Iranian Journal of Pharmacology and Therapeutics Razi Institute for Drug Research (RIDR) of Iran University of Medical Sciences and Health Services (IUMS) ISSN: 1735-2657 Vol. 4, Num. 1, 2005, pp. 36-39

Iranian Journal of Pharmacology & Therapeutics, Vol. 4, No. 1, 2005, pp. 36-39

Anti-Diabetic Activity of Terminalia catappa Linn. Leaf Extracts in Alloxan-Induced Diabetic Rats

SYED MANSOOR AHMED, VRUSHABENDRA SWAMY BM, P GOPKUMAR R DHANAPAL and VM CHANDRASHEKARA

Department of Pharmacology, M.M.U College of Pharmacy (S.M.A.); Department of Pharmacology (V.S.B.M.), Department of Pharmaceutics (R.D.), Rural college of Pharmacy; Department of Pharmaceutical Chemistry, S.A.C College of Pharmacy (P.G.); Department of Pharmacology, H.S.K College of Pharmacy, Bagalkot (V.M.C.), Karnataka, India.

Address correspondence to: Dr. Syed Mansoor Ahmed, De-partment of Pharmacology, M.M.U College of Pharmacy, Karnataka, India. E-mail: mansoor276783@yahoo.com

Received April 4, 2005;
Revised May 6, 2005;
Accepted June 6, 2005

Code Number: pt05009

ABSTRACT

In view of suggested anti diabetic potential, effect of aqueous and cold extracts of Terminalia catappa Linn (Combretaceae) leaves, on fasting blood sugar levels and serum biochemical analysis in alloxan-induced diabetic rats was investigated. All the extracts of Terminalia catappa produced a significant anti diabetic activity at dose levels of 1/5th of their lethal doses. Concurrent histological studies of the pan-creas of these animals showed regeneration by aqueous and cold extracts which were earlier necrosed by alloxan.

Keywords: Anti-diabetic activity, Terminalia catappa, Alloxan, Aqueous extract, Cold extract

Diabetes mellitus (DM) is a chronic disease caused by inherited and/or acquired deficiency in production of insulin by the pancreas, or by ineffectiveness of insulin produced, such a deficiency results in increased concen-tration of glucose in the blood, which in turn damages many of the body’s systems in particular the blood ves-sels and nerves. As the number of the people with dia-betes multiplies world wide, the disease has taken an ever-increasing share of national and international health care budgets. It is projected to become of the world’s main disablers and killers with in the next 25 years. Regions with greatest potential are Asia and Af-rica, where DM rates could rise to two-to-three- folds campened with the present rates. Apart from currently available therapeutic options, many herbal medicines have been recommended for the treatment of diabetes. Traditional plant medicines are used throughout the world for a range of diabetic presentation.

Terminalia catappa Linn (Combretaceae) is found in the warmer parts of India. It is also known as Indian Almond, Malabar Almond, and Tropical Almond [ 1 ]. The various extracts of leaves and bark of the plant have been reported to be anticancer, antioxidant [ 2 ], anti-HIV reverse transcriptase [ 3 ], hepatoprotective [ 4 ], anti in-flammatory [ 5 ], anti-hepatitis [ 6 ], and aphrodisiac [ 7 ]. The phytochemicals of this plant include tannins (puni-calagin, punicalin, terflavins A and B, tergallagin, ter-catain, chebulagic acid, geranin, granatin B, corilagin) [ 8 ] , flavanoids (isovitexin, vitexin, isoorientin, rutin) [ 9 ] and triterpinoids (ursolic acid, 2α, 3β, 23-trihydroxyurs-12-en-28 oic acid) [ 10 ].

Tannins have been reported to posses anti-diabetic activity [ 11 ].

In the light of the above information the present in-vestigation was undertaken to evaluate the anti-diabetic potential of Terminalia catappa Linn. leaves extracts on fasting blood sugar and serum biochemical analysis.

