Evidence based Evaluation of Antidiabetic Potential of Yesaka on Streptozotocin Diabetic Rats

 

Nitin D Deore¹, Shruti Gupta*^3,4, Birendra Shrivastav¹, C. D. Upasni², Kishori G Apte³ , Shaikh A. M⁵

¹School of Pharmaceutical Sciences, Jaipur National University, Jaipur, Rajasthan.

²SNJB's Shriman Sureshdada College of Pharmacy, Nashik, Maharashtra.

³APT Research Foundation, Pune, India.

⁴Faculty of Health and Biomedical Sciences, Symbiosis International University, Pune, India.

⁵AAEMF’S Delight College of Pharmacy, Pune.

 

ABSTRACT:

Yesaka is a known Ayurvedic polyherbal formulation, traditionally employed in the treatment of diabetes mellitus. The current study was performed to evaluate the antidiabetic activity of Yesaka. Antihyperglycemic effects of Yesaka and glipizide were demonstrated in high fat diet-streptozotocin induced diabetic rats. Diabetes was induced by feeding Sprague Dawley rats with high-fat diet (HFD) for two months followed by a single dose of Streptozotocin (STZ, 30mg/kg intraperitoneally). The animals were grouped under normal control and six diabetic subgroups (n=6). All the diabetic rats were orally treated with glipizide as standard (5mg/kg), Yesaka (1.8ml/kg and 3.6ml/kg) and with combination of Yesaka and standard for 4 weeks. The results were assessed by significant restoration of bodyweight, reduction in plasma glucose, total cholesterol (TC), triglycerides (TG), low density lipoprotein-cholesterol (LDL-C) while increased high density lipoprotein cholesterol (HDL-C) levels in all the treatment groups compared to diabetic animals. The antioxidant activity of Yesaka was also evaluated by DPPH. Thus, these findings demonstrated the antidiabetic potential of Yesaka in HFD-fed STZ-treated diabetic rats along with glipizide. Thus, may be a promising candidate in the treatment and management of diabetes with strong antioxidant potential.

 

 

 

1. INTRODUCTION:

As per World Health Organization, Diabetes mellitus (DM) is a defined as systemic endocrine metabolic disorder that results due to deficiency of insulin production by pancreas or inefficient utilization of insulin by the body¹. It is characterized by salient features of chronic hyperglycemia with alteration in carbohydrate, fat and protein metabolism resulting in hyperlipidemia, hyperaminoacidemia, and hypoinsulinaemia².

 

 

Reduction in glycogen synthesis with reduced glucose uptake  by skeletal muscle and uncontrolled hepatic glucose leads to hyperglycemia³. Long term complications with chronic hyperglycemia is associated with retinopathy, nephropathy, peripheral neuropathy with risk of foot ulcers, amputations, charcot joints, autonomic neuropathy causing gastrointestinal, genitourinary, cardiovascular symptoms and sexual dysfunction⁴ in diabetic patients.

 

Diabetes mellitus is a major global health problem that is estimated to affect more than 425 million population worldwide in 2017 and projected to rise to 629 million by 2045⁵. Fortunately, the currently available treatments such as hormone therapy (insulin), oral administration of glucose-lowering agents such as alpha-glucosidase inhibitors, sulfonylureas, biguanides and thiazolidinediones^6,7 lowers the blood sugar levels, but each exhibits its own potential adverse effects. Moreover, none can effectively control the delayed diabetic complications such as nephropathy, neuropathy, retinopathy and cardiovascular diseases. Hence, natural extracts and its constituents derived from medicinal plants with high efficacy but few side effects are urgently required as alternative approach for the prevention and treatment of diabetes compared to their synthetic counterparts. Traditionally, many plants possess marked antidiabetic activity with related ability in restoring pancreatic function via enhanced insulin output,  inhibition of  intestinal glucose absorption or by metabolites facilitation in insulin-dependent processes^8,9. Anacardium Occidentale, Pterocarpus marsupium, Psidium guajava, Mehari choornam and Terminalia arjuna were reported to exhibit significant hypoglycemic activity in alloxan-induced diabetic rats when compared with a standard antidiabetic agent^10–14. It is well documented in the Ayurvedic books that polyherbal drugs are better source in the management of diabetes as compared to the single herb and have extended therapeutic potential.

