Extract of green tea leaves partially attenuates streptozotocin-induced changes in antioxidant status and gastrointestinal functioning in rats

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Nutrition Research 28 (2008) 343 – 349 www.nrjournal.com

Extract of green tea leaves partially attenuates streptozotocin-induced changes in antioxidant status and gastrointestinal functioning in rats Jerzy Juśkiewicz ⁎, Zenon Zduńczyk, Adam Jurgoński, Łucja Brzuzan, Irena Godycka-Kłos, Ewa Żary-Sikorska Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Division of Food Science, 10-747 Olsztyn, Poland Received 9 October 2007; revised 20 December 2007; accepted 7 March 2008

Abstract Rats with severe streptozotocin (STZ)-induced diabetes were subjected to dietary green tea extract supplementation at 2 doses (0.01% and 0.2%; GTL and GTH groups, respectively) to evaluate their effects on antioxidant, gastrointestinal, and renal parameters of experimental animals. The lower dietary supplementation reflects daily consumption of 3 cups of green tea for an average adult weighing 70 kg. Supplementation of a diet with green tea extract had no influence on elevated food intake, body weight loss, increased glucose concentration, or declined antioxidant capacity of water-soluble substances in plasma in the diabetic rats. In cases of intestinal maltase activity, attenuation of liver and kidney hypertrophy, triacylglycerol concentration, and aspartate aminotransferase activity in the serum, both dietary treatments normalized metabolic disorders caused by STZ injection to a similar extent. Unlike the GTL group, the GTH treatment significantly ameliorated development of diabetes-induced abnormal values for small intestinal saccharase and lactase activities, renal microalbuminuria, thiobarbituric acid-reactive substance content in kidney tissue, as well as total antioxidant status in the serum of rats. The GTH group was also characterized by higher antioxidant capacity of lipid-soluble substances in plasma and superoxide dismutase activity in the serum. Although the higher dose of green tea extract did not completely protect against STZ-induced hyperglycemia and oxidative stress in experimental rats, this study suggests that green tea extract ingested at high amounts may prove to be a useful therapeutic option in the reversal of diabetic dysfunction. © 2008 Elsevier Inc. All rights reserved. Keywords: Abbreviations:

Green tea; Polyphenols; Streptozotocin diabetes; Gastrointestinal tract; Oxidative stress; Rat ACL,integral antioxidant capacity of lipophilic substances; ACW, integral antioxidant capacity of water substances; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GTH, the experimental treatment with higher (0.2%; of a diet) dose of green tea extract; GTL, the experimental treatment with lower (0.01%; of a diet) dose of green tea extract; GPx, glutathione peroxidase; SCFA, short chain fatty acid; SOD, superoxide dismutase; STZ, streptozotocin; TAG, triacylglycerol; TAS, total antioxidant capacity; TBARS, thiobarbituric acid-reactive substances; UAE, urinary albumin excretion.

1. Introduction Tea is the most widely consumed beverage in the world, and its polyphenolic compounds have been found to possess ⁎ Corresponding author. Tel.: +4889 523 46 73; fax: +4889 524 01 24. E-mail address: [email protected] (J. Juśkiewicz). 0271-5317/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2008.03.004

widespread biologic functions and health benefits [1]. There is a body of scientific evidence that indicates that green tea (Camellia sinensis) and its catechins, especially (−)epigallocatechin gallate, exhibit antiobesity and antidiabetic effects [2,3]. Green and white teas have the highest concentration of all types of polyphenols because they are minimally processed. It has been reported that a typical European tea

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Table 1 Ingredient composition of experimental diets fed to rats (g/100g) STZ a ± green tea extract

Control

b

Casein Cellulose DL-methionine Soya bean oil Mineral mix c Vitamin mix d Green tea extract Cornstarch e

