Anti-diabetic and antihyperlipidemic effect of allopolyherbal formulation in OGTT and STZ-induced diabetic rat model

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1 Indian Journal of Experimental Biology Vol. 51, September 2013, pp Anti-diabetic and antihyperlipidemic effect of allopolyherbal formulation in OGTT and STZ-induced diabetic rat model Swati Manik, Vinod Gauttam & A N Kalia* Department of Pharmacognosy, ISF College of Pharmacy, Moga, , India Received 11 October 2012; revised 3 June 2013 The present study was undertaken to evaluate the antidiabetic and antihyperlipidemic activities of Allopolyherbal formulation (APHF) consisting of combinations of three well known medicinal plants used in traditional medicines (Trigonella foenum graceum, Momordica charantia, Aegle marmelos) and synthetic oral hypoglycaemic drug (Glipizide-GL). The optimized combination of lyophilized hydro-alcoholic extracts of drugs was 2:2:1 using OGTT model. The optimized PHF was simultaneously administered with GL and optimized using OGTT model in diabetic rats and further studied in STZ-induced diabetic rats for 21 days. The results (serum glucose level, lipid profile, hepatic enzymes and body weight) were compared with the standard drug GL (10 mg/kg body wt). The optimized APHF (500+5 mg/kg body wt) has shown significant antihyperglycemic and antihyperlipidemic activities. The results were comparable with the standard; even better than the GL (10 mg/kg body wt) alone. The proposed hypothesis has reduced the no. of drug components from eight to three and dose almost 50 % of both PHF and GL which fulfil the FDA requirements for export. Thus the developed APHF will be an ideal alternative for the existing hypoglycemic formulations in the market with an additional advantage of hypolipidemic effect and minimizing the cardiovascular risk factors associated with diabetes. Keywords: Allopolyherbal formulation, Diabetes mellitus, OGTT model, Polyherbal formulation, Streptozotocin Diabetes mellitus (DM) is one of the most severe, incurable metabolic disorders characterized by hyperglycaemia as a result of a relative, or an absolute, lack of insulin, or the action of insulin on its target tissue or both 1. Besides hyperglycaemia, several other symptoms, including hyperlipidemia, are involved in the development of microvascular complication of diabetes, which are the major causes of morbidity and death 2. It is a global public health problem, now emerging as a world over epidemic. The number of people with diabetes in India is currently around 40.9 million which is expected to rise to 69.9 million by 2025 unless urgent preventive steps not taken 3. Recently there has been a shift in universal trend from synthetic to herbal medicine which can Return to Nature as modern oral hypoglycaemic agents produce undesirable side effects and herbal drugs/formulations being preferred because of their effectiveness, minimal side effects and relatively low cost 4. Management of diabetes without dyslipidemia and various other associated side effects is still a challenge to the medical community. Combination of allopathic *Correspondent author Telephone: (O); (M) ankalia_47@rediffmail.com and herbal drugs can assist to overcome the resistance due to insulin and/or oral hypoglycaemic therapy in case of uncontrolled diabetes by reducing the dose of allopathic drugs and their associated side effects. There are so many oral hypoglycaemic drugs and polyherbal formulations (PHFs) available in the market but still diabetes is an uncontrolled disease, so there is a need for alternative therapy for the management of diabetes. An attempt has been made to counteract the risk factors associated with chronic oral hypoglycaemic drug by the new concept of allopolyherbal formulation (APHF). The literature survey also reveals that more than 50% of polyherbal antidiabetic formulations available in the market contain Trigonella foenum-graceum (Fenugreek, Methi in Hindi), Momordica charantia (Bitter gourd, Karela in Hindi) and Aegle marmelos (Wood apple, Bael in Hindi) as one of their major components. Moreover, these plants have been proved scientifically for their antihyperglycemic and antihyperlipidemic, spasmolytic, and digestive activities. They have different mechanisms of action, therefore selected for the proposed study 5-7. Glipizide (GL) is one of the newest oral hypoglycemic, effective and safe compound with unique properties 8.

