1. INTRODUCTION:

Diabetes mellitus is a serious complex chronic condition that is a major source of ill health worldwide. This metabolic disorder is characterized by hyperglycemia and disturbances in carbohydrate, protein and fat metabolisms, secondary to an absolute or relative lack of the hormone insulin. Besides hyperglycemia, several other factors including dyslipidemia or hyperlipidemia are involved in the development of micro and macrovascular complications of diabetes, which are the major causes of morbidity and death. According to World Health Organization (WHO) projections, the prevalence of diabetes is likely to increase 35% by 2020. Currently, there are over 150 million diabetics worldwide and this is likely to increase to 300 million or more by the year 2025. Statistical projection about India suggests that the number of diabetics will rise from 15 million in 1995 to 57 million in the year 2025, the highest number of diabetics in the world.

Reasons for this rise include increase in sedentary lifestyle, consumption of energy rich diet, obesity, higher life span, etc. Other regions with greatest number of diabetics are Asia and Africa, where diabetes mellitus rates could rise to 2–3 folds than the present rates. Evaluation of plant products to treat diabetes mellitus is of growing interest as they contain many bioactive substances with therapeutic potential. In recent years, several authors have reported the antidiabetic potential of traditionally used Indian medicinal plants using experimental animals. Although a large number of medicinal plants have been already tested for their antidiabetic effects, several other Indian medicinal plants remain to be investigated.

Peptic ulcer disease broadly refers to a group of disorders characterized by the presence of ulcers in any portions of g.i. tract, exposed to acid in sufficient duration and concentration although these ulceration most commonly occurs in the small intestine (duodenal ulcer) or stomach (gastric ulcer). The most common forms of peptic ulcers are gastric ulcer, duodenal ulcer, esophageal ulcer, meckel’s diverticulum ulcer. The majority of gastric ulcer can be attributed to either H. pylori or NSAIDs induced mucosal damage. Gastric ulcer that occurs in the pyloric area or those in the body associated with a duodenal ulcer. Gastric acid output tends to be normal or decrease in gastric acid animals. When gastric acid develops in the presence of minimal acid level, impairment of mucosal defense factor may be present .Pathogenesis of gastric ulcer usually normal to low acid levels; hyper acidity if present is due to high serum gastrin, damage to mucus barrier.

Jasminum sambac Linn. (Family-Oleaceae) commonly known as Motia or lily jasmine is a scandent or sub-erect shrub with young pubescent branches, broadly ovate or elliptic, opposite leaves, white, very fragrant flowers cultivated nearly throughout the tropical and sub-tropical parts of the world³. Traditionally leaves are used in fever or cough, indolent ulcer, abdominal distension, diarrhoea, lowering the blood glucose level, regulating menstrual flow, to clean kidney waste, inflamed and blood shot eyes^{1, 2}. The plant is reported to have antitumor³, antimicrobia4, antioxidant⁵, anti-acne⁶, suppression of puerperal lactation⁷, A.N.S stimulating effect. The present study was planned to evaluate the antidiabetic potential as well as antiulcer activity of flower extract of J. sambac.

MATERIALS AND METHODS:

Animals:

Experiments were performed on albino rats of either six (Albino Wistar strain) weighing about 200-250 g, divided into group of 6 animals per cage and maintained under standard laboratory conditions (12-h light/dark cycle, 24hrs). The food in the form of dry pellets and water are provided ad libitum. Young male chicks, 4-6 days of age, weighing from 32- 48 gm was obtained from poultry Traders. After 24 hrs fasting, the antiemetic activity was evaluated. All chicks were kept under laboratory conditions at room temperature with 12h light and dark cycles. All the animals were approved by the ethics committee of the institute. Screenings of Isolated compounds were done under Geethanjali College of Pharmacy, Cherryal, Keesara, Ranga Reddy District, Andhra Pradesh, India. Reg no: 1648/PO/A/12/CPCSEA.

Chemicals:

Alloxan monohydrate marketed sample of Qualikems fine chemical Pvt. Ltd, Streptozotocin marketed sample of Cayman chemicals (USA), Glibenclamide marketed sample of Sanofi India Ltd (Mumbai), Dextrose marketed sample of Triveni Chemicals, Jeecon Food Pvt Ltd (Delhi) Glucometer, Blood Gluco-strips one touch- horizon, marketed by Johnson and Johnson Ltd. All other chemicals and reagents used were of analytical grade.