20 5 0.3 7 3.5 1 — 63.2

0%

0.01%

0.2%

20 5 0.3 7 3.5 1 — 63.2

20 5 0.3 7 3.5 1 0.01 63.19

20 5 0.3 7 3.5 1 0.2 63.0

a Diabetes was induced by a single intraperitoneal injection of 65 mg/kg BW STZ. b Casein preparation (g/100g): crude protein, 89.70; crude fat, 0.3; ash, 2.0; and water, 8.0. c AIN-93G-MX according to Reeves [11], per kilogram mix: 357 g of calcium carbonate anhydrous (40.04% Ca), 196 g of potassium phosphate monobasic (22.76% P, 28.73% K), 70.78 g of potassium citrate and tripotassium monohydrate (36.16% K), 74 g of sodium chloride (39.34% Na, 60.66% Cl), 46.6 g of potassium sulfate (44.87% K, 18.39% S), 24 g of magnesium oxide (60.32% Mg), 6.06 g of ferric citrate (16.5% Fe), 1.65 g of zinc carbonate (52.14% Zn), 1.45 g of sodium metasilicate ∙ 9H2O (9.88% Si), 0.63 g of manganous carbonate (47.79% Mn), 0.3 g of cupric carbonate (57.47% Cu), 0.275 g of chromium potassium sulfate ∙ 12H2O (10.42% Cr), 81.5 mg of boric acid (17.5% B), 63.5 mg of sodium fluoride (45.24% F), 31.8 mg of nickel carbonate (45% Ni), 17.4 mg of lithium chloride (16.38% Li), 10.25 mg of sodium selenate anhydrous (41.79% Se), 10 mg of potassium iodate (59.3% I), 7.95 mg of ammonium paramolybdate ∙ 4H2O (54.34% Mo), 6.6 mg of ammonium vanadate (43.55% V), and 221.026 g of powdered sucrose. d AIN-93G-VM [11], grams per kilogram mix: 3.0 nicotinic acid, 1.6 Ca pantothenate, 0.7 pyridoxine-HCl, 0.6 thiamin-HCl, 0.6 riboflavin, 0.2 folic acid, 0.02 biotin, 2.5 vitamin B12 (cyanocobalamin, 0.1% in mannitol), 15.0 vitamin E (all-rac-α-tocopheryl acetate, 500 IU/g), 0.8 vitamin A (all-transretinyl palmitate, 500 000 IU/g), 0.25 vitamin D3 (cholecalciferol, 400 000 IU/g), 0.075 vitamin K1 (phylloquinone), 974.655 powdered sucrose. e Cornstarch preparation (g/100 g): crude protein, 0.6; crude fat, 0.9; ash, 0.2; total dietary fiber, 0; and water, 8.8.

consumer delivers 140 mg of total flavonoids per average serving (235-mL cup) [4]. Fujiki et al [5] suggested drinking green tea in large amounts (ie, 10 or more cups per day) as a potential form of cancer prevention. Prior studies using animal rodent models of diabetes showed a therapeutic effect of green tea on oxidative stress connected with diabetic disorders; however, well-documented experiments addressing the in vivo effects of dietary green tea flavonoids are scarce [1,3]. Clinical and experimental studies have suggested that the extent of absorption of dietary polyphenols in the small intestine is relatively small (10% to 20%), and most ingested polyphenols reach the large intestine where they encounter microflora, which possess an enormous catalytic and hydrolytic potential [6]. Numerous reports have addressed the effects of dietary polyphenols on the large-bowel ecosystem [7,8], but there is no information on their influence when the host's metabolism is disturbed by the diabetic state. In the present study on streptozotocin (STZ)-induced diabetic rats, we determined the potential beneficial anti-

