Journal of Ethnopharmacology 81 (2002) 155 /160 www.elsevier.

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Anti-diabetic activity of medicinal plants and its relationship with their antioxidant property
M.C. Sabu, Ramadasan Kuttan *
Amala Cancer Research Centre, Amala Nagar, Trichur, Kerala 680 553, India Accepted 31 January 2002

Abstract Methanolic extract (75%) of Terminalia chebula , Terminalia belerica , Emblica officinalis and their combination named Triphala (equal proportion of above three plant extracts) are being used extensively in Indian system of medicine. They were found to inhibit lipid peroxide formation and to scavenge hydroxyl and superoxide radicals in vitro. The concentration of plant extracts that inhibited 50% of lipid peroxidation induced with Fe2 /ascorbate were food to be 85.5, 27, 74 and 69 mg/ml, respectively. The concentration needed for the inhibition of hydoxyl radical scavenging were 165, 71, 155.5 and 151 mg/ml, and that for superoxide scavenging activity were found to be 20.5, 40.5, 6.5 and 12.5 mg/ml, respectively. Oral administration of the extracts (100 mg/kg body weight) reduced the blood sugar level in normal and in alloxan (120 mg/kg) diabetic rats significantly within 4 h. Continued, daily administration of the drug produced a sustained effect. # 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Herbal extracts; Antioxidants; Normal rats; Diabetes; Anti-hyperglycemic drug

1. Introduction Free radicals have been implicated in the causation of several diseases such as liver cirrhosis, atherosclerosis, cancer, diabetes, etc. and compounds that can scavenge free radicals have great potential in ameliorating these disease processes (Wilson, 1988). Antioxidants thus play an important role to protect the human body against damage by reactive oxygen species (Lollinger, 1981). Increased oxidative stress has been postulated in the diabetic state. Oxygen free radical activity can initiate peroxidation of lipids, which in turn stimulates glycation of protein, inactivation of enzymes and alterations in the structure and function of collagen, basement and other membranes and play a role in the long team complications of diabetes (Boynes, 1991). Oxidative stress in diabetes coexists with a reduction in the antioxidant status (Collier et al., 1990), which can increase the deleterious effects of free radicals. It has also been known that alloxan induces its diabetogenic activity mainly by inducing oxygen free radicals and thereby
* Corresponding author. Fax: /91-487-211020. E-mail address: amalaresearch@hotmail.com (R. Kuttan).

damaging the pancreas (Halliwell and Gutteridge, 1985). Supplementation with non-toxic antioxidants may have a chemoprotective role in the diabetes (Logani and Davis, 1979). Antioxidants, vitamins C and E have been shown to reduce the oxidative stress in experimental diabetes (Madhu and Devi, 2000). Supplimentation of antioxidant vitamin C has also been shown to lower glycosylated hemoglobin is diabetic patient (Davie et al., 1992). Many plant extracts and plant products have been shown to have significant antioxidant activity (Nagarajan et al., 1987; Jain and Sharma, 1967; Anjali and Manoj, 1995). Role of plant antioxidants in diabetes has not been undertaken. In the present study, we have evaluated Terminalia chebula , Terminalia belerica , Emblica officinalis , their combination Triphala for their antidiabetic activity and their relation with their antioxidant activity.

2. Materials and methods Fruits of T. chebula , T. belerica and E. officinalis were purchased from the local market and authenticated and voucher specimen has been kept at Amala Ayurve-

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 0 3 4 - X

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dic Hospital and Research Centre. Nitroblue tetrazolium (NBT) was purchased from Sisco Research Laboratories Pvt. Ltd., Bombay. 2-Deoxy-D-ribose and alloxan monohydrate were obtained from Sigma, St. Louis, MO. All other chemicals and reagents used were of analytical grade. 2.1. Preparation of plant extracts The authenticated dried fruits of T. chebula , T. belerica , and E. officinalis were powdered separately and 100 g of powder was extracted twice in 500 ml of 75% methanol by stirring overnight and centrifuged at room temperature. The supernatant was collected and evaporated to dryness under reduced pressure in a rotary evaporator. The combination drug Triphala was also extracted in the same manner. The yields of the methanolic extracts were 7.5, 10, 13.5 and 10.5%, respectively. The extracts were solublized in water and used for the in vivo and in vitro experiments. 2.2. Animals Male Wistar albino rats (180 /200 g) were used for the study. They were fed with a standard diet (Sai Foods, Bangalore) and water ad libitum. Animals described as fasted had been deprived of food for 16 h but had been allowed free access to water. 2.3. Inhibition of superoxide radicals by riboavin photoreduction method Superoxide scavenging was determined by the Nitro blue tetrasolium reduction method (McCord and Fridovich, 1969). The reduction mixture contained EDTA (6 mM) containing 3 mg NaCN, riboflavin (2 mM), Nitro blue tetrazolium (50 mM), various concentrations (10 / 160 mg) of the extracts and phosphate buffer (67 mM, pH 7.8) in a final volume of 3 ml. The tubes were uniformly illuminated with an incandescent lamp 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. Hydroxyl radical-scavenging activity Hydroxly 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 (Fenton reaction). The hydroxyl radical attack deoxyribose, which eventually results in thiobarbituric acid reacting substance (TBARS) formation (Elizabeth and Rao, 1990).

