- Email: [email protected]
Anti-diabetic activity of green tea polyphenols and their role in reducing oxidative stress in experimental diabetes
Journal of Ethnopharmacology 83 (2002) 109 /116 www.elsevier.com/locate/jethpharm
Anti-diabetic activity of green tea polyphenols and their role in reducing oxidative stress in experimental diabetes Sabu M.C., Smitha K., Ramadasan Kuttan * Amala Cancer Research Centre, Amala Nagar, Trichur 680 553, Kerala, India Accepted 5 August 2002
Abstract An aqueous solution of green tea polyphenols (GTP) was found to inhibit lipid peroxidation (LP), scavenge hydroxyl and superoxide radicals in vitro. Concentration needed for 50% inhibition of superoxide, hydroxyl and LP radicals were 10, 52.5 and 136 mg/ml, respectively. Administration of GTP (500 mg/kg b.wt.) to normal rats increased glucose tolerance significantly (P B/0.005) at 60 min. GTP was also found to reduce serum glucose level in alloxan diabetic rats significantly at a dose level of 100 mg/kg b.wt. Continued daily administration (15 days) of the extract 50, 100 mg/kg b.wt. produced 29 and 44% reduction in the elevated serum glucose level produced by alloxan administration. Elevated hepatic and renal enzymes produced by alloxan were found to be reduced (P B/0.001) by GTP. The serum LP levels which was increased by alloxan and was reduced by significantly (P B/0.001) by the administration of 100 mg/kg b.wt. of GTP. Decreased liver glycogen, after alloxan administration showed a significant (P B/ 0.001) increase after GTP treatment. GTP treated group showed increased antioxidant potential as seen from improvements in superoxide dismutase and glutathione levels. However catalase, LP and glutathione peroxidase levels were unchanged. These results indicate that alterations in the glucose utilizing system and oxidation status in rats increased by alloxan were partially reversed by the administration of the glutamate pyruvate transaminase. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Green tea; Polyphenols; Diabetes; Alloxan; Antioxidant
1. Introduction Many indigenous Indian medicinal plants have been found to be useful to successfully manage diabetes (Rajasekharan and Tuli, 1976; Bhaskaran Nair and Santhakumari, 1985; Nagarajan et al., 1987; Ponnachan et al., 1993; Subramoniam et al., 1996; Joy and Kuttan, 1999). Despite the introduction of hypoglycemic agents from natural and synthetic sources diabetes and its complications continue to be a major problem in the world population. Green tea is produced by enzymatic inactivation of the leaves of Camellia sinensis followed by rolling or comminution and drying. In the manufacturer of green * Corresponding author. Fax: /91-487-307020 E-mail address: [email protected] (R. Kuttan).
tea, the enzymatic inactivation achieved by steam or pan firing treatment to preserve natural polyphenols with respect to the health promoting properties. Green tea derived products are mainly extracts of green tea in liquid or powder form varying in the proportion of polyphenols (45 /90%) and caffeine content (0.4 /10%). The polyphenolic fraction of green tea, has been reported to have multiple pharmacological actions (Mukhtar et al., 1992; Sano et al., 1995). The antioxidative potency of crude catechin powder and individual catechins were tested in experiments using active oxygen method. Crude catechins reduced the formation of peroxides for more effectively than dl -a-tocopherol (Hara, 1990). In this study we have investigated the effect of crude polyphenolic fraction of green tea in reducing the alloxan-induced oxidative damage and diabetes in rats.
0378-8741/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 2 1 7 - 9
110
S. M.C. et al. / Journal of Ethnopharmacology 83 (2002) 109 /116
2. Materials and methods 2.1. Animals Male Wistar albino rats (250 /300 g) were obtained from the Veterinary College, Mannuthy, India. They were housed in ventilated cages and fed with a pelleted diet (Lipton, India Ltd) and water ad libitum. 2.2. Chemicals Green tea was a gift from Dr J.I.X. Antony, Kancor Flavours and Extracts Limited, India. Alloxan monohydrate was obtained from Sigma, St. Louis, MO. 1chloro-2,4-dinitrobenzene, glutathione and 5-5-dithiobis (2-nitrobenzoic acid) were purchased from Sisco Research Laboratory, Mumbai, India. Thiobarbituric acid (TBA) was obtained from E-Merck, Germany. All other chemicals used were of analytical and reagent grade. 2.3. Preparation of polyphenolic fraction of green tea Dried green tea powder (250 g) was extracted with water and water soluble fraction was dissolved in hot water and washed with chloroform. Aqueous layer was extracted with ethyl acetate and ethyl acetate layer containing polyphenol was separated out and evaporated to dryness (Hara, 1994). It was resuspended in water and used for the experiments. The percentage yield of crude polyphenols is 5.5% w/w. 2.4. In vitro antioxidant activity 2.4.1. Superoxide radicals scavenging activity Superoxide scavenging was determined by the Nitroblue tetrazolum (NBT) reduction method (McCord and Fridovich, 1969). The reaction mixture contained EDTA (6 mM) containing NaCN (3 mg), riboflavin (2 mM), NBT (50 mM), various concentrations of the extracts (5 /50 mg/ml) and phosphate buffer (67 mM, pH 7.8) in a final volume of 3 ml. The tubes were uniformly illuminated with an incandescent visible light (Philips, 40 W) for 15 min, and the optical density was measured at 530 nm before and after the illumination. The percentage inhibition of superoxide generation was evaluated by comparing the absorbance values of the control and experimental tubes. 2.4.2. Hydroxyl radical scavenging activity Hydroxyl radical scavenging was measured by studying the competition between deoxyribose and the test compounds for hydroxyl radicals generated from the Fe3/ascorbate/EDTA/H2O2 system. The hydroxyl radical attack deoxyribose, which eventually results in TBA reacting substance (TBARS) formation (Elizabeth and Rao, 1990). The reaction mixture contained deox-
yribose (2.8 mM), FeCl3 (0.1 mM), EDTA (0.1 mM), H2O2 (1 mM), ascorbic acid (0.1 mM), KH2PO4 /KOH buffer (20 mM, pH 7.4), and various concentrations of the extract (25 /400 mg/ml) in a final volume of 1 ml. The reaction mixture was incubated for 1 h at 37 8C. Deoxyribose degradation was measured as TBARS and percentage inhibition was calculated. 2.4.3. Lipid peroxide scavenging activity Reaction mixture (0.5 ml) containing rat liver homogenate (0.1 ml, 25% w/v) in Tris /HCl buffer (40 mM, pH 7.0), KCl (30 mM), ferrous iron (0.16 mM) and ascorbic acid (0.06 mM) was incubated for 1 h at 37 8C in the presence and absence of the extract (20 /180 mg/ ml). The lipid peroxide formed was measured by TBARS formation (Ohkawa et al., 1979). For this incubation mixture 0.4 ml was treated with sodium dodecyl sulphate (8.1%, 0.2 ml), TBA (0.8%, 1.5 ml) and acetic acid (20%, 1.5 ml, pH 3.5). The total volume was then made upto 4 ml by adding distilled water and kept in a water bath at 100 8C for 1 h. After cooling, 1 ml of distilled water and 5 ml of a mixture of n -butanol and pyridine (15:1 v/v) were added and shaken vigorously. After centrifugation, the absorbance of the organic layer was measured at 532 nm. The percentage inhibition of lipid peroxidation (LP) was determined by comparing the results of the test compounds with those of controls not treated with the extracts. 2.5. Anti-diabetic activity 2.5.1. Glucose tolerance test Normal Wistar rats were fasted overnight. They were divided into three groups containing six animals each. Control rats (Group I) were given 1 ml distilled water orally. Green tea polyphenols (GTP) at a concentration of 100, 500 mg/kg b.wt. were administered orally using a syringe to second and third groups. Glucose (2 g/kg b.wt.) was given orally using a syringe to all groups immediately after the GTP administration. Blood samples were collected from the tail vein just prior to and 30, 60, 120 and 240 min after the glucose loading and serum glucose levels were measured by GOD/POD method (Trinder, 1976). 2.6. Effect of polyphenols on serum glucose levels in alloxan-induced diabetic rats (single and multidose study) Diabetes was induced in male rats by single intraperitonial injection of 120 mg/kg b.wt. of alloxan monohydrate (Cooperstien and Walkins, 1981). Serum glucose level was checked after 72 h. Animals with serum glucose levels /250 mg/dl were considered diabetic and were used for the study (Perfumi and Tacconi, 1996). The rats were divided into four groups of six rats each. Both group I control normal rats (no
S. M.C. et al. / Journal of Ethnopharmacology 83 (2002) 109 /116
alloxan treatment) and group II diabetic animals were given 1 ml of distilled water. Group III and IV were given aqueous suspension of GTP orally at a dose level of 50 and 100 mg/kg b.wt. on 3rd day after alloxan treatment. Overnight fasted blood samples were collected from the tail vein on 3rd day of alloxan treatment prior to and at 2, 4, 6 and 8 h intervals after the administration of 100 mg/kg b.wt. of the extract orally. Serum was separated and glucose levels were estimated as before. In the multidose study, these animals received the same dose of the extract once daily for 15 days. Blood was collected in non fasted rats on 6, 9, 12 15 and 18th day after alloxan administration and serum glucose levels were measured.
