Evaluation of In vivo Antidiabetic activity of extract of herbal formulation obtained from Hathbandh dist. Baloda Bazaar on streptozotozcin-induced diabetic rats

Ramu Singh, Dr. Pushpa P. Gupta*, Dr. Amit Roy, Dr. Shashikant Chandrakar

Columbia Institute of Pharmacy, Raipur (C.G.).

*Corresponding Author E-mail: pushpaprasad81@gmail.com

ABSTRACT:

Herbal formulations are traditionally used in Chhattisgarh, India for the management of diverse diseases along with hyperglycemia i.e. diabetes. The healers of different region use their own formulations claiming to treat the disease but their claims have not being properly investigated and verified scientifically. We therefore collected formulations from a healer of Hathbandh district Baloda Bazaar. We prepared hydroalcoholic extract (70:30, HAE) of a formulation and used it for our study. At first acute oral toxicity study was performed to determine the safety of the formulation, thereafter antidiabetic study was performed on streptozotocin (STZ) induced diabetes in wistar rats. The mean blood glucose levels were examined on 0^th, 7^th, 14^th and 28^th day in normal, untreated diabetic, diabetic treated with standard and test groups. Data was statistically analyzed by anova software SPSS 14.0. P-value < 0.05 was treated as statistically significant. Toxicity studies showed that extract is not toxic and no behavioral change was observed at a dose of 2000mg/kg body wt. The HAE was administered in 200mg/kg and 400 mg/kg body weight for our studies. The extract at both the doses reduced elevated blood glucose level when compared to diabetic control group. It was also noted that in extract treated group body weight increased in comparison to diabetic control group. HAE at dose of 400 mg/kg body weight showed highest reduction in blood glucose level. The biochemical studies of HAE also supported the claim of traditional healers of Chhattisgarh.

KEYWORDS: Antidiabetic, hydroalcoholic extract, toxicity, herbal formulation, streptozotocin, metformin.

INTRODUCTION:

Diabetes mellitus is a metabolic disorder that alters the normal metabolic process of the body. Type II diabetes generally occurs in adults; however the children are also affected because of obesity as well as any other underlying physiological and pathological conditions. The diabetes is responsible for several complications due to its effect on small blood vessels that supply to kidney, nerves and eyes thereby develop nephropathy, neuropathy and retinopathy. It also affects larger blood vessels resulting in angina pectoris, myocardial infarction, transient ischemic attacks, stroke and peripheral arterial diseases.(1)

According to International Diabetes Federation South East Asia (IDF SEA) 2017 report, 425 million people are suffering from diabetes in the world and 82 million are in the SEA region. 72,946,400 cases of diabetes were found in India. (2)

The treatment of type II diabetes includes allopathic as well as herbal drugs along with physical exercise, diet control and change in lifestyle. Traditional medicines have been used since ages for the treatment and management of diabetes throughout the world and in different communities. These traditional medicines are obtained from mostly plants and suppose to have less or no side effects and toxicity. However very few have been studied scientifically for their safety and efficacy. (3)(4)(5)

In this study we performed antidiabetic study of a formulation that was obtained from a traditional healer of village Hathbandh, District Baloda Bazaar, Chhattisgarh.

MATERIAL AND METHOD:

Collection of formulation

Herbal formulation was collected from a traditional healers practicing in village Hathbandh, district Baloda Bazaar, Chhattisgarh, India in July 2017. The formulation was further ground and passed through a no. 10-mesh sieve to obtain a uniform sized powdered material. The powder was kept in sealed jar for further work.

Chemicals and Instruments

Streptozotocin (Himedia chemicals, India) was used to induce non insulin dependent diabetes mellitus in rats and Metformin (Norris medicines, Gujarat) was used as standard drug. Ethanol (Kedia, kumhari, C.G.) and distilled water were used in the ratio 70:30 to perform extraction. ACCU CHEK ACTIVE glucometer ( Roche Diagnostics, India) was used to check blood glucose level in experimental rats. Vacuum rotary evaporator was used to concentrate the extract (BUCHI R-100). Ethanol was procured from Kedia Company, Kumhari, Chhattisgarh (India).