 

Yesaka is a traditionally known marketed polyherbal formulation, comprised of unique comprehensive combination of different time-tested herbs to control excess sugar level. Herbs being formulated stimulate the pancreatic secretion which acts as agents to normalize blood sugar and give good effect in lowering both blood sugar as well as urine sugar levels. The polyherbal syrup consists of Phyllanthus Emblica¹⁵, Terminalia Chebula¹⁶, Terminalia Bellerica¹⁷, Eugena Jambolana^18,19, Picrorhiza Kurroa²⁰, Swerita Chirata²¹, Tinospora Cordifolia²², Gymnena Sylvester²³, Momordica Charantia^24,25, Curcuma Longa^26–28, Salacia Chinesis Linn²⁹ and Melia Azadirachta. The present study systematically researched the effects Yesaka as compared to glipizide in combination or alone. We hypothesized that Yesaka possess more antidiabetic activity as compared glipizide as depicted by reduced plasma sugar and OGTT assay. Hence, polyherbal formulation is safe with no adverse effect when taken with the standard marketed preparation as glipizide.

 

MATERIALS AND METHODS:

Chemicals Reagents and Diagnostic kits:

All chemicals and reagents of analytical grade were purchased from Sigma–Aldrich (St Louis, MO, USA). Ascorbic acid; 1,1-diphenyl-2-picrylhydrazine (DPPH) were obtained from Sigma–Aldrich (St Louis, MO, USA). Glucose, cholesterol, HDL, LDL kits were purchased from Coral Biosystems, India. Glipizide was procured from Alembic Ltd. (Vadodara, India)

 

 

Polyherbal formulated Syrup:

The polyherbal syrup was purchased from Simandhar Herbal Private Limited in a packing of 600ml per bottle in brown colored liquid form.

 

In vitro antioxidant activity:

The free-radical scavenging effect of Yesaka was measured by 1,1-diphenyl-2-picrylhydrazine (DPPH) radical scavenger with minor modification³⁰. Serially diluted concentrations (2-20µl/ml) of Yesaka were prepared in distilled water. Ascorbic acid taken as standard was prepared with different concentrations (1-100 µg/ml). 1 ml DPPH solution (0.1 mM in methanol) was mixed with 1ml of serially diluted Yesaka and standard solution separately and kept in dark for 30 min and absorbance was measured at 517 nm. The degree of discoloration of DPPH-purple to DPPH-yellow indicated the scavenging efficiency of the sample. The percentage scavenging activity was determined by formula

 

% Scavenging = [(A-B)/A] X100,

Where, A was the absorbance of control (DPPH solution without the sample), B was the absorbance of DPPH solution in the presence of the sample.

 

In vivo studies:

The study was initiated after obtaining approval (Research Project No.10/1617 from Institutional Animal Ethical committee (IAEC, APT Testing and Research Private Ltd., Pune; Reg.No. 40/CO/ReBiRc/S/99/CPCSEA) and as per the guidance of Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. Based on the known human dose of Yesaka, rat dose was calculated as 3.2 ml/kg equivalent to human dose and 1.8 ml/kg as half of human dose was selected for the diabetic study. Forty two Male Sprague-Dawley rats of 6-8 weeks old (200-250 g) were procured from National Toxicology Centre, Pune. Initially, all animals were kept on high fat diet for two months.  Animals were housed and acclimatized under standard conditions of 12 h of light/dark illumination cycles in air conditioned room at ambient temperature (22±2°C) and controlled relative humidity (50-60%). Animals were caged in polypropylene cages with metallic grilled tops with maximum of three animals per cage. Animals have free access to standard pellet food (Pranav Agro Industries, Maharashtra) and water ad libitum throughout the experimental period.

 

Induction of Diabetes:

All the obesed rats were injected with 30mg/kg of the Streptozotocin (Sigma-Aldrich) intraperitoneally (i.p) for the diabetes induction^31–33. Overnight fasted animals, on 3^rd day of induction which were intolerant to glucose by OGTT and fasting blood glucose levels >300 mg/dL were selected for the study³⁴.

 

Animal experimentation:

The diabetic animals were randomly distributed into 6 groups (n=6) and normal control and orally treated for 30 days.

