**6. Health benefits**

#### **6.1. Health promotion**

The potential health effects of green tea catechins depend not only on the amount consumed, but also on their bioavailability, which appears to be substantially varied. Following the oral administration of tea catechins to rats [38] and mice [39], the four principal catechins have been identified in the portal vein, indicating that tea catechins are absorbed intestinally. There appear to be species-related differences in the bioavailability of EGCG compared to other tea

acid levels from 4.54 to 4.22 mg/dl (*p*<0.05) within 7 days [59]. Our group has currently found that the consumption of GTE (2, 4 and 6 g/day), of which the 2 g/day dose was found to be the most effective, lowered levels of serum uric acid (3.53%) and also increased serum troloxequivalent antioxidant capacity (TEAC) values in healthy volunteers (n=11) (unpublished data), suggesting an hypouricemic effect by XO inhibition or an increase of urinary uric acid

Green Tea: Just a Drink or Nutraceutical http://dx.doi.org/10.5772/57519 371

In consideration of the evidence for its iron chelating properties, strong antioxidant capacity, low toxicity, and orally available administration, green tea has potential to be used as a pharmacological agent in the management of diseasesrelated to iron overload. Hypothetically, the removal of iron could reduce oxidative damage to the cellular biomolecules, including lipids, proteins, carbohydrates and nucleic acids, and this can consequently prevent, as well as improve the vital organ dysfunctions. DFP, known to readily permeate and mobilize nonheme iron from β-thalassemic patient erythrocytes, serves as a benchmark against which orally effective agents; catechins in GTE and purified EGCG products have been compared. Our studies have paid particular attention to the efficacy of GTE and EGCG in terms of the aspects of anti-oxidation and the potential for iron chelation in order to alleviate oxidative stress and iron overload in β-thalassemic mice in the past experiments, and in Thai β-thalas‐

Though the pathological role of redox iron in the hemosiderosis (such as hereditary hemochromatosis and thalassemia) is well characterized, excessive accumulation of the iron can result in significant organ dysfunction and abnormality. Recognition of the role of iron in conditions beyond transfusion-dependent iron overload may bring significant implica‐ tions for the management of metabolic, infectious, and degenerative diseases. New findings obtained during the past years, especially in terms of the discovery of mutations in the genes associated with brain iron metabolism, have provided key insights into the mecha‐ nisms of brain iron homeostasis and the pathological mechanisms responsible for neurode‐ generative diseases. Increased iron in the brain, which is rich in oxygen and fatty acids, provides an ideal environment for oxidative stress and possible irreparable tissue dam‐ age. Oxidative stress, resulting from increased brain iron levels, and possibly also from defects in antioxidant defense mechanisms, is widely believed to be associated with

The interaction between iron overload and dietary antioxidants has been well characterized; especially, with respect to vitamins E and C. Vitamin E has been extensively studied with respect to its capacity to protect molecules from the *in vitro* and *in vivo* effects of iron toxicity [62, 63]. Elevated levels of ROS tended to normalize in response to oral therapy with vitamin E, with patients exhibiting improvement in the antioxidant–oxidant balance in plasma and decreased lipid peroxidation in erythrocytes [64]. However, prolonged administration of vitamin E did not result in any significant changes in Hb levels in patients with β-thalassemia intermedia [65]. Therefore, vitamin E by itself is probably insufficient in inducing major

excretion.

**6.2. Hematologic disorders**

semia patients with regard to the near future.

neuronal death in brain disorders.

**Figure 2.** Possible mechanism for the hepaticgalloyl ring metabolism (Redrawn from [36]). Abbreviations: COMT=cat‐ echol-*O*-methyltransferase; GSH=reduced glutathione;NAD(P)+=nicotinamide adenine dinucleotide phosphate; NAD(P)H=reduced nicotinamide adenine dinucleotide phosphate; NQO1=NAD(P)H:quinone oxidoreductase-1; SAM=*S*-adenosyl methionine

catechins [7]. The epicatechin isomers purified from green tea were effective agents in protecting human low-density lipoproteins (LDL) and red blood cell (RBC) membranes from oxidative modification [40]. These effects are partly due to their free-radical scavenging abilities [20] and their iron-chelating properties, which are capable of binding any available iron, thus greatly reducing their bioavailability. They also have many pharmacological properties, including anti-hypertensive [41], anti-atheroslcerotic [42], anti-carcinogenic [43-45] and hypocholesterolemic effects [46, 47]. Health benefits of green tea consumption were achieved in the rodents within relatively short periods: three weeks [48], four weeks [49, 50], five weeks [51, 52] and eight weeks [53, 54].

Surprisingly, catechins inhibited xanthine oxidase (XO) activity *in vitro* [55– 58] and the XO inhibition constant (*Ki* ) of EGCG was comparable to that of the common XO inhibitor allopur‐ inol (0.76 versus 0.30 µmol) [56]. GTE consumption was found to decrease levels of serum uric acid concentrations observed in human subjects [59-61]; for instance, by decreasing serum uric acid levels from 4.54 to 4.22 mg/dl (*p*<0.05) within 7 days [59]. Our group has currently found that the consumption of GTE (2, 4 and 6 g/day), of which the 2 g/day dose was found to be the most effective, lowered levels of serum uric acid (3.53%) and also increased serum troloxequivalent antioxidant capacity (TEAC) values in healthy volunteers (n=11) (unpublished data), suggesting an hypouricemic effect by XO inhibition or an increase of urinary uric acid excretion.

