IL-1β, RAGE, adhesion

VEGF has been identified as a primary initiator of proliferative diabetic retinopathy and as a potential mediator of nonproliferative retinopathy. It is also involved in the development of nephropathy and neuropathy. Thus VEGF seems to play an important role in the etiology of several complications of diabetes (Ishii *et al.*, 2001; Evans *et al.*, 2002; Esposito *et al.*, 2002;

molecules like vascular cell adhesion molecule-1, and many others).

Ceriello, 2003; Ceriello, 2006; Negrean *et al.*, 2007)

2004).

production (Maiese *et al.*, 2007).

cardiovascular disorders.

hyperglycemia leads to the downstream events.

#### **3.5 Oxidative stress and antioxidants**

Several free radical species are normally produced in the body to perform specific functions. O2 -., H2O2 and NO are three free radical reactive oxygen species (ROS) that are essential for

The question as to when the man first consumed tea is unanswered as well. According to Chinese mythology, it was the emperor Shen Nung who discovered tea for the first time in 2737 B.C.; but this is not in consistence with the first credible documentary reference on tea

It is probable that our forbears used tea in response to their instinctive seek for a material to calm them; because tea is rich in an alkaloid called caffeine which acts as an opioid in the

On tropical and subtropical climates and regions on which precipitation is coordinate according to months and where summers and winters are lukewarm, tea production is realized. Sour and humid land structure is crucial to growing tea as well (Alkan *et al*., 2009). Based on the data generated by the food and agriculture organization (FAO) of the united nations as of January 2010, China was the leading country in tea production in 2006, 2007 and 2008, followed by India, Kenya, Sri Lanka, Turkey, Vietnam and Indonesia. Other main tea producing countries are Japan, Argentina, Iran, Bangladesh, Malawi and Uganda Figure (3). The global tea production growth rate in 2006 extended more than 3% to reach an estimated 3.6 million tons, China, Viet Nam and India being the main counties to have contributed to this rise. It is predicted that world black tea production rate decreases in the current century, due to slowing down of production growth in Africa. India followed by Kenya and Sri Lanka are projected to be the main contributors to black tea production by 2017 which is estimated to

which was made in 59 B.C. (Hara, 2001; Gupta *et al*., 2002).

reach 3.1 million tons (http://en.wikipedia.org/wiki/Tea; Hicks, 2009).

nervous system, relaxing the consumer.

Fig. 3. The tea producing regions in 2007

normal physiology, but are also believed to accelerate the process of aging and to mediate cellular degeneration in disease states. These agents together produce highly active singlet oxygen, hydroxyl radicals, and peroxynitrite that can attack proteins, lipids, and DNA. Antioxidants are defined as any compound that can donate at least one hydrogen atom to a free radical, resulting in the termination of radical chain reactions. An alternative type of antioxidant is defined by its ability to prevent the initiation of a free radical chain reaction rather than to terminate them. This latter type of antioxidant is usually dependent upon the ability to bind metal ions and includes ceruloplasmin, transferrin and albumin. Cells must maintain the levels of antioxidants, often defined as antioxidant potential, through dietary uptake or *de novo* synthesis. Excess production of free radicals can reduce the intracellular antioxidants, resulting in oxidative stress. In brief, acute hyperglycemic episodes such as an oral glucose tolerance test or a meal can decrease the antioxidant capacity of plasma in both normal and diabetic subjects and increase oxidative stress in diabetic patients. As a type 2 diabetic patient ages, increased basal levels of free radical production and decreased antioxidants are even further intensified by elevated plasma glucose. Analysis of individual vitamin and enzyme components of the antioxidant system in man reveals significant changes in diabetes. The levels of vitamins A and E and catalase activity are decreased in both type 1 and 2 patients compared with controls. Whereas GSH-metabolizing enzymes are decreased in type 1 but not type 2 patients, SOD activity is lower in type 2 but not type 1 (Vincent *et al*., 2004).

#### **4. Tea**

The scientific name given to tea, in the first volume of the book "Species Plantarum" by Carl Linnaeus, was "Thea Sinensis"; but in the second volume of the very book, the tea tree is addressed as "Camelia". Later in 1762, Linnaeus assuming black and green tea to be obtained from two different shrubs, chose the names "Thea bohea" and "Thea vividis" for black and green tea respectively. Now it is revealed that it is "Thea bohea" from which, both black and green tea are attained. Also the scientists have merged the two genuses "Camelia" and "Thea". Today the international scientific expression for tea is "*Camelia Sinensis (L) O.kuntze*", Camelia and Sinensis indicating the genus and the variety respectively, (L) regarding Linneaus, the first botanist to give tea a scientific name and O.kuntze being the one who combined the names used for black and green tea. Camelia Sinensis is an evergreen plant which can grow into a tree of up to 30 meters if left undisturbed; but cultivated plants usually have a height around 50-70 centimeters (Hara, 2001; Moxham, 2009).

#### **4.1 Historical background**

It may always remain in mist, when tea first stepped into man's life. General consensus attributes the birth of the tea bush to the area we now call eastern China. But the discovery of a tea bush deep in Assam, India with leaves much larger than the Chinese one, caused controversy, as far as it concerns the birthplace of Camelia Sinensis. Today it is assumed that the tea bush was first found in the southwestern China, centered in the Yunnan district (Hara, 2001). Tea was first carried westwards during 5th century by Turkish traders (Alkan *et al*., 2009).

normal physiology, but are also believed to accelerate the process of aging and to mediate cellular degeneration in disease states. These agents together produce highly active singlet oxygen, hydroxyl radicals, and peroxynitrite that can attack proteins, lipids, and DNA. Antioxidants are defined as any compound that can donate at least one hydrogen atom to a free radical, resulting in the termination of radical chain reactions. An alternative type of antioxidant is defined by its ability to prevent the initiation of a free radical chain reaction rather than to terminate them. This latter type of antioxidant is usually dependent upon the ability to bind metal ions and includes ceruloplasmin, transferrin and albumin. Cells must maintain the levels of antioxidants, often defined as antioxidant potential, through dietary uptake or *de novo* synthesis. Excess production of free radicals can reduce the intracellular antioxidants, resulting in oxidative stress. In brief, acute hyperglycemic episodes such as an oral glucose tolerance test or a meal can decrease the antioxidant capacity of plasma in both normal and diabetic subjects and increase oxidative stress in diabetic patients. As a type 2 diabetic patient ages, increased basal levels of free radical production and decreased antioxidants are even further intensified by elevated plasma glucose. Analysis of individual vitamin and enzyme components of the antioxidant system in man reveals significant changes in diabetes. The levels of vitamins A and E and catalase activity are decreased in both type 1 and 2 patients compared with controls. Whereas GSH-metabolizing enzymes are decreased in type 1 but not type 2 patients, SOD activity is lower in type 2 but not type 1