2.7. Liver and kidney functions test Normal and diabetic animals were treated with GTP for 15 days at dose levels of 50 and 100 mg/kg body weight. They were sacrificed on 18th day after alloxan treatment. Blood was collected and serum was separated and LP (Satoh, 1978), alkaline phosphatase (ALP; King and Armstrong, 1980), glutamate pyruvate transaminase (GPT; Bergmeyer and Bernt, 1980), blood urea nitrogen (BUN; Haslam, 1966) and creatinine (Brod and Sirota, 1948) were estimated. Protein was determined by the method of Lowry et al. (1951). The initial and final body weights were determined. Total white blood cell count (WBC) was estimated using haemocytometer on 1st and 18th day after alloxan administration. Liver glycogen was estimated by the method of Hassid and Abraham (1957).
2.8. In vivo antioxidant activity All the groups of animals were sacrificed immediately after the experimental period and liver was taken, washed with normal saline and one part was preserved in 10% formalin for histopathological studies. The other part was homogenized by Tris /HCl buffer and used for measuring LP and activities of superoxide dismutase (SOD; Minami and Yoshikawa, 1979), catalase (Aebi, 1974), glutathione (GSH; Beutler et al., 1983) and glutathione peroxidase (GPx; Helen and Vijayammal, 1997).
2.9. Statistical analysis Data were expressed as mean9/standard error of mean. Statistical comparisons with animals of untreated diabetic group with normal and treated with different concentrations of GTP were performed with Student’s ttest for unpaired observations.
111
3. Results The polyphenols of green tea, was found to scavenge the superoxide generated by photoreduction of riboflavin in a concentration dependent manner. The concentration needed for 50% scavenging of superoxides was 10 mg/ml. The concentration needed for 50% inhibition of hydroxyl radical generation was 52.5 mg/ ml. LP induced by Fe2/ascorbate in rat liver homogenate was found to be inhibited and the concentration needed for 50% inhibition was 136 mg/ml (Fig. 1). Administration of 2g glucose/kg b.wt. to normal rats increased serum glucose levels from 77.39/13.8 to 176.49/20.5 at 60 min but returned to normal at 240 min. Administration of GTP 500 mg/kg b.wt. suppressed the elevation of serum glucose level significantly (P B/0.005) at 60 min (Fig. 2). The effect of polyphenols in alloxan diabetic rat is shown in Table 1. Single administration of the extract, 50 mg/kg b.wt. on 3rd day after alloxan administration did not produce a significant reduction in serum glucose level. At a dose level of 100 mg/kg b.wt., there was 17.3% reduction in glucose level (P B/0.001) at 6th hour and the activity was found to be reduced in following hours (Table 1). Continuous administration of the GTPs (50 mg/kg b.wt.) produced 29.8% reduction in the elevated glucose level on 18th day. At dose level of 100 mg/kg b.wt., a significant (P B/0.001) reduction in blood glucose was seen from 12th day by 25.5% and at 18th day after alloxan treatment the percent reduction in serum glucose was 44.1%. Blood glucose levels in the control (alloxan treated) and normal (alloxan non-treated) group was remained unchanged over this period (Table 2). Liver function markers like ALP, GPT and LP in serum were significantly (P B/0.001) elevated in alloxaninduced diabetes when compared with normal animals (Table 3). Animals treated with 50 and 100 mg/kg b.wt. of polyphenols showed significant (P B/0.001) reduction in the elevated level of ALP, GPT and LPO. Renal function indicators like creatinine and BUN were also elevated in the alloxan diabetic rats when compared with normal rats. The creatinine level of 2.81 mg/dl was found to be reduced to 1.98 mg/dl (P B/0.005) by the administration of 100 mg/kg b.wt. of the GTP. When compared with diabetic control group the elevated levels of BUN was significantly (P B/0.001) reduced in animals treated at both the dose levels of GTP. Normal animals treated with GTP at dose level of 100 mg/kg b.wt. did not produce any significant weight loss (Table 4). A significant (P B/0.001) decrease in rat body weight by 19% was noted in alloxan-induced diabetic rats. But, when the animals were treated with 100 mg/kg b.wt. of GTP suppressed the weight loss significantly
112 S. M.C. et al. / Journal of Ethnopharmacology 83 (2002) 109 /116
Fig. 1. Effect of GTP on superoxide, hydroxyl radical generation and LP in vitro. (A) Superoxide; (B) Hydroxyl radical; (C) LP.
S. M.C. et al. / Journal of Ethnopharmacology 83 (2002) 109 /116
113
Fig. 2. Effect of GTP on glucose tolerance in normal rats ( /m /) Control; ( /j /) GTP 100 mg/kg b.wt.; ( /^ /) GTP 500 mg/kg b.wt.;*P B/0.005.
(P B/0.001) when compared with initial body weight of the same group. Effect of polyphenols on total WBC in alloxan diabetic animals is shown in Table 4. Total WBC was found to be 14 650 in normal rats and in alloxan diabetic rat it was 9112, on 18th day of the experiment. Administration of GTP (100 mg/kg b.wt.) considerably reversed alloxan-induced cellular damage as seen from the increased number of total WBC (P B/0.001) when compared with diabetic control group. Lower dose had also a comparable effect. When compared with normal animals, the liver glycogen level was low in alloxan treated rats was increased after the administration of GTP (not significant). In vivo antioxidant activity after administration of GTP for 18 days is shown in Table 5. In diabetic control group, the SOD and GSH levels were significantly (P B/ 0.001) decreased when compared with normal group. The decreased SOD level of diabetic control group were
found to be increased from 9.699/0.72 to 16.439/0.10 at a dose level of 100 mg/kg b.wt. significantly (P B/0.001). There was only marginal difference (P B/0.001) in the values of glutathone peroxidase and LP. The LP was significantly (P B/0.001) decreased by the administration of 100 mg/kg b.wt. of GTP. There was no significant change in the catalase values.
