V Rasal, B Shetty, A Sinnathambi, S Yeshmaina, P Ashok
antioxidant, brassica oleracea, hyperglycaemia, streptozotocin
V Rasal, B Shetty, A Sinnathambi, S Yeshmaina, P Ashok. Antihyperglycaemic And Antioxidant Activity Of Brassica Oleracea In Streptozotocin Diabetic Rats. The Internet Journal of Pharmacology. 2005 Volume 4 Number 2.
The present work is carried out to study the effect of
Brassica or cruciferous vegetables are the “Wonder Kids” of the vegetable world. This includes cabbage, cauliflower, broccoli and kholrabi. They are known as Knol-Khol in Indian vernacular languages. These vegetables are rich in the antioxidant vitamins C, E and carotene and are good sources of dietary fibre. They also contain sulphoraphanes and other isothiocyanates, which are believed to stimulate the production of protective enzymes in the body1,2.
Reactive oxygen species (ROS) are an important part of the defence mechanisms against infection, but excessive generation of free oxygen radicals may damage tissue3. The role of ROS in tissue damage in various human diseases such as cancer, ageing, neurodegenerative disease, diabetes and atherosclerosis has been recognized4.
Materials and Methods
Fresh stem of the plant was collected from the local market, Belgaum, Karnataka, India in the month of November and authenticated as
Preparation of extract
The fresh stem (10 Kg) was cut into small pieces and dried under shade for about a week. The dried material was ground to coarse powder. This powder was then subjected to extraction with petroleum ether solvent (40-60°C) by maceration. The solvent was then evaporated at room temperature and dried mass was collected and weighed (4.217 g).
Healthy Albino rats (150-180gms) of either sex obtained from central animal house, Jawaharlal Nehru Medical College, Belgaum were used for the study. Ethical clearance was obtained from Institutional Animal Ethics Committee, K.L.E.S's College of Pharmacy, Belgaum. Animals were housed individually under standard laboratory conditions and fed with commercial pellet rodent diet and water
Streptozotocin was obtained from Hi-media Ltd., Mumbai. Glutathione standard and Glutathione reductase from Sigma Chemical Company, St Louis, MO, USA. Thiobarbituric acid, Meta phosphoric acid and all other chemicals were of AR grade.
Diabetes was induced by 65 mg/kg of streptozotocin (STZ) administered i.p. in citrate buffer (pH 4.2) (Krishnakumar et al, 1999). After 7 days blood glucose levels were measured to confirm the induction of diabetes. Rats with glucose level above 200 mg/dL were selected as diabetic rats and were included in the experiment.
The dose of the petroleum ether extract was calculated taking into consideration that an average human being consumes 400 g of the stem per day. Thus the petroleum ether soluble fraction in this will be 0.168 g. This dose was then converted to an equivalent dose in rats using the dose conversion table7.
The animals were divided into 4 groups of 6 animals each – normal control, diabetic control, diabetic rats treated with 15.12 mg/Kg of petroleum ether extract of Knol-Khol and diabetic rats treated with Pioglitazone (10 mg/kg. p.o. daily) for 60 days.
On 60th day, blood glucose level and antioxidant enzymes levels were measured in blood samples collected by retro-orbital puncture under light ether anaesthesia.
Estimation of glucose
Blood glucose was measured by using commercial Glucomonitor kit (Pulsatum glucomonitor)8.
Estimation of peroxidation product and antioxidant enzymes
The level of peroxidation product viz. Malondialdehyde (MDA) was measured in blood9 where the reaction depends on the formation of a coloured complex between malondialdehyde (MDA) and thiobarbituric acid (TBA) having an absorption maximum at 532 nm.
Similarly the level of glutathione (GSH) content in blood was measured10 where 5-51-Dithiobis 2-nitro benzoic acid (DTNB) is reduced by glutathione, forming highly coloured yellow anion. The optical density of this yellow substance was measured at 412 nm.
After estimating MDA and GSH level in blood the remaining blood was used for the preparation of RBC haemolysate11. Then the haemoglobin content of this haemolysate was determined12. Haemolysate was further used to check the activities of antioxidant enzymes.
Superoxide dismutase (SOD) activity was measured in haemolysate13. Epinephrine can be autooxidised to adrenochrome by superoxide radicals. The ability of SOD to inhibit the autooxidation of epinephrine to adrenochrome has been used as the basis for the assay of this enzyme.
Catalase (CAT) and glutathione peroxidase (GSHPxase ) were measured in haemolysate10 where the rate of decomposition of hydrogen peroxide by catalase was measured spectrophotometricaly at 230 nm and in case of glutathione peroxidase, the rate of reduction of oxidized glutathione by glutathione reductase was measured.
At the end of the study, animals from each group were sacrificed, pancreas excised and sent for histopathological examination. The staining was done using H&E stain.