The scientific name given to tea, in the first volume of the book "Species Plantarum" by Carl Linnaeus, was "Thea Sinensis"; but in the second volume of the very book, the tea tree is addressed as "Camelia". Later in 1762, Linnaeus assuming black and green tea to be obtained from two different shrubs, chose the names "Thea bohea" and "Thea vividis" for black and green tea respectively. Now it is revealed that it is "Thea bohea" from which, both black and green tea are attained. Also the scientists have merged the two genuses "Camelia" and "Thea". Today the international scientific expression for tea is "*Camelia Sinensis (L) O.kuntze*", Camelia and Sinensis indicating the genus and the variety respectively, (L) regarding Linneaus, the first botanist to give tea a scientific name and O.kuntze being the one who combined the names used for black and green tea. Camelia Sinensis is an evergreen plant which can grow into a tree of up to 30 meters if left undisturbed; but cultivated plants usually have a height around 50-70 centimeters (Hara,

It may always remain in mist, when tea first stepped into man's life. General consensus attributes the birth of the tea bush to the area we now call eastern China. But the discovery of a tea bush deep in Assam, India with leaves much larger than the Chinese one, caused controversy, as far as it concerns the birthplace of Camelia Sinensis. Today it is assumed that the tea bush was first found in the southwestern China, centered in the Yunnan district (Hara, 2001). Tea was first carried westwards during 5th century by Turkish traders (Alkan *et* 

(Vincent *et al*., 2004).

2001; Moxham, 2009).

*al*., 2009).

**4.1 Historical background** 

**4. Tea** 

The question as to when the man first consumed tea is unanswered as well. According to Chinese mythology, it was the emperor Shen Nung who discovered tea for the first time in 2737 B.C.; but this is not in consistence with the first credible documentary reference on tea which was made in 59 B.C. (Hara, 2001; Gupta *et al*., 2002).

It is probable that our forbears used tea in response to their instinctive seek for a material to calm them; because tea is rich in an alkaloid called caffeine which acts as an opioid in the nervous system, relaxing the consumer.

On tropical and subtropical climates and regions on which precipitation is coordinate according to months and where summers and winters are lukewarm, tea production is realized. Sour and humid land structure is crucial to growing tea as well (Alkan *et al*., 2009). Based on the data generated by the food and agriculture organization (FAO) of the united nations as of January 2010, China was the leading country in tea production in 2006, 2007 and 2008, followed by India, Kenya, Sri Lanka, Turkey, Vietnam and Indonesia. Other main tea producing countries are Japan, Argentina, Iran, Bangladesh, Malawi and Uganda Figure (3). The global tea production growth rate in 2006 extended more than 3% to reach an estimated 3.6 million tons, China, Viet Nam and India being the main counties to have contributed to this rise. It is predicted that world black tea production rate decreases in the current century, due to slowing down of production growth in Africa. India followed by Kenya and Sri Lanka are projected to be the main contributors to black tea production by 2017 which is estimated to reach 3.1 million tons (http://en.wikipedia.org/wiki/Tea; Hicks, 2009).

Fig. 3. The tea producing regions in 2007

Epigallocatechin gallate (EGCG), being the greatest in amount in tea compared to the other catechins, makes up to 50% of its catechins. EGCG is more abundant in green tea and its quantity is negatively correlated with the age of the leaves (Hara, 2001; Leung *et al*., 2001). *Vitamins and minerals*: Vitamin C was discovered in 1924 in fresh tea leaves. Tea is a great source of fluoride too (Hara, 2001). Other vitamins and minerals may be present in tea at

*Enzymes*: The enzymes in tea which catalyze the oxidation processes are called Thease. During fermentation in which tea pectins are demetylated, polyphenolic compounds are decomposed which as a result of the quinone appearance, turn into some colorful agents including theaflavin and thearubigin, both of which are plentiful in black tea (Figure 4)

*Volatile oils*: More than 600 volatile agents have been established in tea, most of which have a yellow color and a characteristic scent. Linalool is the main essence in tea, other of lesser importance ones being dihydroactinide iolido paravinile phenol, hexenol, hexenal,

Based upon the preparation method, the degree to which it is fermented and the steps it goes under during the production, different types of tea consumed all over the world are classified into at least six categories (Figure 5). The less processed the tea, the greater the polyphenols

content will be, which the extent of oxidation accounts for (Santana-Rios *et al.*, 2001).

aldehydes, phenyl ethyl alcohols, phenols and geraniols (Hara, 2001).

little amounts as well.

(Hara, 2001; Leung *et al*., 2001; Cadenas, 2002).

Fig. 4. Major polyphenols in green and black tea

#### **4.2 Chemical compounds in tea**

In contrast to the history of tea drinking which is ancient, the chemical components of tea have quite recently been investigated. Teas acquired from different regions may have different chemical components in different amounts. The agents found in tea are classified as primary or subordinate (Table 1):


Table 1. The classification of primary and subordinate tea agents

The quality of a tea is related to its content of alkaloids (caffeine), flavonoids (catechins), phenolic acids (gallic acid, coumaric acid, caffeic acid and chlorogenic acid) and volatile oils (essences) (Table 1) (Bendini *et al*., 1998; Wang *et al*., 2000).

*Alkaloids*: In 1827, caffeine which is present in a few other plants was discovered in tea. By then it was given the name "Theine" which was dropped as its structure was proven to be exactly the same as that of caffeine, in 1820. The mean content of caffeine in tea ranges between 1.9 and 4.5 and is negatively correlated with the age of the leaves (Wanger *et al*., 1996; Hara, 2001).

*Polyphenols*: Theanine and flavonoids (catechins in particular) are the main polyphenols found in tea constituting 30% of its agents.

