Preparation, Standardisation and Evaluation of Hypoglycaemic effect of Herbal Formulation containing five Ethnomedicinal plants in Alloxan-Induced Hyperglycemic Wistar Rats

 

Archana Sharma¹*, Prosanta Pal², Bapi Ray Sarkar², J. P Mohanty¹, Sonam Bhutia³

¹Himalayan Pharmacy Institute, Majhitar, Sikkim University, East Sikkim-737136.

²Department of Pharmaceutical Technology, University of North Bengal, West Bengal, India-734013.

³Government Pharmacy College, Government of Sikkim, Sikkim University, Sajong,

Rumtek East Sikkim-737135.

 

ABSTRACT:

The present study was undertaken to evaluate the pharmacognostical parameters, formulation, standardization and hypoglycaemic effect of herbal preparation in alloxan induced diabetic rats. The physicochemical properties of polyherbal finished product were: Specific gravity (1.03 at 25˚C), pH of 10%v/v solution (4.543), solid content (7% w/v), viscosity (4.59 cp), surface tension (43.37 dynes/cm) and refractive index (1.37). The colour (brown), odour (sweet aromatic), and taste (sweet) of polyherbal formulation was satisfactory in physical appearance. The study was also aimed to evaluate the antidiabetic activity of herbal formulation in alloxan induced diabetic rats model. The results of the body weight and blood glucose level of control, diabetic control group, standard group (Glibenclamide 5mg/kg)-standard drug used and two different doses of trial formulation (600mg and 300mg/kg)-summarised in Table No 1 and Table No 2 respectively. The initial body weight of Herbal formulation low dose and high dose were 123.33±5.78 and 185±8.38 respectively and after 11 days of treatment the bodyweight was decreased to 119.33±8.45 and1 70.33±7.79 respectively. Two different doses of the herbal formulation (low dose and high dose) i,e 123.33±5.78 and 185±8.38 blood glucose level were studied in two different groups of animals. Both groups showed a significant decrease of blood glucose level on alloxan induced diabetic rats when compared to control group. The low dose result shows for respective day 3: 5.01±0.21, day 7: 33.23±1.70 and day11: 63.20±2.46 and for high doses for day 3: 6.59±0.34, day 7: 35.26±1.84 and day 11: 65.63±2.46. Both groups showed a significant decrease of blood glucose level on alloxan induced diabetic rats when compared to control group. The present study showed that the investigation of the polyherbal formulation containing five herbs has a potent anti diabetic effect which could be used in the management of diabetes mellitus effectively.

 

 

 

INTRODUCTION:

India is one of the most significant nations on the planet as far as floristic decent variety. About 54% of the nation's property is under development of nourishment, fancy and restorative plant crops and roughly 19% area has fluctuating level of woods vegetation spread.

 

Land based formative exercises give methods for occupation, wellbeing and open door for business^[1]. India has worldwide situation in the field of conventional meds. About 90% home grown crude medications utilized in the assembling of vegetable medications are acquired from the wild source which is restricted. There are around 45,000 plants species in India which are in copious in the locales of Eastern Himalaya, Western Ghats and Andaman and Nicobar Islands. The rich legacy of verdure is because of broadened and differed agro-climatic conditions. The authority recorded plants with restorative potential are 3,000 however conventional expert use around 8,000 vegetable medications^[2]. India is the biggest maker of conventional restorative herbs and around called the professional flowerbed of the world. In Indian restorative framework the most specialists plan and administer their own plans; henceforth, this requires appropriate documentation and research. There are as of now around 250000 enrolled restorative specialists of Ayurvedic framework; the aggregate for every customary framework being almost 291000 as practically identical to 700000 of the cutting edge drugs^[3]. In rustic India 70% of the populace is reliant on the customary arrangement of medication (Ali 2009). Diabetes mellitus (DM), usually alluded to as diabetes, is a gathering of metabolic ailments where there are high glucose levels over a delayed period. Side effects of high glucose incorporate continuous pee, expanded thirst, and expanded yearning^[4]. Whenever left untreated, diabetes can cause numerous entanglements. Intense difficulties can incorporate diabetic ketoacidosis, nonketotic hyperosmolar trance like state, or passing. Genuine long haul entanglements incorporate coronary illness, stroke, constant kidney disappointment, foot ulcers, and harm to the eyes^[5-6].