Plant material:

Flowers of J.sambac were collected in local market of Hyderabad. The collected flowers were dried under shade at room temperature (25°C) for 10 days and powdered to a coarse consistency in a grinder mill. The powder was passed through 40 # mesh particle size and stored in an airtight container at room temperature.

Preparation of plant extract¹⁷

2.5 kg of the fresh air-dried powered crude drug of J.sambac was extracted with 95% ethanol by soxhelet apparatus at room temperature for 7 days. The extract was filtered and concentrated to dryness at room temperature to avoid the decomposition of the natural metabolites. The yield of the extracts was ethanol .The extract was preserved in a refrigerator till further use. Preliminary phytochemical analysis was carried out in by different methods of phytochemical analysis.

Experimental models:

Oral glucose tolerance test^14-16

Fasted rats were divided into six groups of six rats in each. Group I served as normal control and After 30 min of extract administration, the rats of all groups were orally treated with 2 g/kg of glucose. Blood samples were collected from the rat tail vein just prior to glucose administration and at 30, 60, 90 and 120 min after glucose loading. Blood glucose levels were measured immediately by using Glucometer.

Alloxan-induced diabetic model^7-13

Alloxan monohydrate was first weighed individually for each animal according to its weight and then solubilized with 0.2 ml saline just prior to injection. Diabetes was induced by injecting it at a dose of 150 mg/kg b. wt. intraperitonially. After 1 hr of alloxan administration, the animals were given feed ad libitum and 5% dextrose solution was also given in feeding bottle for a day to overcome the early hypoglycemic phase. The animals were kept under observation, and after 48 hr, blood glucose was measured by glucometer. One group served as a control which received vehicle alone. The diabetic rats (glucose level > 300 mg/dl) were separated and divided into six different groups for experimental study, with each group containing six animals. Group II were left untreated and served as diabetic controls. Group III received Glibenclamide 600 μg/kg, Group IV rats were treated with ethanolic extract of J.sambac at a dose of 200 mg/kg b. wt., Group V rats were treated with ethanolic extract of J.sambac 400 mg/kg b. wt. for 7 days.

Streptozotocin induced diabeteic model^{1, 4}

After 2 weeks of feeding with high fat food the rats were fasted for a period of 18 hours before induction of diabetes, and were injected intra-peritonially with a single dose of Streptozocin 60 mg/kg, freshly dissolved in normal saline solution. After the administration, the rats had free access to food (normal pellet diet) and water ad libitum. Diabetes in rats was identified by moderate polydipsia and marked polyuria. After 3 days i.e. 72hrs of injection blood glucose was measured by glucometer. Animal with blood glucose level >140mg/dl were separated and used for the study. One group served as a control which received vehicle alone. The diabetic rats (glucose level > 300 mg/dl) were separated and divided into five different groups for experimental study, with each group containing six animals. Group II were left untreated and served as diabetic controls. Group III received Glibenclamide 600 μg/kg, Group IV rats were treated with ethanolic extract of J.sambac at a dose of 200 mg/kg b. wt., Group V rats were treated with ethanolic extract of J.sambac 400 mg/kg b. wt.The selected rats were treated with similar test samples as above, but the blood glucose level was measured on 1, 3, 5, 7, 9 and 14 days of treatment.

ANTI ULCER ACTIVITY ^18-21

Pyloric ligation method:

In this method, albino rats were fasted in individual cages for 36 hr. Care was being taken to avoid coprophagy. Rats of either sex were randomly divided in to 5 groups of 6 animals in each group. Group-I: Normal control (Normal saline), Group-II: Ulcerated control (1% CMC, 5 ml/kg, p.o.)., Group-III: Ranitidine (30 mg/kg,i.p.)., Group-IV: Test extract 1(200mg/kg i.p) Group-V:Test extract 2 (400mg/kg i.p). Test extracts or reference drug or control vehicle was administered 30 min prior to pyloric ligation. Under light ether anesthesia, the abdomen was opened and the pylorus was ligated. The abdomen was then sutured. At the end of 19 h after ligation, the animals were sacrificed with excess of anesthetic ether, and the stomach was dissected out. Gastric juice was collected and its volume was measured. The glandular portion was then exposed and examined for ulceration. Ulcer index was determined.