4. Discussion Green tea has become very popular neutraceutical as an antioxidant and a health promoting traditional drug preparation. In the present study we report anti-diabetic and free radical scavenging activity of GTP. GTP contains gallocatechin (GC), epigallocatechin (EGC), epicatechin (EC), epigallocatechin gallate (EGCg) and epicatechin gallate (ECg). Tea components possess antioxidant, antimutagenic and anticarcinogenic effects
Table 1 Effect of polyphenlic fraction of Green tea on serum glucose levels in alloxan-induced diabetic rats (single dose short term study) Group
I II III IV
Treatment (dose/kg b.wt.)
Normal Control (alloxan) GTPs (50 mg) GPTs (100 mg)
Serum Glucose level in mg/dl (h) 0
2
4
6
8
73.494.4 252.7916.0* 246.6951.1 254.5924.7
65.394.3 255.3916.4* 247.1952.4 251.7923.4
58.393.9 257.1915.4* 232.3953.1 237.5919.6
59.793.5 261.6916.1* 233.7952.9 210.9919.4**
68.892.8 260.0915.1* 236.4953.4 216.8923.9
Values represent the fasting sugar level on 3rd day after alloxan administration in presents and absence of polyphenol extract (values are mean9SD, n 6). * P B 0.001 (compared to normal group). ** P B 0.001 (compared to control group).
S. M.C. et al. / Journal of Ethnopharmacology 83 (2002) 109 /116
114
Table 2 Effect of polyphenolic fraction of Green tea on serum glucose level in alloxan-induced diabetic rats (multidose long term study) Group
I II III IV
Treatment (dose/kg b.wt.)
Serum glucose level in mg/dl (days)
Normal Control (alloxan) GTPs (50 mg) GTPs (100 mg)
3
6
9
12
15
18
73.494.4 252.7916.0* 246.6951.1 254.5924.7
70.793.7 265.2915.2* 234.5952.7 240.1919.4
68.993.8 265.4912.9* 222.7949.8 225.1920.0
67.193.8 275.7912.1* 216.6950.3 189.6919.6**
65.193.5 269.998.5* 193.0954.3 167.4923.0**
63.693.6 256.398.6* 173.2947.9 142.3929.6**
Values represent serum glucose level in alloxan treated animals with or without GTP from 3rd day to 18th day. (Values are mean9SD, n 6.) * P B 0.001 (compared to values of normal group with the corresponding days). ** P B 0.001 (compared to values on 3rd day after alloxan treatment of the same group).
Table 3 Effect of GTP on liver and kidney biochemical parameters in normal and diabetic rats Treatment (dose/kg b.wt.)
Normal Control (Alloxan) GTPs (50 mg) GTPs (100 mg)
Liver
Kidney
ALP (KA/dl)
GPT (U/mg protein)
LPO (U/ml)
BUN (mg/dl)
Creatinine (mg/dl)
33.190.87 46.491.78* 40.392.11** 38.190.68***
141.0911.92 307919.67* 250.598.06*** 225.896.40***
1.690.09 3.3390.22* 2.4790.17*** 1.7490.17***
9.490.28 20.190.97* 16.490.72*** 13.590.39***
0.990.13 2.390.14* 2.390.32*** 1.990.25**
Biochemical parameters were determined at the end of the experiment (18 days after alloxan administration) (values are mean9SD, n 6). * P B 0.001 (compared to normal group). ** P B 0.005 (compared to control group). *** P B 0.001 (compared to control group).
and could protect humans against the risk of cancer by environmental agents (Mukhtar et al., 1992). Sano et al. (1995) studied the inhibitory effects of green tea leaves against tert-butyl hydroperoxide induced LP and a similar antioxidant effect on the kidney was observed after oral administration of a major tea polyphenols (/)-EGCg. Shim et al. (1995) studied the chemopreventive effect of green tea among cigarette smokers and found that it can block the cigarette-induced increase in sisterchromatid exchange frequency. Anti-hyperglycemic effect of black tea has been reported earlier by Gomes et al. (1995). EGCg was found to inhibit intestinal glucose uptake by sodium dependent glucose
transporter, SGLT1 indicating its increase in controlling blood sugar (Kobayashi et al., 2000). Streptozotocindiabetic rats showed increased sensitivity to platelet aggregation and thrombosis and this abnormality could be improved by dietary catechin of green tea (Yang et al., 1999; Choi et al., 1998). Alloxan produces oxygen radicals in the body, which cause pancreatic injury (Halliwell and Gutteridge, 1985) which is responsible for increased blood sugar seen in the animals. However, it is found that action is not specific to pancreas as other organs such as liver, kidney and haemopoietic system also affected by alloxan administration as seen from the elevation of marker
Table 4 Effect of GTP on total leucocyte counts, body weight and liver glycogen in normal and diabetic rats Treatment (dose/kg b.wt.)
Normal (untreated) Control (alloxan) GTPs (50 mg) GTPs (100 mg)
Total leucocyte count (mm3)
14650.09445.4 9112.591134.6a 9387.591781.6 140259290.1*
Body weight (g)
Liver glycogen (g/g tissue)
Initial
Final
282.5910.4 277.5910.4 274.099.2 269.5912.8
293.0910.2 224.5915.7b 243.598.1b 258.0913.3
76.197.4 54.692.7a 56.191.8 61.993.4**
a P B 0.001 (compared to normal group). bP B 0.001 (compared to initial body weight of the same group). Values are determined from day 18 after alloxan administration. (Values are mean9SD, n 6.) * P B 0.001 (compared to control group). ** P B 0.005 (compared to control group).
S. M.C. et al. / Journal of Ethnopharmacology 83 (2002) 109 /116
115
Table 5 Effect of GTP on in vivo antioxidant activity in normal and diabetic rats Treatment (dose/kg b.wt.)
SODa
Catalaseb
LPOc
GSHd
GPxe
Normal (untreated) Control (alloxan) GTPs (50 mg) GTPs (100 mg)
14.691.03 9.790.72* 9.390.49 16.490.10***
8.591.50 6.691.37 6.390.58 7.891.75
0.6690.10 0.8990.02** 0.8590.04 0.7290.03***
3.2490.29 2.0690.04* 2.0690.07 2.3690.10***
63.699.93 46.894.9** 44.796.5 53.7915.9
Values are determined from day 18 after alloxan administration, (Values are mean9SD, n 6). a Units (Enzymes concentration required to inhibit chromogen production by 50% in 1 min)/mg protein. b Values 10 3 units (velocity constant/s/mg protein). c mM/100 g wet tissue. d mM/g wet tissue. e U/mg protein. * P B 0.001 (compared to control group). ** P B 0.005 (compared to normal group). *** P B 0.001 (compared to control group).
enzymes and reduction of haemtological parameters. This was reversed by the continued administration of GTP extract. The present study indicates that alloxaninduced diabetes and subsequent elevation of blood sugar was reversed by simultaneous administration of GTP. Under in vivo condition, glutathione (GSH) acts as an antioxidant and its decrease was reported in diabetes mellitus (Illing et al., 1951). The increased GSH content in the liver of the rats treated with GTP may be one of the factors responsible for the inhibition of LP. SOD and catalase are the two major scavenging enzymes that remove the toxic free radicals in vivo. Vucic et al. (1997) reported that the activity of SOD is low in diabetes mellitus. The GTP treated rats showed decreased LP associated with increased activity of SOD and GSH (Table 5). The antioxidant enzymes catalase and GPx showed non-significant differences in GTP treated groups. In conclusion, oral administration of GTP was found to reduce the serum glucose tolerance in alloxan diabetic rats. It could also increase the antioxidant potential in the rats.