Theanine: A unique substance in tea is theanine which is a kind of amino acid comprising more than half of the amino acids present in tea. It has an "umami" or sweet taste and constitutes 2% of tea (Hara, 2001).

Flavonoids: Flavanols and their derivatives including flavan-3-ols (catechins and epicatechins) and flavonols are the chief flavonoids in tea. Under mild oxidation, flavan 3-4 diol derivatives of flavonoids are converted to catechins and its isomers. Green tea is a great source of catechins and thus exerts antioxidant properties. These catechins change into oligomeric quinones under the fermentation process of black tea which reduces its antioxidant capacity by 2-6 times in comparison to the green tea (Hara, 2001). Each gram of green tea contains 123.8- 206.3 milligrams of catechins which is 10-30 percent of the dry weight of the green leaves. In black tea, 79.3 milligrams of catechins is found in one gram (Wang *et al*., 2000; Bronner *et al*., 1998; Keys, 1976). All catechins have 2 asymmetric carbons, thus there are four isomers of them: catechin (C)(+), catechin gallate (CG)(-), gallocatechin (GC) and gallocatechin gallate (GCG) (-). The number of hydroxyl group on the B ring differs for the derivatives of catechins. Like catechins, epicatechins are the monomers of the condensed thanines, are derived from flavan 3-4 diols and have two asymmetric carbons in their structure resulting in four isomers. These isomers include: epicatechin (EC)(-), epigallocatechin (EGC)(-), Epicatechin gallate (ECG)(-) and epigallocatechin gallate (EGCG)(-). Catechins and epicatechins are the major polyphenols found particularly in green tea (Figure 4).

In contrast to the history of tea drinking which is ancient, the chemical components of tea have quite recently been investigated. Teas acquired from different regions may have different chemical components in different amounts. The agents found in tea are classified

> **Primary components subordinate components**  Alkaloids (xanthines) Mineral acids Polyphenols Organic acids Vitamins Proteins Enzymes Pectin Volatile oils (essence) Lignans

The quality of a tea is related to its content of alkaloids (caffeine), flavonoids (catechins), phenolic acids (gallic acid, coumaric acid, caffeic acid and chlorogenic acid) and volatile oils

*Alkaloids*: In 1827, caffeine which is present in a few other plants was discovered in tea. By then it was given the name "Theine" which was dropped as its structure was proven to be exactly the same as that of caffeine, in 1820. The mean content of caffeine in tea ranges between 1.9 and 4.5 and is negatively correlated with the age of the leaves (Wanger *et al*.,

*Polyphenols*: Theanine and flavonoids (catechins in particular) are the main polyphenols

Theanine: A unique substance in tea is theanine which is a kind of amino acid comprising more than half of the amino acids present in tea. It has an "umami" or sweet taste and

Flavonoids: Flavanols and their derivatives including flavan-3-ols (catechins and epicatechins) and flavonols are the chief flavonoids in tea. Under mild oxidation, flavan 3-4 diol derivatives of flavonoids are converted to catechins and its isomers. Green tea is a great source of catechins and thus exerts antioxidant properties. These catechins change into oligomeric quinones under the fermentation process of black tea which reduces its antioxidant capacity by 2-6 times in comparison to the green tea (Hara, 2001). Each gram of green tea contains 123.8- 206.3 milligrams of catechins which is 10-30 percent of the dry weight of the green leaves. In black tea, 79.3 milligrams of catechins is found in one gram (Wang *et al*., 2000; Bronner *et al*., 1998; Keys, 1976). All catechins have 2 asymmetric carbons, thus there are four isomers of them: catechin (C)(+), catechin gallate (CG)(-), gallocatechin (GC) and gallocatechin gallate (GCG) (-). The number of hydroxyl group on the B ring differs for the derivatives of catechins. Like catechins, epicatechins are the monomers of the condensed thanines, are derived from flavan 3-4 diols and have two asymmetric carbons in their structure resulting in four isomers. These isomers include: epicatechin (EC)(-), epigallocatechin (EGC)(-), Epicatechin gallate (ECG)(-) and epigallocatechin gallate (EGCG)(-). Catechins and epicatechins are the major polyphenols found particularly in

Table 1. The classification of primary and subordinate tea agents

(essences) (Table 1) (Bendini *et al*., 1998; Wang *et al*., 2000).

found in tea constituting 30% of its agents.

constitutes 2% of tea (Hara, 2001).

Amino acids

**4.2 Chemical compounds in tea** 

as primary or subordinate (Table 1):

1996; Hara, 2001).

green tea (Figure 4).

Epigallocatechin gallate (EGCG), being the greatest in amount in tea compared to the other catechins, makes up to 50% of its catechins. EGCG is more abundant in green tea and its quantity is negatively correlated with the age of the leaves (Hara, 2001; Leung *et al*., 2001).

*Vitamins and minerals*: Vitamin C was discovered in 1924 in fresh tea leaves. Tea is a great source of fluoride too (Hara, 2001). Other vitamins and minerals may be present in tea at little amounts as well.

*Enzymes*: The enzymes in tea which catalyze the oxidation processes are called Thease. During fermentation in which tea pectins are demetylated, polyphenolic compounds are decomposed which as a result of the quinone appearance, turn into some colorful agents including theaflavin and thearubigin, both of which are plentiful in black tea (Figure 4) (Hara, 2001; Leung *et al*., 2001; Cadenas, 2002).

*Volatile oils*: More than 600 volatile agents have been established in tea, most of which have a yellow color and a characteristic scent. Linalool is the main essence in tea, other of lesser importance ones being dihydroactinide iolido paravinile phenol, hexenol, hexenal, aldehydes, phenyl ethyl alcohols, phenols and geraniols (Hara, 2001).