The reaction mixture contained deoxyribose (2.8 mM), FeCl3 (0.1 mM), EDTA (0.1 mM), H2O2 (1 mM), ascorbate (0.1 mM), KH2PO4 /KOH buffer (20 mM, pH 7.4), and various concentrations (25 /400 mg) of the extract 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 the percentage inhibition was calculated.

2.5. Inhibition of lipid peroxide formation induced by Fe2-ascorbate system 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) and various concentration (10 / 90 mg) of the extracts were incubated for 1 h at 37 8C. The lipid peroxide formed was measured by TBARS (Ohkawa et al., 1979). The incubated reaction mixture (0.4 ml) was treated with sodium dodecyl sulphate (SDS-0.2 ml, 8.1%), thiobarbituricacid (TBA-1.5 ml, 0.8%) and acetic acid (1.5 ml, 20%, pH 3.5). The total volume was then made up to 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 was determined by comparing the results of the test compounds with those of controls not treated with the extracts.

2.6. Effect of plant extracts on blood glucose levels in normal rats (single and multi dose study) Male Wistar rats were divided into five groups of six rats per group. Group-I animals were given distilled water through oral intubation, which served as control. Group II /V were given aqueous suspension of the extract of T. chebula , T. belerica , E. officinalis or Triphala orally at a dose level of 100 mg/kg body weight, respectively. In single dose study, after administration of a single dose of extracts, blood samples were collected from the tail vein just prior to and 1, 2, 4 and 6 h intervals. Serum was separated and glucose levels were estimated by enzymatic glucose oxidase-method (Varley et al., 1976). For multi dose study, the same groups of normal animals were continued with the same dose level once daily, up to 11 days. The glucose levels of all the animals were measured on 3, 5, 7, 9 and 11th day, respectively.

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2.7. Effect of plant extracts on serum glucose levels in alloxan-induced diabetic rats (single and multi dose study) Male rats were induced diabetes by injecting a single i.p. injection of 120 mg/kg body weight of alloxan monohydrate (Cooperstein and Watkins, 1981). Serum glucose level was checked after 72 h. Animals with serum glucose levels /300 mg/100 ml were considered diabetic and were used for the study (Perfumi and Tacconi, 1996). The diabetic rats were divided into five groups of six rats per group. Control diabetic rats (Group-I) were given distilled water. Groups II /V were given aqueous suspension of T. chebula , T. belerica , E. officinalis or Triphala orally at a dose level of 100 mg/ kg body weight, respectively. In single dose study, after administration of a single dose, blood samples were collected from the tail vein just prior to 1, 2, 4 and 6 h intervals. Serum was separated and glucose levels were estimated. For multi dose study, the same groups of diabetic animals were continued with the same dose level once daily, up to 11 days. The blood glucose levels of all the animals were measured on 3, 5, 7, 9 and 11th day, respectively. 2.8. Data and statistical analysis Data were expressed as mean9/standard error of mean (SEM). Statistical comparisons between all groups were performed by using ANOVA-1.

Table 1 Effect of in vitro antioxidant activity of T. chebula , T. belerica , E. ofcinalis and Triphala extracts Treatment Concentration needed for 50% inhibition of oxygen radicals (mg/ml) Superoxide T. chebula 20.593.2 T. belerica 40.596.5 E. officinalis 6.591.5 Triphala 12.592.2 Hydroxyl radical 165.598.5 71.092.7 155.098.1 151.097.2 Lipid peroxidation 85.596.5 27.093.2 74.095.8 69.095.1

Values are mean9SD; n  3.