References Aebi, H., 1974. Catalase estimation. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Verlag chemic, Weinheim, New York, pp. 673 /684. Bergmeyer, H.W., Bernt, E., 1980. In: Varley, H., Gowenlock, A.H., Bell, M. (Eds.), Practical Clinical Biochemistry. William Heinemann Medical Books Ltd, London, p. 741. Bhaskaran Nair, R., Santhakumari, G., 1985. Antidiabetic activity of the seed kernal of Syzygium cumini Linn. Ancient Science of Life 6, 80 /84. Beutler, E., Duron, O., Kelly, B.M., 1983. Improved method for the determination of blood glutathione. Journal of Laboratory and Clinical Medicine 61, 882 /888. Brod, Sirota, 1948. In: Varley, H., Gowenlock, A.H., Bell, M. (Eds.), Practical Clinical Biochemistry. William Heinemann Medicinal Books Ltd, London, p. 190. Choi, J.H., Cha, B.K., Rhee, S.J., 1998. Effect of green tea catechin on hepatic microsomal phospholipase A2 activities and changes of
hepatic phospholipid species in streptozotocin-induced diabetic rats. Journal of Nutrinitiol Science and Vitaminol 44, 673 /683. Cooperstien, S.J., Walkins, D., 1981. Action of toxic drugs on islet cells. In: The Islets of Langerhans. Academic press, New York, pp. 387 /390. Elizabeth, K., Rao, M.N.A., 1990. Oxygen radical scavenging activity of curcumin. International Journal of Pharmacy 58, 237 /240. Gomes, A., Vedasiromoni, J.R., Das, M., Sharma, R.M., Ganguly, D.K., 1995. Anti-hyperglycemic effect of black tea (Camellia sinensis ) in rat. Journal of Ethnopharmacology 45, 223 /226. Halliwell, B., Gutteridge, J.M.C., 1985. Free Radicals in Biology and Medicine. Clarendon press, Oxford, London, pp. 22 /35. Hara, Y., 1994. Prophylatic functions of tea polyphenols. In: Chi-tang, H., Toshihiko, O., Mou-Tuan, H., Robert, T.R. (Eds.), Food Phytochemicals for Cancer Prevention. American Chemical Society, Washington, pp. 34 /35. Hara, Y., 1990. Advances in Food Science and Technology. Nippon Shokuhin Kogyo Gakkai, Korin, Tokyo, pp. 21 /39. Haslam, 1966. In: Varley, H., Gowenlock, A.H., Bell, M. (Eds.), Practical Clinical Biochemistry. William Heinemann Medicinal Books Ltd, London, p. 459. Hassid, W.Z., Abraham, S., 1957. Chemical procedures for analysis of polysaccharides. In: Colowick, S.P., Kaplan, N.O. (Eds.), Methods in Enzymology. Academic press, New York, London, pp. 34 /36. Helen, A., Vijayammal, P.L., 1997. Vitamin C supplementation on hepatic oxidative stress induced cigarette smoke. Journal of Applied Toxicology 17, 289 /295. Illing, E.K.B., Gray, C.H., Lawrence, R.D., 1951. Blood glutathione and non-glucose reducing substances in diabetes. Journal of Biochemistry 48, 637 /640. Joy, K.L., Kuttan, R., 1999. Anti-diabetic activity of Picorrhiza kurroa extract. Journal of Ethnopharmacology 167, 143 /148. King, E.J., Armstrong, A.R., 1980. In: Varley, H., Gowenlock, A.H., Bell, M. (Eds.), Practical Clinical Biochemistry. William Heinemann Medicinal Books Ltd, London, p. 897. Kobayashi, Y., Suzuki, M., Satsu, H., Arai, S., Hara, Y., Suzuki, K., Miyamoto, Y., Shimizu, M., 2000. Green tea polyphenols inhibit the sodium-dependent glucose transportre of intestinal epithelial cells by a competitive mechanism. Journal of Agricultural and Food Chemistry 48, 5618 /5623. Lowry, O.H., Rosebrough, N.F., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265 /273. McCord, J.M., Fridovich, I., 1969. Superoxide dismutase: an enzymatic function of erythrocuprein. Journal of Biological Chemistry 244, 6049 /6055.
116
S. M.C. et al. / Journal of Ethnopharmacology 83 (2002) 109 /116
Minami, M., Yoshikawa, H., 1979. A simplified assay method of superoxide dismutase activity for clinical use. Clinica Chimica Acta 92, 337 /342. Mukhtar, H., Wang, Z.Y., Katlya, S.K., Agarwal, R., 1992. Tea components: antimutagenic and anticarcinogenic effects. Preventive Medicine 21, 351 /360. Nagarajan, S., Jain, H.C., Aulakh, G.S., 1987. Indigenous Plants used in the Control of Diabetes. Publication and information directorate CSIR, New Delhi, p. 586. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thio-barbituric acid reaction. Analytical Biochemistry 95, 351 /358. Perfumi, M., Tacconi, R., 1996. Anti hyperglycemic effect of fresh Opuntia dillenii fruit from Tenerife (Canry islands). International Journal of Pharmacology 34, 41. Ponnachan, P.T.C., Paulose, C.S., Panikkar, K.R., 1993. Effect of leaf extract of Aegle marmelos in diabetic rats. Indian Journal of Experimental Biology 31, 345 /347. Rajasekharan, S., Tuli, S.N., 1976. Vijaysar’ (Pterocarpus marsupium ) in the treatment of ‘Madhumeha’ (diabetes mellitus) */a clinical trial. Journal of Research in Indian Medicine and Yoga homeopathy 9, 76 /78. Sano, M., Takahashi, Y., Yoshino, K., Shimoi, K., Nakamura, Y., Tomita, I., Oguni, I., Konomoto, H., 1995. Effect of tea (Camellia
sinensis L.) on lipid peroxidation in rat liver and kidney: a comparison of green and black tea feeding. Biological Pharmacy Bulletin 18, 1006 /1008. Satoh, K., 1978. Serum lipid peroxide in cerebrovascular disorders determined by a new calorimetric method. Clinica Chimica Acta 90, 37 /43. Subramoniam, A., Pushpangadan, P., Rajasekharan, S., Evans, D.A., Latha, P.G., valsaraj, R., 1996. Effects of Artemisia pallens Wall on blood glucose levels in normal and alloxan-induced diabetic rats. Journal of Ethnopharmacology 50, 13 /17. Shim, J.S., Kang, M.H., Kim, Y.H., Roh, J.K., Roberts, C., Lee, I.P., 1995. Chemopreventive effect of green tea (Camellia sinensis ) among cigarette smokers. Cancer Prevention and Epidemiological Biomarkers 4, 387 /391. Trinder, 1976. In: Varley, H., Gowenlock, A.H., Bell, M. (Eds.), Practical Clinical Biochemistry. William Heinemann Medicinal Books Ltd, London, p. 389. Vucic, M., Gavell, M., Bozikov, V., Ashcroft, J.H., Rocic, B., 1997. Superoxide dismutase activity in lymphocytes and polymorphonuclear cells of diabetic patients. European Journal of Clinical Chemistry Biochemistry 35, 517 /521. Yang, J.A., Choi, J.H., Rhee, S.J., 1999. Effects of green tea catechin on phospholipase A2 activity and antithrombus in streptozotocin diabetes rats. Journal of Nutrition Science Vitaminol 45, 337 /346.