diabetic effects of a dietary supplementation with 0.01% of lyophilized green tea extract changed upon its dietary increase of up to 20 times (up to 0.2%) of their diet. The lower dietary treatment reflects a daily consumption of 3 cups of green tea by an adult weighing 70 kg. The effect of a 4-week administration of green tea extract on several antioxidant, gastrointestinal, and renal parameters was evaluated in the diabetic rats. 2. Methods and materials 2.1. Plant material Plant material used in this study consisted of the leaves of green tea purchased in a local market. Polyphenols were extracted into 75% (vol/vol) acetone/water solution at solid material to solvent ratio of 1:8, with the addition of 200 ppm of SO2. After 4 hours of extraction, the procedure was repeated. After centrifugation, the combined extract was evaporated in a rotary evaporator and freeze-dried. The total content of polyphenols in the green tea extract was 850 mg/g, and it consisted mainly of (−)epicatechin (365.2 mg/g) and epigallocatechin gallate (385.0 mg/g). Also, small amounts of epicatechin gallate (58.5 mg/g), (+)catechin (9.0 mg/g), and procyanidins (32.1 mg/g) were noted. The total phenolic content of the green tea extract was determined photometrically using the Folin-Ciocalteu method [9]. The extract was characterized using the high-performance liquid chromatography method described elsewhere [10]. 2.2. Animals and diets The animal protocol used in this study was approved by the University of Warmia and Mazury Institutional Animal Care and Use Committee. A 4-week experiment was conducted on 64 male adult Wistar rats maintained individually in cages of glass under a 12-hour light/dark cycle, controlled temperature of 21°C to 22°C, relative humidity of 50% to 70%, and intensive ventilation of rooms (15 times per hour). The experiment was conducted on rats weighing 365.5 ± 5.1 g. Diabetes was induced by a single intraperitoneal injection of 65 mg/kg STZ (Sigma Chemical Co, St Louis, Mo) freshly dissolved in 0.05 mol/L sodium citrate buffer (pH 4.5). Control rats received an injection of citrate buffer. Blood glucose levels were measured 48 hours after STZ injection. Rats with blood glucose levels of 300 mg/dL or greater were considered diabetic and were used for the study. To prevent hypoglycemia, rats were kept on a 5% glucose solution for 24 hours. All treatments of the rats were started at 9 AM, and the killing was also carried out at 9 AM. Three days after the administration of STZ, diabetic rats were randomly assigned to intake of green tea. Rats were divided into 4 groups of 8 rats each: group 1, the healthy (C) rats fed with a casein diet; group 2, the diabetic (STZ) rats fed with the casein diet; group 3, the diabetic rats fed with the casein diet supplemented with 0.01% of the green tea extract (GTL;

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Table 2 Effects of dietary green tea extract on internal organ weights, intestinal disaccharidases activity, and cecal parameters of STZ-treated rats Control a

STZ + green tea extract 0.01% c

0.2% d

371.0 ± 3.1 369.9 ± 4.2⁎⁎ 29.32 ± 0.77⁎ 4.178 ± 0.090⁎ 6.66 ± 0.13⁎,⁎⁎

364.2 ± 3.0 366.1 ± 4.0⁎⁎ 30.60 ± 0.72⁎ 3.868 ± 0.094⁎⁎ 6.56 ± 0.18⁎,⁎⁎

362.4 ± 2.8 360.3 ± 4.1⁎⁎ 30.04 ± 0.68⁎ 3.912 ± 0.064⁎⁎ 6.43 ± 0.09⁎⁎

21.60 ± 1.61⁎ 101.1 ± 9⁎ 4.70 ± 0.43⁎

18.04 ± 1.72⁎,⁎⁎ 83.30 ± 6.5⁎⁎ 4.33 ± 0.38⁎

13.99 ± 1.61⁎⁎ 75.5 ± 5.1⁎⁎ 3.16 ± 0.21⁎⁎

6.95 ± 0.11⁎ 0.308 ± 0.022⁎ 0.888 ± 0.092⁎⁎ 24.23 ± 0.52⁎,⁎⁎ 0.394 ± 0.020⁎,⁎⁎

6.74 ± 0.06⁎ 0.324 ± 0.021⁎ 1.094 ± 0.121⁎,⁎⁎ 24.40 ± 0.62⁎,⁎⁎ 0.427 ± 0.011⁎

6.42 ± 0.14⁎⁎ 0.347 ± 0.024⁎ 1.350 ± 0.141⁎ 23.54 ± 0.55⁎⁎ 0.366 ± 0.022⁎⁎

71.06 ± 2.23 22.61 ± 1.12⁎⁎ 0.80 ± 0.11⁎⁎,⁎⁎⁎ 12.10 ± 1.1⁎ 1.20 ± 0.18⁎ 1.67 ± 0.06 109.44 ± 3.40

80.57 ± 3.63 28.51 ± 1.64⁎ 0.60 ± 0.10⁎⁎⁎ 12.13 ± 1.4⁎ 0.72 ± 0.11⁎⁎ 1.93 ± 0.21 124.46 ± 5.33