Gastric cytoprotection methods (Ethanol induced ulcers):

Albino rats (Wistar) of 200-250 g are maintained under standard conditions (room temperature 24- 27^oC and humidity 60-65%) with 12 h light and dark cycle. The food in the form of dry pellets (Amrut Lab., Pune) and water were available ad libitum. Rats of either sex were randomly divided in to 5 groups of 6 animals in each group. Group-I: Normal control (Normal saline), Group-II: Ulcerated control (1 ml ethanol, p.o.), Group-III: Sucralfate (400 mg/kg,p.o.), Group-IV: Test extract 1 (200 mg/kg i.p), Group-V: Test extract 2 (400 mg/kg i.p). Thirty minutes after the test or reference drug or the control vehicle treatment, 1 ml of ethanol was orally administered to each rat. After 1 h the rats were euthanized with excess of anesthetic ether and stomach was cut open along the greater curvature, cleared of residual matter with saline and the inner surface was examined for ulceration. Ulcer index and % ulcer protection were calculated.

Determination of various parameters:

Determination of ulcer index (UI):

The ulcerative index was calculated by severity of gastric mucosal lesions and graded as Erosions 1mm or less,1-2mm and more than 2mm they were scored as 1,2,3 respectively.

Then the UI was calculated by using the formula:

UI = 1 (no. of lesions of grade 1) + 2 (no. of lesions of grade 2) + 3 (no. of lesions of grade 3).

Then the overall score was divided by a factor 10, which was designed as ulcer index. % gastro protection was calculated according to

% Gastro protection = (UIC-UIT)/UIC×100

Where, UIC-ulcer index of control, UIT-ulcer index of test.

Collection of gastric juice:

Gastric juice was collected from pylorus ligated rats. The gastric juice collected was centrifuged at 60 rpm for 10 min. and the volume of gastric juice was measured.

Determination of free acidity and total acidity:

Gastric juice (1 ml) was taken in to a 100 ml conical flask, to this 2-3 drops of Topfer’s reagent was added and titrated with 0.01 N NaOH until all traces of red colour disappears and the colour of the solution turns yellowish orange (end point). The volume of alkali added was noted. This volume corresponds to free acidity. Then 2-3 drops of phenolphthalein solution was added and titration was continued until a definite red tinge reappears. The volume of alkali added was noted which corresponds to total acidity. Acidity was calculated by using the formula:

Acidity (mEq/litre) = Volume of NaOH× Normality of NaOH×100/0.1

Statistical analysis:

The results of the study were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's t-test for multiple comparisons. Values with P <0.05 were considered significant.

RESULTS AND DISCUSSION:

ANTI DIABETIC ACTIVITY:

The data obtained from normal, glucose-hyperglycaemic and diabetic rats were shown in Table 1-5. The ethanolic extract of J. sambac, but no significant effects were observed. Glibenclamide, as used reference, showed significant activity in the same condition (Table1). Extract treated groups shown significant effects.

Table: 2 Effect of J.sambac extract on blood glucose levels (mg /dl) of alloxan induced diabetic rats

Group	Treatment	Blood glucose level mg/dl
Group	Treatment	0 hr	1hr	3 hr	5 hr	3^rdday	5^th day	7^th day
Group I	Normal control(Saline)	80.00±1.693	80.83±1.721	80.83±1.424	79.83±1.376	81.33±0.988	79.83±0.833	81.16±0.7923
Group II	Diabetic Control	322.33±7.775	327.50±7.945	329.50±7.388	336.67±6.515	369.00±6.110	388.33±16.591	413.50±4.752
Group III	Glibenclamide	277.33±7.923	206.66±6.280	174±7.095	154.83±5.043	125.33±6.960	114.33±5.251	105.66±5.097***
Group IV	Test extract 1 (200mg/kg)	348.5±11.730	311±10.517	295±12.757	287.3±12.236	259±15.908	242.16±14.437	320.83±13.516*
Group V	Test extract 2 (400mg/kg)	297.5±10.388	245.33±14.769	212±18.475	206.83±18.757	166.83±15.993	137.5±12.066	126.66±13.012**

Anti Diabetic effect of J.sambac extract Values are mean ± SD, n = 6 in each group, *P<0.05, **P<0.01 when compared with diabetic control group (alloxan induced)

Table:3 Effect of J.sambac extract on blood glucose levels (mg /dl) of streptozotocin induced diabetic rats