Group 1: Normal control

Group 2: Disease (Diabetic) Control

Group 3: Diabetic rats+ Glipizide (5 mg/kg)

Group 4: Diabetic rats+ 3.2 ml/kg Yesaka

Group 5: Diabetic rats+ 1.8 ml/kg Yesaka

Group 6: Diabetic rats+ Glipizide (5 mg/kg) + 3.2 ml/kg Yesaka

Group 7: Diabetic rats+ Glipizide (5 mg/kg) + 1.8 ml/kg Yesaka

 

Animals were weekly weight and the study was conducted for 30 days to evaluate the potential of the Yesaka to lower blood glucose level.

 

Biochemical evaluation:

The blood was withdrawn by retro orbital plexus under anaesthesia of the fasted animals. The fasting blood glucose levels were estimated at 0, 15^th and 30^th day by GOD/POD method by semi automatic analyzer (Pathozyme Smart-7, India) using diagnostic kits (Coral Biosystems, India). Total cholesterol, triglycerides, HDL and LDL were estimated by semi automatic analyzer (Pathozyme Smart-7, India) using diagnostic kits (Coral Biosystems, India).

 

Oral glucose tolerance test:

Oral Glucose Tolerance Test (OGTT) was estimated in overnight fasted rats and the baseline blood glucose was determined next day with the automated glucometer (AccucheckactiveR)³⁵. The rats were loaded with 2gm/kg of glucose 30 minutes after administration of drug at their respective doses. The blood glucose was monitored for 30, 60 and 120 minutes thereafter.

 

Histomorphometric Analysis:

At the end of 30 days, animals were sacrificed and the pancreas were dissected, cleaned and fixed in 10% formalin for monitoring necropsy or adverse effect of the treatment. The tissues were washed, dehydrated in gradient alcohol, defatted in xylene and embedded in paraffin wax. It was sectioned (5-μm thickness) longitudinally on a rotary microtome (Leica RM22, Germany) and processed for hematoxylin and eosin staining. Microarchitectural changes were observed under microscope (Nikon H550S Eclipse Ci-L, Japan) with NIS Elements Imaging Software version 4.

 

 

 

Statistical analysis:

All data were expressed as Mean±SD and analyzed using one-way analysis of variance (ANOVA) followed by post hoc Dunnett’s test to compare all treatment groups with normal and Diabetic control.  Group means were considered to be significantly different at 5% level of significance, 𝑃 <0.05. All statistical analyses were performed using the “GraphPad Prism V-5.03”.

 

RESULTS:

DPPH radical scavenging assay:

The DPPH radical scavenging effect of YESAKA are shown in Fig 1and 2. The results demonstrated that there was a significant (p<0.05) difference of mean percentage scavenging between all the tested concentrations of the Yesaka and standard. The antioxidant content in YESAKA was revealed by the gradient decolorization of the purple DPPH radicals in a dose dependent manner. The inhibition concentration (IC₅₀) values of YESAKA and ascorbic acid were calculated as 7.38μg/ml and 8.52µl respectively, which were inversely proportional to the antioxidant capacity.

 

 

Fig- 1: Ascorbic acid

 

 

Fig- 2: Yesaka

 

Effect on Bodyweight:

The initial and final body weights of all the animals in each group are shown in Table 1. The control group showed linear growth in bodyweight with 13% gain whereas the diabetic control group showed a 3% weight gain. However, administration of Yesaka with different doses, standards and combination to diabetic rats significantly restored the body weight to a normal level (p<0.005) with 5-10% increase in bodyweight.

 

Table -1: Effect of Yesaka on the body weight of the diabetic animals.

The results were expressed as Mean ±SD, n=6 in each group. Statistical significant-  ^*p< 0.05, ^**p< 0.01, ^***p< 0.001 versus Group 1. ^#p< 0.05, ^##p< 0.01, ^###p< 0.001 versus Group 2 (Disease control).

 

Effect of Yesaka on the plasma glucose of the diabetic animals:

As observed from Fig 3 streptozotocin induced rats showed significant 73% rise in the plasma glucose levels. Daily oral administration of Yesaka alone and combination with glipizide significantly reduced the plasma glucose (p<0.05) by ~30% as compared to hyperglycemic rats. However, diabetic animals without treatment continued to show high plasma glucose throughout the study.