#### **6.2. Hematologic disorders**

catechins [7]. The epicatechin isomers purified from green tea were effective agents in protecting human low-density lipoproteins (LDL) and red blood cell (RBC) membranes from oxidative modification [40]. These effects are partly due to their free-radical scavenging abilities [20] and their iron-chelating properties, which are capable of binding any available iron, thus greatly reducing their bioavailability. They also have many pharmacological properties, including anti-hypertensive [41], anti-atheroslcerotic [42], anti-carcinogenic [43-45] and hypocholesterolemic effects [46, 47]. Health benefits of green tea consumption were achieved in the rodents within relatively short periods: three weeks [48], four weeks [49, 50],

**Figure 2.** Possible mechanism for the hepaticgalloyl ring metabolism (Redrawn from [36]). Abbreviations: COMT=cat‐ echol-*O*-methyltransferase; GSH=reduced glutathione;NAD(P)+=nicotinamide adenine dinucleotide phosphate; NAD(P)H=reduced nicotinamide adenine dinucleotide phosphate; NQO1=NAD(P)H:quinone oxidoreductase-1;

O O OH


COOH

NAD(P)H

*o*-quinone

OCH3 HO OH

COOH

Surprisingly, catechins inhibited xanthine oxidase (XO) activity *in vitro* [55– 58] and the XO

inol (0.76 versus 0.30 µmol) [56]. GTE consumption was found to decrease levels of serum uric acid concentrations observed in human subjects [59-61]; for instance, by decreasing serum uric

) of EGCG was comparable to that of the common XO inhibitor allopur‐

OH HO OH

> GALLIC ACID GSH CONJUGATE

COOH

GS

five weeks [51, 52] and eight weeks [53, 54].

inhibition constant (*Ki*

OH HO OH

GALLIC ACID

O HO OH

Phenoxyl radical

SAM=*S*-adenosyl methionine

COOH

O2

O2

O2

H2O2

COOH

SAM

370 Pharmacology and Nutritional Intervention in the Treatment of Disease

COMT

NQO1

NAD(P)+

In consideration of the evidence for its iron chelating properties, strong antioxidant capacity, low toxicity, and orally available administration, green tea has potential to be used as a pharmacological agent in the management of diseasesrelated to iron overload. Hypothetically, the removal of iron could reduce oxidative damage to the cellular biomolecules, including lipids, proteins, carbohydrates and nucleic acids, and this can consequently prevent, as well as improve the vital organ dysfunctions. DFP, known to readily permeate and mobilize nonheme iron from β-thalassemic patient erythrocytes, serves as a benchmark against which orally effective agents; catechins in GTE and purified EGCG products have been compared. Our studies have paid particular attention to the efficacy of GTE and EGCG in terms of the aspects of anti-oxidation and the potential for iron chelation in order to alleviate oxidative stress and iron overload in β-thalassemic mice in the past experiments, and in Thai β-thalas‐ semia patients with regard to the near future.

Though the pathological role of redox iron in the hemosiderosis (such as hereditary hemochromatosis and thalassemia) is well characterized, excessive accumulation of the iron can result in significant organ dysfunction and abnormality. Recognition of the role of iron in conditions beyond transfusion-dependent iron overload may bring significant implica‐ tions for the management of metabolic, infectious, and degenerative diseases. New findings obtained during the past years, especially in terms of the discovery of mutations in the genes associated with brain iron metabolism, have provided key insights into the mecha‐ nisms of brain iron homeostasis and the pathological mechanisms responsible for neurode‐ generative diseases. Increased iron in the brain, which is rich in oxygen and fatty acids, provides an ideal environment for oxidative stress and possible irreparable tissue dam‐ age. Oxidative stress, resulting from increased brain iron levels, and possibly also from defects in antioxidant defense mechanisms, is widely believed to be associated with neuronal death in brain disorders.

The interaction between iron overload and dietary antioxidants has been well characterized; especially, with respect to vitamins E and C. Vitamin E has been extensively studied with respect to its capacity to protect molecules from the *in vitro* and *in vivo* effects of iron toxicity [62, 63]. Elevated levels of ROS tended to normalize in response to oral therapy with vitamin E, with patients exhibiting improvement in the antioxidant–oxidant balance in plasma and decreased lipid peroxidation in erythrocytes [64]. However, prolonged administration of vitamin E did not result in any significant changes in Hb levels in patients with β-thalassemia intermedia [65]. Therefore, vitamin E by itself is probably insufficient in inducing major changes in the rate of RBC hemolysis and in prolonging their survival, resulting in increased Hb levels.

**Mice**

WT (n = 24)

BKO (n = 16)

DH (n = 10)

WT (n = 10)

BKO (n = 10)

DH (n = 10)

WT (n = 8)

BKO (n = 8)

DH (n = 8)

WT (n = 8)

BKO (n = 8)

DH (n = 8)

ND=not done.

14.80±1.14

9.45 ±0.56

38.46±9.17

48.29±8.01

43.08±4.38

ND ND

ND ND

ND ND

15.19 ±1.11

9.35 ±0.40

38.29 ±10.93

53.92 ±9.69

44.38 ±3.78

9.28 ±0.31

2.80 ±0.52

2.56 ±0.22

2.76 ±0.18

9.55 ±0.59

**Fe diet (0.2% ferrocene, w/w)**

Month 0 Month 1 Month 2 Month 0 Month 1 Month 2 Month 0 Month 1 Month 2 **Blood Hb concentration (g/dl)**

> 9.60 ±0.45

15.97±1.09 15.80±1.01 15.85±0.88 16.11±0.84 16.25±0.85 16.17±0.77 16.33±0.76 16.41±0.72 16.29±0.71

**Erythrocyte ROS (Fluorescent intensity unit)**

**Erythrocyte MDA (pmol/g Hb)**

ND ND 48.23±6.15 ND ND 36.80±3.23† ND ND 36.82±2.71†

ND ND 60.63±3.28 ND ND 44.26±2.87† ND ND 44.74±2.79†

ND ND 50.86±1.90 ND ND 42.00±2.85† ND ND 40.36±2.83†

**Erythrocyte GSH (**μ**mol/g Hb)**

ND ND

ND ND

ND ND

†*p* <0.05 compared to DW.

