INTRODUCTION:

Diabetes mellitus is a set of metabolic diseases characterized by hyperglycaemia starting from defects in insulin secretion, insulin action, or together. The chronic hyperglycaemia of diabetes is related with long-term damage, dysfunction, and malfunction of different organs, particularly the eyes, kidneys, nerves, heart, and blood vessels. A number of pathogenic processes are involved in the progress of diabetes. These range from autoimmune destruction of the β-cells of the pancrease with subsequent insulin deficiency to abnormalities that effect resistance to insulin action. The beginning of the abnormalities in carbohydrate, fat, and protein metabolism in diabetes is deficient action of insulin on target tissues. Symptoms of marked hyperglycaemia consist of polyuria, polydipsia, weight loss, occasionally polyphagia, and blurred vision¹.

Over the past 40 years, the prevalence of diabetes mellitus has increased worldwide and the tendency for the future is a continuous increase of all ethnic groups, men or women, for all age groups^2,3. This increase was observed above all in type 2 diabetes mellitus (T2DM)^4,5. In 1995, about 135 million people were affected by diabetes and an increase of 300 million cases was estimated for the year 2025⁶. The various complications of diabetes are coronary artery disease (CAD) including obesity, physical inactivity, hypertension (HT), and dyslipidemia⁷. Obesity increases the risk of CAD in adults and has been strongly associated with insulin resistance in normoglycemic persons and in individuals with type 2 DM.⁸

The treatment of diabetes depends upon the type of Diabetes mellitus. People who have Type 1 diabetes must plan and carefully follow their meals, meals and activity to check their blood sugar levels (sugar). It is important to measure blood sugar levels because even low blood sugar can be dangerous.⁹Patients with type 2 diabetes can monitor their blood sugar levels by following a diet, exercise program and excess weight loss. An oral drug can be added to the treatment plan, often with metformin first with other drugs if necessary. Patients with type 2 diabetes may also need injected insulin and in some circumstances may be used as the first medication.¹⁰

Allopathic drugs used for the treatment of diabetes have their own side effect & adverse effect like hypoglycaemia, nausea, vomiting, hyponatremia, flatulence, diarrhoea or constipation, alcohol flush, headache, weight gain, lactic acidosis, pernicious anaemia, dyspepsia, dizziness, joint pain. So instead of allopathic drugs, herbal drugs are a great choice which is having more or less no side effect & adverse effects.¹¹ There are many herbal plants which produce Antidiabetic activity. Ethyl acetate leaf extract of Azima tetracant shows antidiabetic activity against Streptozotacin induced diabetic rat.¹²

Coccinia grandis L. (Cucurbitaceae) is a well known medicinal herb, termed ivy gourd in English, Vimboshta, Vimbaja in Sanskrit, ban-kundri in Odia, kundree in Hindi. It is widely distributed throughout the world mainly in England, China, Bangladesh and India. In India it is mostly found in Odisha, Delhi, Tamilnadu, Karnatak, Maharastra, Gujrat, West bengal, Assam, Rajasthan. The fruit is ovoid, glabrous, 3.5 - 4.5 cm long and 1.5-2 cm thick, greenish-brown to yellowish-brown with white linings; characteristic odour and taste^,. Epicarp single layered; mesocarp composed of a wide zone of thin-walled parenchymatous cells differentiated into two regions, outer 5-6 layers rectangular to polygonal, smaller in size, while inner region composed of oval to polygonal cells of larger size; a few fibro-vascular bundles present in this region. Fruits of Coccinia grandis L. contains Alkaloid, Steroids, Tannins, Saponin, Ellagic acid, Phenols, Glycoside, Triterpenoids, Flavonoids, Taraxerone, Taraxerol (24R)-24- ethylcholest- 5- en- 3β- ol glucoside, Β- carotene, Lycopene, Cryptoxanthin, apo- 6’- lycopenal, Β- sitosterol, taraxerol. The extract of fruit of Coccinia grandis L. is traditionally used as hypoglycaemic agent. Green fruit cures sores on tongue and dried fruit removes eczema. Fruit juice is useful in treatment of infections caused by helminthes. Fruit is used in treatment of fever, leprosy, infective hepatitis, jaundice, asthma, ulcer, Tuberculosis and cough. Fruit is also used as Analgesic, Anti-inflammatory.¹³

The present study aimed to evaluate antidiabetic activity of ethanolic extract of fruit of Coccinia grandis in normoglycemic, adrenaline induced and alloxan induced diabetic rats.