#

***

 

Fig-3 Effect of YESAKA on the plasma glucose of the diabetic animals

Results were expressed as Mean ±SD, n=6 in each group.  ^*p< 0.05, ^**p< 0.01, ^***p< 0.001 versus Group 1. ^#p< 0.05, ^##p< 0.01, ^###p< 0.001 versus Group 2

 

Effect of Yesaka on Biochemical Parameters:

Lipid profile was assessed by investigating triglycerides, total cholesterol, HDL and LDL cholesterol. It is reported that hyperglycemia leads to hypertriglyceridemia and hypercholesterolemia. As depicted from the results, diabetic rats showed significant increase in total cholesterol, triglyceride and LDL levels, whereas reduced HDL cholesterol values. Additionally, treatment with standard and Yesaka alone and combination restore the lipid profile towards normal (p<0.05) (Table 2).

 

Table- 2 Effect of Yesaka on Lipid Profile

	Total Cholesterol (mg/dl)	Triglyceride(mg/dl)	HDL (mg/dl)	LDL (mg/dl)
Group 1	88.3± 11.7	100.9± 10.6	48.7± 5.4	24.4± 8.3
Group 2	197.0± 22.4***	267.8± 57.8***	29.8± 4.7***	113.9± 27.4***
Group 3	152.5± 12.3	139.8± 25.3###	43.8± 7.7#	74.8± 16.4#
Group 4	121.8± 13.7#	163.6± 33.4##	46.6± 12.5##	54.7± 5.4###
Group 5	124.7± 14.6	96.7± 10.9###	34.4± 4.4	57.3± 5.2##
Group 6	144.8± 19.6	173.6± 26.5##	43.8± 6.7##	65.5± 7.4##
Group 7	118.7± 12.8##	116.2± 26.2###	38.0± 5.5	56.7± 10.4###

Results were expressed as Mean ±SD, n=6 in each group.  ^*p< 0.05, ^**p< 0.01, ^***p< 0.001 versus Group 1. ^#p< 0.05, ^##p< 0.01, ^###p< 0.001 versus Group 2

 

Effect of Yesaka on Oral Glucose Tolerance Test:

After 30 minutes of the glucose load all the groups showed gradual increase, this later came to the baseline during after 2hrs but the diabetic group showed persistent elevated levels of blood glucose even after 2 hours indicating inadequacy of the pancreatic function (Fig 4).

 

 

Fig- 4:  Effect of Yesaka on Oral Glucose Tolerance Test

 

Histopathological Evaluation:

Histological observation of the pancreatic tissue showed normal histomorphology of endocrine and exocrine pancreas with adequate number, size and architecture of β cells in the Islets of Langerhans. The diabetic rats exhibited reduction in number and size of β cells in the Islets of Langerhans. However, all the treatment groups showed significant restoration of the pancreatic islets cells from the damage caused by streptozotocin (Fig 5).

 

 

Group 1                                   Group 2  

 

 

Group 3                                   Group 4

 

 

Group 5                                       Group 6

 

 

Group 7

Fig- 5:  Effect of Yesaka on Histopathology of Pancreas.

 

SUMMARY AND CONCLUSION:

The presented study is an attempt to investigate the effect of Yesaka on streptozotocin induced diabetic in albino rats. As investigated by reduced fasting plasma sugar and OGTT assay, the Yesaka showed more antidiabetic activity as compared to glipizide alone and combination. The free radical DPPH scavenging assay of Yesaka significantly correlates scavenging activity and potency of the antioxidants in the Yesaka in dose dependent manner due to its stronger proton-donating abilities and higher concentrations of flavonoids and phenols. Hence, polyherbal formulation is safe, cost effective, easily available and when taken with the standard marketed preparation like Glipizide showed no adverse effect.

 

CONFLICT OF INTEREST:

The authors assert no conflict of interest associated with this project.

 

ACKNOWLEDGEMENT:

The authors are grateful to Dr. Birendra Shrivastav and Dr. C. D. for continuous guidance and encouragement. Dr Kishori G Apte for providing necessary facilities to carry out the research project and support.

 

FUNDING:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

 

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Accepted on 14.08.2018           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(11): 4965-4970.