**Table 3.** Levels of blood Hb, erythrocyte ROS, GSH and MDA (mean±SD) in WT, BKO and DH mice fed with an Fe diet (iron content 780 mg/kg) and treated with DW, GTE and EGCG for 2 months.†*p* <0.05 compared to DW treatment.

**+DW +GTE (90 mg/kg/day) +EGCG (50 mg/kg/day)**

15.15±1.06 15.22±1.23 15.37±1.23 15.32±1.14 15.29±1.25 15.40±1.13 15.41±1.00

9.54 ±0.48

42.62±9.63 31.74±9.14 23.29±3.99† 22.45±3.39† 33.07±8.08 26.87±3.72† 23.26±3.02†

59.81±8.96 35.07±7.68 33.69±7.83† 38.50±6.90† 33.88±7.08 31.47±8.93† 36.67±5.16†

49.60±4.25 31.97±4.23 31.57±4.31 27.88±3.65† 31.51±5.38 31.50±4.38 28.17±4.64†

3.61 ±0.32†

3.31 ±0.40†

3.49 ±0.22† ND ND

ND ND

ND ND

3.73 ±0.29†

3.24 ±0.36†

3.46 ±0.19†

9.45 ±0.56

9.53 ±0.36

Green Tea: Just a Drink or Nutraceutical http://dx.doi.org/10.5772/57519

> 9.56 ±0.39

373

The interaction of another dietary antioxidant, vitamin C (ascorbic acid) and iron is less clear. Ascorbic acid can reduce 'free iron' (ferric) to a ferrous form, promoting the initiation and propagation of free radical reactions [66, 67]. In people under a risk of iron overload, in which the elevated levels of iron could lead to higher 'free iron' concentrations, an excess of vitamin C could have deleterious effects. Plant flavonoids (including rutin and curcumin) are another group of antioxidants, which may have therapeutic potential in thalassemia. However, despite their apparent salutary effects on erythrocytes, antioxidants have not yet been shown to ameliorate the anemia of the patients [68]. Antioxidants may be more effective if used in combination with an iron chelator. This approach, if successful, could be particularly useful in countries with limited financial resources.

The potential of green tea to prevent or ameliorate chronic disease is currently the subject of considerable scientific investigation. Although a number of mechanisms have been proposed as being responsible for green tea's beneficial effects, the radical scavenging and antioxidant properties of green tea catechins are frequently cited as important contributors. Emerging evidence has shown that catechins and their metabolites have many additional mechanisms of action [69] by affecting numerous sites, potentiating endogenous antioxidants and eliciting dual actions during oxidative stress. Much of the evidence supporting the antioxidant function for green tea catechins has been derived from assays of their antioxidant activity *in vitro*. However, evidence that green tea catechins are acting directly or indirectly as antioxidants *in vivo* is more limited [14]. Interestingly, green tea catechins can play a double role in reducing the rate of oxidation, as they can participate in: i) iron chelation [19]; and ii) trapping radicals [2, 20]. The catechins have been shown to protect culture cells from iron-mediated damage [21, 22]. In animal models of iron overload, they have been reported to ameliorate iron accumula‐ tion [17] and inhibit iron-induced lipid oxidation in the liver [23]. They also play a dual effect in decreased labile iron in the plasma and consequently depleting oxidative stress in ironloaded rats [18]. Animal studies offer a unique opportunity to assess the contribution of the antioxidant properties of the catechins in terms of the physiological effects of tea administra‐ tion in different models of oxidative-related diseases. Combining free-radical scavenging with iron-chelating properties, green tea catechins may have a capacity of chelating an excess of iron under iron-overloaded conditions and play the important preventive and/or protective roles in this unpleasant condition. None of the treatments over 2 months affected the levels of blood hemoglobin (Hb) in the WT, BKO and DH mice challenged by iron overload, there were fed a high iron-diet. Nonetheless, iron-induced oxidative stress as well as GSH content in the RBC cytoplasm, and lipid-peroxidation in the RBC plasma membrane were reversed by the GTE that was used in the testing, as well as the EGCG, when compared to the DW treatment (Table 3). We also found that these two green tea products increased the survival rate or halflife of the RBC of the WT and BKO mice significantly (Sakaewan Ounjaijen and Somdet Srichairatanakool, unpublished data). Our results suggest that GTE and EGCG treatment might directly increase the erythropoietic rate, either in normal mice or in thalassemic mice, but probably do protect red cells from ROS-induced hemolysis.


changes in the rate of RBC hemolysis and in prolonging their survival, resulting in increased

The interaction of another dietary antioxidant, vitamin C (ascorbic acid) and iron is less clear. Ascorbic acid can reduce 'free iron' (ferric) to a ferrous form, promoting the initiation and propagation of free radical reactions [66, 67]. In people under a risk of iron overload, in which the elevated levels of iron could lead to higher 'free iron' concentrations, an excess of vitamin C could have deleterious effects. Plant flavonoids (including rutin and curcumin) are another group of antioxidants, which may have therapeutic potential in thalassemia. However, despite their apparent salutary effects on erythrocytes, antioxidants have not yet been shown to ameliorate the anemia of the patients [68]. Antioxidants may be more effective if used in combination with an iron chelator. This approach, if successful, could be particularly useful