2 MANIK et al: ANTIDIABETIC AND ANTIHYPERLIPIDEMIC EFFECT OF ALLOPOLYHERBAL FORMULATION 703 Lyophilized extracts of Methi, Karela and Bael were combined in different ratios to find out the best antihyperglycemic combination and then the optimized formulation based on preliminary screening using Oral glucose tolerance test (OGTT) model, was combined with GL as APHF in different doses and were used to investigate their effect on blood glucose, body weight, lipid profile, serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvate transaminase (SGPT) in streptozotocin (STZ) induced diabetic rat model. Materials and Methods Chemicals STZ was purchased from Sigma Aldrich Co. (St Louis, MO, USA), GL was from USV Limited (Mumbai, India) and the kits for glucose determination; total cholesterol, triglycerides, high density lipoprotein cholesterol (HDL-c), SGOT and SGPT were purchased from Coral Company (Goa, India). All other chemicals and reagents used were of laboratory grade and were purchased from LDH, Ranbaxy and MERCK etc. Plant material The plant materials i.e. Methi seeds, Karela fruits and Bael leaves were procured locally, in the month of August and were authenticated by Dr. Adarsh Pal, Head, Department of Botanical and Enviornmental Sciences, Guru Nanak Dev University, Amritsar (Punjab) and voucher specimens were deposited in the department for future reference. Extraction and lyophilization All the drugs were dried, coarsely powdered and stored in a closed container. Dried powder of Methi was defatted with petroleum ether and then extracted by triple maceration with 70% ethanol whereas dried powder of fruit and leaves of Karela and Bael were extracted with 50 and 70% ethanol respectively using triple maceration. These extracts were concentrated under vacuum, deep freezed for overnight and were lyophilized. Development and optimization of polyherbal (PHF) and allopolyherbal (APHF) formulation The PHF were developed by combining the lyophilized extracts of selected plant materials in different experimental ratios keeping Methi at constant ratio (Table 1) and were optimized on the basis of OGTT study in normal rats. The APHF were developed by mixing the optimized PHF and GL in different experimental ratios and optimized using OGTT studies in diabetic rats. The optimized APHF was further evaluated by OGTT in STZ-induced diabetic rat model. Animals Wistar rats (either sex) weighing g were procured from the animal house of I.S.F. College of Pharmacy, Moga (Reg. No. 816/PO/a/04/CPCSEA). The animals were kept in polypropylene cages (3 in each cage) at an ambient temperature of 25±2 C and 55-65% RH. A h light and dark schedule was maintained in the animal house. The rats had free access to water and were fed with commercially available feed. The protocol of the experiment (IAEC/CPCSEA/2011/17) was approved by Institutional Animal Ethics Committee and were conducted in accordance with guidelines as per Guide for the care and use of laboratory animal and with permission from Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Pharmacological screening Optimization of PHF Antihyperglycemic study in oral glucose tolerance (OGTT) model in normal rats 9 Overnight fasted normal rats were divided into 6 groups of 6 animals in each. Gr. I Normal control (0.5% CMC solution); Gr. II to IV were of different combination (PHF-A, PHF-B, PHF-C)-1000 mg/kg b. wt; Gr. V PHF-B (500 mg/kg body wt) and Gr. VI standard GL (10 mg/kg body wt). The single dose of each prepared combination was administered orally in the rats. All animals received glucose (2 g/kg) orally after an hour of drug treatment. The blood samples were withdrawn by retro-orbital plexus under mild anaesthesia before administration of the test drug-basal and at 0, 60 and 120 min after glucose administration, the serum glucose was estimated by glucose oxidase-peroxidase method 10. Induction of diabetes Diabetes was induced by a single intra-peritoneal injection of freshly prepared STZ (50 mg/kg body wt) in 0.1M citrate buffer (ph 4.5) to overnight fasted rats. STZ injected rats were allowed to drink 20% glucose solution for 24 h to prevent STZ-induced hypoglycaemic mortality 11. The development of diabetes was confirmed after 1 week of STZ injection, the animals with fasting blood Table 1 Preparation of the polyherbal combination Name of the formulation Polyherbal formulation A (PHF-A)-1000 mg/kg body wt. Polyherbal formulation B (PHF-B)-1000 mg/kg body wt. Polyherbal formulation C (PHF-C)-1000 mg/kg body wt. Ratio of the plant material Methi Karela Bael