Fig. 4. Major polyphenols in green and black tea

Based upon the preparation method, the degree to which it is fermented and the steps it goes under during the production, different types of tea consumed all over the world are classified into at least six categories (Figure 5). The less processed the tea, the greater the polyphenols content will be, which the extent of oxidation accounts for (Santana-Rios *et al.*, 2001).

activity of enzymes involved in glutathione and quinone synthesis and remove the free radicals of hydrogen peroxide and superoxide anions. Tea consumption also inhibits metastasis of human lymphoid leukemia cells through stimulation of apoptosis and hindrance of platelet aggregation (Bronner *et al*., 1998; Dulluge *et al*., 1998; Integrative

Studies have shown that tea is beneficial in delaying cardiovascular disorders. Some mechanisms described are: inhibiting the progression of atherosclerosis and thrombosis, preventing hypertension by either exerting effects similar to those of beta-blockers or stimulating diuresis, decreasing postprandial blood cholesterol and triglycerides, inhibition of LDL oxidation and improvement of endothelial function. Also, decreasing the activity of lipoxygenase enzymes and stimulating central nervous system, tea can improve heart muscle function, circulation in coronary vessels and respiration (Yamamoto, 1997; Robbers *et al*., 1999; Leung *et al*., 2001; Alipoor et al., 2008). Hypertension is another disorder which can be corrected by tea and its polyphenols. This has been attributed to its role in regulating renin-angiotensin system (RAS) and improving endothelial function (Adlercreutz, 1991).

Since tea and its polyphenols have been observed to reduce digestion and absorption of fats and carbohydrates, and due to their role in controlling food intake, increasing energy expenditure, modifying the activity of liver, muscle, gastrointestinal tract and fat cells, weight loss and prevention of diabetes mellitus could be one advantage of drinking appropriate amounts of tea (Watanabe *et al*., 1998; Kuo *et al*., 2005; Ynng *et al*., 2006). How tea can play a major role in prevention and treatment of many complications of diabetes

Other disorders which tea can play a role in prevention or treatment of, includes inflammation, migraine, nausea, diarrhea, maldigestion, sore throat, depression, prostatitis, hemochromatosis, neurodegenerative diseases like Parkinson and Alzheimer, cataract, dental carries and some viral and bacterial infections including influenza, polio, herpes simplex and AIDS (Duke, 1985; Robertson *et al*., 1991; Hertog *et al*., 1993; Cummings *et al*., 1995; Tavani *et al*., 1996; Van Het Hof *et al*., 1997; Integrative Medicine, 2000; Mills *et al*., 2000; McKay *et al*., 2002; Wright, 2005; Kao *et al*., 2006; Sasso *et al*., 2006; Alipoor et al., 2011).

There is a considerable amount of evidence indicating the benefits of tea consumption to prevent diabetes and reducing its resulting complications. The less processed the tea, the more its antioxidant content; which may explain why most studies have been conducted using green tea as the supplement. Recently, white tea which is not fermented either, has been studied for its impact on diabetes too. Some ways through which tea and its bioactive compounds affect diabetes are not related to the antioxidant properties of tea, thus not

Many studies have shown that different types of tea are potentially effective in reducing oxidative stress and related diseases. Attempts have been made to manufacture products containing tea bioactive compounds for prevention and treatment of mentioned diseases. In order to design such product, the effective compounds of tea and the safe dose of them must be first identified. For instance, EGCG has been shown to act as a prooxidant when administered in high doses and lead to apoptosis. Furthermore compounds other than

mellitus will be presented more precisely in the next section.

within the scope of this chapter; and won't be discussed here.

**4.4 Tea antioxidants and oxidative stress** 

Medicine, 2000; Springhouse, 2001).


Fig. 5. The production of different types of tea

#### **4.3 Tea and diseases**

Tea being a great source of phytoestrogens and fluoride, both of which play a major role in bone health, is reported to prevent osteoporosis and the lower prevalence of the very disease in Japanese postmenopausal women in comparison with American and European ones, is attributed to greater amounts of tea consumed by Japanese (Adlercreutz *et al*., 1991; Adlercreutz *et al*., 1992; Johnell *et al*., 1995; Kanis *et al*., 1999).

Tea due to its content of polyphenols has been found to be effective in preventing many types of cancer including liver, small intestine and lung. Polyphenols increase the catalytic

1. White tea: White tea is manufactured only from the buds or first leaves of *C.sinensis*. It is the least processed type of tea and is simply steamed and dried without a prior withering stage; therefore the concentrations of EGCG and also methylxanthines (like

2. Yellow tea: It usually implies a special tea processed in a similar way to green tea; but the drying process takes place at a slower rate. The damp tea leaves are allowed to sit

3. Green tea: To manufacture green tea, first the fresh leaves are steamed, then primary drying-rolling, rolling, secondary drying-rolling, final drying-rolling and at last drying

4. Oolong tea: Fresh leaves undergo solar withering at the first step, indoor withering and rolling, pan firing, rolling, mass breaking and drying are the steps to be taken, to produce oolong tea. In this kind of tea, partial fermentation occurs after the rolling. 5. Black tea: The manufacturing process for black tea includes withering of fresh leaves,

6. Pu-erh: Pu-erh is applied to old tea with extreme fermentation in it (Hara, 2001; Santana-Rios *et al.*, 2001; Kuo *et al*., 2005; Lin *et al*., 2006; Sohle *et al*. 2009; Wang *et al*.,

Tea being a great source of phytoestrogens and fluoride, both of which play a major role in bone health, is reported to prevent osteoporosis and the lower prevalence of the very disease in Japanese postmenopausal women in comparison with American and European ones, is attributed to greater amounts of tea consumed by Japanese (Adlercreutz *et al*., 1991;

Tea due to its content of polyphenols has been found to be effective in preventing many types of cancer including liver, small intestine and lung. Polyphenols increase the catalytic

rolling, fermenting and drying. Thorough fermentation is done in black tea.

2008; http://en.wikipedia.org/wiki/Tea; http://www.tea-of-chinese.com).

caffeine) are enriched in white tea compared with green and black tea.

and yellow. Its taste resembles that of green and white teas.

are performed. No fermentation takes place in this type of tea.

Fig. 5. The production of different types of tea

Adlercreutz *et al*., 1992; Johnell *et al*., 1995; Kanis *et al*., 1999).

**4.3 Tea and diseases** 

activity of enzymes involved in glutathione and quinone synthesis and remove the free radicals of hydrogen peroxide and superoxide anions. Tea consumption also inhibits metastasis of human lymphoid leukemia cells through stimulation of apoptosis and hindrance of platelet aggregation (Bronner *et al*., 1998; Dulluge *et al*., 1998; Integrative Medicine, 2000; Springhouse, 2001).