The potential of green tea to prevent or ameliorate chronic disease is currently the subject of considerable scientific investigation. Although a number of mechanisms have been proposed as being responsible for green tea's beneficial effects, the radical scavenging and antioxidant properties of green tea catechins are frequently cited as important contributors. Emerging evidence has shown that catechins and their metabolites have many additional mechanisms of action [69] by affecting numerous sites, potentiating endogenous antioxidants and eliciting dual actions during oxidative stress. Much of the evidence supporting the antioxidant function for green tea catechins has been derived from assays of their antioxidant activity *in vitro*. However, evidence that green tea catechins are acting directly or indirectly as antioxidants *in vivo* is more limited [14]. Interestingly, green tea catechins can play a double role in reducing the rate of oxidation, as they can participate in: i) iron chelation [19]; and ii) trapping radicals [2, 20]. The catechins have been shown to protect culture cells from iron-mediated damage [21, 22]. In animal models of iron overload, they have been reported to ameliorate iron accumula‐ tion [17] and inhibit iron-induced lipid oxidation in the liver [23]. They also play a dual effect in decreased labile iron in the plasma and consequently depleting oxidative stress in ironloaded rats [18]. Animal studies offer a unique opportunity to assess the contribution of the antioxidant properties of the catechins in terms of the physiological effects of tea administra‐ tion in different models of oxidative-related diseases. Combining free-radical scavenging with iron-chelating properties, green tea catechins may have a capacity of chelating an excess of iron under iron-overloaded conditions and play the important preventive and/or protective roles in this unpleasant condition. None of the treatments over 2 months affected the levels of blood hemoglobin (Hb) in the WT, BKO and DH mice challenged by iron overload, there were fed a high iron-diet. Nonetheless, iron-induced oxidative stress as well as GSH content in the RBC cytoplasm, and lipid-peroxidation in the RBC plasma membrane were reversed by the GTE that was used in the testing, as well as the EGCG, when compared to the DW treatment (Table 3). We also found that these two green tea products increased the survival rate or halflife of the RBC of the WT and BKO mice significantly (Sakaewan Ounjaijen and Somdet Srichairatanakool, unpublished data). Our results suggest that GTE and EGCG treatment might directly increase the erythropoietic rate, either in normal mice or in thalassemic mice,

Hb levels.

in countries with limited financial resources.

372 Pharmacology and Nutritional Intervention in the Treatment of Disease

but probably do protect red cells from ROS-induced hemolysis.

**Table 3.** Levels of blood Hb, erythrocyte ROS, GSH and MDA (mean±SD) in WT, BKO and DH mice fed with an Fe diet (iron content 780 mg/kg) and treated with DW, GTE and EGCG for 2 months.†*p* <0.05 compared to DW treatment. ND=not done.

The inhibition of the growth of blast cells from patients with acute myelocytic leukemic (AML) by EGCG affected hematopoietic growth factors (HGF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF) or interleukin-3 (IL-3), was useful in terms of the stimulation of leukemic cell proliferation. EGCG dosage and time dependently reduced the survival of NSF60 leukemic cell lines [69, 70]. These factors can lead to an induction of cell apoptosis, including endonuclease activation, p53 induction, caspase 3 protease activation *via* a Bcl-2-insensitive pathway, free-radical production and sphinganine accumulation. Chemopreventive effects in multi-step carcinogenesis by EGCG have also been mentioned [71]. From in vitro studies, green tea and EGCG (dose 9-27 µg/ml) displayed a significant level of inhibition of the peripheral blood T-lymphocytes of adult T-lymphocytes patients, along with a moderate level of inhibition of human T-cell lymphotropic virus type I (HTLV-I)-infected T-cell line, but not of T-lymphocytes in the healthy controls. This was possibly due to a suppression of HTLV-I pX gene expression and induction of cell apoptosis [72].Consistently, Japanese HTLV-1 carriers who had taken green tea extract powder (9 capsules/day equivalent to 10 cups of regular green tea drinking) for 5 months showed a significant decrease in the HTLV-1 provirus load, suggesting that green tea drinking could inhibit the proliferation of HTLV-1-infected lymphocytes [73].

fat absorption, plasma levels of triglycerides, free fatty acids, cholesterol, glucose, insulin and

Green Tea: Just a Drink or Nutraceutical http://dx.doi.org/10.5772/57519 375

Yang and Koo reported that the ECG and EGCG that are present in Chinese green tea reduced lipid deposition-caused weight, as well as the cholesterol content of the liver, importantly lowered levels of serum cholesterol levels and the atherogenic index in the rats fed with a cholesterol-enriched diet [54]. Rats fed with the green tea powder (20 g/kg)-enriched diet showed a significant increase in the lag-phase oxidation of plasma with very low density in lipoproteins (VLDL) and LDL by 33% when compared to the control diet [48]. EGCG was proposed to be a competitive inhibitor of β-ketoacyl reductase of the FAS enzyme complex in the liver [79]. EGCG and ECG lowered FAS and malate enzymes in rat liver cytosol, resulting in a significant decrease in visceral fat deposition and hepatic triglyceride content [80]. Nonetheless, experiments showed that GTE lowered serum cholesterol and triglyceride concentrations in the hamsters fed with high fat diet (200 g lard and 1 g cholesterol/kg) in a concentration-dependent manner, for which the mechanism was most likely due to its influence on the absorption of dietary fat and cholesterol, but not on the inhibition of the synthesis of cholesterol or fatty acid [49]. Another study supports the evidence that Chinese green tea lowered plasma cholesterol levels by increasing fecal bile acids and cholesterol excretion, but not by inhibiting activities of three major lipid metabolizing enzymes, including 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-Co A) reductase, cholesterol 7α-hydroxylase and FAS [53]. More importantly, high plasma cholesterol levels, fatty liver, renal dysfunctions and severe atherosclerotic aorta were observed in the LDL knockout mice fed with a normal diet, but not in those fed with a 4% green tea catechins-enriched diet for 35 weeks [81].