0% Initial BW (g) 364.1 ± 2.9 Final BW (g) 459.6 ± 5.8⁎ Diet intake (g/d) 23.51 ± 0.63⁎⁎ Liver e 3.026 ± 0.101⁎⁎⁎ pH ileum 6.99 ± 0.15⁎ Mucosal disaccharidase activity Saccharase f 8.32 ± 0.84⁎⁎⁎ Maltase f 31.2 ± 4.2⁎⁎⁎ Lactase f 2.56 ± 0.25⁎⁎⁎ Cecal parameters pH 7.00 ± 0.08⁎ Tissue e 0.221 ± 0.010⁎⁎ Digesta e 0.522 ± 0.041⁎⁎⁎ DM (%) 25.47 ± 0.26⁎ Ammonia 0.398 ± 0.014⁎,⁎⁎ (mg/g) SCFA (μmol/g) C2 81.00 ± 5.62 C3 20.58 ± 1.73⁎⁎ C4i 1.27 ± 0.13⁎ C4 10.18 ± 0.80⁎,⁎⁎ C5i 1.45 ± 0.16⁎ C5 2.01 ± 0.18 Total 116.48 ± 11.04

b

72.56 ± 4.31 21.69 ± 1.20⁎⁎ 1.09 ± 0.11⁎,⁎⁎ 8.89 ± 0.72⁎⁎ 1.09 ± 0.12⁎,⁎⁎ 2.01 ± 0.16 107.33 ± 5.41

All values are expressed as means ± SEM (n = 8). Means within a row with asterisks (⁎, ⁎⁎, ⁎⁎⁎) are statistically different (P b .05) as determined by 1-way ANOVA and post hoc Duncan multiple range test. a Healthy rats fed with a casein diet for 28 days (the C group). b Diabetic rats fed with a casein diet for 28 days (the STZ group). c Diabetic rats fed for 28 days with a casein diet supplemented with 0.01% of the green tea extract (the GTL group). d Diabetic rats fed for 28 days with a casein diet supplemented with 0.2% of the green tea extract (the GTH group). e g/100 g BW. f μmol hydrolyzed disaccharide/g protein per minute.

this dietary treatment reflected a daily consumption of 3 cups of green tea by an adult weighing 70 kg); and group 4, the diabetic rats fed with the casein diet supplemented with 0.2% of the green tea extract (GTH). Diet composition (Table 1) was based on recommendations of AIN-1993 [11]. 2.3. Procedures At termination of the feeding experiment, the rats were anesthetized with sodium pentobarbitone, and after laparotomy, blood samples were taken from the tail vein; then, the liver, heart, kidneys, small intestine, and cecum with content were removed and weighed. The cecal and ileal pH was measured using a microelectrode and a pH/Ion meter (model 301; Hanna Instruments, Vila do Conde, Portugal). Samples of fresh digesta were weighed and used for immediate analysis of dry matter (at 105°C), ammonia, and short chain fatty acids (SCFAs). Ammonia extracted and trapped in a solution of boric acid was determined by direct titration with sulfuric acid [12].The amount of SCFA were measured using gas chromatography under conditions described previously [13]. The cecum wall was flushed clean with water, blotted on filter paper, and weighed as the cecal tissue mass. The small intestine was divided on 4 equal parts; the second part

(jejunum) from the stomach side was rinsed with ice-cold physiological saline and cut open. The mucosal samples were collected by scraping with glass slides on an iced glass plate, weighed, and subsequently stored at −40°C. Disaccharidase activities (saccharase, maltase, and lactase) were assayed by the method of Messer and Dahlqvist [14], with modifications. The amount of liberated glucose was measured spectrophotometrically, and enzyme activity was expressed as micromole disaccharide hydrolyzed per minute per gram of protein. The protein content of the supernatant was estimated as described by Lowry et al [15] using bovine serum albumin as a standard. In heparinized blood, the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) was determined with the aid of enzymatic kits (Randox Laboratories Ltd, Crumlin, United Kingdom). Integral antioxidant capacity of water soluble substances in plasma (ACW) and integral antioxidant capacity of lipophilic substances in plasma (ACL) were determined by photochemiluminescence detection using a Photochem (Analytik Jena AG, Jena, Germany) according to the method described by Popov and Lewin [16]. In the photochemiluminescence assay, the antioxidant potential was assayed by means of the lag phase (ACW) or by means of the area under