 

MATERIALS AND METHOD:

Collection, Identification and Authentication of the plant material:

The plants Ocimum sanctum, Azadirachta indica, Trigonella foenum-graecum, Tinospora cordifo and Gymnema sylvestre. were collected in the period of august – September or during stormy season from the neighborhoods Sikkim Himalayan district. The leaves were recognized by standard literature^[7-10].

 

PHARMACOGNOSTICAL STUDIES:

Macroscopical examination:

The macroscopical observation were carried out as per by the standard methods to determine the shape, size, taste, colour, odour^[11-13].

 

PHYSIOCHEMICAL EVALUATION:

The different parameters in this aspect like ash values such as total ash, acid insoluble ash, sulphated ash and extractive values like water soluble extractives, alcohol soluble extractives were carried out as method described by Pharmacopoeia of India. Moisture content was carried out as method described by Khandelwal K R. Determination of fluorescence analysis was followed as method described by Nisha Raj RS et al^[14-16].

 

PREPARATION OF HERBAL FORMULATION:

PREPARATION OF PLANT EXTRACTS:

The collected crude drugs were washed completely with water to expel any undesirable material. These were additionally dried in shade. After complete drying it was powdered^[17-19].

PREPARATION OF AQUEOUS EXTRACT:

20gms of each powdered plant material was cold macerated with 100ml of distilled water for 7 days. Additionally 5 drops of chloroform were added to it.

 

DRYING OF EXTRACT:

The aqueous extract obtained from the process of extraction i.e. maceration was concentrated by using water bath. After complete evaporation, dried extract was stored in a well closed container to prevent the growth of moulds and microorganisms.

 

PREPARATION OF HERBAL LIQUID FORMULATION:

At first methyl parabin 0.25gm, propyl parabin 0.05g and sodium saccharin 0.1gm were dissolved in 10ml of water. After that 0.5gm of EDTA was added in it. In another beaker 0.1gm of vaniline was added in 25ml of ethanol and 25ml of propylene glycol. Both the mixture was mixed together. In this mixture the extracts of Ocimum sanctum 16gm, Azadirachta indica 6gm, Trigonella foenum-graecum 10gm, Tinospora cordifolia 1.3gm, Gymnema sylvestre 1.3gm respectively was dissolved. Then the volume was made up to 100ml with distilled water.

 

EVALUATION OF PHYSICAL PARAMETER OF HERBAL FORMULATION:

The determination of physical parameters such as colour, odour, taste, pH, surface tension, specific gravity, total solid content, viscosity and refractive index were carried out following method described by Bitasta, M. 2011 and Prasad S.K. 2012.^[20-22]

 

PHARMACOLOGICAL ACTIVITY:

Selection of Animal Species:

Wistar Albino rat of either sex weighing between 100-180g were used in the experiment. Animals were maintained under uniform laboratory conditions (12hr. light and 12hr. dark cycle, 25+30) in standard stainless steel cages and provided food and water ad libitum. The animals were fasted for 18 hours prior to experimentation but allowed free access to water. The animal study was permitted by the Institutional Animal Ethics Committee.

 

Animals:

Male albino rats (100-180g) were selected and animals were housed at room temperature (37^oc) with 12 hours light dark cycle between humidity 40% and 60% RH. The animals were deprived of food for 18 hours, before the commencement of the experiment, but water allowed at ad libitum.

 

Acute Toxicity Studies:

Determination of oral acute toxicity of herbal formulation was determined following OECD guideline No. 421^[23-24].

 

Determination of OGTT activity:

The blood glucose concentrations of animals were measured at the beginning of the study. Then the rats were orally treated with 2g/kg body weight glucose solution after 30 minutes of the product and drug treatment. The measurements were repeated after 30, 90 and 150 minutes after the glucose load.

 

ANTIDIABETIC STUDY:

Animals:

Albino rats (100-180g) of either sex were selected and animals were housed at room temperature (37^oc) with 12 hours light dark cycle between humidity 40% and 60% rH. The animals were deprived of food for 18 hours, before the commencement of the experiment, but water allowed at ad libitum.