Experimental animals

The experiment was performed on adult albino wistar rats, weighing 180-240 gm. Wistar rats were obtained from Columbia Institute of Pharmacy, Raipur, (C.G), India. The experimental procedure was performed after getting permission from the Institutional Animal Ethical Committee of Columbia Institute of Pharmacy, Raipur (C.G), Reg.No. 1321/Po/ReBi/s/10/ CPCSEA/ Dated-22-10-2014. The animals were maintained at 23±2 ºC and 12 hrs light-dark cycles. Animal had free access to diet and water (6)

Preparation of hydroalcoholic extract

100 gm of coarse powder of the formulation was accurately measured and was extracted with 500 milliliters of hydroalcoholic (70:30) solution in a soxhlet extraction unit. After the extraction was complete, the extract was taken out and filtered to discard any trace of solid particles. Thereafter the extract was dried initially in rotary vacuum evaporator. A viscous solution was obtained which was further subjected to lyophilization in a freeze dryer. The resultant solid mass was kept in refrigerator for further work.(7)(8)

Acute oral toxicity of the extract

Acute oral toxicity of the extract was assessed according to OECD 423 (Organization Economics and co-operation developments) guidelines at a limit dose of 2000 mg/kg body weight p.o. as a single dose. According to the guideline, 3 male and 3 female rats were selected randomly for this test. The rats were kept fasted overnight but had access to water ad libitum. Rats were administered solution of HAE at dose of 2000mg/kg body weight. The rats were closely observed for first 30 minutes and then at an interval of 4 hours. Standard pellet and water was provided to rats after 4 hrs of dosing. The rats were observed daily for 14 days for any change in their behavior and/ or physical activities. Observations were taken timely and systematically maintained. Observation recorded includes physical appearance (changes in skin and fur, eyes) and behavior pattern (tremors, salivation, lethargy, sleep, diarrhea and coma were also observed during these two weeks. (9)(10)

Induction of type II diabetes in experimental rats

Rats were weighed and their blood glucose determined and then fasted overnight (12-14hours) before inducing diabetes. Streptozotocin was dissolved in citrate buffer (pH-4.5). Te rats were fed with nicotinamide in saline solution (120mg/kg body weight, i.p.) and STZ solution was at the dose of 60 mg/kg body weight (i.p.). The blood glucose level was recorded after 72 hrs of administering streptozotocin and the raised glucose level was measured for confirming hyperglycemic condition in rat.(11)(12)

Experimental

Antidiabetic activity

The animals were randomly divided into five groups, containing six animals each (n = 6). The groups were treated as mentioned in table 1.

Table 1: Treatment on Experimental Rats

S.No.	Group	Treatment	Route
1	Group-I	Normal control (vehicle treated)	Oral
2	Group-II	Diabetic control (Untreated)	--
3	Group-III	Treated with metformin, 50mg/kg (Standard)	Oral
4	Group-IV	Treated with HAE (200mg/kg)	Oral
5	Group-V	Treated with HAE (400mg/kg)	Oral

The blood glucose was determined on 0^th, 7^th, 14^th and 28^th day of treatment. Body weight of animals was taken daily during this tenure and mean change was calculated. (12)

Collection of serum sample

The blood sample was withdrawn from the rat’s eye from retro orbital plexus for biochemical analysis on 28^th day. The biochemical investigation was performed in Shri Ram Pathology laboratory, Raipur (C.G), India.(13)(14)

RESULTS:

Extraction of formulation

The yield of hydroalcoholic extracts (HAE) of herbal formulation was 8.36 %.

Acute toxicity study

In acute toxicity study no mortality was observed during the observation period of two weeks. Also there was no sign of physical or psychological abnormality visible during this period. This result proves that the median lethal dose is greater than 2000mg/kg body weight in treated animals.(12)

Antidiabetic activity

Effect on blood glucose level

The glucose level of diabetic control group significantly increased compared to normal control group. The groups treated with standard drug and HAE (200 mg/kg and 400 mg/kg) showed significant reduction of glucose level. The highest reduction of glucose level was observed on 28^th days. The results are shown in table 2.

Effect on body weight

The result of diabetic control group showed significant decrease in body weight. Whereas HAE (400mg/kg) and metformin treated rats were found to gain body weight, compared to diabetic control group as shown in Table 3.