MATERIAL AND METHODS:

Plant material:

Fresh fruits of C. grandis L. were collected in the month of September from Laumunda village, Bargarh, Odisha, India. The plant was identified with the help of available Indian literature.¹⁴A voucher specimen was deposited at Gayatri College of Pharmacy, Sambalpur.

Preparation of plant powder and extracts:

Ether and ethanol extracts of fruits were prepared sequentially by standard procedures in soxhelation apparatus. Matured unripe fruits were shade dried until properly dried, crushed in a mechanical grinder into fine powder. The powder (500g) was extracted sequentially with 1 litres of ether, 1 litres of ethanol in a Soxhlet apparatus at 65°C until the powder became exhausted totally. The resulting extracts were filtered, concentrated and dried in vacuo (yield 7.60, 8.25 and 8.75% w/w, respectively). The extracts were stored in desiccators for use in subsequent experiments.

Qualitative phytochemical analysis:

Preliminary phytochemical analysis was carried out for the extract as per standard methods described by Brain and Turner (1975) and Evans (1996).^15,16,17

Animals:

Healthy adult wistar albino rats weighing 180-240g were used for this study. Animals were allowed to acclimatize for a period of 15 days in the laboratory environment prior to the experiment. Rats were housed in standard polypropylene cages (three animals per cage), maintained under standard laboratory conditions (i.e. 12:12 h light and dark cycle; at an ambient temperature of 25±5°C, 35-60% of relative humidity)¹⁸, the animals were fed with standard rat pellet diet (Hindustan Lever Ltd. Mumbai) and water ad libitum. Animal House of Gayatri College of Pharmacy, Sambalpur was used for the study after prior scrutinization and approval from Institutional Animal Ethical Committee (No. 1339/Po/Re/S/10/CPCSEA).

Chemicals:

Adrenaline (Vasocon) was procured from Neon Laboratories ltd, Andheri (East), Mumbai. Alloxan monohydrate was procured from Loba Chemie, Mumbai. Other reagents used in the experiment were of analytical grade. Glipizide (Dibizide) a standard antidiabetic agent was purchased from Micro Labs Ltd (India).

Antihyperglycaemic studies:

Induction of hyperglycaemia was carried out in overnight fasted adult rats weighing 180-240g by a single intraperitoneal injection of Adrenaline at a dose of 0.8mg/kg body weight for Adrenaline induced hyperglycaemia. For Alloxan induced hyperglycaemia, Alloxan was used in the dose of 150mg/kg body weight. The hyperglycaemic rats were used for antihyperglycaemic study.

Experimental design:

1. Study of Antihyperglycaemic effect of ethanolic extract of Coccinia grandis fruit on normoglycemic rat

2. Study of Antihyperglycaemic effect of ethanolic extract of Coccinia grandis fruit on Adrenaline induced hyperglycemic rat

3. Study of Antihyperglycaemic effect of ethanolic extract of Coccinia grandis fruit on alloxan induced diabetic rat

Experimental design no 1:

Animals are divided into three groups of six rats per group. Test groups are administered Ethanol extracts at a dose of 250mg/kg body wt by oral route. Control groups are administered Normal saline at a dose 2ml/kg bodyweight. Standard groups were administered Glipizide orally at a dose of 1 mg/kg body wt.

The experimental design is as follows:

Group I – Normal Control (saline)

Group II – Test drug group (ethanol extracts of C. Indica fruit (250 mg/kg body wt)).

Group III – Standard drug group (Glipizide (1 mg/kg body wt)).

Blood glucose levels are estimated at 1, 3, 5 and 7 h after administration of drugs.