Anti-diabetic activity of green tea polyphenols and their role in reducing oxidative stress in experimental diabetes Sabu M.C., Smitha K., Ramadasan Kuttan * Amala Cancer Research Centre, Amala Nagar, Trichur 680 553, Kerala, India Accepted 5 August 2002
Abstract An aqueous solution of green tea polyphenols (GTP) was found to inhibit lipid peroxidation (LP), scavenge hydroxyl and superoxide radicals in vitro. Concentration needed for 50% inhibition of superoxide, hydroxyl and LP radicals were 10, 52.5 and 136 mg/ml, respectively. Administration of GTP (500 mg/kg b.wt.) to normal rats increased glucose tolerance significantly (P B/0.005) at 60 min. GTP was also found to reduce serum glucose level in alloxan diabetic rats significantly at a dose level of 100 mg/kg b.wt. Continued daily administration (15 days) of the extract 50, 100 mg/kg b.wt. produced 29 and 44% reduction in the elevated serum glucose level produced by alloxan administration. Elevated hepatic and renal enzymes produced by alloxan were found to be reduced (P B/0.001) by GTP. The serum LP levels which was increased by alloxan and was reduced by significantly (P B/0.001) by the administration of 100 mg/kg b.wt. of GTP. Decreased liver glycogen, after alloxan administration showed a significant (P B/ 0.001) increase after GTP treatment. GTP treated group showed increased antioxidant potential as seen from improvements in superoxide dismutase and glutathione levels. However catalase, LP and glutathione peroxidase levels were unchanged. These results indicate that alterations in the glucose utilizing system and oxidation status in rats increased by alloxan were partially reversed by the administration of the glutamate pyruvate transaminase. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Green tea; Polyphenols; Diabetes; Alloxan; Antioxidant
1. Introduction Many indigenous Indian medicinal plants have been found to be useful to successfully manage diabetes (Rajasekharan and Tuli, 1976; Bhaskaran Nair and Santhakumari, 1985; Nagarajan et al., 1987; Ponnachan et al., 1993; Subramoniam et al., 1996; Joy and Kuttan, 1999). Despite the introduction of hypoglycemic agents from natural and synthetic sources diabetes and its complications continue to be a major problem in the world population. Green tea is produced by enzymatic inactivation of the leaves of Camellia sinensis followed by rolling or comminution and drying. In the manufacturer of green * Corresponding author. Fax: /91-487-307020 E-mail address: [email protected] (R. Kuttan).
tea, the enzymatic inactivation achieved by steam or pan firing treatment to preserve natural polyphenols with respect to the health promoting properties. Green tea derived products are mainly extracts of green tea in liquid or powder form varying in the proportion of polyphenols (45 /90%) and caffeine content (0.4 /10%). The polyphenolic fraction of green tea, has been reported to have multiple pharmacological actions (Mukhtar et al., 1992; Sano et al., 1995). The antioxidative potency of crude catechin powder and individual catechins were tested in experiments using active oxygen method. Crude catechins reduced the formation of peroxides for more effectively than dl -a-tocopherol (Hara, 1990). In this study we have investigated the effect of crude polyphenolic fraction of green tea in reducing the alloxan-induced oxidative damage and diabetes in rats.
0378-8741/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 2 1 7 - 9
110
S. M.C. et al. / Journal of Ethnopharmacology 83 (2002) 109 /116
2. Materials and methods 2.1. Animals Male Wistar albino rats (250 /300 g) were obtained from the Veterinary College, Mannuthy, India. They were housed in ventilated cages and fed with a pelleted diet (Lipton, India Ltd) and water ad libitum. 2.2. Chemicals Green tea was a gift from Dr J.I.X. Antony, Kancor Flavours and Extracts Limited, India. Alloxan monohydrate was obtained from Sigma, St. Louis, MO. 1chloro-2,4-dinitrobenzene, glutathione and 5-5-dithiobis (2-nitrobenzoic acid) were purchased from Sisco Research Laboratory, Mumbai, India. Thiobarbituric acid (TBA) was obtained from E-Merck, Germany. All other chemicals used were of analytical and reagent grade. 2.3. Preparation of polyphenolic fraction of green tea Dried green tea powder (250 g) was extracted with water and water soluble fraction was dissolved in hot water and washed with chloroform. Aqueous layer was extracted with ethyl acetate and ethyl acetate layer containing polyphenol was separated out and evaporated to dryness (Hara, 1994). It was resuspended in water and used for the experiments. The percentage yield of crude polyphenols is 5.5% w/w. 2.4. In vitro antioxidant activity 2.4.1. Superoxide radicals scavenging activity Superoxide scavenging was determined by the Nitroblue tetrazolum (NBT) reduction method (McCord and Fridovich, 1969). The reaction mixture contained EDTA (6 mM) containing NaCN (3 mg), riboflavin (2 mM), NBT (50 mM), various concentrations of the extracts (5 /50 mg/ml) and phosphate buffer (67 mM, pH 7.8) in a final volume of 3 ml. The tubes were uniformly illuminated with an incandescent visible light (Philips, 40 W) for 15 min, and the optical density was measured at 530 nm before and after the illumination. The percentage inhibition of superoxide generation was evaluated by comparing the absorbance values of the control and experimental tubes. 2.4.2. Hydroxyl radical scavenging activity Hydroxyl radical scavenging was measured by studying the competition between deoxyribose and the test compounds for hydroxyl radicals generated from the Fe3/ascorbate/EDTA/H2O2 system. The hydroxyl radical attack deoxyribose, which eventually results in TBA reacting substance (TBARS) formation (Elizabeth and Rao, 1990). The reaction mixture contained deox-
yribose (2.8 mM), FeCl3 (0.1 mM), EDTA (0.1 mM), H2O2 (1 mM), ascorbic acid (0.1 mM), KH2PO4 /KOH buffer (20 mM, pH 7.4), and various concentrations of the extract (25 /400 mg/ml) in a final volume of 1 ml. The reaction mixture was incubated for 1 h at 37 8C. Deoxyribose degradation was measured as TBARS and percentage inhibition was calculated. 2.4.3. Lipid peroxide scavenging activity Reaction mixture (0.5 ml) containing rat liver homogenate (0.1 ml, 25% w/v) in Tris /HCl buffer (40 mM, pH 7.0), KCl (30 mM), ferrous iron (0.16 mM) and ascorbic acid (0.06 mM) was incubated for 1 h at 37 8C in the presence and absence of the extract (20 /180 mg/ ml). The lipid peroxide formed was measured by TBARS formation (Ohkawa et al., 1979). For this incubation mixture 0.4 ml was treated with sodium dodecyl sulphate (8.1%, 0.2 ml), TBA (0.8%, 1.5 ml) and acetic acid (20%, 1.5 ml, pH 3.5). The total volume was then made upto 4 ml by adding distilled water and kept in a water bath at 100 8C for 1 h. After cooling, 1 ml of distilled water and 5 ml of a mixture of n -butanol and pyridine (15:1 v/v) were added and shaken vigorously. After centrifugation, the absorbance of the organic layer was measured at 532 nm. The percentage inhibition of lipid peroxidation (LP) was determined by comparing the results of the test compounds with those of controls not treated with the extracts. 2.5. Anti-diabetic activity 2.5.1. Glucose tolerance test Normal Wistar rats were fasted overnight. They were divided into three groups containing six animals each. Control rats (Group I) were given 1 ml distilled water orally. Green tea polyphenols (GTP) at a concentration of 100, 500 mg/kg b.wt. were administered orally using a syringe to second and third groups. Glucose (2 g/kg b.wt.) was given orally using a syringe to all groups immediately after the GTP administration. Blood samples were collected from the tail vein just prior to and 30, 60, 120 and 240 min after the glucose loading and serum glucose levels were measured by GOD/POD method (Trinder, 1976). 2.6. Effect of polyphenols on serum glucose levels in alloxan-induced diabetic rats (single and multidose study) Diabetes was induced in male rats by single intraperitonial injection of 120 mg/kg b.wt. of alloxan monohydrate (Cooperstien and Walkins, 1981). Serum glucose level was checked after 72 h. Animals with serum glucose levels /250 mg/dl were considered diabetic and were used for the study (Perfumi and Tacconi, 1996). The rats were divided into four groups of six rats each. Both group I control normal rats (no
S. M.C. et al. / Journal of Ethnopharmacology 83 (2002) 109 /116
alloxan treatment) and group II diabetic animals were given 1 ml of distilled water. Group III and IV were given aqueous suspension of GTP orally at a dose level of 50 and 100 mg/kg b.wt. on 3rd day after alloxan treatment. Overnight fasted blood samples were collected from the tail vein on 3rd day of alloxan treatment prior to and at 2, 4, 6 and 8 h intervals after the administration of 100 mg/kg b.wt. of the extract orally. Serum was separated and glucose levels were estimated as before. In the multidose study, these animals received the same dose of the extract once daily for 15 days. Blood was collected in non fasted rats on 6, 9, 12 15 and 18th day after alloxan administration and serum glucose levels were measured.