Studies have shown that tea is beneficial in delaying cardiovascular disorders. Some mechanisms described are: inhibiting the progression of atherosclerosis and thrombosis, preventing hypertension by either exerting effects similar to those of beta-blockers or stimulating diuresis, decreasing postprandial blood cholesterol and triglycerides, inhibition of LDL oxidation and improvement of endothelial function. Also, decreasing the activity of lipoxygenase enzymes and stimulating central nervous system, tea can improve heart muscle function, circulation in coronary vessels and respiration (Yamamoto, 1997; Robbers *et al*., 1999; Leung *et al*., 2001; Alipoor et al., 2008). Hypertension is another disorder which can be corrected by tea and its polyphenols. This has been attributed to its role in regulating renin-angiotensin system (RAS) and improving endothelial function (Adlercreutz, 1991).

Since tea and its polyphenols have been observed to reduce digestion and absorption of fats and carbohydrates, and due to their role in controlling food intake, increasing energy expenditure, modifying the activity of liver, muscle, gastrointestinal tract and fat cells, weight loss and prevention of diabetes mellitus could be one advantage of drinking appropriate amounts of tea (Watanabe *et al*., 1998; Kuo *et al*., 2005; Ynng *et al*., 2006). How tea can play a major role in prevention and treatment of many complications of diabetes mellitus will be presented more precisely in the next section.

Other disorders which tea can play a role in prevention or treatment of, includes inflammation, migraine, nausea, diarrhea, maldigestion, sore throat, depression, prostatitis, hemochromatosis, neurodegenerative diseases like Parkinson and Alzheimer, cataract, dental carries and some viral and bacterial infections including influenza, polio, herpes simplex and AIDS (Duke, 1985; Robertson *et al*., 1991; Hertog *et al*., 1993; Cummings *et al*., 1995; Tavani *et al*., 1996; Van Het Hof *et al*., 1997; Integrative Medicine, 2000; Mills *et al*., 2000; McKay *et al*., 2002; Wright, 2005; Kao *et al*., 2006; Sasso *et al*., 2006; Alipoor et al., 2011).

#### **4.4 Tea antioxidants and oxidative stress**

There is a considerable amount of evidence indicating the benefits of tea consumption to prevent diabetes and reducing its resulting complications. The less processed the tea, the more its antioxidant content; which may explain why most studies have been conducted using green tea as the supplement. Recently, white tea which is not fermented either, has been studied for its impact on diabetes too. Some ways through which tea and its bioactive compounds affect diabetes are not related to the antioxidant properties of tea, thus not within the scope of this chapter; and won't be discussed here.

Many studies have shown that different types of tea are potentially effective in reducing oxidative stress and related diseases. Attempts have been made to manufacture products containing tea bioactive compounds for prevention and treatment of mentioned diseases. In order to design such product, the effective compounds of tea and the safe dose of them must be first identified. For instance, EGCG has been shown to act as a prooxidant when administered in high doses and lead to apoptosis. Furthermore compounds other than

transcription factors that bind to ARE, NF-E2-related factor 2 (Nrf2) was identified (Zhang, 2006). Nrf2 binds to Kelch-like ECH-associated protein 1 (Keap1) under nonstressed conditions. Keap1 in complex with cullin3, Roc1 and E2 proteins provides ubiquitination followed by proteasomal degradation. When oxidative stress occurs, oxidation of Keap1 leads to inability to bind Nrf2 protein by forming intramolecular disulfide bonds. Then Nrf2 migrates into the nucleus and binds a protein of Maf family (like sMaf) and CBP/p. This complex is formed on ARE promoter region of certain genes leading to transcription activation. Phosphorylation of by protein kinases which may be activated by oxidants is one

Tea polyphenols have been demonstrated to improve lipid profile in diabetic and nondiabetic models. In this section, we follow the antioxidant properties of tea. Improved glucose tolerance and increased plasma insulin concentrations by tea, have been found in some studies which may be, in part, explained by the effect of tea antioxidants on insulin resistance and β-cell function. EGCG has also been shown to suppress cytokine-induced βcell damage; this may also contribute to glucose lowering effect of tea (Gomes *et al*., 1995; Han *et al*., 1999; Kao *et al.*, 2000; Sabu *et al*., 2002; Wu *et al.*, 2004; Tsuneki *et al.*, 2004; Babu *et al*., 2006; Wolfram *et al*., 2006; Igarashi *et al*., 2007; Badawoud *et al*., 2007; Ostad Rahimi *et al*.,

Lipid peroxidation is an indicator of oxidative stress and plays major role in development of some complications of diabetes. Animal studies have shown that green tea administration can reduce lipid peroxidation in diabetic animals (Yamaguchi *et al*., 1991; Tijburg *et al*., 1997; Vinson *et al*., 1998; Miura *et al*., 2001; Guleria *et al*., 2002; Kasaoka *et al*., 2002; Nakagawa *et al*., 2002; Liuji *et al*., 2002; Sabu *et al*., 2002; Skrzydlewska *et al*., 2002; Babu *et al*., 2006). Black tea has been reported to be an efficient reducer of peroxidation of lipoproteins as well (Tijburg *et al*., 1997; Vinson *et al*., 1998; Sur-Altiner *et al*., 2000; Yokozawa *et al*., 2002; Vinson *et al*., 2005; Alipoor et al., 2008). Some studies have investigated the effects of purified tea

MDA is another important indicator of oxidative stress that is usually measured in diabetics. Based on the results of studies, tea seems to affect this factor too and reduce its plasma concentration (Durate *et al*., 2001; Skrzydlewska *et al.*, 2002; Chander *et al*., 2003; Sürmen-Gür

The activity of antioxidant enzymes such as catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase has been shown to increase by supplementation of tea or its polyphenols as well. Actually some enzymes showed greater activity after the very supplementation in one study but not the other which seems to be due to different doses of supplementation, design and duration of the study (Khan *et al*., 1992; Lin *et al*., 1998; Durate *et al*., 2001; Sabu *et al*., 2002; Skrzydlewska *et al.*, 2002; Chander *et al*., 2003; Kuo *et al*., 2005;

Glutathione is another parameter which has been measured in some studies and seems to increase in diabetics receiving tea intervention (Sohn *et al*., 1994; Lin *et al*., 1998; Durate *et al*.,

2001; Sabu *et al*., 2002; Skrzydlewska *et al.*, 2002; Babu *et al*., 2006; Alipoor *et al*., 2009).

polyphenols and drawn similar results (Quine *et al*., 2005; Yamabe *et al*., 2006).

way to provide Nrf2 migration in nucleus (Lushchak, 2011).