Results were expressed as mean ± SEM and evaluated for statistical significance by ANOVA followed by Dunnet's ‘t' test. Values of P< 0.05 were considered to be statistically significant.
The hyperglycaemic animals showed significant (F(3/20) = 1104, P<0.001) decrease in the blood glucose level on long term treatment for 60 days with Knol-Khol extract and Pioglitazone, when compared to diabetic control rats. (Table 1).
Lipid peroxide concentration
The concentration of malondialdehyde (MDA) was significantly ((F(3/20) = 3266, P<0.001) decreased in the Knol-Khol extract and Pioglitazone treated groups as compared to diabetic control. (Table 1).
The glutathione content was significantly (F(3/20) = 174.5, P<0.0001) increased in the Knol-Khol extract and Pioglitazone treated groups as compared to diabetic control (Table 1).
Antioxidant enzymes levels
The levels of superoxide dismutase, catalase and glutathione peroxidase was significantly (F(3/20) =118.3, P<0.0001, F(3/20) = 143.3, P<0.0001, F(3/20) = 95.03, P<0.0001 respectively) increased in Knol-Khol extract and Pioglitazone treated groups when compared to diabetic control (Table 1).
Section from the nondiabetic rats showed normal acini and islets while the section from diabetic control rats showed minute and reduced number of islets. Section from Knol-Khol extract treated diabetic rats showed good number of regenerating tiny islets, which could be comparable to that of nondiabetic rats (Fig 1,2,3,4).
Diabetes mellitus and its complications are mainly due to impaired pancreatic, antioxidants function and lack curative treatment. Oxidative stress resulting from enhanced free radical formation and/or defects antioxidants defense caused severe tissue damage and may lead to number of diseases like coronary artery disease, atherosclerosis, cancer and diabetes. Increased oxidative stress in streptozotocin diabetic rats has been reported3. This oxidative stress is also implicated in the development of diabetic complications14.
An elevated level of lipid peroxides in the plasma of streptozotocin diabetic rats and lipid peroxidation is one of the characteristic features of chronic diabetes15. The increased levels of thiobarbituric acid reactive substances (TBARS), conjugated dienes (CD), malondialdehyde and hydroperoxides are indices of lipid peroxidation16.
In the present study we found that the levels of lipid peroxidation product viz. MDA was decreased significantly in diabetic rats given Knol-Khol extract as compared to diabetic control where the levels were significantly high. This indicates that Knol-Khol extract may inhibit lipid peroxidation and thereby oxidative damage to tissues and organs in diabetes.
Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSHPxase) are the three major scavenging enzymes that remove the toxic free radicals
The activities of SOD, CAT and glutathione peroxidase are low in diabetes mellitus18. Erythrocytes were probably affected more due to the higher vulnerability thus causing inhibition of erythrocyte SOD, CAT and GSHPxase activity in diabetic rats. Antidiabetic treatment neutralized oxidative stress and increased erythrocyte SOD, CAT and GSHPxase activities19.
The activities of SOD, CAT and GSHPxase were significantly lowered in the erythrocytes of diabetic control rats. However the treatment with Knol-Khol extract significantly increased the SOD, CAT and GSHPxase activities in erythrocytes of diabetic rats. This indicates that treatment with Knol-Khol extract increases the antioxidant enzymes activities in STZ induced diabetic rats and thereby scavenges the toxic free radical which are responsible for tissue damage
Increased oxidative stress in chronic diabetic state has also been reported. Further, supplementation with Vitamin C and Vitamin E are reported to protect STZ diabetic rats against oxidative stress3. Treatment with Knol-Khol extract lowered oxidative stress in STZ diabetic rats by lowering glucose level in blood and by increasing the antioxidant enzyme activities. This activity of the extract in the present study is probably due to the presence of Vitamin E, carotene and other antioxidant constituents in it. Vitamin C may not play any role here as it is a water-soluble vitamin, which may not be isolated with petroleum ether. The reason for decrease in the blood glucose level in Knol-Khol extract treated group could be increased responsiveness of the tissues to insulin or increased release of insulin and possibly due to regeneration of islets of langerhans in the pancreas. In the present study, pioglitazone was used as reference drug to compare the activity of Knol-Khol. The result suggests that the antihyperglycaemic activity of Knol-Khol extract was almost comparable to that of the pioglitazone.
From these results, we conclude that petroleum ether extract of Knol-Khol regenerated the pancreas, lowered hyperglycemia and oxidative stress. Thus, it may be useful for the treatment of diabetes and associated complications. However, the present study strongly recommends clinical trials with this extract to establish its role in the treatment of diabetes and its complications, if not an alternative to insulin or oral antihyperglycaemic agent, at least as an adjuvant.
The authors wish to thank Prof. F. V. Manvi, Principal, K.L.E. Society's college of Pharmacy, Belgaum and Prof. B.G. Desai, Principal, K.L.E. Society's college of Pharmacy, Bangalore for the facilities provided for the study.