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Table 3 Effects of dietary green tea extract on the kidneys and serum parameters, as well as on blood and plasma antioxidant concentrations in STZ-treated rats Control a

STZ + green tea extract 0%

Kidneys Tissue e UAE (mg/24h) TBARS f Serum Glucose g Cholesterol g TAG g ALT (U/L) AST (U/L) Antioxidants GPx (U/mL) SOD (U/mL) TAS (mmol/L) ACW h ACL i

b

0.01% c

0.2% d

0.528 ± 0.014⁎⁎⁎ Trace⁎⁎⁎ 9.80 ± 0.14⁎⁎

1.046 ± 0.030⁎ 130.6 ± 19.2⁎ 10.77 ± 0.70⁎

0.896 ± 0.041⁎⁎ 125.7 ± 20.4⁎,⁎⁎ 10.04 ± 0.45⁎,⁎⁎

0.906 ± 0.013⁎⁎ 85.3 ± 9.3⁎⁎ 9.26 ± 0.31⁎⁎⁎

196.5 ± 16.2⁎⁎ 79.6 ± 5.8 146.9 ± 9.7⁎⁎ 35.2 ± 2.6⁎⁎ 224 ± 32⁎⁎

794.6 ± 79.4⁎ 86.4 ± 4.2 194.4 ± 29.1⁎ 46.7 ± 4.1⁎ 285 ± 25⁎

733.6 ± 56.2⁎ 90.7 ± 6.6 132.2 ± 12.2⁎⁎ 46.1 ± 5.9⁎ 200 ± 36⁎⁎

746.9 ± 77.1⁎ 87.9 ± 4.7 146.5 ± 23.3⁎⁎ 52.2 ± 4.3⁎ 202 ± 45⁎⁎

64.0 ± 2.7⁎⁎ 343 ± 15⁎⁎ 0.868 ± 0.033⁎ 0.062 ± 0.007⁎ 0.104 ± 0.004⁎⁎

73.6 ± 2.2⁎ 325 ± 19⁎⁎ 0.705 ± 0.052⁎⁎ 0.039 ± 0.001⁎⁎ 0.108 ± 0.005⁎,⁎⁎

75.7 ± 2.8⁎ 339 ± 11⁎⁎ 0.739 ± 0.030⁎⁎ 0.039 ± 0.003⁎⁎ 0.107 ± 0.003⁎,⁎⁎

76.3 ± 2.8⁎ 426 ± 31⁎ 0.762 ± 0.024⁎,⁎⁎ 0.038 ± 0.002⁎⁎ 0.114 ± 0.002⁎

All values are expressed as means ± SEM (n = 8). Means within a row with asterisks (⁎, ⁎⁎, ⁎⁎⁎) are statistically different (P b .05) as determined by 1-way ANOVA and post hoc Duncan multiple range test. a Healthy rats fed with a casein diet for 28 days (the C group). b Diabetic rats fed with a casein diet for 28 days (the STZ group). c Diabetic rats fed with a casein diet supplemented with 0.01% of the green tea extract (the GTL group) for 28 days. d Diabetic rats fed with a casein diet supplemented with 0.2% of the green tea extract (the GTH group) for 28 days. e g/100 g BW. f µmol per 100 g of kidney tissue. g mg/dL. h mmol ascorbic acid equivalent per milliliter of plasma. i mmol trolox equivalent per milliliter of plasma.

the curve (ACL) at different concentrations. From the remaining nonheparinized blood, serum was obtained after solidification. The concentrations of glucose, cholesterol, and triacylglycerol (TAG) in the serum, as well as activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were determined with commercial diagnostic kits (Alpha Diagnostics, Warsaw, Poland). Total antioxidant status (TAS) in the serum was estimated using a kit from Randox Laboratories Ltd. The extent of lipid peroxidation in the kidneys was measured by quantifying the malondialdehyde formed in terms of thiobarbituric acid-reactive substances (TBARS) [17]. Microalbuminuria was estimated at the end of the experiment in rats housed in individual metabolic cages for 48 hours. The first 24 hours was composed of an acclimation period, after which, a 24-hour urine sample was collected. Urinary albumin concentration was determined using an Alpha Diagnostics kit. 2.4. Statistics All data are presented as means ± SE. The results were calculated statistically using 1-way analysis of variance (ANOVA) and the Duncan multiple range test. Differences were considered to be significant at P b .05 [18]. The calculations were made using the STATISTICA software package version 6.0 (StatSoft Corp, Krakow, Poland).