**4.4.1 Evidence from animal studies** 

2007; Potenza *et al*., 2007).

*et al*., 2006; Alipoor *et al*., 2009).

Babu *et al*., 2006; Alipoor *et al*., 2009).

catechins may exert the desired effects as well (Liao *et al.*, 2001; Mandel *et al.*, 2004; Kao *et al.*, 2006). To determine the very compounds acting as antioxidants in black tea, Alipoor et al., (2009) performed a study in which diabetic rats were supplemented total extract of black tea and its fractions. Total extract and fractions were attained by hydromethanol method and solid phase extraction using Sep-Pak respectively. Results of this study showed that injection of total extract and 20% fraction of black tea decreased malondialdehyde (MDA) and increased total antioxidant, Super oxide Dismotase (SOD), Glutathione Peroxides (GPX) and Glutathione in diabetic rats. To find out the major substances in the 20% fraction, Analytical HPLC, Preparative HPLC (High Performance Liquid Chromatography) and NMR (Nuclear Magnetic Resonance) (CNMR and HNMR) were employed. Caffeine, Epicatechin Gallate, Quercetin and Kampferol were the main compounds capable of combating oxiadative stress, to be determined in 20% fraction of tea (Figure 6) (Alipoor *et al.*, 2010).

Fig. 6. Major antioxidants in 20% fraction of black tea

Caffeine is a strong antioxidant and its activity being equal to that of glutathione and exceeding that of vitamin C (Devasagayam *et al.*, 1996; Kamat *et al.*, 2000; Nikolic *et al.*, 2003). The free radical scavenging capacity of flavonoids is due to the 3', 4' dihydroxyl and 3' hydroxy in the β ring (Amic *et al.*, 2003). The 20% fraction of black tea has been shown to be more effective than the other fractions which may be explained by the high concentration of the aforementioned compounds in it and absence of polyphenol antagonists in the very extract prepared (Alipoor *et al.*, 2009).

Tea polyphenols have been found to induce expression of phase II enzymes and endogenous antioxidants that defend cells from oxidative stress. The promoter regions of the phase II genes contain specific DNA sequences, termed the antioxidant response elements (AREs) or the electrophile response elements (EREs) that are required for induction by chemopreventive compounds, oxidative stress or electrophiles. In an attempt to find the

catechins may exert the desired effects as well (Liao *et al.*, 2001; Mandel *et al.*, 2004; Kao *et al.*, 2006). To determine the very compounds acting as antioxidants in black tea, Alipoor et al., (2009) performed a study in which diabetic rats were supplemented total extract of black tea and its fractions. Total extract and fractions were attained by hydromethanol method and solid phase extraction using Sep-Pak respectively. Results of this study showed that injection of total extract and 20% fraction of black tea decreased malondialdehyde (MDA) and increased total antioxidant, Super oxide Dismotase (SOD), Glutathione Peroxides (GPX) and Glutathione in diabetic rats. To find out the major substances in the 20% fraction, Analytical HPLC, Preparative HPLC (High Performance Liquid Chromatography) and NMR (Nuclear Magnetic Resonance) (CNMR and HNMR) were employed. Caffeine, Epicatechin Gallate, Quercetin and Kampferol were the main compounds capable of combating oxiadative stress, to be

O

OH

**Epicatechin Gallate**

OH OH

CH2OH

Caffeine is a strong antioxidant and its activity being equal to that of glutathione and exceeding that of vitamin C (Devasagayam *et al.*, 1996; Kamat *et al.*, 2000; Nikolic *et al.*, 2003). The free radical scavenging capacity of flavonoids is due to the 3', 4' dihydroxyl and 3' hydroxy in the β ring (Amic *et al.*, 2003). The 20% fraction of black tea has been shown to be more effective than the other fractions which may be explained by the high concentration of the aforementioned compounds in it and absence of polyphenol antagonists in the very

Tea polyphenols have been found to induce expression of phase II enzymes and endogenous antioxidants that defend cells from oxidative stress. The promoter regions of the phase II genes contain specific DNA sequences, termed the antioxidant response elements (AREs) or the electrophile response elements (EREs) that are required for induction by chemopreventive compounds, oxidative stress or electrophiles. In an attempt to find the

HO

O

O

OH

OH

OH

OH

OH

**3** R=OH **4** R=H

**R: OH Quercetin R: H Kampferol**

determined in 20% fraction of tea (Figure 6) (Alipoor *et al.*, 2010).

O

O

OH

Fig. 6. Major antioxidants in 20% fraction of black tea

O

R

**<sup>1</sup> <sup>2</sup> Caffeine**

O

HO

OH

N

O

H3C

O

N N

CH3

HO

extract prepared (Alipoor *et al.*, 2009).

N

CH3

transcription factors that bind to ARE, NF-E2-related factor 2 (Nrf2) was identified (Zhang, 2006). Nrf2 binds to Kelch-like ECH-associated protein 1 (Keap1) under nonstressed conditions. Keap1 in complex with cullin3, Roc1 and E2 proteins provides ubiquitination followed by proteasomal degradation. When oxidative stress occurs, oxidation of Keap1 leads to inability to bind Nrf2 protein by forming intramolecular disulfide bonds. Then Nrf2 migrates into the nucleus and binds a protein of Maf family (like sMaf) and CBP/p. This complex is formed on ARE promoter region of certain genes leading to transcription activation. Phosphorylation of by protein kinases which may be activated by oxidants is one way to provide Nrf2 migration in nucleus (Lushchak, 2011).

#### **4.4.1 Evidence from animal studies**

Tea polyphenols have been demonstrated to improve lipid profile in diabetic and nondiabetic models. In this section, we follow the antioxidant properties of tea. Improved glucose tolerance and increased plasma insulin concentrations by tea, have been found in some studies which may be, in part, explained by the effect of tea antioxidants on insulin resistance and β-cell function. EGCG has also been shown to suppress cytokine-induced βcell damage; this may also contribute to glucose lowering effect of tea (Gomes *et al*., 1995; Han *et al*., 1999; Kao *et al.*, 2000; Sabu *et al*., 2002; Wu *et al.*, 2004; Tsuneki *et al.*, 2004; Babu *et al*., 2006; Wolfram *et al*., 2006; Igarashi *et al*., 2007; Badawoud *et al*., 2007; Ostad Rahimi *et al*., 2007; Potenza *et al*., 2007).