3. Results Extracts of T. chebula , T. belerica , E. officinalis and Triphala were found to scavenge the superoxides generated by photoreduction of riboflavin. The concentration of above plant extracts needed for 50% scaven-

ging of superoxides by these extracts were found to be 20.5, 40.5, 6.5 and 12.5 mg/ml, respectively. Degradation of deoxyribose mediated by hydroxyl radicals generated by the Fe3/ascorbate/EDTA/H2O2 system was also found to be inhibited and the concentration needed for 50% inhibition were 165.5, 71, 155 and 151 mg/ml, respectively. Generation of lipid peroxidase by Fe2/ ascorbate in rat liver homogenates were found to be inhibited and the concentration needed for 50% inhibition were 85.5, 27, 74 and 69 mg/ml, respectively (Table 1). Hydroxyl radicals, as well as lipid peroxidation were better inhibited by T. belerica as compared with other two extracts. However, superoxide generation was inhibited maximally by E. officinalis followed by T. chebula and T. belerica (Fig. 1). Administration of these plant extracts were found to reduce serum glucose level in normal rats both in single and multi dose study. In single dose study the maximum reduction in serum glucose level was noted at second hour by T. belerica extract was 52.74% followed by T. chebula 50.98%, E. officinalis 29.52% (Table 2). In multi dose study from 7th to 11th day, all the extracts significantly (P B/0.001) reduced the serum glucose level, but drugs comparison E. officinalis and Triphala showed homogenous effect with control group. Tri-

Fig. 1. Effect of Terminalia chebula, Terminalia belerica, Emblica ofcinalis and Triphala on superoxide, hydroxyl radical generation and lipid peroxidation in vitro.

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Table 2 Effect of plant extracts on serum glucose level in fasted normal rats (single dose) Groups Treatment (100 mg/kg) Serum glucose level (mg/100 ml) Fasting I II III IV V Control T. chebula T. belerica E. officinalis Triphala 75.792.8 81.495.6 80.596.1 81.397.3 77.696.4 1h 76.192.2A 47.094.6B* 49.197.3B* 60.197.1AB* 65.196.3A 2h 75.192.3A 39.993.3B** 38.196.5B** 57.396.2AB** 64.196.1A 4h 73.393.2A 43.293.2B* 41.395.2B* 62.295.2A 67.495.2A 6h 73.293.1A 63.194.5AB* 52.594.4B* 64.195.1A 71.294.3A

Values are mean9SD; n  6. * P B 0.05, ** P B 0.001 are significant as compared to control: capital letters indicate between group comparison.

Table 3 Effect of continued administration of plant extracts on serum glucose level in normal rats Groups Treatment (100 mg/kg) Serum glucose level (mg/100 ml) 3rd day I II III IV V Control T. chebula T. belerica E. officinalis Triphala 75.692.9 55.995.4 62.998.8 72.896.5 72.598.6 5th day 76.293.2 57.796.9 59.198.1 69.695.8 68.998.1 7th day 75.793.3A 55.495.7B* 55.796.4B* 68.895.3A 69.697.7A 9th day 75.092.9A 55.895.9C* 56.695.5C* 66.694.2ABC* 67.896.1AB* 11th day 76.392.1A 57.795.3BC* 54.194.8C* 62.594.6ABC* 66.295.5AB*

Values are mean9SD; n  6. * P B 0.05 are significant as compared to control: capital letters indicate between group comparison.

Table 4 Effect of plant extracts on serum glucose level in fasted diabetic rats (single dose) Groups Treatment (100 mg/kg) Serum glucose level (mg/100 ml) Fasting I II III IV V Control T. chebula T. belerica E. officinalis Triphala 342.5922.4 397.8939.9 381.8954.9 398.8922.9 390.5914.3 1h 347.3916.1 363.0934.5 367.3951.3 389.1921.2 373.7913.2 2h 359.3915.0 349.1931.0 343.9950.2 365.9921.5 350.8911.3 4h 366.5913.6 347.1945.1 331.9949.2 345.1920.5 332.4917.7 6h 375.796.9B 438.5971.1A** 334.7953.1B 354.9919.3B 339.6914.5B

Values are mean9SD; n  6. ** P B 0.001 are significant as compared to control: capital letters indicate between group comparison.

