Streptozotocin Diabetic Mice
Richa Shri* and Disha Arora
Department of Pharmaceutical Sciences and Drug Research,
Punjabi University, Patiala, Punjab, India
ABSTRACT
The popular and commonly used fruit–amla (Emblica officinalis), is called a maharasaayana in the Ayurveda and is traditionally used for the management of diabetes. Diet plays a major role in the management of diabetes. Hence in the present study diet containing various doses of amla fruit powder (5, 10 and 15 per cent) was fed to normal and diabetic mice (Blood glucose levels ? 250mg/dl induced by STZ 200mg/kg IP) for 21 days. Results show that amla diet showed hypoglycemic effect that was most significant with diet containing 20 per cent amla i.e. there was a 6 per cent reduction in the blood glucose level as compared to the basal value. After induction of diabetes amla diets were continued in one set of animals and significant antihyperglycemic effect was observed. Feeding 5, 10 and 20 per cent diet produced a reduction in blood glucose by 20.7, 31 and 38.9 per cent respectively when compared with diabetic control on day 32 of the study. In another set of experimental animals after pretreatment with amla diets, there was induction of diabetes following which animals were fed normal diet. In this group negligible antihyperglycemic effect (4 per cent reduction of blood glucose was observed in group which had pretreatment with 20 per cent amla diet) was observed. Also serum TBARS levels among the groups treated with amla diet were lower with respect to diabetic control. This shows that diet containing Emblica officinalis has hypoglycemic and antihyperglycemic effect and reduces the oxidative stress in experimental diabetes.
Keywords:Emblica officinalis, Diet, Antioxidant, Diabetes, Streptozotocin.
Introduction
Diabetes mellitus is one of the most common endocrine disorders that affect more than 100 million people worldwide (6 per cent of the population) and in the next 10 years it may affect about five times more population (King et al., 1998; Grover et al., 2002). The effects of diabetes are devastating and well documented. Diet, exercise and medical intervention with drugs like insulin, thiazolidinediones etc are used for the management of diabetes mellitus (White and Campbell, 1996; Tripathi, 2003).
Oxidative stress is involved in the pathogenesis of diabetes and its complications. Use of antioxidants reduces oxidative stress and alleviates diabetic complications (Sabu and Kuttan, 2002). Antioxidants, vitamins C and E have been shown to reduce the oxidative stress in experimental diabetes (Madhu and Devi, 2000; Rahimi et al., 2005). Antioxidant-enriched dietary strategy using specific traditional plant food combinations can generate a whole food profile that has the potential to reduce hyperglycemia-induced pathogenesis and also associated complications (Ruhe and McDonald, 2001). Hence dietary supplementation with antioxidant nutrients may be a safe and simple complement to traditional therapies for preventing and treating diabetic complications (Tapsell et al., 2006; Kwon et al., 2007).
Emblica officinalis (family Euphorbiaceae) which is used traditionally to manage diabetes (Kirtikar and Basu, 1993; Ghosal et al., 1996), possesses potent antioxidant activity because of its rich vitamin C and polyphenol contents (Ghosal et al., 1996; Bhattacharya et al., 1999; Prakash et al., 2000; Khopde et al., 2001; Sabu and Kuttan, 2002; Jain and Khurdiya, 2004; Rao et al., 2005) but so far its role in diabetes has not been investigated. Hence the present study has been designed to investigate the effect of pretreatment with amla (Emblica officinalis) diet on blood glucose levels, body weight and TBARS (Thiobarbituric acid reactive substances) levels of normal and diabetic mice.
Materials and Methods
Plant Material
Fruits of Emblica officinalis were purchased from the local market (Patiala, Punjab, India) in the month of March and April, 2007 and were authenticated by Dr. H. B. Singh, National Institute of Science Communication and Information Resources, New Delhi, India (vide voucher number NISCAIR/RHM/F-3/2006/Conslt/662/143).