Experimental design no 2:

Animals are divided into four groups of six rats per group. Test groups are administered Ethanol extracts at a dose of 250mg/kg body wt by oral route. Control groups are administered Normal saline at a dose 2ml/kg bodyweight. Hyperglycaemic control groups are administered Adrenaline at a dose of 0.8mg/kg body weight. Standard groups were administered Glipizide orally at a dose of 1mg/kg body wt.

The experimental design is as follows:

Group I – Normal Control (saline)

Group II – Hyperglycemic control (Adrenaline 0.8 mg/kg body wt)

Group III – Test drug group (Hyperglycemic + ethanol extracts of C. Indica fruit (250 mg/kg body wt)).

Group IV – Standard drug group (Hyperglycemic + Glipizide (1 mg/kg body wt)).

Blood glucose levels are estimated at 1, 3, 5 and 7 h after administration of drugs

Experimental design no 3:

Animals are divided into four groups of six rats per group. Test groups are administered Ethanol extracts at a dose of 250mg/kg body wt by oral route. Control groups are administered Normal saline at a dose 2ml/kg bodyweight. Hyperglycaemic control groups are administered Alloxan at a dose of 150mg/kg body weight. Standard groups were administered Glipizide orally at a dose of 1mg/kg body wt.

The experimental design is as follows:

Group I – Normal Control (saline)

Group II – Hyperglycemic control (Alloxan 150mg/kg body wt)

Group III – Test drug group (Hyperglycemic + ethanol extracts of C. Indica fruit (250mg/kg body wt)).

Group IV – Standard drug group (Hyperglycemic + Glipizide (1mg/kg body wt)).

Blood glucose levels are estimated at 1, 3, 5 and 7 h after administration of drugs during acute study and are estimated at 1^st, 5^th, 10^th and 15^th day after administration of drugs during chronic study.

Collection of blood and determination of serum glucose:

Blood was withdrawn from the tail vein and glucose levels were estimated using glucose oxidaseperoxidase reactive strips and a glucometer (One touch select simple Glucometer (white))

RESULT:

Table no-1: Phytochemical analysis from alcoholic extract of Coccinia grandis L. Fruit

Sl. No	Test	Inference	Result
1	Alkaloids	Yellow cream precipitate	Present
2	Flavonoids	Yellow colour Precipitate	Present
3	Steroids	Violet to blue or green colour formation	Present
4	Terpenoids	Reddish brown precipitate	Present
5	Anthroquinone	No pink colour	Absent
6	Phenols	Deep bluish to black colour formation	Present
7	Saponins	Frothing Formation	Present
8	Tannins	Dark green formation	Present
9	Carbohydrates	No Brick color precipitate was formed.	Absent
10	Oil and Resin	No transparent in filter Paper	Absent

Table no-2: Effect of ethanolic extract of Coccinia indica fruits on blood glucose level of Normoglycemic albino rats

Group Number	Group	Dose (mg/kg body wt)	Blood glucose level (mg/dl) (mean ± SEM)
Group Number	Group	Dose (mg/kg body wt)	Initial	1h	3h	5h	7h
I	Normal Control (saline)	2 ml saline/kg body weight	96.56 ± 3.19	95.25± 3.09	94.22± 3.29	91.41± 3.40	89.23 ± 3.35
II	Ethanol extract	250 mg/kg b. wt.	94.17± 3.14	90.50 ± 3.40	87.42 ± 3.31	79.92 ± 3.42*	75.01 ± 4.19*
III	Standard drug (Glipizide)	1 mg/kg b. wt.	98.42 ± 3.51	88.38 ± 3.49	79.61 ± 4.15*	75.10± 4.53*	68.92 ± 5.19*

*P < 0.05–Significant; SEM–Standard Error of Mean, n–Number of animals in each group (6).Group II,III compared with I.