GTE, which had greater effects than oolong tea and black tea, decreased high serum trigly‐ ceride (195 vs. 119 mg/dl) and cholesterol (104 vs. 73 mg/dl) levels to normal levels, and also decreased hepatic triglyceride content in sucrose-induced hyperlipidemic rats (8.4±1.5 vs. 27.7±7.9 mg/g wet weight (*p*<0.05); possibly by limiting the intestinal absorption of dietary fat and storage of fat in the liver [52]. EGCG (dose of 0.5 g/kg for 4 weeks) can interfere with the micellar solubilization of dietary cholesterol in the gastrointestinal tract and subsequently

Green tea catechins functioned against membrane phospholipid peroxidation [82], showed a strong synergistic anti-oxidative activity with α-tocopherol in micelles [83],as well as in human LDL levels [84], and had a protective effect of high-density lipoprotein (HDL) on endothelial cell-dependent vasodilatation [85]. They have many pharmacological activities, including antihypertensive [41] and anti-atherosclerotic [86, 87] effects. Clearly, green tea catechins; espe‐ cially EGCG, has a potential hypolipidemic effect, possibly by interfering with the emulsification, digestion, and micellar solubilization of lipids in the critical steps of intestinal absorption of dietary lipids [88]. Bursill and coworkers found that GTE (2% catechins, *w*/*w*) decreased the levels of total cholesterol (-60%), VLDL-and intermediate density lipoprotein (IDL)-cholesterol (-70%), LDL-cholesterol (-80%) in plasma, total cholesterol (622 versus 800 µmol/g,-10%) and unesterified cholesterol (323 versus 399 µmol/g,-15%) in the liver, and cholesterol (-25%) in the thoracic aorta (0.73 versus 0.98 µmol/g,-25%) and aortic arch fatty streak (1.93 versus 2.35 µmol/g) of the rabbits fed with a high cholesterol (0.25%, *w*/*w*) diet

reduce cholesterol absorption in the rats fed with a high cholesterol diet [50].

leptin; and increases in β-oxidation and thermogenesis.

Surprisingly, EGCG diminished the phosphorylation of vascular endothelial growth factor (VEGF) receptors, potent angiogenic cytokines that are essential for the survival of tumor cells in the autocrine pathway, in chronic lymphocytic leukemia B cells that have been isolated from leukemic patients, leading to cell apoptosis [74]. EGCG decreased human telomerase reverse transcriptase (hTERT) promoter methylation and histone H3 Lys9 acetylation, but increased hTERT repressor E2F-1 binding at the promoter of MCF-7 breast cancers and HL60 promye‐ locytic leukemia cell cultures [75]. EGCG treatment induced death-associated protein kinase 2 (DAPK2) in multiple myeloma cells and dose dependently increased levels of the DAPK2 in HL60 and NB4 AML cells, while a combined treatment of EGCG (0 – 40 µM) with all-*trans* retinoic acid (ATRA) (1 µM) cooperatively induced and potentially differentiated the DAPK2 enzyme [76]. After ten patients with stage-0 chronic lymphocytic leukemia (CLL) had been orally given GTE for 6 months; eight patients showed decreases in lymphocytosis and circulating regulatory T cells (Treg) numbers, while one patient revealed stable lymphocytosis with decreased Treg numbers, and one patient showed increased lymphocytosis and Treg numbers [77].

The results of the phase 2 clinical study demonstrated that patients with early stage CLL consumed GTE (Polyphenon E preparation, dose of 2000 mg/day, twice daily) for 6 months showed decreases in the absolute lymphocyte counts and lymphadenopathy [78].

#### **6.3. Cardiovascular diseases**

Epigenetics, hypercholesterolemia, diabetes, hypertension, heavy smoking, physical inactivi‐ ty, stress and obesity are all risk factors for atherosclerosis and cardiovascular diseases (CVD). Green tea catechins have been reported to exert anti-obesity effects, including a reduction of adipocyte differentiation and proliferation; decreases in lipogenesis, fat mass, body weight, fat absorption, plasma levels of triglycerides, free fatty acids, cholesterol, glucose, insulin and leptin; and increases in β-oxidation and thermogenesis.

The inhibition of the growth of blast cells from patients with acute myelocytic leukemic (AML) by EGCG affected hematopoietic growth factors (HGF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF) or interleukin-3 (IL-3), was useful in terms of the stimulation of leukemic cell proliferation. EGCG dosage and time dependently reduced the survival of NSF60 leukemic cell lines [69, 70]. These factors can lead to an induction of cell apoptosis, including endonuclease activation, p53 induction, caspase 3 protease activation *via* a Bcl-2-insensitive pathway, free-radical production and sphinganine accumulation. Chemopreventive effects in multi-step carcinogenesis by EGCG have also been mentioned [71]. From in vitro studies, green tea and EGCG (dose 9-27 µg/ml) displayed a significant level of inhibition of the peripheral blood T-lymphocytes of adult T-lymphocytes patients, along with a moderate level of inhibition of human T-cell lymphotropic virus type I (HTLV-I)-infected T-cell line, but not of T-lymphocytes in the healthy controls. This was possibly due to a suppression of HTLV-I pX gene expression and induction of cell apoptosis [72].Consistently, Japanese HTLV-1 carriers who had taken green tea extract powder (9 capsules/day equivalent to 10 cups of regular green tea drinking) for 5 months showed a significant decrease in the HTLV-1 provirus load, suggesting that green tea drinking could

Surprisingly, EGCG diminished the phosphorylation of vascular endothelial growth factor (VEGF) receptors, potent angiogenic cytokines that are essential for the survival of tumor cells in the autocrine pathway, in chronic lymphocytic leukemia B cells that have been isolated from leukemic patients, leading to cell apoptosis [74]. EGCG decreased human telomerase reverse transcriptase (hTERT) promoter methylation and histone H3 Lys9 acetylation, but increased hTERT repressor E2F-1 binding at the promoter of MCF-7 breast cancers and HL60 promye‐ locytic leukemia cell cultures [75]. EGCG treatment induced death-associated protein kinase 2 (DAPK2) in multiple myeloma cells and dose dependently increased levels of the DAPK2 in HL60 and NB4 AML cells, while a combined treatment of EGCG (0 – 40 µM) with all-*trans* retinoic acid (ATRA) (1 µM) cooperatively induced and potentially differentiated the DAPK2 enzyme [76]. After ten patients with stage-0 chronic lymphocytic leukemia (CLL) had been orally given GTE for 6 months; eight patients showed decreases in lymphocytosis and circulating regulatory T cells (Treg) numbers, while one patient revealed stable lymphocytosis with decreased Treg numbers, and one patient showed increased lymphocytosis and Treg