 

Induction of diabetes:

Animals were fasted for 24 hrs then a single interperitoneal injection of freshly prepared alloxan (110mg/kg dissolved in 0.9% saline) was injected. After the animals were left aside for 4 hrs and then 10% glucose solution was placed in the cages for 24 hrs. The diabetes was confirmed by estimation of blood glucose level (BGL) on the third day. Rats having BGL>200 mg/dl were used for study and during the experiment the animals were divided into five groups of six animals in each group.\

 

1.     Control-   Vehicle (normal saline)

2.     Diseased (Diabetic control)-   Alloxan

3.     Standard-   Glibenclamide

4.     Test-I-   Herbal preparation low dose (300mg/kg)

5.     Test-2-   Herbal preparation high dose (600mg/kg)

 

Method for collection of blood sample:

The rat was placed into the rat holder, such that the tail was pulled out and was depilated for collection of blood sample. Tail vein was dilated by focusing a low voltage electric lamp. The tip of the tail was thin sliced (0.05mm) using a sharp scissors. The blood drops were collected into a 0.5ml of centrifuge tube containing a pinch of anticoagulant powder (Heparin). The tail was gently pressed with fingers to enhance the blood flow. Later dry cotton was applied for few minutes to stop the blood flow and the tail was sterilized by spirit.

 

Determination of Antidiabetic activity:

The blood glucose concentrations of the animals were measured at the beginning of the study and measurements were repeated on 3rd, 7th, and 11th day of the experiment. The blood sample was taken by pricking the rat’s tail. Polyherbal preparation was administered with glass syringe and micro suction canula.

 

STATISTICAL ANALYSIS:

The results were represented as Mean±Standard Error Mean. The statistical significance was computed using One Way ANOVA followed by Dunnet’s multiple comparison test and compared with diabetic untreated control group with Standard, low dose and high dose where the n=3 animals in each one group were used. P<0.05 was considered statistically significant.^[25-26]

 

RESULTS AND DISCUSSION:

Pharmacognostical evaluation:

Table 1: Macroscopic examination: Comparative study of different macroscopic parameters of five Ethnomedicinal plants:

Sl. No.	Plant name	Plant part used	Macroscopic parameters
			Length	Taste	Thickness	Odour	Colour
1.	Ocimum sanctum	Leaf	3cm	characteristic	0.1cm	Aromatic	Blackish green
2.	Azadirachta indica	Leaf	20cm	Bitter	0.2cm	Indistinct	Dark green
3.	Trigonella foenum-graecum	Seed	0.3cm	Bitter	0.3cm	Pleasant	Dull yellow
4.	Tinospora cordifolia	Stem	30cm	Bitter	10cm	Pleasant	Brown
5.	Gymnema sylvestre	Leaf	5cm	Bitter	0.1cm	Pleasant	Light green

 

 

Table 2: Moisture content of Ocimum sanctum, Azadirachta indica, Trigonella foenum-graecum, Tinospora cordifolia and Gymnema sylvestre.

SL No	PLANTS	FRESH WEIGHT (gm)	DRY WEIGHT (gm)	MOISTURE CONTENT (%W/W)
1	Ocimum sanctum	80.25	80.01	12%
2	Azadirachta indica	79.13	79.04	4.5%
3	Trigonella foenum-graecum	79.23	79.10	6.47%
4	Tinospora cordifolia	80.05	79.93	6%
5	Gymneva sylvestre	68.06	67.89	8.46%

 

 

Table 3: Ash value of Ocimum sanctum, Azadirachta indica, Trigonella foenum-graecum, Tinospora cordifolia and Gymnema sylvestre.