Table no-3: Effect of ethanolic extract of Coccinia indica fruits on blood glucose level of Adrenaline-induced acute hyperglycemic albino rats

Group number	Group	Dose (mg/kg body wt)	Blood glucose level (mg/dl ) (mean ± SEM)
Group number	Group	Dose (mg/kg body wt)	Initial	1h	3h	5h	7h
I	Normal Control (saline)	2 ml saline/kg body weight	90.56 ± 3.21	88.32 ± 3.19	86.27 ± 3.25	83.72 ± 3.37	80.74 ± 3.40
II	Hyperglycemic control	0.8mg Adrenaline/kg body weight	94.51± 3.28	208.31 ± 3.51	213.05 ± 3.83	201.89 ± 3.72	172.09 ± 4.01
III	Ethanolic extract	250 mg/kg b. wt.	93.12 ± 3.39	206.42 ± 3.38	199.21 ± 3.52	193.28 ± 4.15*	154.82 ± 4.89*
IV	Standard drug (Glipizide)	1 mg/kg b. wt.	91.41 ± 3.40	201.67 ± 3.51	180.83 ± 3.89	165.51 ± 4.52*	146.67 ± 5.59*

*P < 0.05–Significant; SEM–Standard Error of Mean, n–Number of animals in each group (6). Group III, IV compared with Group II.

Table no-4: Effect of ethanolic extract of Coccinia indica fruits on blood glucose level on Alloxan induced acute hyperglycaemic albino rats

Group number	Group	Dose (mg/kg body wt)	Blood glucose level (mg/dl) (mean ± SEM)
Group number	Group	Dose (mg/kg body wt)	Initial	1h	3h	5h	7h
I	Normal Control (saline)	2 ml saline/kg body weight	96.56 ± 3.29	88.32 ± 3.38	86.27 ± 3.19	83.72 ± 3.28	80.74 ± 3.32
II	Hyperglycemic control	Alloxan 150mg/kg body weight	98.51± 3.37	208.31 ± 3.49	213.05 ± 3.79	217.89 ± 3.82	222.09 ± 4.03
III	Ethanolic extract	250 mg/kg b. wt.	99.12 ± 3.21	202.42 ± 3.38	199.21 ± 3.52	193.28 ± 4.29*	174.82 ± 4.87*
IV	Standard drug (Glipizide)	1 mg/kg b. wt.	97.41 ± 3.40	200.67 ± 3.50	173.83 ± 3.85	159.51 ± 4.51*	146.67 ± 5.57*
	*P < 0.05–Significant; SEM–Standard Error of Mean, n–Number of animals in each group (6). Group III, IV compared with II.

Table no-5: Effect of ethanolic extract of Coccinia indica fruits on blood glucose level on Alloxan induced chronic hyperglycaemic albino rats

Group number	Group	Dose (mg/kg body wt)	Serum Glucose Level (mg/dl) on different Days of treatment
Group number	Group	Dose (mg/kg body wt)	Initial	5^th Day	10^th Day	15^th Day
I	Normal Control (saline)	2 ml saline/kg body weight	95.16 ± 3.21	93.52 ± 3.14	91.21 ± 3.39	91.09 ± 3.32
II	Hyperglycemic control	Alloxan 150mg/kg body weight	308.25 ± 6.28	306.21 ± 6.19	310.20 ± 6.34	316.3 ± 7.41
III	Ethanolic extract	250 mg/kg b. wt.	296.14 ± 5.39	284.2 ± 5.88	214.8 ± 4.16*	150.5 ± 5.67*
IV	Standard drug (Glipizide)	1 mg/kg b. wt.	317.15 ± 6.29	233.01 ± 6.64	167.71 ± 5.54*	113.81 ± 4.39*

*P < 0.05–Significant; SEM–Standard Error of Mean, n–Number of animals in each group (6). Group III, IV compared with Gr II.

Statistical analysis:

Data were statistically analysed using one-way ANOVA and expressed as mean ± S.E.M. followed by Dunnett’s t-test using computerized Graph pad instate version 3.05, Graph pad software, U.S.A.

DISCUSSION:

The preliminary phytochemical analysis revealed that the presence of some bioactive compounds which include Alkaloids, Flavonoids, Steroids, Terpenoids, Phenols, Saponins and Tannins.

The effect of ethanolic extracts of fruits on blood glucose levels of normoglycaemic rats are shown in Table no 2. The blood glucose level was reduced maximum in ethanol extract at 5^th and 7^th h after treatment. Blood glucose was depressed by 8.2% (P<0.05) in normoglycaemic rats after treatment which was comparable to standard drug, Glipizide.