The results of the phase 2 clinical study demonstrated that patients with early stage CLL consumed GTE (Polyphenon E preparation, dose of 2000 mg/day, twice daily) for 6 months

Epigenetics, hypercholesterolemia, diabetes, hypertension, heavy smoking, physical inactivi‐ ty, stress and obesity are all risk factors for atherosclerosis and cardiovascular diseases (CVD). Green tea catechins have been reported to exert anti-obesity effects, including a reduction of adipocyte differentiation and proliferation; decreases in lipogenesis, fat mass, body weight,

showed decreases in the absolute lymphocyte counts and lymphadenopathy [78].

inhibit the proliferation of HTLV-1-infected lymphocytes [73].

374 Pharmacology and Nutritional Intervention in the Treatment of Disease

numbers [77].

**6.3. Cardiovascular diseases**

Yang and Koo reported that the ECG and EGCG that are present in Chinese green tea reduced lipid deposition-caused weight, as well as the cholesterol content of the liver, importantly lowered levels of serum cholesterol levels and the atherogenic index in the rats fed with a cholesterol-enriched diet [54]. Rats fed with the green tea powder (20 g/kg)-enriched diet showed a significant increase in the lag-phase oxidation of plasma with very low density in lipoproteins (VLDL) and LDL by 33% when compared to the control diet [48]. EGCG was proposed to be a competitive inhibitor of β-ketoacyl reductase of the FAS enzyme complex in the liver [79]. EGCG and ECG lowered FAS and malate enzymes in rat liver cytosol, resulting in a significant decrease in visceral fat deposition and hepatic triglyceride content [80]. Nonetheless, experiments showed that GTE lowered serum cholesterol and triglyceride concentrations in the hamsters fed with high fat diet (200 g lard and 1 g cholesterol/kg) in a concentration-dependent manner, for which the mechanism was most likely due to its influence on the absorption of dietary fat and cholesterol, but not on the inhibition of the synthesis of cholesterol or fatty acid [49]. Another study supports the evidence that Chinese green tea lowered plasma cholesterol levels by increasing fecal bile acids and cholesterol excretion, but not by inhibiting activities of three major lipid metabolizing enzymes, including 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-Co A) reductase, cholesterol 7α-hydroxylase and FAS [53]. More importantly, high plasma cholesterol levels, fatty liver, renal dysfunctions and severe atherosclerotic aorta were observed in the LDL knockout mice fed with a normal diet, but not in those fed with a 4% green tea catechins-enriched diet for 35 weeks [81].

GTE, which had greater effects than oolong tea and black tea, decreased high serum trigly‐ ceride (195 vs. 119 mg/dl) and cholesterol (104 vs. 73 mg/dl) levels to normal levels, and also decreased hepatic triglyceride content in sucrose-induced hyperlipidemic rats (8.4±1.5 vs. 27.7±7.9 mg/g wet weight (*p*<0.05); possibly by limiting the intestinal absorption of dietary fat and storage of fat in the liver [52]. EGCG (dose of 0.5 g/kg for 4 weeks) can interfere with the micellar solubilization of dietary cholesterol in the gastrointestinal tract and subsequently reduce cholesterol absorption in the rats fed with a high cholesterol diet [50].

Green tea catechins functioned against membrane phospholipid peroxidation [82], showed a strong synergistic anti-oxidative activity with α-tocopherol in micelles [83],as well as in human LDL levels [84], and had a protective effect of high-density lipoprotein (HDL) on endothelial cell-dependent vasodilatation [85]. They have many pharmacological activities, including antihypertensive [41] and anti-atherosclerotic [86, 87] effects. Clearly, green tea catechins; espe‐ cially EGCG, has a potential hypolipidemic effect, possibly by interfering with the emulsification, digestion, and micellar solubilization of lipids in the critical steps of intestinal absorption of dietary lipids [88]. Bursill and coworkers found that GTE (2% catechins, *w*/*w*) decreased the levels of total cholesterol (-60%), VLDL-and intermediate density lipoprotein (IDL)-cholesterol (-70%), LDL-cholesterol (-80%) in plasma, total cholesterol (622 versus 800 µmol/g,-10%) and unesterified cholesterol (323 versus 399 µmol/g,-15%) in the liver, and cholesterol (-25%) in the thoracic aorta (0.73 versus 0.98 µmol/g,-25%) and aortic arch fatty streak (1.93 versus 2.35 µmol/g) of the rabbits fed with a high cholesterol (0.25%, *w*/*w*) diet compared with the placebo group, possibly due to a decrease in cholesterol synthesis and an upregulation of hepatic LDL receptor gene expression [89, 90]. Controversial data has indi‐ cated that the consumption of high doses of green tea polyphenols (714 mg/day) for 3 weeks resulted in decreases in the total cholesterol:HDL-cholesterol ratio, but had no effects on the risk biomarkers of CVD [91].