Effect on lipid profile

The animals treated with standard drug and HAE (200 and 400 mg/kg body weight), showed significant reduction in levels of triglycerides, total cholesterol and LDL in diabetic rats compared to control group ; and at the same time the level of HDL in the treated group showed significant increase compared to untreated control group. The result are shown in table no.4.

Table no.2 Effect on blood glucose level

Group	Blood glucose level (mg/dl) on days
Group	0^th	7^th	14^th	28^th
Normal Control	78.5±3.53	77.6±4.84	75.3±3.02	76.53±4.45
Diabetic Control (Streptozotocin)	144.1±9.10^a	177.8±11.35^a	208.3±7.32^a	237.1±4.18^a
Diabetic + Standard Metformin 50 mg/kg	128.1±8.62	103.5±6.35*	91.5±6.82*	79.7±7.7*
Diabetic + HAE (200mg/kg)	125.5±6.50	154.7±7.30*	114.8±5.41*	94.8±6.06*
Diabetic + HAE (400mg/kg)	126.6±5.43	139.9±5.31*	98.2±5.07	80.05±6.83*

Values are expressed as mean ± SEM (Number of animals, n=6); significantly different at ^aP<0.05 when compared with normal control group, *P<0.05 when compared with diabetic control group

Table no.3 Effect on body weight

Group	Change in Body weight (gm)
Group	Before Induction	After Induction	After Treatment
Group-I (normal)	220±1.1	222.26±1.47	225±1.5
Group-II (diabetic control)	220.32±3.14	193.82±2.65	158.67±1.72^a
Group-III (rats treated with standard)	215.68±2.69	210.54±3.25	219.92±2.43*
Group-IV ( HAE 200mg/kg)	214.25±2.52	181.24±5.64	200.41±4.72
Group-V ( HAE 400mg/kg)	216.65±4.62	198.62±2.51	211.17±2.68*

Values are expressed as mean ± SEM (Number of animals, n=6); significantly different at ^aP<0.05 when compared with normal control group, *P<0.05 when compared with diabetic control group

Table no.4. Determination of biochemical parameters after treatment with HAE

Group	Lipid Profile (mg/dl)
Group	Triglyceride	Total cholesterol	HDL	LDL
Normal control	83.1±3.32	85.6±5.03	54.6±5.56	44.8±5.70
Diabetic control (Streptozotocin)	152.6±6.90^a	162.4±6.74^a	31.6±4.57^a	142.2±6.59^a
Diabetic + Standard metformin (50 mg/kg)	86.5±4.93*	83.4±8.38*	55.7±5.44*	56.7±4.51*
Diabetic + HAE (200 mg/kg)	95.4±5.87*	101.5±6.49*	49.0±3.11*	58.2±4.76*
Diabetic + HAE (400 mg/kg)	87.2±6.40*	89.4±5.60*	53.7±4.85*	48.6±7.20*

Values are expressed as mean ± SEM (Number of animals, n=6); significantly different at ^aP<0.05 when compared with normal control group, *P<0.05 when compared with diabetic control group

DISCUSSION AND CONCLUSION:

In the present work the acute toxicity study that was performed to determine the LD50 values of HAE showed that the animals were safe upto a maximum dose of 2000 mg/kg body weight. There were no changes in normal behavior pattern and at the same time there was no mortality or no symptoms of any toxicity.

In the STZ induced antidiabetic activity of HAE at dose levels of 200 and 400 mg/kg body weight in the result showed that there was significant reduction in blood glucose level. The results were more prominent at the dose of 400 mg/kg body weight and was comparable to the group that received standard drug on 28^th day (80.05±6.83* and 79.7±7.7* respectively).

In case of change in body weight in the diabetic rats that were treated with standard drug and HAE (200 and 400 mg/kg body weight), it was observed that groups of animals treated with standard drug and HAE recovered significantly and their body weight was regained after treatment for 28 days, which may be attributed to improved glycemic control achieved by the administration of the extract and the drug. Here also the results at the dose of 400 mg/kg body weight and was comparable to the group that received standard drug on 28^th day.

