EFFECT of AQUEOUS EXTRACT of BITTER LEAF (Vernonia Amygdalina Del) on CARBON TETRA-CHLORIDE

HYPOGLYCEMIC EFFECT OF AQUEOUS EXTRACT OF MISTLETOE (Tapinanthus Bengwensis) ON STREPTOZOTOCIN-INDUCED DIABETIC RATS

GEORGE, G.S; IBEH, G.O. and *UWAKWE, A.A.

department of Biochemistry,

University of Port Harcourt, Nigeria.

ABSTRACT

The hypoglycemic effect of mistletoe (Tapinanthus bengwensis) extract on streptozotocin (STZ)-induced diabetic rats was studied. Male Wistar Albino rats weighing between 200-350g were used for the study. The study groups comprised of eight (8) rats in each group. Group 1 was (Normal rats), Group 2 (Diabetic rats), Group 3 and 4 (Diabetic test rats), Group 5 Rats on drug, Daonil, (0.02mg) and Group 6 (Normal rats on 5% of extact). After induction with STZ, the course of hyperglycaemia was monitored by the estimation of fasting blood glucose (FBG).

After fifteen (15) days of administration of T. bengwensis extract, comparison of the changes in glucose concentration of the Normal Rats against Diabetic test rats treated with extract were evaluated using one way of variance (ANOVA).

When compared the glucose level of the Normal and Diabetic test rats varied significantly. (P 0.05). Whereas the mean glucose level for the Group 1 was 4.4 + 0.13 mmol/l, those for groups 2-6 were 17.7 + 0.97, 12.2 + 0.71; 12.8 + 0.69, 11.5 + 0.81 and 4.2 + 0.27 mmol/l respectively. On administration of 10% and 5% concentration of extract to the Diabetic test rats (Groups 3 and 4), statistically significant decrease in the glucose concentration was observed (P 0.05).

The glucose level of Diabetic test rats given 10% of extract dropped from 15.8 + 0.18 to 5.7 + 0.22 mmol/l while those on 5% dropped from 16.0 + 0.17 to 7.1 + 0.23. Normal rats on 5% extract showed glucose level of 5.7 + 0.25 falling to 2.3 + 0.12 mmol/l. There was moderate to marked reduction in the weight of the rats, a significant elevation of the WBC which decreased at the end of the study period. Histological examination of the liver, kidney and pancreas showed some effect on the kidney and pancreas. This result demonstrated a potent factor in the extract that is responsible for the reduction of the glucose concentration and it showed that induced diabetes is reversible with mistletoe treatment. The implications of these findings are discussed in this work.

Key words: Streptozotocin; hyperglycaemia; hypoglycaemia, T. bengwensis,

Diabetic- rats, fasting blood glucose.

* Corresponding author.

Introduction

The use of indigenous plants in the management of diseases has been a common practice over the years. Few of the plants have received adequate scientific scrutiny. The study and understanding of ethno-botanical information, chemical constituents of plants and the therapeutic application of the plant drug will help in understanding efficiency of the plant as a potent remedy, extend our knowledge of their pharmacological activity, active principles, dosage and administration (1, 2).

This research effort is on Mistletoe (Tapinanthus bengwensis). The study investigated the hypoglycemic effect of aqueous extract of Tapinanthus bengwensis on experimental diabetic rats induced with streptozotocin (STZ).

Materials And Methods:

Animals: Wistar Albino rats aged (12-15 weeks), weighing between 200-350g derived from a colony maintained at the animal house of the department of Biochemistry, University of Port Harcourt were put in separate cages within a temperature of (25 + 2oC), divided into Normal group, normal treated with 5% of extract, Diabetic control group, Diabetic test rats treated with 5% and 10% extract of T. bengwensis per 200g body weight of rat and group 5 treated with Daonil (0.02mg/200g body weight). The animals were fed with standard laboratory chow (Pfizer Feeds Plc, Nigeria) and water ad libitum.

Preparation of Plant Extract:

The specimen of Tapinanthus bengwensis was collected at Delta Park, University of Port Harcourt, Rivers State, Nigeria. They were dried under shade in the house. 50g of the dried leaves was weighed and homogenized (grounded) into fine powder. Aqueous extract of the pulverized plant was prepared by weighing out 50g of the pulverized leaves and boiled in 1 litre of distilled water for 15 minutes after which the suspension was filtered. The filtrate was evaporated to dryness. A 5% and 10% portion of the resulting material were weighed out into aliquots and were stored at 4oC. It was reconstituted and used as the stock crude drug.

Experimental Procedure:

Induction of diabetes was achieved by the intraperitoneal injection of 70mg/kg body weight of streptozotocin for 2 days. The animals were considered diabetic when the blood glucose level exceeded 10mmol/l (range 10mml/l – 18mmol/l) 2 weeks after STZ treatment. The administration of the drugs to the rats was by intubations. Blood samples were collected from the tail vein into fluoride oxalate containers. It was then spun and the resulting serum used for assay for the fasting blood glucose (FBG) level.