#### **6.4. Neurological diseases**

The errors in brain iron metabolism found in neurological disorders are found to be multifac‐ torial, however treatment conditions seem to be particularly important. Epilepsy, an oxidative related disorder of the brain, has served as a model for the investigation in the role of iron, especially NTBI in neurological disorders. The effect of antiepileptic drug treatment on changes of iron status and oxidative stress parameters were determined [92]. Recent genetic and biochemical manipulations of iron overload have placed iron at the centre of research into neurodegenerative diseases. This is supported by the discovery of genetic and non-genetic misregulations of the iron metabolism in these disorders. In many neurodegenerative disor‐ ders, abnormally high levels of iron in specific regions of the brain have been reported [93]. Evidence for iron contribution to diseases by augmentation of oxidative stress have led to an examination of the contribution of iron to other diseases, in which oxidative stress may be involved, including epilepsy [94]. It has not been possible to determine whether the accumu‐ lated iron is in the labile iron pool (LIP), which can participate in the Fenton reaction to generate the reactive hydroxyl radical. The reduction in GSH during disease progression and in response to neurotoxins, together with increased iron accumulation and ROS generation, might be taken as evidence for the presence of free iron [95].

Oxidative stress, resulting from increased brain iron levels, and possibly also from defects in antioxidant defensive mechanisms, is widely believed to be associated with neuronal death in these disorders [96, 97], along with a reduced availability of GSH and other antioxidant substances in the brain. Changes in the integrity of the blood brain barrier (BBB) due to altered vascularization of the tissue or inflammatory events could be another initial cause. However, neuronal death by any initial cause could lead to large amounts of iron release and increased ROS formation [98]. Therefore, iron and iron-induced oxidative stress could possibly be a common mechanism involved in the development of neurodegeneration [99] (Figure 3). Available data strongly support this hypothesis [100, 101]

would raise the possibility of monitoring iron changes as a marker of disease progression, and perhaps even in a pre-clinical diagnosis in conditions where iron misregulation is an early

**Ferritin**

Released iron Neurotoxic event,

+ -synuclein + A

Fenton reaction

OH + OH

Vitamin E GSH GSH/ GSSG Glutathioneperoxidase Lipid peroxidation DNA damage

genetic-environment

Green Tea: Just a Drink or Nutraceutical http://dx.doi.org/10.5772/57519 377


**Oxidativestress N euronal death**

Protein oxidation and misf olding

Fe2+ Fe3+ Fe3+

**Labileironpool (I RPs)**

H2O2

Other oxidative processes

H2O + 1/ 2O2

**Figure 3.** Mechanism of iron-induced neurodegeneration and its prevention [95]. Abbreviations: DMT1=divalent met‐ al ion transporter-1; GSH=reduced glutathione; GSSG=oxidized glutathione; IRPs=iron regulatory proteins; MAO=monoamine oxidase; MPTP=1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine;NADP+=nicotinamide adenine dinu‐ cleotide phosphate; NADPH=reduced nicotinamide adenine dinucleotide phosphate; 6-OHDA=6-hydroxydopamine

More *in vitro* and *in vivo* studies should be done to investigate how iron misregulation/ accumulation synergizes with common endogenous and environmental toxins. Iron misregulation/accumulation alone can kill neurons only in genetic disorders where iron imbalance occurs rapidly and is extensive. However, in most cases, the amount of iron build-up is relatively low and the neurotoxic mechanism might involve a combination of iron and other toxins. Future investigations of iron in the brain, including how to best monitor iron deposition *in vivo*, the clinical relevance of excessive iron deposition, and the mechanistic relationship between iron deposition and disease pathophysiology, hold the promise of advancements in

Topical applications of green tea polyphenol fractions (such as EGCG, EGC and ECG) inhibitedbenz[a]-pyrene (BP)-and 7,12-dimethylbenz[a]-anthracene (DMBA)-initiated and 12- *O*-tetradecanoylphorbol-13-acetate (TPA)-promoted tumorigenesis of mouse skin, possibly by inhibiting ROS production, inflammation and hyperplasia [105]. Green tea catechins can

event.

NADP+

NADPH+H<sup>+</sup>

the field of neurotherapeutics.

**6.5. Mutagenicity and cancinogenesis**

Fe Serum iron 2+

2GSH

Dopamine MAO R-CHO + NH3+

GSSG

6-OHDA MPTP

DMT1

Based on this hypothesis, therefore, therapeutic efforts should be devoted to reducing brain iron levels and inhibiting the generation of ROS. A few recent studies have shown that the iron chelator, deferrioxamine, may be a significant factor in an effective therapy for the prevention and treatment of brain disorders [102]. One possible non-toxic approach with natural metal chelators could make use of green tea catechins, especially EGCG. This compound comprising antioxidant, iron chelating and anti-inflammatory activities; has been shown to be neuropro‐ tective in animal models of Parkinson's disease (PD) and Alzheimer's disease (AD), and also regulates the processing of amyloid precursor protein (APP) through a non-amyloidogenic pathway [103, 104]. Understanding the timing of iron mismanagement in relation to the progression of neuronal losses would provide important information on pathogenesis, and

**Figure 3.** Mechanism of iron-induced neurodegeneration and its prevention [95]. Abbreviations: DMT1=divalent met‐ al ion transporter-1; GSH=reduced glutathione; GSSG=oxidized glutathione; IRPs=iron regulatory proteins; MAO=monoamine oxidase; MPTP=1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine;NADP+=nicotinamide adenine dinu‐ cleotide phosphate; NADPH=reduced nicotinamide adenine dinucleotide phosphate; 6-OHDA=6-hydroxydopamine

would raise the possibility of monitoring iron changes as a marker of disease progression, and perhaps even in a pre-clinical diagnosis in conditions where iron misregulation is an early event.

More *in vitro* and *in vivo* studies should be done to investigate how iron misregulation/ accumulation synergizes with common endogenous and environmental toxins. Iron misregulation/accumulation alone can kill neurons only in genetic disorders where iron imbalance occurs rapidly and is extensive. However, in most cases, the amount of iron build-up is relatively low and the neurotoxic mechanism might involve a combination of iron and other toxins. Future investigations of iron in the brain, including how to best monitor iron deposition *in vivo*, the clinical relevance of excessive iron deposition, and the mechanistic relationship between iron deposition and disease pathophysiology, hold the promise of advancements in the field of neurotherapeutics.