Table 5 Effect of continued administration of plant extracts on serum glucose level in fasted diabetic rats Groups Treatment (100 mg/kg) Serum glucose level (mg/100 ml) 3rd day I II III IV V Control T. chebula T. belerica E. officinalis Triphala 410.2910.4 403.0941.7A 302.5963.2C** 368.4921.4AB** 347.7911.2BC**
A

5th day 428.8913.5 337.2928.3B** 271.9953.5C** 349.4921.7B** 308.998.4BC**

A

7th day 459.6922.1 290.7929.9BC** 262.4955.4C** 312.5915.2B** 274.599.2BC**

A

9th day 381.3923.6 252.6926.4BC** 253.8951.9BC** 280.3913.3B** 233.899.3C**

A

11th day 321.3918.3A 239.3922.9B** 240.6950.0B** 228.3914.0B** 183.5910.4C**

Values are mean9SD; n  6. ** P B 0.001 are significant as compared to control: capital letters indicate between group comparison.

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phala did not reduce the serum glucose level below normal both in single and multi dose study (Table 3). In order to find out whether the extract interfered with glucose determination, a known quantity of the serum from extract treated animals were mixed with assay mixture. It was found that the serum from animals treated the extract did not inhibit the glucose determination indicating that reduction in the blood glucose was not due to an artifact of glucose estimation. The effect of plant extracts in alloxan induced diabetic rats is shown in Table 4 and Table 5. The fasting blood glucose levels in alloxan induced diabetic rats were 340 / 400 mg/100 ml. During the single dose study, the results were found to be similar to that of normal animals. Maximum effect was seen within 2 /4 h with T. belerica showing maximum effect followed by T. chebula and E. officinalis that had similar activity. There was tendency to increase serum glucose after 6 h. Between group comparison, all other groups are homogenous at sixth hour. There was a significant fall in glucose level in these rats 4 h after administration of T. chebula (12.7%), T. belerica (13.1%), E. officinalis (13.5%) and Triphala extract reduced the serum glucose level by 14.9% in single dose study. Similar results could be seen upon continuous administration of all the extracts. The serum glucose levels of alloxan alone treated group significantly increased from 75.7 to 459.6 mg/100 ml on day 7 after the alloxan injection. From 5th to 11th day, all the extract treated groups were heterogeneously significant (P B/0.001) when compared with alloxan treated control group. Between group comparison by ANOVA test on 11th day, Triphala showed highly significant action and between the groups all the three plants showed similar activity as they were homogeneously significant (Table 5).

ifested by these drug. T . belerica was the most active as an antioxidant followed by E. officinalis and T. chebula. Major ingredients in T. belerica is ellagic and gallic acid. E . officinalis has several gallic acid derivatives including epigallocatachin gallate. Major ingredient in the T. chebula is gallic acid, which is also found to be an antioxidant. It was found that potential of the extracts to acavenge oxygen radicals depend upon the types of radicals encountered. For example concentration needed for 50% inhibition of superoxide by E. officinalis was six times less than that of T. belerica , but the hydroxyl radicals were effectively scavenged by T. belerica than the other two extracts. Hydroxyl radicals are more potent than superoxides and as such T. belerica could effectively scavenge these radicals together with the inhibition of lipid peroxidation makes it as the best antioxidant of the three. Results also suggest that these plant materials together as Triphala could produce significant inhibition of alloxan induced diabetes in rats. T. belerica which was maximally active as an antioxidant was found to be most active to reduce serum glucose level followed by E. officinalis and T. chebula. Triphala which is a combination of all the three produced a significant action in reducing the alloxan induced diabetic and ANOVA test indicated that of the different groups, animals taking Triphala produced maximum significant reduction in the serum glucose level. Mechanism of action of these drugs in reducing the diabetic is not known. Infact, it was found that traditional medicines used in human diabetes also have a significant antioxidant activity. It may be possible that these extracts may reduce the effect of inflammatory cytokine release during diabetes which may be one of the causative agents for the tissue distraction and insulin resistance (Saghizadeh et al., 1996).

4. Discussion Diabetes mellitus of long duration is associated with several complications such as atherosclerosis, myocardial infarction, neuropathy, nephropathy etc. These complications have long been assumed to be related to chronically elevated glucose levels and subsequent oxidative stress. Mechanisms that contribute to increased oxidative stress in diabetes include non-enzymatic glycosylation, autooxidative glycosylation and metabolic stress. Oxidative stress in diabetes may partially be reduced by antioxidants and as seen antioxidants have been prescribed to reduce the longterm complications seen in diabetes. The present study indicated a strong antioxidant activity of T. chebula , T. belerica , E. officinalis and their combination Triphala, which may be partially responsible for many of the biological properties man-

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