MATERIAL AND METHODS

Plant Material

Fresh tender leaves of Terminalia catappa were col-lected from Mysore botanical garden and were authenti-cated by botanist of M.M.U college of Pharmacy, Ra-managar. A voucher specimen (RTCL) is kept in our laboratory for future reference.

Preparation of Extracts

The leaves were shade dried at room temperature. The dried leaves were subjected to size reduction to coarse powder by using dry grinder and passed through sieve no 40.

Aqueous extraction. The powder of Terminalia catappa leaves were packed in a Soxhlet apparatus and extracted with distilled water for 18 hours. The obtained extract (7.5%) was dried at 45°C in hot air oven till solid/semisolid mass was obtained.

Cold extraction. Approximately about 100 g of the powdered leaves is taken in a 2000 ml conical flask with 500 ml of distilled water and 10 ml of chloroform added as a preservative (maceration process). It was extracted up to one week with daily 2 hours stirring with a me-chanical stirrer. After 7 days the extract was filtered through muslin cloth and marc was discarded and its filtrate (8.2%) dried in a hot air oven at 45°C till solid/semisolid mass, was produced.

Both the extracts were stored in air tight container in refrigerator below 10°C. The suspensions of aqueous and cold extracts were prepared by using normal saline as solvent for administration to experimental animals.

Animals Used

Wistar albino rats (150-200 g) and Wistar albino mice (20-25 g) of both sexes were procured from Indian Institute of Sciences, Bangalore, India. Before and dur-ing the experiment rats were fed with standard diet (Gold Mohr, Lipton India Ltd). After randomization to various groups and before initiation of experiment, the rats were acclimatized for a period of 7 days under stan-dard environmental conditions of temperature, relative humidity, and dark/light cycle. Animals described as fasting were deprived of food and water for 16 hours ad libitum.

Sample Collection

Blood samples were collected by the retro-orbital plexus puncture method and blood glucose levels were estimated using an electronic glucometer (Miles Inc., USA) and glucostix (Bayer diagnostic India Ltd., Baroda).

Experimental Design

All the animals were randomly divided in the five groups with six in each group. Group I, II and III were administered saline, diabetic, and standard drug (gliben-clamide, 10 mg/kg per day p.o) control, respectively. Preliminary oral LD₅₀ doses of aqueous and cold extract of Terminalia catappa in mice were found to be 215, 230 mg/kg respectively. Group IV and V were treated with leaves extracts in one-fifth of LD₅₀ doses of aque-ous extract (43g/kg per day p.o) and cold extract (46 mg/kg per day p.o), respectively.

Assessment of Extracts on Alloxan-Induced Diabetic Animals

Rats were made diabetic by a single intraperitoneal injection of alloxan monohydrate (Loba Chemie, Bom-bay: 150 mg/kg) [ 12 ]. Alloxan was first weighed indi-vidually for each animals according to the weight and solubilized with 0.2ml saline (154 mM NaCl) just prior to injection. Two days after alloxan injection, rats with plasma glucose levels of >140 mg/dl were included in the study. Treatment with plant extracts was started 48 hours after alloxan injection. Blood sample were drawn at weekly intervals till end of study (i.e. 3 weeks). Fast-ing blood glucose estimation and body weight meas-urement were done on day of 1, 7 and 21 of the study.

On day 21, blood was collected by cardiac puncture under mild ether anesthesia from overnight fasted rats and fasting blood sugar was estimated [ 13 ]. Serum was separated and analyzed for serum cholesterol [ 14 ], se-rum triglycerides by enzymatic DHBS colorimetric method [ 15 ], serum HDL [ 16 ], serum LDL [ 17 ], serum creatinine [ 18 ], serum urea [ 19 ] and serum alkaline phosphatase by hydrolyzed phenol amino antipyrine method [ 20 ].

The whole pancreas from each animal was removed after killing the animals, was placed in 10% formaline solution, and immediately processed by the paraffin technique. Sections of 5µm thickness were cut and stained by haematoxylin and eosin (H&E) for histologi-cal examination. The photomicrographs of histological studies are presented in Fig 2 (A-E).