Adrenaline causes a prompt increase in blood glucose concentration in the postabsorptive state. This effect is mediated by a transient increase in hepatic glucose production and an inhibition of glucose disposal by insulin-dependent tissues. Adrenaline augments hepatic glucose production by stimulating glycogenolysis and gluconeogenesis. Although its effect on glycogenolysis rapidly wanes, hyperglycemia continues because the effects of epinephrine on gluconeogenesis and glucose disposal persist. Adrenaline -induced hyperglycemia is markedly accentuated by concomitant elevations of glucagon and cortisol or in patients with diabetes. In both cases, the effect of epinephrine on hepatic glucose production is converted from a transient to a sustained response, thereby accounting for the exaggerated hyperglycemia. During glucose feeding, mild elevations of Adrenaline that have little effect on fasting glucose levels cause marked glucose intolerance. This exquisite sensitivity to the diabetogenic effects of Adrenaline is accounted for by its capacity to interfere with each of the components of the glucoregulatory response, i.e., stimulation of splanchnic and peripheral glucose uptake and suppression of hepatic glucose production. Adrenaline is an important contributor to stress-induced hyperglycemia and the susceptibility of diabetics to the adverse metabolic effects of stress²⁵.

The effect of ethanolic extracts of fruits on blood glucose levels of Adrenaline induced acute hyperglycaemic rats are shown in Table no 3. The blood glucose level was reduced maximum in ethanol extract at 5^th and 7^th h after treatment. Blood glucose was depressed by 8.2% (P<0.05) in adrenaline induced diabetic rats after treatment which was comparable to standard drug, Glipizide. This may be due to decrease in blood glucose concentration in the postabsorptive state by ethanolic extract. C. grandis fruit extract decrease hepatic glucose production by inhibiting hepatic glycogenolysis and gluconeogenesis.

Alloxan has two distinct pathological effects: it selectively inhibits glucose-induced insulin secretion through specific inhibition of glucokinase, the glucose sensor of the beta cell, and it causes a state of insulin-dependent diabetes through its ability to induce ROS formation, resulting in the selective necrosis of beta cells. These two effects can be assigned to the specific chemical properties of alloxan, the common denominator being selective cellular uptake and accumulation of alloxan by the beta cell²⁶.

The effect of ethanolic extracts of fruits on blood glucose levels of alloxan-induced acute hyperglycaemic rats is shown in Table no 4. The blood glucose level was reduced maximum in ethanol extract at 5^th and 7^th h after treatment. Blood glucose was depressed by 8.2% (P<0.05) in adrenaline induced diabetic rats after treatment which was comparable to standard drug, Glipizide.

The effect of ethanolic extracts of fruits on blood glucose levels of alloxan-induced chronic hyperglycaemic rats is shown in Table no 5. The blood glucose level was reduced maximum in ethanol extract at 10^th and 15^th day after treatment. Blood glucose was depressed by 8.2% (P < 0.05) in alloxan-induced diabetic rats after treatment which was comparable to the standard drug, Glipizide.

These effects may be due to the activation of the existing pancreatic cells in diabetic rats by the ethanolic extract. The pectin isolated from the fruits of C. grandis showed a significant reduction in blood glucose levels by decreasing the absorption level of glucose from the intestine, increasing the rate of liver glycogen and decreasing the glycogen phosphorylase.

CONCLUSION:

From the above experiment, it is concluded that Coccinia grandis L. fruit possess Antidiabetic activity. It produces hypoglycaemic effect in normoglycaemic rat as well as in both Adrenaline and Alloxan induced diabetic rat. It also produces antidiabetic effect in chronic hyperglycaemia in Alloxan induced chronic hyperglycaemic rat. It needs to investigate the underlying molecular mechanism of action, also long term effect on other animals.

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Received on 31.05.2019 Modified on 27.06.2019

Research J. Pharm. and Tech. 2019; 12(12): 5917-5922.

DOI: 10.5958/0974-360X.2019.01026.6