3 704 INDIAN J EXP BIOL, SEPTEMBER 2013 glucose level more than 200 mg/dl were selected for the present study 12. Optimization of the APHF Antihyperglycemic study in oral glucose tolerance test (OGTT) model in diabetic rats 9 Overnight fasted diabetic rats were divided into 4 groups of 6 animals in each. Grouping of the animals was done as mentioned below: Gr. I Diabetic control (0.5% CMC solution); Gr. II APHF A (500+5 mg/kg body wt); Gr. III APHF B (750+5 mg/kg body wt) and Gr. IV GL (10 mg/kg body wt). Antihyperglycemic studies of optimized APHF in STZ induced diabetic rat model On the basis of OGTT studies in normal and diabetic rats dose was selected for the STZ-induced diabetic rat model. Experimental design All diabetic rats were randomly divided into following 5 groups of 6 each: Gr. I : Normal control (0.5% w/v CMC sol.) Gr. II : Diabetic control (0.5% w/v CMC sol.) Gr. III : APHF A (500 mg/kg b. wt PHF-B+ 5 mg/kg Gr. IV body wt GL) : APHF B (750 mg/kg b. wt PHF-B+ 5 mg/kg body wt GL) Group V : GL 10 mg/kg body wt All the test drugs were suspended in a vehicle containing 0.5% (w/v) CMC in distilled water and administered orally using an intra-gastric canula once daily for 20 days. The body weight of the animals was measured at the onset of the study and at the regular intervals of every week up to 21 days 13. The blood samples were collected on 0, 7, 14, 21 days of the study by puncturing the retro-orbital plexus under mild anaesthesia. Serum was separated by centrifugation at 3000 rpm for 15 min and used for the estimation of glucose (GOD/POD method) 10, total cholesterol (CHOD/PAP method), total triglycerides (TG) by GPO/PAP method, LDL-c, VLDL-c (Freidewald s formula), and HDL-c levels (PEG Precipitation method) Hepatic enzymes SGOT and SGPT were determined using UV spectrometer 18. Postprandial studies The dose of APHF showing the best antidiabetic activity in STZ-induced diabetic rat model was selected for this study. Diabetic rats fasted overnight were divided into four groups of six rats each. Group I and III served as control (0.5% w/v CMC sol.) and group II and IV received APHF B (750 mg + 5 mg/ kg body wt). In group I and III dose was given after meal and to group II and IV it was given before meal. SGL was taken initially and then food was given to the rats for 1 h and then dose was given after meal and before meal to the respective groups and SGL was checked at 2, 3 and 5 hrs after food administration. Statistical analysis Results are presented as mean±sd. The statistical analysis involving two groups was evaluated by means of two-way ANOVA followed by Bonferroni post-test whereas one-way ANOVA followed by Tukey post-test was used for column analysis and P values (P<0.001 and P<0.05) were considered to be significant. Results and Discussion Optimization of the formulation using OGTT normal rat model The results showed and 17.66% rise in serum glucose level (SGL) after one hour of glucose administration in vehicle control and GL (10 mg/kg b. wt) pretreated groups, respectively. Whereas, group of animals pretreated with the developed formulation (PHF-A, PHF-B and PHF-C at the dose of 1000 mg/kg b. wt, have shown rise in SGL by 28.26, and 24.82%, respectively in comparison to 0 h (Table 2), Table 2 Effect of PHFs on serum glucose levels in OGTT model in normal rats [Values are mean±sd from 6 rate in each group. Figure in parentheses are % increase over O min values] Serum glucose levels (mg/dl Basal 0 min 60 min 120 min Normal control 91.33± ± ±3.49 (44.64) 99.25±3.16 PHF A (1000 mg/kg body wt) 92.15± ± ±4.08 (28.26) a 96.66±3.85 PHF B (1000 mg/kg body wt) 91.33± ± ±3.01 (18.44) a 93.75±3.75 PHF C (1000 mg/kg body wt) 90.13± ± ±4.78 (24.82) a 94.00±2.16 PHF B (500 mg/kg body wt) 89.45± ± ±4.15 (25.13) a 94.37±3.75 GL (10 mg/kg body wt) 93.15± ± ±3.69 (17.66) a 95.00±2.63 a P< vs normal vehicle control group. GL = Glipizide.