Statistical Analysis

All the values of body weight, fasting blood sugar, and biochemical estimations were expressed as mean ± standard error of mean (SEM) and analyzed using Stu-dent ‘t’ test.

RESULTS

The anti diabetic effects of the extracts on the fasting blood sugar levels of diabetic one shown in Fig 1. Ad-ministration of alloxan (150 mg/kg, i.p) led to 1.5-fold elevation of fasting blood glucose levels, which was maintained for period of 3 weeks. Three weeks of daily treatment of extracts led to a dose-dependent fall in blood sugar levels by 25-62%. Effect seems to reach maximum after 15 days of treatment and remained con-stant in third week.

Vehicle control animals were found to be stable in their body weight while diabetic rats showed significant reduction in body weight during 21 days (Table 1). Al-loxan caused weight reduction, which was reversed by aqueous and cold extracts of Terminalia catappa after 7 days of treatment.

Serum cholesterol, serum triglycerides, serum LDL, serum creatinine, serum urea, and serum alkaline phos-phatase levels were decreased significantly by gliben-calmide (p < 0.001), aqueous extract (p < 0.001) and cold extract (p < 0.01) of Terminalia catappa, after 21 days of treatment compared with diabetic control. HDL levels were increased by glibenclamide (p < 0.001), aqueous extract (p < 0.001) and cold extract (p < 0.01) compared with diabetic control (Table 2).

Photomicrographs (Fig 2), showed normal acini, and normal cellular population in the islets of langerhans in pancreas of vehicle-treated rats (A). Extensive damage to the islets of langerhans and reduced dimensions of islets (B), restoration of normal cellular population size of islets with hyperplasia by glibenclamide (C) was also shown. The partial restoration of normal cellular population and enlarged size of β-cells with hyperplasia was shown by aqueous and cold extracts (Fig 2D-E).

DISCUSSION AND CONCLUSION

In the light of the results, our study indicates that Terminalia catappa leaves extracts have anti-diabetic activity. Aqueous and cold extracts of Terminalia catappa exhibited significant anti- hyperglycemic activities in alloxan-induced hyperglycemic rats without significant change in body weight. They also improved conditions of DM as indicated by parameters like bodyweight, and lipid profiles along with serum creatinine, serum urea, and serum alkaline phosphatase. The number of functionally intact β-cells in the islet organ is of decisive importance the development course and outcome of DM. The renewal of β-cells in diabetes has been studied in several animal models. The total β-cell mass reflects the balance between the renewal and loss of these cells. It was also suggested that regenera-tion of islet β-cells following destruction by alloxan may be the primary cause of the recovery of alloxan –injected guinea pigs from the effects of the drug [ 21]. In alloxan-induced diabetes, (-)-epicatechin [ 22] and Vinca rosea extract [ 23] have also shown to act by β-cell re-generation. Similar effect in streptozotacin treated diabetic animals were reported by pancreas tonic [ 24], ephedrine [ 25], and Gymnema Sylvestre leaf extracts [ 26].

In our studies, damage to pancreas in alloxan-treated diabetic control (Fig 2B), and regeneration of β-cells by glibenclamide (Fig 2C) was observed. A comparable regeneration was also shown by aqueous and cold ex-tracts of Terminalia catappa (Fig 2D and Fig 2E). This effect may be due to β-carotene, which was reported to be constituents of Terminalia catappa [ 27]. The benefi-cial role of β-carotene in reducing diabetic complica-tions like glycosylation in alloxan-induced diabetic rats [ 12] had been reported previously. Photomicrographical data in our studies confirne healing of pancreas by Ter-minalia catappa leaves extracts, as a plausible mecha-nism of their anti diabetic activity.

Aqueous and cold extract of Terminalia catappa leaves exhibited significant anti hyperglycemic activi-ties in alloxan-induced diabetic rats. These extracts showed improvement in parameters like body weight and lipid profile as well as regeneration of β-cells of pancreas and so might be of value in diabetes treatment.

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