3. Results 3.1. Body weight and gastrointestinal parameters The effect of STZ and feeding green tea extract on body weight and gastrointestinal parameters is shown in Table 2. Whereas control rats (C group) gained significant weight in the 28-day period, there was no appreciable increase in the diabetic controls (STZ group) over the same period. The addition of green tea to a diet did not improve weight gain of rats compared with the STZ group. Diet intake was significantly lower in the C group than in the experimental treatments. Liver weight expressed as a percentage of body weight was significantly increased in all diabetic patients (P b .05) vs healthy controls. However, that alternation was significantly reduced (P b .05) by feeding green tea extract when compared with the STZ rats. The ileal pH was insignificantly decreased by the STZ treatment. The lowest pH of ileal digesta was in the GTH group (P b .05 vs C group). The activity of intestinal disaccharidases (saccharase, maltase, and lactase) was significantly increased in the diabetic control rats when compared with the healthy control group. Treatment with 0.01% of GT insignificantly decreased, whereas that with 0.2% of GT significantly decreased saccharase activity in the small intestine compared with the STZ group. Intestinal maltase activity was significantly decreased by both levels of green tea extract

J. Juśkiewicz et al. / Nutrition Research 28 (2008) 343–349

supplementation, but the activity of lactase was reduced (P b .05) only by 0.2% of green tea addition when compared with the diabetic control rats. The STZ treatment did not change cecal pH, dry matter, ammonia, or SCFA concentrations, whereas the weight of cecum tissue and digesta was enhanced, when compared with the untreated rats. The addition of 0.2% green tea extract was accompanied by a significant decrease in pH value of the cecal digesta (6.42 vs 6.74-7.00 in the remaining groups). The GTH group was also characterized by the highest digesta weight (P b .05 vs C and STZ groups) and propionic acid concentration (P b .05 vs remaining treatments), as well as the lowest ammonia (P b .05 vs GTL group) and dry matter concentrations (P b .05 vs C group) among all dietary treatments. 3.2. Kidney, serum, and antioxidant parameters Significant hypertrophy of kidneys was observed in the diabetic rats (Table 3). Similarly, the incorporation of the green tea extract in the diet (at 0.01% and 0.2%) significantly reduced the diabetes-induced hypertrophy of kidneys. The control diabetic rats exhibited urinary albumin excretion (UAE) at the end of the experiment; this state was significantly ameliorated by the GTH diet. The content of TBARS in the kidney tissue was significantly enhanced by STZ treatment; at the same time, it was significantly decreased in the GTH group, in comparison with the control rats. A significantly increased concentration of serum glucose was observed in the diabetic rats compared with the control group. Feeding the green tea extract at either 0.01% or 0.2% did not affect the blood glucose level in rats. Total cholesterol remained unaffected, whereas TAG concentration in the serum was significantly higher in the diabetic rats than in the nondiabetic control rats. Both green tea treatments markedly decreased TAG concentration to the level observed in the C group. Alanine aminotransferase and AST activities in the serum were significantly increased in the STZ group. None of the green tea treatments changed ALT level, but AST activity was significantly reduced compared with the diabetic control group. The activity of blood GPx was found to be significantly elevated (P b .05 vs C group) in the diabetic rats. Glutathione peroxidase activity was not influenced by the supplementation of a diet with green tea extract. The SOD activity was not observed to be changed in the STZ group when compared with the untreated rats, but it was found to be significantly higher in the GTH group than in the remaining groups. Rat serum TAS was significantly higher in the C group than in the C and GTL groups. Level of integral antioxidant capacity of water substances was significantly lower in all diabetic treatments (C, GTL, and GTH) when compared with that in the healthy rats. Although ACL was not affected by STZ injection, its level was found to be increased (P b .05 vs C group) in the rats fed with the diet containing a higher content of green tea extract.