4 MANIK et al: ANTIDIABETIC AND ANTIHYPERLIPIDEMIC EFFECT OF ALLOPOLYHERBAL FORMULATION 705 on the basis of results PHF-B was administered at the dose of 500 mg/kg b. wt and have shown an increase of 25.49% rise in SGL just at par with the 1000 mg/kg b. wt dose of PHF-A & PHF-C, hence selected for further study in STZ induced diabetic rat model. However, all groups of animals almost normalize the SGLs within two hours indicating that the pancreas of animals was healthy to clear out the glucose load from the body. The result of OGTT study had shown that PHF-B consisting of Methi, Karela and Bael (2:2:1) have maximum antihyperglycemic activity even at par with the standard drug. This is because of Methi and Karela in the higher ratio, possessing insulin modulation action and extra-pancreatic effects such as the regulation of glucose uptake from the intestinal lumen by the inhibition of carbohydrate digestion by Methi 19 ; acting like insulin or promoting insulin release by Karela 20 as well as the synergistic effect of the Bael for its extra-pancreatic effect 7. Therefore, PHF-B was selected for further study in combination with GL in diabetic rats. Optimization of the APHF using OGTT diabetic rat model Vehicle treated group and GL (10 mg/kg body wt) showed and 33.6% increase, respectively in SGL at 1 h after glucose administration whereas, APHF A (500 mg+5 mg/kg body wt) and APHF B ( mg/kg body wt) showed and 27.52% rise, respectively in SGLs at 1 h compared to 0 h (Table 3). The results showed that APHF combinations (A, B) have significantly improved glucose tolerance in glucose induced hyperglycaemia in comparison to PHF-B (500 and 1000 mg/kg body wt). This is because of the presence of GL, as it has rapid absorption and onset of action, it further have potentiated the insulin action. Its insulin action is associated with an increase in plasma membrane insulin receptor number, involves some post receptor events, and is significantly greater on peripheral uptake of glucose than suppression of hepatic glucose production. 21 However, when compared with standard drug its activity was at par with GL (10 mg/kg body weightt) Hence, our concept of the development of the APHF has been achieved and it was selected for further chronic study in STZ induced diabetic rats. Effect of APHF on serum glucose level and other parameters of diabetic rats Diabetic control rats showed consistent and gradual rise in the fasting SGL during the study. GL, (10 mg/kg body wt), APHF-A (500+5 mg/kg body wt) and APHF-B (750+5 mg/kg body wt) treated rats showed a reduction in SGL by 20.07, and %; 23.35, and %; 27.56, and % on 7 th, 14 th and 21 st day of the study, respectively as compared to onset of the study and the results were found to be statistically significant (P<0.001) as compared to diabetic control group (Table 4). The effect was found to be time dependent up to 21 day of the study. Decrease in SGL was more significant (P<0.001) on 21 st day in comparison to 14 th and 7 th days. The effect of developed APHF was significant (P<0.001) when compared with standard drug. This effect of APHF on SGL was due to the synergistic effect of PHF and of GL. The PHF might be due to the regeneration of β cell in presence of Methi 22, Bael 23 and preventing the death of β cell by Karela 24. However, no significant difference was observed when the results were compared between high and low doses of APHF (Table 4). Hence, the lower dose of APHF ( mg/kg body wt) is recommended for the management of SGL. Body weight Table 5 shows the average body weights of animals on 21 st day. Reduction in body weight was observed in diabetic animals. However, animals treated with APHF A, APHF B and GL registered the significant (P<0.001) check on the loss of body weight on 21 st day in comparison to onset day of the study. This effect may be attributed to increased Table 3 Effect of APHF on serum glucose levels in OGTT in diabetic rats [Values are mean±sd from 6 rats in each group. Figures in parentheses are % increase of serum glucose lever over O min values] Serum glucose levels (mg/dl) Basal 0 min 60 min 120 min. Diabetic control ± ± ±3.81 (62.02) a ±2.34 APHF A (500 mg+5 mg/kg body wt) ± ± ±3.55 (30.52) a ±4.32 APHF B (750 mg+5 mg/kg body wt) ± ± ±3.01 (27.52) a ±4.01 GL (10 mg/kg body wt) ± ± ±2.84 (33.60) a ±3.75 a P< vs diabetic control group.