SL No.	PLANTS	TOTAL ASH (%w/w)	ACID INSOLUBLE ASH (%w/w)	WATER SOLUBLE ASH (%w/w)	SULPHATED ASH (%w/w)
1	Ocimum sanctum	8.6	0.8	3.6	0.8
2	Azadirachta indica	5.2	0.6	2.4	0.7
3	Trigonella foenum-graecum	3.3	0.4	1.6	0.5
4	Tinospora cordifolia	5.9	0.7	1.4	0.4
5	Gymneva sylvestre	8.22	3.39	1.08	1.2

 

Table 4: EVALUATION OF PHYSICAL PARAMETERS AND ORGANOLEPTIC CHARACTERISTICS OF HERBAL FORMULATION

SL. No.	TEST	RESULT
1	pH of 10% v/v solution	4.543
2	Surface tension	43.37 dynes/cm
3	Total solid content	7% w/v
4	Specific gravity	1.03 at 25○ C
5	Viscosity	4.59 cp
6	Refractive index	1.37
7	Physical appearance	Slightly viscous liquid
8	Colour	Brownish
9	Taste	Sweet
10	Odour	Sweet aromatic

SAFE DOSE:

The LD₅₀ of the herbal formulation was found to be 3000mg/kg from the acute toxicity study. So from the observed LD₅₀ dose of herbal formulation 1/5^th and 1/10^th doses were selected for the present study.

 

Determination of OGTT activity:

The OGTT activity was performed and the results were satisfactory. The entire animal pass the OGTT test (results has not been displayed).

 

Effect of herbal formulation on body weight in Alloxan induced diabetic animal:

Table 5: Body weight in 11 days (gm) (Mean _­± SEM)

Groups	Initial	Day 3	Day 7	Day 11
Control	161.33 ±7.53	163±7.50	170.66±8.66	175.66±8.09
Diabetic control	182.66±8.66	178.33±7.79	166.66±6.93	162.66±6.93
Standard (5mg/kg)	181.66±7.83	178.33±7.53	174.33±7.51	169.66±7.21
Low dose (300mg/kg)	123.33±5.78	121.33±6.06	119±6.35	119.33±8.45
High Dose (600mg/kg)	185±8.38	180.66±6.93	176.66±7.83	170.33±7.79

Values are MEAN ± SEM (n=3) one way ANOVA followed by Dunnet’s multiple comparison test. Where the values are P<0.05; when compared with diabetic control group.

 

 

Graph 1: Showing the diagrammatic approach of body weight of the animal groups

 

Table 6: Anti diabetic activity of herbal formulation against Alloxan induced diabetic rats. Blood sugar level in 11 days mg/dl (Mean ± SEM). Values are MEAN ± SEM (n=3) one way ANOVA followed by Dunnet’s multiple comparison test. Where the values are P<0.05; when standard, low dose and high dose compared with diabetic control group.

Groups	Initial	Day 3	Day 7	Day 11
Control	69±2.88	72.33±3.18	70.66±3.18	71.73±3.47
Diabetic control	363.66±18.47	367.33±21.09	376.33±19.34	374.66±19.63
Standard (5mg/kg)	351.66±19.34	300±17.34	210.66±10.39	96.33±4.33***
Low dose (300mg/kg)	280.33±14.72	266.33±13.56	187.33±8.95	106.33±6.96**
High dose (600mg/kg)	288.66±15.01	268.66±14.14	187±8.38	101±6.96*

 

Graph 2: Showing the diagrammatic approach of blood glucose level of the animal groups

 

Table 7: % Glycemic change (Mean ± SEM)

Groups	Day 3	Day 7	Day 11
Glibenclamide (5mg/kg) Treated	15.47±0.63	40.24±1.81	73.18±2.26
Low dose (300mg/kg)	5.01±0.21	33.23±1.70	63.20±2.46
High dose (600mg/kg)	6.59±0.34	35.26±1.84	65.63±2.46

 

DISCUSSION:

One of the most commonly used chemical agent predominantly in laboratories to induce diabetes in animal is alloxan which is an oxidized product of uric acid that tends to destroy the islet cells of the pancreas by oxidation mechanism and producing Type 2 diabetes known as alloxan Induced diabetes. The present study was aimed to evaluate the antidiabetic activity of polyherbal formulation in alloxan induced diabetic rats. Glibenclamide is the standard drug used to stimulate insulin from β cells of islets of Langerhans for many years. Hence Glibenclamide (5mg/Kg) was selected as standard drug. Acute toxicity study showed that the herbal formulation was safe up to the dose of 3000mg/kg in rats. So two doses i.e.1/5^th and 1/10^th of LD₅₀ were used for the present study. The continuous treatment of the herbal formulation was done for the period of 11 days in two different doses high dose (600mg/kg) and low dose (300mg/kg). The results of the body weight and blood glucose level of control, diabetic control group, standard group (Glibenclamide 5mg/kg) and two different doses of trial formulation (600mg and 300mg/kg) are summarized in Table No 1 and Table No 2 respectively. Data are statistically obtained by paired ‘t’ test to analyze the significance of standard and trial drug between the test and control groups. The % Glycemic change in the blood sugar level is shown in Table No 3. The body weight of the control group was increased from 161.33±7.53 to 175.66±8.09 after 11 days. In standard drug group the initial body weight was 181.66±7.83 and after 11 days of treatment it was 169.66±7.21. The initial body weight of Herbal formulation low dose and high dose were 123.33±5.78 and 185±8.38 respectively and after 11 days of treatment the bodyweight was decreased to 119.33±8.45 and1 70.33±7.79 respectively. When compared between the two different doses, low dose and high dose, high dose reduced more weight than the low dose group. Two different doses of the herbal formulation (low dose and high dose) blood glucose level were studied in two different groups of animals. Both groups showed a significant decrease of blood glucose level on alloxan induced diabetic rats when compared to control group. The initial readings of blood glucose level of low dose and high dose were 280.33±14.72 and 288.66±15.01 respectively. After the trial period both doses produced marked reduction in the blood glucose level in 11 days (106.33±6.96 and 101±6.96). In standard group initial blood glucose level was 351.66±19.34 and the post test was 96.33±4.33 which showed that the standard drug produced maximum hypoglycemic effect and the statistical analysis was extremely significant and slightly higher than that of trial drug groups. Diabetic control group showed increase in blood glucose level throughout the entire study period. Initially blood glucose level of diabetic control group was 363.66.50±18.47and after 11 days of trial period the blood glucose level was increased to 374.66±19.63. The % Glycemic change was also found to be higher in the Glibenclamide treated group as compared to the test group.

 

Based on the results it can be observed that the trial poly herbal formulation which includes extracts of the five crude drugs has the ability to regulate pancreatic insulin secretion. Thus the mechanism of the hypoglycemic activity of poly herbal formulation may be due to the ability to regulate pancreatic insulin secretion and stimulatory effects on physiological pathways of insulin secretion.

 

CONCLUSION:

The result obtained from present study showed that the poly herbal formulation had a beneficial effect on blood glucose level in alloxan induced diabetic rats. Thus this herbal formulation can be an attractive material for further studies, leading to possible drug development for diabetes. In conclusion the present study showed that the investigation of the polyherbal formulation containing five herbs has a potent antidiabetic effect which could be used in the management of diabetes mellitus effectively. The antidiabetic activity may be due to the ability to regulate pancreatic insulin secretion and stimulatory effects on physiological pathways of insulin secretion. Thus further investigation is required to know the exact mechanism of the poly herbal formulation.

 

REFERENCES:

1.      Ali, M. (2009). "Present status of herbal medicines in India." Journal of herbal medicine and toxicology 3(2): 1-7.

2.      Avise, J. C. (2004). The hope, hype, and reality of genetic engineering: Remarkable stories from agriculture, industry, medicine, and the environment, Oxford University Press 5(3):56-59.

3.      Bussmann, R. W. (2002). Ethnobotany and biodiversity conservation. Modern trends in applied terrestrial ecology, Springer 6(4): 343-360.

4.      Casey, G. (2011). "The sugar disease—understanding type 2 diabetes mellitus." Kai Tiaki Nursing New Zealand 17(2): 16-21.

5.      Jamal, A. R. M. (2011). Ocimum sanctum L.: A review of phytochemical and pharmacological profile 2(3):76-84.

6.      Pandey, G. and S. Madhuri (2010). "Pharmacological activities of Ocimum sanctum (tulsi): a review." Int J Pharm Sci Rev Res 5(1): 61-66.

7.      Ashafa, A. O. T. (2012). Toxicity profile of ethanolic extract of Azadirachta indica stem bark in male Wistar rats. Asian Pacific Journal of Tropical Biomedicine 811-817.