2.7. Liver and kidney functions test Normal and diabetic animals were treated with GTP for 15 days at dose levels of 50 and 100 mg/kg body weight. They were sacrificed on 18th day after alloxan treatment. Blood was collected and serum was separated and LP (Satoh, 1978), alkaline phosphatase (ALP; King and Armstrong, 1980), glutamate pyruvate transaminase (GPT; Bergmeyer and Bernt, 1980), blood urea nitrogen (BUN; Haslam, 1966) and creatinine (Brod and Sirota, 1948) were estimated. Protein was determined by the method of Lowry et al. (1951). The initial and final body weights were determined. Total white blood cell count (WBC) was estimated using haemocytometer on 1st and 18th day after alloxan administration. Liver glycogen was estimated by the method of Hassid and Abraham (1957).
2.8. In vivo antioxidant activity All the groups of animals were sacrificed immediately after the experimental period and liver was taken, washed with normal saline and one part was preserved in 10% formalin for histopathological studies. The other part was homogenized by Tris /HCl buffer and used for measuring LP and activities of superoxide dismutase (SOD; Minami and Yoshikawa, 1979), catalase (Aebi, 1974), glutathione (GSH; Beutler et al., 1983) and glutathione peroxidase (GPx; Helen and Vijayammal, 1997).
2.9. Statistical analysis Data were expressed as mean9/standard error of mean. Statistical comparisons with animals of untreated diabetic group with normal and treated with different concentrations of GTP were performed with Student’s ttest for unpaired observations.
111
3. Results The polyphenols of green tea, was found to scavenge the superoxide generated by photoreduction of riboflavin in a concentration dependent manner. The concentration needed for 50% scavenging of superoxides was 10 mg/ml. The concentration needed for 50% inhibition of hydroxyl radical generation was 52.5 mg/ ml. LP induced by Fe2/ascorbate in rat liver homogenate was found to be inhibited and the concentration needed for 50% inhibition was 136 mg/ml (Fig. 1). Administration of 2g glucose/kg b.wt. to normal rats increased serum glucose levels from 77.39/13.8 to 176.49/20.5 at 60 min but returned to normal at 240 min. Administration of GTP 500 mg/kg b.wt. suppressed the elevation of serum glucose level significantly (P B/0.005) at 60 min (Fig. 2). The effect of polyphenols in alloxan diabetic rat is shown in Table 1. Single administration of the extract, 50 mg/kg b.wt. on 3rd day after alloxan administration did not produce a significant reduction in serum glucose level. At a dose level of 100 mg/kg b.wt., there was 17.3% reduction in glucose level (P B/0.001) at 6th hour and the activity was found to be reduced in following hours (Table 1). Continuous administration of the GTPs (50 mg/kg b.wt.) produced 29.8% reduction in the elevated glucose level on 18th day. At dose level of 100 mg/kg b.wt., a significant (P B/0.001) reduction in blood glucose was seen from 12th day by 25.5% and at 18th day after alloxan treatment the percent reduction in serum glucose was 44.1%. Blood glucose levels in the control (alloxan treated) and normal (alloxan non-treated) group was remained unchanged over this period (Table 2). Liver function markers like ALP, GPT and LP in serum were significantly (P B/0.001) elevated in alloxaninduced diabetes when compared with normal animals (Table 3). Animals treated with 50 and 100 mg/kg b.wt. of polyphenols showed significant (P B/0.001) reduction in the elevated level of ALP, GPT and LPO. Renal function indicators like creatinine and BUN were also elevated in the alloxan diabetic rats when compared with normal rats. The creatinine level of 2.81 mg/dl was found to be reduced to 1.98 mg/dl (P B/0.005) by the administration of 100 mg/kg b.wt. of the GTP. When compared with diabetic control group the elevated levels of BUN was significantly (P B/0.001) reduced in animals treated at both the dose levels of GTP. Normal animals treated with GTP at dose level of 100 mg/kg b.wt. did not produce any significant weight loss (Table 4). A significant (P B/0.001) decrease in rat body weight by 19% was noted in alloxan-induced diabetic rats. But, when the animals were treated with 100 mg/kg b.wt. of GTP suppressed the weight loss significantly
112 S. M.C. et al. / Journal of Ethnopharmacology 83 (2002) 109 /116
Fig. 1. Effect of GTP on superoxide, hydroxyl radical generation and LP in vitro. (A) Superoxide; (B) Hydroxyl radical; (C) LP.
S. M.C. et al. / Journal of Ethnopharmacology 83 (2002) 109 /116
113
Fig. 2. Effect of GTP on glucose tolerance in normal rats ( /m /) Control; ( /j /) GTP 100 mg/kg b.wt.; ( /^ /) GTP 500 mg/kg b.wt.;*P B/0.005.
(P B/0.001) when compared with initial body weight of the same group. Effect of polyphenols on total WBC in alloxan diabetic animals is shown in Table 4. Total WBC was found to be 14 650 in normal rats and in alloxan diabetic rat it was 9112, on 18th day of the experiment. Administration of GTP (100 mg/kg b.wt.) considerably reversed alloxan-induced cellular damage as seen from the increased number of total WBC (P B/0.001) when compared with diabetic control group. Lower dose had also a comparable effect. When compared with normal animals, the liver glycogen level was low in alloxan treated rats was increased after the administration of GTP (not significant). In vivo antioxidant activity after administration of GTP for 18 days is shown in Table 5. In diabetic control group, the SOD and GSH levels were significantly (P B/ 0.001) decreased when compared with normal group. The decreased SOD level of diabetic control group were
found to be increased from 9.699/0.72 to 16.439/0.10 at a dose level of 100 mg/kg b.wt. significantly (P B/0.001). There was only marginal difference (P B/0.001) in the values of glutathone peroxidase and LP. The LP was significantly (P B/0.001) decreased by the administration of 100 mg/kg b.wt. of GTP. There was no significant change in the catalase values.