5 706 INDIAN J EXP BIOL, SEPTEMBER 2013 insulin secretion and food consumption 25, 26 ; increased GLUT-4 transporter protein of muscles and increased glucose utilization in the liver and muscle by Karela 20. Lipid profile and hepatic enzymes After 21 days of the study, animals of the diabetic control group showed a significant (P<0.05) rise in serum cholesterol, TG, LDL-c, VLDL-c, SGOT and SGPT levels, whereas, significant reduction (P<0.05) was seen in serum HDLc in comparison to normal rats. The animals treated with GL, APHF A and APHF B showed significant (P<0.05) check on these abnormal levels of cholesterol, TG, LDL-c, VLDL-c, SGOT and SGPT levels and HDL-c levels in comparison to diabetic animals (Table 5). The developed APHF has proved to be more effective in improving lipid metabolism as compared to GL because Bael exerts its action by interfering with the biosynthesis of cholesterol and utilization of lipids 27, hypo-cholesterolemic effect of Karela 20 and Methi seeds increase biliary cholesterol excretion in liver due to sapogenins 28 and the lipotropic effect of the lecithin 22. These results implied that developed formulation can reduce the complications of lipid metabolism and associated cardiovascular risk factors during diabetes. Parameters Table 4 Effect of 20 days treatment of APHF on serum glucose levels of STZ-induced diabetic rats. [Values are mean±sd from rats in each group. Figures in parentheses are % increase of serum glucose level over O day values] Serum glucose levels (mg/dl) 0 day 7 day 14 day 21 day Normal group 85.16± ± ± ±6.07 Diabetic control ± ±6.13 a ±5.23 a ±6.53 a APHF A (500 mg+5 mg/kg body wt) ± ±6.99 (23.35) abc ±6.03 (39.39) abc ±7.90 (49.20) abc APHF B (750 mg+5 mg/kg body wt) ± ±7.13 (27.56) abc ±5.74 (42.15) abc ±6.45 (54.40) abc GL (10 mg/kg body wt) ± ±6.30 (20.07) ab ±8.71 (34.49) ab ±6.08 (47.08) ab Pvlaues: <0.001; Vs a normal control group; b diabetic control group; cgl c treated group Figures in parentheses are % decrease of serum glucose level over 0 day values. Table 5 Effect of APHF treatment on body weight, serum lipids and hepatic enzymes in STZ-induced diabetic rats on 21 day [Values are mean±sd from 6 rats in each group. Figures in parentheses are % increase/decrease] Normal control Diabetic control APHF-A (500 mg+5 mg/kg bdy wt) APHF-B (750 mg+5 mg/kg body wt) GL (10 mg/kg body wt) Body weight (g) ± ± ± ± ±5.01 (45.12% ) * (17.54% ) *#$ (15.18) *#$ (19.87) *# Cholesterol (mg/dl) 96.45± ± ± ± ±7.01 (151.79% ) a (49.80% ) ab (54.10% ) abc (46.27% ) ab TG (mg/dl) 61.53± ± ± ± ±7.34 (118.34% ) a (32.71% ) ab (40.91% ) abc (30.37% ) ab HDL-c (mg/dl) 42.35± ± ± ± ±5.34 (45.25% ) a (48.77% ) ab (72.12% ) bc (29.66% ) a VLDL-c (mg/dl) 12.45± ± ± ± ±3.25 (93.37% ) a (28.72% ) ab (36.23% ) b (26.10% ) ab LDL-c (mg/dl) 42.72± ± ± ± ±4.10 (337.76% ) a (64.55% ) abc (71.93% ) abc (59.24% ) ab SGOT (U/L) 25.47± ± ± ± ± % ) a 68.69% ) bc (76.71% ) abc (59.84% ) b SGPT (U/L) 27.38± ± ± ± ± % ) a 65.53% ) b 74.99% ) abc (59.36% ) b Pvalues: *<0.001, a <0.05 vs normal group #<0.001, b <0.05 vs diabetic group $ <0.001, c <0.05 vs Glipizide group