8.      Pandey, G. (2014). Evaluation of phytochemical, antibacterial and free radical scavenging properties of Azadirachta indica (Neem) leaves. International Journal of Pharmacy and Pharmaceutical Sciences 6(2): 444-447.

9.      Omezzine, F., et al. (2012). Induction and flow cytometry identification of mixoploidy through colchicine treatment of Trigonella foenum-graecum L. African Journal of Biotechnology 11(98): 16434-16442.

10.   Yadav, R., et al. (2011). A pharmacognostical monograph of trigonella foenum- graecum seeds. International Journal of Pharmacy and Pharmaceutical Sciences 3(5): 442-445.

11.   Grover, J.K. (2000). Anti-hyperglycemic effect of Eugenia jambolana and Tinospora cordifolia in experimental diabetes and their effects on key metabolic enzymes involved in carbohydrate metabolism. Journal of Ethnopharmacology 73: 461–470.

12.   Nasreen, S. (2010). Assessment of quality of Tinospora cordifolia (wild.) miers. (Menispermaceae): pharmacognostical and phyto-physicochemical profile. International Journal of Comprehensive Pharmacy 5(03) 1-4.

13.   Baskaran, K. et al., (1990). Antidiaretic effect of a leaf extract from Gymnema sylvestre in non-insulin-dependent diabetes mellitus patients. Journal of Ethnopharmacology 30: 295–305.

14.   Patil, R. (2011). Current status of Indian medicinal plants with antidiabetic potential: a review. Asian Pacific Journal of Tropical Biomedicine. S291-S298.

15.   Kumar, G.,(2013). "Natural Product and Health-A Review on All Aspects." Current Research in Pharmaceutical Sciences 3(3): 68-79.

16.   Akbar, S. (2014). Pharmacognostic studies of stem, roots and leaves of Malva parviflora L. Asian Pacific Journal of Tropical Biomedicine 4(5): 410-415.

17.   Kumar D, Kumar K, Kumar S, Kumar T, Kumar A, Prakash O. Pharmacognostic evaluation of leaf and root bark of Holoptelea integrifolia Roxb. Asian Pac J Trop Biomed 2012; 2(3): 169–175.

18.   Anonymus. Pharmacopoeia of India. Vol. 2. New Delhi: The Controller of Publications; 1996. P. A47- A54.

19.   Bhutia S, Uriah T, Mohanty J P, Pal P, Kakoti B B. Physicochemical evaluation, gc-ms and antibacterial activity of essential oil of the fruit peels of Citrus macroptera montr. of Meghalaya north east India. World J. Ph Life Sci 2019; 5(4): 108-115.

20.   Stanley Irobekhian Reuben Okoduwa, Ismaila A Umar, Dorcas B James, Hajara M Inuwa, and James D Habila, Evaluation of extraction protocols for anti-diabetic phytochemical substances from medicinal plants, 2016 ; 7(20): 605–614.

21.   Bitasta, M. (2011). Comparative Standardization and Physicochemical Evaluation of the Leaves of Stevia rebaudiana Bertoni from Different Geographical Sources. Pharmacognosy Journal 3(25): 21-26.

22.   Prasad S.K. (2012). Physicochemical standardization and evaluation of in-vitro antioxidant activity of Aconitum heterophyllum Wall. Asian Pacific Journal of Tropical Biomedicine. S526-S531.

23.   Lipnick, R.L. et al., (1995). Comparison of Conventional LDs0, Toxicity the Up-and -Down and Fixed-Dose Acute Procedures. Food and Chemical Toxicology 33(3): 223-23.

24.    BRUCE R. D. (1985). An Up-and-Down Procedure for Acute Toxicity Testing. Fundamental and applied toxicology 5: 15 1- 157.

25.   Oliveira, H.C. et al., (2008). Antidiabetic activity of Vatairea macrocarpa extract in rats. Journal of Ethnopharmacology 115: 515–519.

26.   Esampally Sucharitha, Mamidala Estari, Evaluation of antidiabetic activity of medicinal plant extracts used by tribal communities in rural areas of Warangal district, Andhra Pradesh, India, Biology and Medicine, 5: 20–25, 2013

 

 

 

Received on 13.02.2020           Modified on 14.04.2020

Accepted on 09.05.2020         © RJPT All right reserved