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4. Discussion The present study was conducted to determine the effect of feeding 2 doses of green tea extract on the gastrointestinal and antioxidant parameters in STZ (65 mg/kg) rats. Streptozotocin-induced diabetes is characterized by severe loss in body weight [19]. Although the healthy rats registered approximately 25% growth in body weight, the diabetic rats showed no gain, and the green tea extract treatments did not improve the gain of STZ-treated animals. In another study, the administration of a green tea extract containing a similar amount of polyphenols (832 mg/g of total polyphenols) caused a significant increase in body weight and reduction in food and water intake in STZ-diabetic rats [2]. However, those authors administered several dozen more green tea extract orally for 4 weeks, 300 mg/kg body weight per day, than in the present study. Numerous authors have observed an abnormal increase of both liver and kidney weights in diabetic rats [19,20]. In the present study, the significant hypertrophy of both liver (38%) and kidneys (98%) was also observed in the diabetic rats. The incorporation of the green tea extract into the diet (at 0.01% and 0.2%) significantly reduced the diabetes-induced hypertrophy of liver (7.5% and 6.4%, respectively) and kidneys (14.3% and 13.4%, respectively) vs the STZ group. It indicated that, compared with the GTL group (lower dose of GT extract), the elevated amount of green tea extract did not exert an additional effect on a reduction in liver and kidney hypertrophy. Results from the present study showed a significant increase in the activities of disaccharidases in the intestinal mucosa of diabetic rats, contributing to the observed elevated glucose level in the blood. The increased levels of disaccharidases activity in diabetic state have been postulated to result from the lack of insulin (which has an inhibitory effect on disaccharidase activity) [21]. In this study, supplementation of the diet with green tea extract significantly reduced maltase (both levels of GT extract) as well as saccharase and lactase (at higher dose) activities, which may be indicative of a reduced level of absorbable glucose and, thus, may be beneficial in the amelioration of the diabetic state. Surprisingly, the higher dose of green tea extract (20 times higher than that in the GTL group) did not change the maltase activity at all, and the saccharase activity was decreased only insignificantly when compared with the 0.01% GT treatment. The glucose level in the serum of the green tea–treated diabetic rats was reduced insignificantly by 8% and 6% in the case of the GTL and GTH groups, respectively. On the other hand, many authors have reported that green tea catechins possess a strong antihyperglycemic effect by enhancing basal- and insulinstimulated glucose uptake [22], inhibiting intestinal glucose uptake by sodium-dependent glucose transporter [23] and mimicking insulin by decreasing the expression of genes that control gluconeogenesis [24]. The STZ injection did not deteriorate the cecal ecosystem. It is worth mentioning, however, that dietary supplementation

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with green tea extract, especially at higher doses, positively influenced the cecal ecosystem that was indicated by reduced pH value of digesta and decreased concentration of branchedchain fatty acids (isobutyrate and isovalerate). A decline in pH of cecal/colonic digesta is considered beneficial to health status because such conditions of the ecosystem enhance the proliferation of beneficial bacteria and inhibit the formation of detrimental fermentation products [13]. The concentrations of branched-chain fatty acids are known to be increased by bacterial proteolytic degradation [25]. The elevated concentration of propionic acid in the GTH group is consistent with the study of Aura et al [26], in which C-ring fission metabolism in the lower intestine was shown to occur in flavonols, flavanols, and flavones, and hydroxyphenylacetic and corresponding propionic acids were formed. Renal damage is a well-known consequence of diabetes, and the early identification of microalbuminuria is considered to be clinically relevant [27]. A therapeutic intervention should start early enough to be effective or to delay the development of end-stage renal disease [28]. In the present study, we have shown that, although both green tea treatments significantly reduced the diabetic-induced hypertrophy of kidneys, only the higher dose of the extract ameliorated renal microalbuminuria and effectively reduced TBARS level in the kidney tissue. The increase in TBARS, an index of lipid peroxidation in the STZ rats, might be due to an increased level of oxygen free radicals [2]. Green tea polyphenols can act as scavengers of free radicals caused by reactive oxygen species and prevent radical damage [1]. It has been reported that epigallocatechin gallate, the main constituent of the extract used in the present study, is the most potent tea antioxidant with 4 dihydroxy groups [2]. In diabetes, several authors have reported increases in AST and ALT activities as well as changes in lipid concentration in the serum of diabetic patients [19,29]. Aspartate aminotransferase and ALT levels act as indicators of liver function; hence, restoration of the normal level of AST may indicate the normalizing effect of both 0.01% and 0.2% of dietary green tea extract. Similarly, the lower level of dietary green tea extract was sufficient for TAG restoration to the control nondiabetic level. Available data have pointed to green tea constituents as hypolipidemic agents in normal and diabetic conditions [1,30]. Oxidative stress, which results from an imbalance in the oxidant/antioxidant system favoring the former, is implicated in diabetes. This is thought to be due to either overproduction of oxidants or a decrease in antioxidant defenses [31]. Superoxide dismutase (scavenges superoxide anions) and GPx (removes H2O2 and lipid peroxides) are considered primary antioxidant enzymes involved in the direct elimination of reactive oxygen species. According to our results, green tea extract supplementation did not have any significant effects on the activity of GPx in the blood of diabetic animals. In the case of SOD, only the GTH treatment (0.2% of a diet) significantly increased its activity, despite the fact that the STZ injection had no affect on SOD