Results

Fasting blood glucose (FBG) values obtained during the 15 days of study for each group is shown in Table 1. Values in group one did not show significant changes within the study. The mean was 4.4 + 0.13mmol/lGroup 2 rats were induced with streptozotocin but were not given extract. Hence the glucose level rose consistently and was sustained until day 15 when the value was within the hyperglycemic range 23.2mmol/l. Group 3, 4, 6 were subjected to extract and showed significant decrease in the blood glucose consistent with the concentration. (Table 2) Group 6 rats were normal but subjected to 5% concentration of the aqueous extract. The level of glucose fell to 2.3mmol which is compatible with hypoglycemia.

Discussion:

Induction of diabetes was successfully achieved following the administration of streptozotocin (STZ) to the rats. In the study, 70mg/kg body weight of STZ was used. Diabetes mellitus was confirmed by evaluation of the fasting blood glucose level of the rats. Only rats with fasting blood glucose more than 10.0mmol were considered diabetic and used for further experiment with the aqueous extract of mistletoe (Tapinanthus bengwensis). This level

of glucose compared with the threshold as previously reported by Alwadi et

al (3).

There was considerable weight loss in the rats, polydipsia and polyuria were evident. The Normal rats maintained a fairly stable level of glucose throughout the study period with mean level of 4.4 + 0.13 mmol/l. There was sustained rise in the level of glucose concentration for rats that were induced with streptozotocin which were not treated with mistletoe (T. bengwensis). Range was between 10.3 + 0.20 mmol/l to 23.2 + 0.10 mmol/l on the last day of the study (day 15).

The effect of T. bengwensis was significant following administration of 5% and 10% aqueous extract. Control rats given water instead of extract did not produce any significant change at P > 0.05 in the plasma fasting glucose level. There was a fall in glucose value from 11.4 + 0.17mmol/l to 5.7 + 0.22 mmol/l for diabetic test rats treated with 10% extract and a fall in glucose value from 8.7 + 0.17 mmol/l to 7.1 + 0.23 for diabetic test rats treated with 5% extract. The reduction in glucose level compared moderately with the reference drug Daonil used. The drop in glucose level gives credence and suggest a possible B-cell recovery as reported by Gray and Flatt (4).

The efficacy of T. bengwensis in diabetes has not been previously tested. However it has been reported (5,6) of the presence of active chemical constituents. The works suggest that the antidiabetic properties of mistletoe is highly dependent on the host plant with that on citrus showing best result. Test tube and animal studies suggest that mistletoe extract can stimulate insulin secretion from pancreas cells and may improve blood sugar level in people with diabetes.

The presence of tannin in mistletoe (T. bengwensis) must have played a role in the observed lowering effect of the extract on the glucose level. It is known that tannins lower the rate of starch digestion which invariably leads to reduction in blood glucose level by the same mechanism which makes them antinutrients. The inactivation of pancreatic amylase by tannin has been reported (8).

Tannins may also bind with calcium and calcium is needed to stabilize amylase activity or with starch to influence the degree of gelatinization, amylase accessibility to the digestive system is hampered.

We observed a major systemic effect of the STZ on the pancreas and the kidney (8, 9, 10) which helped in maintaining the diabetic state (pictures not shown). This was not observed for the liver. The evidence from the study suggests strongly that mistletoe (Tapinanthus bengwensis)
aqueous extract may have blood glucose lowering effect. Our findings suggest that administration of graded concentration of T. bengwensis is an antidote to diabetes and is likely to help patients from diabetic complications.

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TABLE 1: FASTING BLOOD SUGAR (MMOL) OF NORMAL AND DIABETIC RATS.

VALUES ARE MEAN + SEM

TABLE 2: FBG VALUES OF NORMAL/DIABETIC TEST RATS (DTR) TREATED WITH MISTLETOE (Tapinanthus Bengwensis EXTRACT (MML/L)

VALUES ARE MEAN + SEM

VALUES ON THE SAME ROW HAVING THE SAME SUPERSCRIPTS ARE NOT SIGNIFICANTLY DIFFERENT FROM EACH OTHER.

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4.  Gray, A.M. and Flatt P.R. (1998): Insulin-secreting activity of traditional and antidiabetic plant viscum album mistletoe. Journal of Endocrinology. 160:409-141.

5.  George, D. and Pamplona-Roger M.D. (1999): Encyclopedia of Medical Plants pg. 246-7, 227.

6.  Allesi D; Berger, A., Cepko C., Colombet, I; Costigan, M. and Gulunezoglu, A.M. (2003): Mistletoe Complementary Alternative Medicine Review 1-4.

7.  Bell RH; and Jr., Hye R.J. (1983): Physiology and Pathology. J. Surg Res, 35:433-460.

8.  Philips, A. (1999): Progression of diabetic nephropathy, Insight from cell studies and animal models. Kidney Blood Press Res; 22:81-97.

9.  O’Brien, B.A., Harmon B.V., Carmeron D.P., and Allan D.J. (1996): Beta-cell apoptosis is responsible for the development of IDDM in the multi low-dose streptozotocin model. J. Pathol 178:176-181.

10.  Lorenzi M, Montisano D, and Toledo S. (1985): High glucose Induces DNA damage in culture Human endothelial cells. J. Clinic Invest; 72:322-5.

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