#### **6.5. Mutagenicity and cancinogenesis**

compared with the placebo group, possibly due to a decrease in cholesterol synthesis and an upregulation of hepatic LDL receptor gene expression [89, 90]. Controversial data has indi‐ cated that the consumption of high doses of green tea polyphenols (714 mg/day) for 3 weeks resulted in decreases in the total cholesterol:HDL-cholesterol ratio, but had no effects on the

The errors in brain iron metabolism found in neurological disorders are found to be multifac‐ torial, however treatment conditions seem to be particularly important. Epilepsy, an oxidative related disorder of the brain, has served as a model for the investigation in the role of iron, especially NTBI in neurological disorders. The effect of antiepileptic drug treatment on changes of iron status and oxidative stress parameters were determined [92]. Recent genetic and biochemical manipulations of iron overload have placed iron at the centre of research into neurodegenerative diseases. This is supported by the discovery of genetic and non-genetic misregulations of the iron metabolism in these disorders. In many neurodegenerative disor‐ ders, abnormally high levels of iron in specific regions of the brain have been reported [93]. Evidence for iron contribution to diseases by augmentation of oxidative stress have led to an examination of the contribution of iron to other diseases, in which oxidative stress may be involved, including epilepsy [94]. It has not been possible to determine whether the accumu‐ lated iron is in the labile iron pool (LIP), which can participate in the Fenton reaction to generate the reactive hydroxyl radical. The reduction in GSH during disease progression and in response to neurotoxins, together with increased iron accumulation and ROS generation,

Oxidative stress, resulting from increased brain iron levels, and possibly also from defects in antioxidant defensive mechanisms, is widely believed to be associated with neuronal death in these disorders [96, 97], along with a reduced availability of GSH and other antioxidant substances in the brain. Changes in the integrity of the blood brain barrier (BBB) due to altered vascularization of the tissue or inflammatory events could be another initial cause. However, neuronal death by any initial cause could lead to large amounts of iron release and increased ROS formation [98]. Therefore, iron and iron-induced oxidative stress could possibly be a common mechanism involved in the development of neurodegeneration [99] (Figure 3).

Based on this hypothesis, therefore, therapeutic efforts should be devoted to reducing brain iron levels and inhibiting the generation of ROS. A few recent studies have shown that the iron chelator, deferrioxamine, may be a significant factor in an effective therapy for the prevention and treatment of brain disorders [102]. One possible non-toxic approach with natural metal chelators could make use of green tea catechins, especially EGCG. This compound comprising antioxidant, iron chelating and anti-inflammatory activities; has been shown to be neuropro‐ tective in animal models of Parkinson's disease (PD) and Alzheimer's disease (AD), and also regulates the processing of amyloid precursor protein (APP) through a non-amyloidogenic pathway [103, 104]. Understanding the timing of iron mismanagement in relation to the progression of neuronal losses would provide important information on pathogenesis, and

might be taken as evidence for the presence of free iron [95].

Available data strongly support this hypothesis [100, 101]

risk biomarkers of CVD [91].

376 Pharmacology and Nutritional Intervention in the Treatment of Disease

**6.4. Neurological diseases**

Topical applications of green tea polyphenol fractions (such as EGCG, EGC and ECG) inhibitedbenz[a]-pyrene (BP)-and 7,12-dimethylbenz[a]-anthracene (DMBA)-initiated and 12- *O*-tetradecanoylphorbol-13-acetate (TPA)-promoted tumorigenesis of mouse skin, possibly by inhibiting ROS production, inflammation and hyperplasia [105]. Green tea catechins can counteract against tumorigenesis and DNA damage [106]. Mu and colleagues have identified an association between tea consumption and decreased cancer risk [107]. Surprisingly, EGCG in green tea exerted inhibitory effects on skin cancer, hepatocellular carcinoma and duodenal cancer, on metastasis of the melanoma cell line in lung cancer in mice, possibly by blocking the interaction of their tumor promoters with their own membrane receptors called sealing effects. Green tea significantly prevented growth of Namalwa, RAP1-EIO and HS-Sultan tumors transplanted intraperitoneally in the NOD/SCID mice; possibly by impairment of tumor invasion, anti-angiogenesis and by cell apoptosis induction [108]. EGCG decreased ornithine decarboxylase (ODC) and Ras and Jun oncogene levels, and also inhibited tyrosine kinase (TK) as well as mitogen-activated protein kinase (MAPK) activities in the transformed NIH-pATMras fibroblasts [109].

AML=acute myelocytic leukemia APP=amyloid precursor protein

ATRA=all-*trans* retinoic acid

BKO=β-globin gene knockout

CVD=cardiovascular diseases

CLL=chronic lymphocytic leukemia COMT=catechol-*O*-methyltransferase

DAPK2=death-associated protein kinase 2

DMBA=7,12-dimethylbenz[a]-anthracene DMT1=divalent metal ion transporter-1

DH=double heterozygous β-globin gene knockout carrying human β<sup>E</sup> gene

Green Tea: Just a Drink or Nutraceutical http://dx.doi.org/10.5772/57519 379

BBB=blood brain barrier

BP=benz[a]-pyrene

DFP=deferiprone

DW=deionized water

ECG=epicatechin 3-gallate

EGCG=epigallocatechin 3-gallate

G-CSF=granulocyte colony stimulating factor

GM-CSF=granulocyte macrophage colony stimulating factor

EGC=epigallocatechin

FAS=fatty acid synthase

GSH=reduced glutathione GSSG=oxidized glutathione

HDL=high-density lipoprotein

GTE=green tea extract

Hb=hemoglobin

EC=epicatechin

GA=gallic acid

GC=gallocatechin

C=catechin