activity in the blood. It has been reported that green tea catechins act as strong antioxidative substances in normal as well as diabetic conditions [1,32]. On the other hand, some authors have found increases and decreases in SOD and GPx activities in diabetes [31]. These apparently contradictory results could be due to variations in severity, duration, and treatment of the disease [31,33]. In our study, green tea extract supplemented at a higher dose, but not at the lower dose, effectively ameliorating a decline in the TAS was observed in the STZ group. As a result, the difference in TAS level between the C and GTH groups turned out not to be statistically significant. The elevated level of antioxidant capacity of lipid-soluble substances in plasma of rats fed with the GTH diet (significant vs the control group) also confirmed better antioxidant properties of a diet with a higher content of green tea extract. We conclude that the effect of green tea extract, added to the diet at 2 levels of 0.01% and 0.2% for diabetic rats, seems to be dose dependent in the case of the following examined parameters: small intestinal saccharase and lactase activities, UAE, TBARS content in kidneys' tissue, SOD activity in the serum, and levels of TAS and ACL. In cases of intestinal maltase activity, attenuation of liver and kidney hypertrophy, TAG concentration, and AST activity in the serum, both dietary treatments normalized metabolic disorders caused by STZ injection to a similar extent. On the other hand, diet intake, body weight loss, glucose concentration, and antioxidant capacity of watersoluble substances in plasma were not influenced by the addition of dietary green tea extract to the diet. Because of several potential benefits of green tea constituents ingested at high amounts, this study in rats provides the rationale to further investigate the use of dry green tea extracts to attenuate diabetic-induced complications. References [1] Kao Y-H, Chang H-H, Lee M-J, Chen C-L. Tea, obesity, and diabetes. Mol Nutr Food Res 2006;50:188-210. [2] Babu PVA, Sabitha KE, Shyamaladevi CS. Therapeutic effect of green tea extract on oxidative stress in aorta and heart of streptozotocin diabetic rats. Chem Biol Interact 2006;162:114-20. [3] Sabu MC, Simitha K, Kuttan R. Antidiabetic activity of green tea polyphenols and their role in reducing oxidative stress in experimental diabetes. J Ethnopharmacol 2002;83:109-16. [4] Lakenbrink C, Lapczynski S, Maiwald B, Engelhardt UH. Flavonoids and other polyphenols in consumer brews of tea and other caffeinated beverages. J Agric Food Chem 2000;48:2848-52. [5] Fujiki H, Yoshizawa S, Horiuchi T, Suganuma M, Yatsunami J, Hishiwaki S, et al. Anticarcinogenic effects of (–)-epigallo-catechin gallate. Prev Med 1992;21:503-9. [6] Spencer JPE. Metabolism of tea flavonoids in the gastrointestinal tract. J Nutr 2003;133:3255S-61S. [7] Juśkiewicz J, Zduńczyk Z, Wróblewska M, Oszmiański J, Hernandez T. The response of rats to feeding with diets containing grapefruit flavonoid extract. Food Res Intern 2002;35:201-5. [8] Zduńczyk Z, Frejnagel S, Wróblewska M, Juśkiewicz J, Oszmiański J, Estrella I. Biological activity of polyphenol extracts from different plant sources. Food Res Intern 2002;35:183-6.

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