Chemicals
Thiobarbituric acid; streptozotocin; 1,1,3,3-tetraethoxypropane were obtained from Sigma Chemicals; solvents (petroleum ether, methanol, chloroform) were of analytical reagent grade and purchased from Loba Chem. All the other reagents were of analytical reagent grade.
Animals
Swiss albino mice weighing 20-30g of either sex were employed in the present study. Animals were housed in the institutional animal house under standard conditions, with 12 hour light and 12 hour dark cycle and they had free access to food (Kisan feeds Ltd., New Delhi, India) and tap water ad libitum.
Preparation of Treatment Diet
The authenticated fruits of Emblica officinalis were dried under shade at room temperature (<30 ºC). After complete drying, the fruits were grounded into a fine powder using a domestic electric grinder and mixed thoroughly with the already grounded standard chow diet. Binder in the form of 1 per cent starch solution was added to the mixture and kneaded into a dough, with a semisolid consistency. The dough was then spread on a tray and cut into pieces of suitable size. Different dose diets were prepared by mixing fruit powder with chow powder in the ratio of 5 per cent, 10 per cent and 20 per cent separately.
Sample Collection
Blood samples were collected retro-orbitally from inner canthus of eyes using Micro Hematocrit Capillaries from overnight fasted animals.
Induction of Experimental Diabetes
Administration of streptozotocin (200 mg/kg, i.p.) produced hyperglycemia. Three days after administration of the diabetogenic agent fasting blood glucose was determined and mice with blood glucose level more than 250mg/dl were included in the study.
Parameters Studied
- Fasting blood glucose: Blood glucose was estimated by commercially available glucose kit based on glucose oxidase method (Trinder, 1969).
- Body weight: Gravimetrically
- TBARS: Malondialdehyde concentration was measured by estimating thiobarbituric acid reactive substances (TBARS) (Liu et al., 1995).
Experimental Protocol
The experimental animals were divided into five groups:
Normal Control (Group I, n = 5)
Blood samples of the overnight fasted mice were collected retro-orbitally on day 0, just prior to the initiation of various treatment schedules, for the estimation of the three parameters.
Diabetic Control (Group II, n = 5)
Three weeks after the administration of streptozotocin (200 mg/kg, i.p.) i.e. 21st day, blood samples of the overnight fasted mice were collected retro-orbitally for the experimental parameters.
Treated Groups (Group III-V, n = 30)
Three different dose diets containing Emblica officinalis fruit powder (5 per cent, 10 per cent and 20 per cent) were fed to normal mice i.e., groups III–V (each group consisted of 10 animals) for ten days.
On the 10th day, the experimental parameters were determined. This was done to assess the hypoglycemic effect of Emblica officinalis.
On 11th day, the treated groups were rendered diabetic by single streptozotocin injection (200 mg/kg, i.p.). After induction of diabetes, groups III-V were subdivided as:
Group III divided into IIIa (n = 5) and IIIb (n = 5)
Group IV divided into IVa (n = 5) and IVb (n = 5)
Group V divided into Va (n = 5) and Vb (n = 5)
Groups IIIa, IVa and Va were continued on the amla diet (5 per cent,10 per cent and 20 per cent, respectively) for 21 days to assess the antihyperglycemic effect of amla diet.
While groups IIIb, IVb and Vb were fed normal chow diet for 21 days in order to observe prophylactic effect of pretreatment with amla diet.
After 3 weeks of the treatment, blood samples from the overnight fasted mice were collected retro-orbitally from both the subgroups for the estimation of the different parameters.
Statistical Analysis
The data for blood glucose, body weight and thiobarbituric acid reactive substances (TBARS) was expressed as mean±S.D. Data was analysed using one way ANOVA. Multiple range Tukey’s test was employed as post-hoc test for comparison between various groups and with control group. p? 0.05 was considered to be statistically significant.
Results and Discussion
Single dose of streptozotocin (STZ) is well reported to induce insulin dependent diabetes mellitus (IDDM) in mice (200 mg/kg, i.p.) (Anjaneyulu and Chopra, 2003) and in rats (65 mg/kg, i.p.) (Malcangio and Tomlinson, 1998; Utugol et al.,
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