4. Discussion Green tea has become very popular neutraceutical as an antioxidant and a health promoting traditional drug preparation. In the present study we report anti-diabetic and free radical scavenging activity of GTP. GTP contains gallocatechin (GC), epigallocatechin (EGC), epicatechin (EC), epigallocatechin gallate (EGCg) and epicatechin gallate (ECg). Tea components possess antioxidant, antimutagenic and anticarcinogenic effects
Table 1 Effect of polyphenlic fraction of Green tea on serum glucose levels in alloxan-induced diabetic rats (single dose short term study) Group
I II III IV
Treatment (dose/kg b.wt.)
Normal Control (alloxan) GTPs (50 mg) GPTs (100 mg)
Serum Glucose level in mg/dl (h) 0
2
4
6
8
73.494.4 252.7916.0* 246.6951.1 254.5924.7
65.394.3 255.3916.4* 247.1952.4 251.7923.4
58.393.9 257.1915.4* 232.3953.1 237.5919.6
59.793.5 261.6916.1* 233.7952.9 210.9919.4**
68.892.8 260.0915.1* 236.4953.4 216.8923.9
Values represent the fasting sugar level on 3rd day after alloxan administration in presents and absence of polyphenol extract (values are mean9SD, n 6). * P B 0.001 (compared to normal group). ** P B 0.001 (compared to control group).
S. M.C. et al. / Journal of Ethnopharmacology 83 (2002) 109 /116
114
Table 2 Effect of polyphenolic fraction of Green tea on serum glucose level in alloxan-induced diabetic rats (multidose long term study) Group
I II III IV
Treatment (dose/kg b.wt.)
Serum glucose level in mg/dl (days)
Normal Control (alloxan) GTPs (50 mg) GTPs (100 mg)
3
6
9
12
15
18
73.494.4 252.7916.0* 246.6951.1 254.5924.7
70.793.7 265.2915.2* 234.5952.7 240.1919.4
68.993.8 265.4912.9* 222.7949.8 225.1920.0
67.193.8 275.7912.1* 216.6950.3 189.6919.6**
65.193.5 269.998.5* 193.0954.3 167.4923.0**
63.693.6 256.398.6* 173.2947.9 142.3929.6**
Values represent serum glucose level in alloxan treated animals with or without GTP from 3rd day to 18th day. (Values are mean9SD, n 6.) * P B 0.001 (compared to values of normal group with the corresponding days). ** P B 0.001 (compared to values on 3rd day after alloxan treatment of the same group).
Table 3 Effect of GTP on liver and kidney biochemical parameters in normal and diabetic rats Treatment (dose/kg b.wt.)
Normal Control (Alloxan) GTPs (50 mg) GTPs (100 mg)
Liver
Kidney
ALP (KA/dl)
GPT (U/mg protein)
LPO (U/ml)
BUN (mg/dl)
Creatinine (mg/dl)
33.190.87 46.491.78* 40.392.11** 38.190.68***
141.0911.92 307919.67* 250.598.06*** 225.896.40***
1.690.09 3.3390.22* 2.4790.17*** 1.7490.17***
9.490.28 20.190.97* 16.490.72*** 13.590.39***
0.990.13 2.390.14* 2.390.32*** 1.990.25**
Biochemical parameters were determined at the end of the experiment (18 days after alloxan administration) (values are mean9SD, n 6). * P B 0.001 (compared to normal group). ** P B 0.005 (compared to control group). *** P B 0.001 (compared to control group).
and could protect humans against the risk of cancer by environmental agents (Mukhtar et al., 1992). Sano et al. (1995) studied the inhibitory effects of green tea leaves against tert-butyl hydroperoxide induced LP and a similar antioxidant effect on the kidney was observed after oral administration of a major tea polyphenols (/)-EGCg. Shim et al. (1995) studied the chemopreventive effect of green tea among cigarette smokers and found that it can block the cigarette-induced increase in sisterchromatid exchange frequency. Anti-hyperglycemic effect of black tea has been reported earlier by Gomes et al. (1995). EGCg was found to inhibit intestinal glucose uptake by sodium dependent glucose
transporter, SGLT1 indicating its increase in controlling blood sugar (Kobayashi et al., 2000). Streptozotocindiabetic rats showed increased sensitivity to platelet aggregation and thrombosis and this abnormality could be improved by dietary catechin of green tea (Yang et al., 1999; Choi et al., 1998). Alloxan produces oxygen radicals in the body, which cause pancreatic injury (Halliwell and Gutteridge, 1985) which is responsible for increased blood sugar seen in the animals. However, it is found that action is not specific to pancreas as other organs such as liver, kidney and haemopoietic system also affected by alloxan administration as seen from the elevation of marker
Table 4 Effect of GTP on total leucocyte counts, body weight and liver glycogen in normal and diabetic rats Treatment (dose/kg b.wt.)
Normal (untreated) Control (alloxan) GTPs (50 mg) GTPs (100 mg)
Total leucocyte count (mm3)
14650.09445.4 9112.591134.6a 9387.591781.6 140259290.1*
Body weight (g)
Liver glycogen (g/g tissue)
Initial
Final
282.5910.4 277.5910.4 274.099.2 269.5912.8
293.0910.2 224.5915.7b 243.598.1b 258.0913.3
76.197.4 54.692.7a 56.191.8 61.993.4**
a P B 0.001 (compared to normal group). bP B 0.001 (compared to initial body weight of the same group). Values are determined from day 18 after alloxan administration. (Values are mean9SD, n 6.) * P B 0.001 (compared to control group). ** P B 0.005 (compared to control group).
S. M.C. et al. / Journal of Ethnopharmacology 83 (2002) 109 /116
115
Table 5 Effect of GTP on in vivo antioxidant activity in normal and diabetic rats Treatment (dose/kg b.wt.)
SODa
Catalaseb
LPOc
GSHd
GPxe
Normal (untreated) Control (alloxan) GTPs (50 mg) GTPs (100 mg)
14.691.03 9.790.72* 9.390.49 16.490.10***
8.591.50 6.691.37 6.390.58 7.891.75
0.6690.10 0.8990.02** 0.8590.04 0.7290.03***
3.2490.29 2.0690.04* 2.0690.07 2.3690.10***
63.699.93 46.894.9** 44.796.5 53.7915.9
Values are determined from day 18 after alloxan administration, (Values are mean9SD, n 6). a Units (Enzymes concentration required to inhibit chromogen production by 50% in 1 min)/mg protein. b Values 10 3 units (velocity constant/s/mg protein). c mM/100 g wet tissue. d mM/g wet tissue. e U/mg protein. * P B 0.001 (compared to control group). ** P B 0.005 (compared to normal group). *** P B 0.001 (compared to control group).
enzymes and reduction of haemtological parameters. This was reversed by the continued administration of GTP extract. The present study indicates that alloxaninduced diabetes and subsequent elevation of blood sugar was reversed by simultaneous administration of GTP. Under in vivo condition, glutathione (GSH) acts as an antioxidant and its decrease was reported in diabetes mellitus (Illing et al., 1951). The increased GSH content in the liver of the rats treated with GTP may be one of the factors responsible for the inhibition of LP. SOD and catalase are the two major scavenging enzymes that remove the toxic free radicals in vivo. Vucic et al. (1997) reported that the activity of SOD is low in diabetes mellitus. The GTP treated rats showed decreased LP associated with increased activity of SOD and GSH (Table 5). The antioxidant enzymes catalase and GPx showed non-significant differences in GTP treated groups. In conclusion, oral administration of GTP was found to reduce the serum glucose tolerance in alloxan diabetic rats. It could also increase the antioxidant potential in the rats.