Lipid peroxidation is an indicator of oxidative stress and plays major role in development of some complications of diabetes. Animal studies have shown that green tea administration can reduce lipid peroxidation in diabetic animals (Yamaguchi *et al*., 1991; Tijburg *et al*., 1997; Vinson *et al*., 1998; Miura *et al*., 2001; Guleria *et al*., 2002; Kasaoka *et al*., 2002; Nakagawa *et al*., 2002; Liuji *et al*., 2002; Sabu *et al*., 2002; Skrzydlewska *et al*., 2002; Babu *et al*., 2006). Black tea has been reported to be an efficient reducer of peroxidation of lipoproteins as well (Tijburg *et al*., 1997; Vinson *et al*., 1998; Sur-Altiner *et al*., 2000; Yokozawa *et al*., 2002; Vinson *et al*., 2005; Alipoor et al., 2008). Some studies have investigated the effects of purified tea polyphenols and drawn similar results (Quine *et al*., 2005; Yamabe *et al*., 2006).

MDA is another important indicator of oxidative stress that is usually measured in diabetics. Based on the results of studies, tea seems to affect this factor too and reduce its plasma concentration (Durate *et al*., 2001; Skrzydlewska *et al.*, 2002; Chander *et al*., 2003; Sürmen-Gür *et al*., 2006; Alipoor *et al*., 2009).

The activity of antioxidant enzymes such as catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase has been shown to increase by supplementation of tea or its polyphenols as well. Actually some enzymes showed greater activity after the very supplementation in one study but not the other which seems to be due to different doses of supplementation, design and duration of the study (Khan *et al*., 1992; Lin *et al*., 1998; Durate *et al*., 2001; Sabu *et al*., 2002; Skrzydlewska *et al.*, 2002; Chander *et al*., 2003; Kuo *et al*., 2005; Babu *et al*., 2006; Alipoor *et al*., 2009).

Glutathione is another parameter which has been measured in some studies and seems to increase in diabetics receiving tea intervention (Sohn *et al*., 1994; Lin *et al*., 1998; Durate *et al*., 2001; Sabu *et al*., 2002; Skrzydlewska *et al.*, 2002; Babu *et al*., 2006; Alipoor *et al*., 2009).

Animal studies have strongly supported the idea of tea being an efficient suppressor of oxidative stress in diabetic animals but human studies have faced inconsistency which may be rooted in factors like the design and time course of the study, the dose supplemented, the oxidative status of the subjects at baseline, the type of the tea studied, the stage of the disease, confounding factors not considered in some studies. It is recommended that well designed controlled clinical trials be done taking into account all the factors affecting the oxidative status of the patients and using sensitive and specific indicators of oxidative stress.

Adlercreutz H, Hamalaine E, Gorbach S, Goldin B. (1992). Dietary phyto-eostrogens and the

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Alipoor B, Delazar A, Ostad Rahimi A, Alipoor Aghiri S, Mesghary M. (2009). The effect of

Alipoor B, Ostad Rahimi A, Delazar A, Meskary M, Osnaashary S, Vatankhah A.M, Alipoor

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**6. References** 

#### **4.4.2 Evidence from human studies**

Human studies are not as conclusive as animal ones. There is some evidence that in countries with higher tea consumption like Japan, diabetes is less prevalent (Iso *et al*., 2006). Some studies have shown a negative correlation between tea consumption and heart disorders and its consequent death (Stensvold *et al*., 1992; Imai *et al*., 1995; Duffy *et al*., 2001; Geleijnse *et al*., 2002; Hodgson *et al*., 2002; Hakim *et al.*, 2003) which can be, in large part, attributed to the effects of tea on endothelial function through reducing oxidative stress; but there have been studies in which no relation was observed (Brown *et al*., 1993; Sesso *et al*., 2003)

Tea consumption decreased lipid peroxidation in some clinical trials (Klaunig *et al*., 1999), but was not as effective in the others (Van Het Hof *et al*., 1999; Hodgson *et al*., 2000; Rumpler *et al*., 2001; Hodgson *et al*., 2002).

Results for malondialdehyde were inconsistent as well: some investigations indicating a negative relation between tea and MDA (Freese *et al*., 1999; Hirano-Ohmori *et al*., 2005; Nagao *et al*., 2005), others showing no significant relationship (Rumpler *et al*., 2001; Davis *et al*., 2003).

The activity of antioxidant enzymes or oxidative status of the serum were improved by tea intervention in some studies (Serafini *et al*., 1996; Nakagawa *et al*., 1999; Leenen *et al*., 2000; Sung *et al*., 2000; Young *et al*., 2002), but remained unchanged in the others (Van Het Hof *et al*., 1997; Princen *et al*., 1998; Freese *et al*., 1999; Miura *et al*., 2000; Davis *et al*., 2003; Henning *et al*., 2004; Davis *et al*., 2005).

As reviewed above there is some evidence that tea and its fractions can act against development of diabetes and its complications but some studies have shown insignificant results. More detailed and precise clinical trials are essential to better understanding of tea's role in diabetes through its capacity to reduce oxidative stress.

Although animal studies provide great deal of evidence on usefulness of tea and its polyphenols against oxidative stress and its consequences in diabetes, human studies are not conclusive and limited research has not generally revealed significant decreases in biomarkers of *in vivo* oxidative damage. Far wider genetic variations in the response of humans to oxidative stress in comparison with animals may be one important factor obscuring small changes in biomarkers induced by tea and its polyphenols. Another reason may be that, though the dose of tea and its effective compounds used in animal and human studies do not differ much. Much higher doses relative to body weight is used in animal studies (Frei *et al.*, 2003).

#### **5. Conclusion and future trends**

Oxidative stress has been showed to play an important role in initiation and progression of diabetes and its accompanying complications. Thus to prevent the very consequences of oxidative stress, it seems logical to take the necessary steps to reduce it. Antioxidants have been reported to be effective in fulfilling this goal. Tea is a great source of a group of antioxidants so called flavonoids. Animal studies have been done to detect which compounds in tea are responsible for its effects on oxidative stress but human studies are lacking.