Group	Treatment	Blood glucose level mg/dl
Group	Treatment	1^st day	3 ^rdday	5 ^thday	7 ^thday	14^th day
Group I	Normal control(Saline)	111.4 ±6.34	113.3 ±7.43	114.5 ±8.84	112± 9.97	165 ± 7.73
Group II	Diabetic Control	284.9 ±93.55	283.9 ±91.25	279.8 ±88.71	283 ± 43.92	281.8 ±37.44
Group III	Glibenclamide	289.4 ±3.8	258.9 ±4.9	163.3± 6.51	137.3 ± 3.96	125.9 ±5.4***
Group IV	Test extract 1 (200mg/kg)	287.4 ±13.2	263.4 ±18.5	198.2 ±20.8	152.6 ±24.54	133.8 ±29.4*
Group V	extract Test 2 (400mg/kg)	289 ±66.2	261.4 ± 68.2	187.5 ±78.72	104.5 ±86.1	124.8 ±75.1**

Anti Diabetic effect of J.sambac extract Values are mean ± SD, n = 6 in each group, *P<0.05, **P<0.01 when compared with diabetic control group (streptozotocin induced)

Table: 4 Lipid profile in alloxan induced diabetis rats

Group	Treatment	Cholesterol (mg/dL)	TGs (mg/dL)	HDL (mg/dL)
Group I	Normal control(Saline)	56.17±2.358	65.17±1.797	41.33±1.022
Group II	Diabetic Control	127.33±2.246	77.67±1.333	30.67±0.954
Group III	Glibenclamide	64.67±1.801	63.17±1.302	40.67±1.202
Group IV	Test extract 1 (200mg/kg)	120.17±1.973	75.17±1.42	31.17±0.601
Group V	Test extract 2 (400mg/kg)	84±2.033	64.33±1.801	39.17±0.833

Anti Diabetic effect of J.sambac extract Values are mean ± SD, n = 6 in each group, *P<0.05, **P<0.01 when compared with diabetic control group (alloxan induced)

Table: 5 Lipid profile in streptozotocin induced diabetis rats

Group	Treatment	Cholesterol (mg/dL)	TGs (mg/dL)	HDL (mg/dL)
Group I	Normal control(Saline)	60.11±2.134	70.17±1.89	38.33±2.111
Group II	Diabetic Control	135.36±1.786	80.69±2.423	32.72±1.775
Group III	Glibenclamide	65.53±1.713	62.66±1.543	43.66±1.716
Group IV	Test extract 1 (200mg/kg)	115.321±1.973	80.212±1.42	33.211±1.601
Group V	Test extract 2 (400mg/kg)	70±2.112	63.33±2.801	33.441±1.233

Anti Diabetic effect of J.sambac extract Values are mean ± SD, n = 6 in each group, *P<0.05, **P<0.01 when compared with diabetic control group (streptozotocin induced)

ANTI ULCER ACTIVITY:

Pyloric ligation method:

Table:6 Effect of J.sambac flower extract on volume, pH of gastric content, total acidity, free acidity in pylorus ligated rats

Treatment Dose (mg/kg)	Volume of gastric juice in ml	pH	Total acidity	Free acidity
Control	5.5±0.428	3.77±0.03	17.9±0.368	0.198±0.007
Ulcerated control	6.18±0.17	1.67±0.05	45.7 ± 1.21	0.252±0.009
Ranitidine(30mg/kg)	1.73±0.095	3.50±0.06	6.21±0.200	0.075±0.004
Test extract 1(200mg/kg)	4.96±0.240	2.38±0.07	15.58±0.407	0.178±0.006
Test extract 2(400mg/kg)	2.56±0.182	3.34±0.10	7.21±0.302	0.101±0.004

Values are mean ± SEM for 6 rats.