6 MANIK et al: ANTIDIABETIC AND ANTIHYPERLIPIDEMIC EFFECT OF ALLOPOLYHERBAL FORMULATION 707 Table 6 Effect of APHF on serum glucose levels on postprandial studies in diabetic rats [Values are mean±sd from 6 rats in each group. Figures in parentheses are % increase of serum glucose level over O day values] Dose administered after meal Diabetic control group ± ±2.05 (47.08) APHF D (750+5 mg/kg body wt.) Dose administered before meal Diabetic control group ± ±2.75 (40.34) APHF D (750+5 mg/kg body wt.) a P< vs diabetic control group. Serum glucose levels (mg/dl) Basal 2 h 3 h 5 h ±3.01 (38.20) ±2.86 (31.20) ± ± ± ±3.15 (25.28) a (18.95) a (12.50) a ±3.45 (23.53) ±2.86 (15.04) ± ± ± ±2.15 (14.29) a (4.51) a (1.93) a Hepatotoxicity is another risk factor associated with oral hypoglycemics treatment on long term use. This risk factor can be minimized by reducing the dose of oral hypoglycemics in combination with herbal drugs. Karela and Bael have been already proved as hepatoprotective agents 29,30. The APHFs (A, B) exhibited better results than GL in the improvement of hepatic enzymes SGOT and SGPT levels (Table 5) indicating that the liver damage is to a less extent in rats treated with APHFs than with GL alone (10 mg/kg body wt). Effect of APHF on postprandial studies in diabetic rats The postprandial study in diabetic animals have shown that the developed APHF is more effective if it is given before food, which may be due to the medication of enhanced insulin secretion by Bael and the results were found to be significant (P<0.001) as compared to respective diabetic control group (Table 6). This effect may be due to the insulin secretagogue effect by Bael; insulino-mimetic effect of Karela and α glucosidase enzyme inhibitory effect of Methi. Moreover, blood glucose lowering effect of the developed formulation is improved when it was given before meal. Toxicity study Throughout the period of study, animals treated with developed APHFs did not show any behavioural changes and mortality as evident from results showing normal hepatic functions, therefore on this basis no separate toxicity studies were carried out. Conclusion The postprandial study in diabetic animals have shown that the developed APHF is more effective if it is given before food, which may be due to the mediation of enhanced insulin secretion by Bael, inhibition of alpha amylase enzyme by Methi 31 and insulin like effect of Karela. Moreover, blood glucose-lowering effect is improved when GL is given before meal 32 (Table 6). The developed APHFs at lower dose of (500 mg/kg body weight PHF + 5 mg/kg body wt GL) is recommended for the management of diabetes and its complications as it was found to be more effective than standard drug GL (10 mg/kg body wt) alone and the side effects associated with GL can be reduced as its dose is reduced. There was no behavioural toxicity; hepato-toxicity and mortality have been observed during the complete duration of study. Hence, developed APHF may be an ideal alternative for the existing antihyperglycemic formulations with an additional advantage of antihyperlipidemic effect and minimizing the cardiovascular risk factors associated with diabetes mellitus, hence, the concept has been achieved. It may be concluded that these findings will open a new vista in the area of medical science for the development of therapeutic approach for the management of diabetes by simultaneous administration of polyherbal formulation and synthetic drug as this approach has reduced the dose, side effects and adverse biological interaction of synthetic drug. Acknowledgement Thanks are due to Prof. P. L. Sharma, Head, Department of Pharmacology for criticism and Shri Parveen Garg (Chairman, ISFCP, Moga) for infrastructure.

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