The level of lipids in the serum increases in case of type II diabetes, which is usually due to deposits of fats that results because of inactivity or insufficiency of insulin. In the present study also the diabetic rats showed hypercholesterolemia and hypertriglyceridemia. However when the animals were treated with standard drug and HAE it was noted that the lipid and triglycerides level decrease significantly and also the level of HDL increased. This implies that HAE can be useful in correcting the lipid profile associated with type II diabetes.

This work proved that the HAE at the doses of 200 and 400 mg/kg body weight possess significant antidiabetic activity. The result also showed that total cholesterol, triglyceride, low density lipoprotein (LDL) decreases significantly and the level of HDL increased as compared to diabetic control (Table no. 2, 3, 4). The result also shows that the HAE has a dose dependent activity.

We may conclude here that the formulation that was procured from a tribal healer possess promising activity in case of type II diabetes and can be used effectively for its management. However, further studies are required not only to identify and characterize the phytoconstitutents present in the formulation that are responsible for the activity, also an in depth an exhaustive pharmacological study are required to understand the mechanism by which the formulation works for management of type II diabetes.

ACKNOWLEDGEMENT:

We are thankful to the Principal and Management of Columbia Institute of Pharmacy, Raipur (C.G) India for providing necessary equipment, chemicals and assistance for completion of project work.

CONFLICT OF INTEREST:

There is no conflict of interest.

REFERENCES:

1. Mohan H. Textbook of Pathology, Sixth Edition, Jaypee Publisher, Page no. 818-829.

2. South East Asia- International Diabetes Federation 2018-19.

3. Sen P, Sahu K, Prasad P, Chandrakar S, Sahu R, Roy A. Approach to phtochemistry and mechanism of action of plants having antidiabetic activity. UK J Pharm Biosci. 2016;4(1):82–120.

4. Rai R, Nath V. Use of medicinal plants by traditional herbal healers in Central India. Indian For. 2005;131(3):463–8.

5. Grover J, Yadav S, Vats V. Medicinal plants of India with antidiabetic potential. J Ethnopharmacol. 2002;81(1):81–100.

6. Yang Y, Tarabra E, Yang G, Vaitheesvaran B, Palacios G, Kurland I, et al. Alteration of denovo glucose production contributes to fasting hypoglycaemia in Fyn deficient mice. PLoS One. 2013;8(11).

7. Roy A, Prasad P. Study of Physicochemical , Preliminary Phytochemical and Antioxidant activity of Calocybe indica. 2016;9(January):707–9.

8. Begum D, Kumar K, Sravanthi P, Prasad K, Reddy R. Evaluating phytochemical and antidiabetic activity of ethanolic extract of whole plant of Acalypha indica Linn on alloxan induced diabetic Rats. J Pharm Res. 2011;4:1704–6.

9. Vijayanand S. Evaluation of antidiabetic activity of Murraya Koenigii on alloxan induced diabetic rats. Int J Pharma Sci Res. 2015;6(12).

10. Glinsukon T, Stitmunnaithum V, Toskulkao C, Buranawuti T, Tangkrisanavinont V. Acute toxicity of capsaicin in several animal specise. Toxicon. 1980;18(2):215–20.

11. Singh J, Sahu R, Prasad D, Jangde R, Gupta R. Evaluation of antidiabetic potential of Ougeinia oohjeinesis leaves in streptozotocin-induced-diabetic rats. Pharmacologyonline. 2011;2:1046–52.

12. Amit R, Pushpa P. Assessment of Antihyperglycemic Potential of Lyophilized and Oven-Dried Extract of Calocybe indica in Experimentally Streptozotocin-Nicotinamide Induced Diabetic Rats. 2016;82–8.

13. Van herck H, Baumans V, Brandt C, Boere H, Hesp A, Van lith H, et al. Blood sampling from the retro-orbital plexus, the saphenous vein and the tail vein in rats:comparative effects on selected behavioural and blood variable. Lab Anim. 2001;35(2):131–9.

14. Petchi R, Vijaya C, Parasuraman S. Antidiabetic activity of polyherbal formulation in streptozotocin-nicotinamide induced diabetic wistar rats. Med J Tradit Complement. 2014;4(2):108–17.

Received on 09.07.2020 Modified on 28.08.2020

Research J. Pharm. and Tech. 2020; 13(10):4957-4960.

DOI: 10.5958/0974-360X.2020.00869.0