Animal studies have strongly supported the idea of tea being an efficient suppressor of oxidative stress in diabetic animals but human studies have faced inconsistency which may be rooted in factors like the design and time course of the study, the dose supplemented, the oxidative status of the subjects at baseline, the type of the tea studied, the stage of the disease, confounding factors not considered in some studies. It is recommended that well designed controlled clinical trials be done taking into account all the factors affecting the oxidative status of the patients and using sensitive and specific indicators of oxidative stress.

#### **6. References**

240 Oxidative Stress – Environmental Induction and Dietary Antioxidants

Human studies are not as conclusive as animal ones. There is some evidence that in countries with higher tea consumption like Japan, diabetes is less prevalent (Iso *et al*., 2006). Some studies have shown a negative correlation between tea consumption and heart disorders and its consequent death (Stensvold *et al*., 1992; Imai *et al*., 1995; Duffy *et al*., 2001; Geleijnse *et al*., 2002; Hodgson *et al*., 2002; Hakim *et al.*, 2003) which can be, in large part, attributed to the effects of tea on endothelial function through reducing oxidative stress; but there have been

Tea consumption decreased lipid peroxidation in some clinical trials (Klaunig *et al*., 1999), but was not as effective in the others (Van Het Hof *et al*., 1999; Hodgson *et al*., 2000; Rumpler

Results for malondialdehyde were inconsistent as well: some investigations indicating a negative relation between tea and MDA (Freese *et al*., 1999; Hirano-Ohmori *et al*., 2005; Nagao *et al*., 2005), others showing no significant relationship (Rumpler *et al*., 2001; Davis *et al*., 2003). The activity of antioxidant enzymes or oxidative status of the serum were improved by tea intervention in some studies (Serafini *et al*., 1996; Nakagawa *et al*., 1999; Leenen *et al*., 2000; Sung *et al*., 2000; Young *et al*., 2002), but remained unchanged in the others (Van Het Hof *et al*., 1997; Princen *et al*., 1998; Freese *et al*., 1999; Miura *et al*., 2000; Davis *et al*., 2003; Henning

As reviewed above there is some evidence that tea and its fractions can act against development of diabetes and its complications but some studies have shown insignificant results. More detailed and precise clinical trials are essential to better understanding of tea's

Although animal studies provide great deal of evidence on usefulness of tea and its polyphenols against oxidative stress and its consequences in diabetes, human studies are not conclusive and limited research has not generally revealed significant decreases in biomarkers of *in vivo* oxidative damage. Far wider genetic variations in the response of humans to oxidative stress in comparison with animals may be one important factor obscuring small changes in biomarkers induced by tea and its polyphenols. Another reason may be that, though the dose of tea and its effective compounds used in animal and human studies do not differ much. Much higher doses relative to body weight is used in animal

Oxidative stress has been showed to play an important role in initiation and progression of diabetes and its accompanying complications. Thus to prevent the very consequences of oxidative stress, it seems logical to take the necessary steps to reduce it. Antioxidants have been reported to be effective in fulfilling this goal. Tea is a great source of a group of antioxidants so called flavonoids. Animal studies have been done to detect which compounds in tea are responsible for its effects on oxidative stress but human studies are

role in diabetes through its capacity to reduce oxidative stress.

studies in which no relation was observed (Brown *et al*., 1993; Sesso *et al*., 2003)

**4.4.2 Evidence from human studies** 

*et al*., 2001; Hodgson *et al*., 2002).

*et al*., 2004; Davis *et al*., 2005).

studies (Frei *et al.*, 2003).

lacking.

**5. Conclusion and future trends** 


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**12** 

 *India* 

**Flavonoid Treatment for** 

**Mustard Agents' Toxicity** 

Rajagopalan Vijayaraghavan and Anshoo Gautam *Defence Research and Development Establishment, Gwalior* 

The weapons of mass destruction, chemical, biological and nuclear warfare are the most brutal created by the humans. They kill and incapacitate not only the armed forces but also the innocent public, without any mercy. The Chemical Weapons Convention prohibits the production, storage and use of toxic chemicals during warfare. In fact, the use of "Any chemical which through its chemical action on life processes can cause death, temporary incapacitation or permanent harm to humans and animals" as a method of warfare is discouraged by the Chemical Weapons Convention and many of such toxic chemicals are listed in its three Schedules for verification purpose (OPCW). The chemical warfare agents are extremely toxic chemicals. They act in very small quantities and very rapidly, and death may occur in minutes, like the nerve agents and the blood agents (Somani, 1992). Some of them like the blistering agents, though may not cause immediate lethality, but are highly incapacitating (Dacre & Goldman, 1996). The nerve agents are organophosphorous compounds that include tabun, sarin, soman and Vx. They inhibit acetylcholinesterase enzyme resulting in the accumulation of acetylcholine leading to muscarinic and nicotinic receptor stimulation (Bajgar, 2004). The blood agents include the cyanides. They inhibit cytochrome oxidase enzyme leading to cellular hypoxia (Way, 1984). Though the nerve agents and the blood agents are immediately lethal, specific antidotes are available for use in the field as First Aid Kit (Vijayaraghavan et al, 2011). For nerve agent poisoning the recommended antidotes are atropine sulphate and pralidoxime chloride that are administered by autoinjectors (Friedl, 1989; Vijayaraghavan et al, 2007). For cyanide poisoning the recommended antidotes are amyl nitrite inhalation, and sodium nitrite and sodium thiosulphate injection (Chen & Rose, 1952; Bhattacharya & Vijayaraghavan, 2002). The blistering agents are the sulphur mustard (SM) and the nitrogen mustards (NM). They cause severe toxicity with delayed clinical symptom. In the biological system they undergo an intramolecular cyclisation and produce highly reactive electrophiles that have strong affinity for a variety of macromolecules. They are extremely toxic to rapidly dividing cells, resulting in multiorgan failure (Papirmeister et al, 1991). Unlike the nerve agents and the blood agents no specific treatment is available for the mustard agents. A wide variety of molecules are being evaluated as antidote for mustard agent toxicity. Antidote against mustard agents require few major characteristics: (a) molecules should be strong neucleophiles because mustard agents are highly reactive electrophiles, (b) molecules may

**1. Introduction** 