References Aebi, H., 1974. Catalase estimation. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Verlag chemic, Weinheim, New York, pp. 673 /684. Bergmeyer, H.W., Bernt, E., 1980. In: Varley, H., Gowenlock, A.H., Bell, M. (Eds.), Practical Clinical Biochemistry. William Heinemann Medical Books Ltd, London, p. 741. Bhaskaran Nair, R., Santhakumari, G., 1985. Antidiabetic activity of the seed kernal of Syzygium cumini Linn. Ancient Science of Life 6, 80 /84. Beutler, E., Duron, O., Kelly, B.M., 1983. Improved method for the determination of blood glutathione. Journal of Laboratory and Clinical Medicine 61, 882 /888. Brod, Sirota, 1948. In: Varley, H., Gowenlock, A.H., Bell, M. (Eds.), Practical Clinical Biochemistry. William Heinemann Medicinal Books Ltd, London, p. 190. Choi, J.H., Cha, B.K., Rhee, S.J., 1998. Effect of green tea catechin on hepatic microsomal phospholipase A2 activities and changes of
hepatic phospholipid species in streptozotocin-induced diabetic rats. Journal of Nutrinitiol Science and Vitaminol 44, 673 /683. Cooperstien, S.J., Walkins, D., 1981. Action of toxic drugs on islet cells. In: The Islets of Langerhans. Academic press, New York, pp. 387 /390. Elizabeth, K., Rao, M.N.A., 1990. Oxygen radical scavenging activity of curcumin. International Journal of Pharmacy 58, 237 /240. Gomes, A., Vedasiromoni, J.R., Das, M., Sharma, R.M., Ganguly, D.K., 1995. Anti-hyperglycemic effect of black tea (Camellia sinensis ) in rat. Journal of Ethnopharmacology 45, 223 /226. Halliwell, B., Gutteridge, J.M.C., 1985. Free Radicals in Biology and Medicine. Clarendon press, Oxford, London, pp. 22 /35. Hara, Y., 1994. Prophylatic functions of tea polyphenols. In: Chi-tang, H., Toshihiko, O., Mou-Tuan, H., Robert, T.R. (Eds.), Food Phytochemicals for Cancer Prevention. American Chemical Society, Washington, pp. 34 /35. Hara, Y., 1990. Advances in Food Science and Technology. Nippon Shokuhin Kogyo Gakkai, Korin, Tokyo, pp. 21 /39. Haslam, 1966. In: Varley, H., Gowenlock, A.H., Bell, M. (Eds.), Practical Clinical Biochemistry. William Heinemann Medicinal Books Ltd, London, p. 459. Hassid, W.Z., Abraham, S., 1957. Chemical procedures for analysis of polysaccharides. In: Colowick, S.P., Kaplan, N.O. (Eds.), Methods in Enzymology. Academic press, New York, London, pp. 34 /36. Helen, A., Vijayammal, P.L., 1997. Vitamin C supplementation on hepatic oxidative stress induced cigarette smoke. Journal of Applied Toxicology 17, 289 /295. Illing, E.K.B., Gray, C.H., Lawrence, R.D., 1951. Blood glutathione and non-glucose reducing substances in diabetes. Journal of Biochemistry 48, 637 /640. Joy, K.L., Kuttan, R., 1999. Anti-diabetic activity of Picorrhiza kurroa extract. Journal of Ethnopharmacology 167, 143 /148. King, E.J., Armstrong, A.R., 1980. In: Varley, H., Gowenlock, A.H., Bell, M. (Eds.), Practical Clinical Biochemistry. William Heinemann Medicinal Books Ltd, London, p. 897. Kobayashi, Y., Suzuki, M., Satsu, H., Arai, S., Hara, Y., Suzuki, K., Miyamoto, Y., Shimizu, M., 2000. Green tea polyphenols inhibit the sodium-dependent glucose transportre of intestinal epithelial cells by a competitive mechanism. Journal of Agricultural and Food Chemistry 48, 5618 /5623. Lowry, O.H., Rosebrough, N.F., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265 /273. McCord, J.M., Fridovich, I., 1969. Superoxide dismutase: an enzymatic function of erythrocuprein. Journal of Biological Chemistry 244, 6049 /6055.
116
S. M.C. et al. / Journal of Ethnopharmacology 83 (2002) 109 /116
Minami, M., Yoshikawa, H., 1979. A simplified assay method of superoxide dismutase activity for clinical use. Clinica Chimica Acta 92, 337 /342. Mukhtar, H., Wang, Z.Y., Katlya, S.K., Agarwal, R., 1992. Tea components: antimutagenic and anticarcinogenic effects. Preventive Medicine 21, 351 /360. Nagarajan, S., Jain, H.C., Aulakh, G.S., 1987. Indigenous Plants used in the Control of Diabetes. Publication and information directorate CSIR, New Delhi, p. 586. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thio-barbituric acid reaction. Analytical Biochemistry 95, 351 /358. Perfumi, M., Tacconi, R., 1996. Anti hyperglycemic effect of fresh Opuntia dillenii fruit from Tenerife (Canry islands). International Journal of Pharmacology 34, 41. Ponnachan, P.T.C., Paulose, C.S., Panikkar, K.R., 1993. Effect of leaf extract of Aegle marmelos in diabetic rats. Indian Journal of Experimental Biology 31, 345 /347. Rajasekharan, S., Tuli, S.N., 1976. Vijaysar’ (Pterocarpus marsupium ) in the treatment of ‘Madhumeha’ (diabetes mellitus) */a clinical trial. Journal of Research in Indian Medicine and Yoga homeopathy 9, 76 /78. Sano, M., Takahashi, Y., Yoshino, K., Shimoi, K., Nakamura, Y., Tomita, I., Oguni, I., Konomoto, H., 1995. Effect of tea (Camellia
sinensis L.) on lipid peroxidation in rat liver and kidney: a comparison of green and black tea feeding. Biological Pharmacy Bulletin 18, 1006 /1008. Satoh, K., 1978. Serum lipid peroxide in cerebrovascular disorders determined by a new calorimetric method. Clinica Chimica Acta 90, 37 /43. Subramoniam, A., Pushpangadan, P., Rajasekharan, S., Evans, D.A., Latha, P.G., valsaraj, R., 1996. Effects of Artemisia pallens Wall on blood glucose levels in normal and alloxan-induced diabetic rats. Journal of Ethnopharmacology 50, 13 /17. Shim, J.S., Kang, M.H., Kim, Y.H., Roh, J.K., Roberts, C., Lee, I.P., 1995. Chemopreventive effect of green tea (Camellia sinensis ) among cigarette smokers. Cancer Prevention and Epidemiological Biomarkers 4, 387 /391. Trinder, 1976. In: Varley, H., Gowenlock, A.H., Bell, M. (Eds.), Practical Clinical Biochemistry. William Heinemann Medicinal Books Ltd, London, p. 389. Vucic, M., Gavell, M., Bozikov, V., Ashcroft, J.H., Rocic, B., 1997. Superoxide dismutase activity in lymphocytes and polymorphonuclear cells of diabetic patients. European Journal of Clinical Chemistry Biochemistry 35, 517 /521. Yang, J.A., Choi, J.H., Rhee, S.J., 1999. Effects of green tea catechin on phospholipase A2 activity and antithrombus in streptozotocin diabetes rats. Journal of Nutrition Science Vitaminol 45, 337 /346.