Table:7 Effect of J.sambac flower extract on ulcer index and % gastric ulceration in pylorus ligated rats

Treatment Dose (mg/kg)	Ulcer index	% Gastric protection
Control	00.00±0.00	-
Ulcerated control	10.25±0.891	-
Ranitidine(30mg/kg)	1.66±0.088	83.8
Test extract 1(200mg/kg)	5.33±0.494	48.0
Test extract 2(400mg/kg)	2.55±0.199	75.12

Ethanol induced ulcers:

Table 8:Effect of J.sambac flower extract on volume, pH of gastric content, total acidity, free acidity in ethanol induced ulcers in rats

Treatment Dose (mg/kg)	Volume of gastric juice in ml	pH	Total acidity	Free acidity
Control	2.23 ± 0.22	1.89±0.12	73.48±3.33	47.30±2.73
Ulcerated control	3.47±0.28	1.26±0.19	101.59±9.56	81.35±2.56
Sucralfate (400 mg/kg)	1.43±0.09	2.84±0.32	39.99±2.96	24.64±1.08
Test extract 1(200mg/kg)	2.36±0.10	2.32±0.18	53.34±3.59	30.62±1.77
Test extract 2(400mg/kg)	1.82±0.28	2.69±0.20	43.76±2.03	26.45±0.74

Table:9 Effect of J.sambac flower extract on ulcer index and % gastric ulceration in ethanol induced ulcers in rats

Treatment Dose (mg/kg)	Ulcer index	% Gastric protection
Control	00.00±0.00	-
Ulcerated control	18.5±4.86	-
Sucralfate (400 mg/kg)	1.45±0.32	92.16
Test extract 1(200mg/kg)	5.10±1.21	72.43
Test extract 2(400mg/kg)	2.50±0.99	86.49

All values are in mean ± SEM, n=6, VGJ = Volume of gastric juice in ml,

TA = Total acidity in mEq/l., FA = Free acidity in mEq/l.

UI = Ulcer index, P <0.001 = *

The volume of Sodium hydroxide required corresponds to the total acidity. Acidity

(mEq /l/100g) was calculated as; Acidity = {Volume of NaOH X Normality X 100 mEq/l/100g X 0.1}

Effect of J.sambac flower extract in pylorus ligated rats

Pylorus ligation in ulcerated control group had produced ulcer in all animals and the mean ulcer index was 10.25±0.891 indicating the ulcerogenic effect. Another ulcerogenic effect as compared to normal control was measured as follows (Table 6 and 7); mean gastric content volume as 6.18±0.17, pH as 1.67±0.05, free acidity as 0.252±0.009, total acidity as 45.7 ± 1.21, indicating the ulcer production in animals. Pylorus ligation also produced ulcers in all the J.sambac flower extract pretreated animals. However, the ulcer index showed significant dose dependent reduction in the animal pretreated with test extract 200 mg/kg (UI; 5.33±0.494 ) and 400 mg/kg (UI:2.55±0.199 ).It indicated 48.00% gastroprotection at 200 mg/kg and 75.12% gastroprotection at 400 mg/kg as compared with ulcerated control. The results indicate that the higher dose of test extract at 400 mg/kg was effective in protecting ulcers in pylorus ligated rats. Pylorus ligation had produce ulcers in all animals pretreated with Ranitidine 20 mg/kg. However, ulcer index (1.66 ± 0.088) showed significant reduction as compared with ulcerated control and showed 83.80% gastroprotection.

Effect of J.sambac flower extract in ethanol induced gastric ulcer:

Ethanol induced ulcerated control group had produced ulcer in all animals and the mean ulcer index was 18.5±4.86 indicating the ulcerogenic effect. However, the ulcer index showed (Table 7 and 9) significant dose dependent reduction in the animal pretreated with test extract 200 mg//kg (UI; 5.10±1.21) and 5 mg/kg (UI; 2.50±0.99). It indicated 72.43% gastro protection at 200 mg/kg and 86.49% gastro protection at 400 mg/kg as compared with ulcerated control. The results indicate that the higher dose of test extract i.e. 400 mg/kg was effective in protecting ulcers in ethanol induced gastric ulcers in rats. Ethanol had produced ulcers in all animals pretreated with Sucralfate 400 mg/kg. However, ulcer index (1.45±0.32) showed significant reduction as compared with ulcerated control and showed 92.16 % gastro protection.

CONCLUSION:

The blood glucose levels of Jasminum sambac extract treated animals was found to be significantly less in oral glucose tolerance test, alloxan induced diabetes, streptozotocin induced diabetes models compared to diabetic control. Similarly the ulcer index in the Jasminum sambac extract t treated animals was found to be significantly less in all the models compared to vehicle control animals. Our results suggest that J.sambac extract possesses significant antidiabetic property as well as antiulcer property.

ACKNOWLEDGEMENTS:

I am very much thankful to Dr. M. Ravi Kumar, Principal, Geethanjali College of Pharmacy, For his guidance, kind help and constant encouragement at every step during the progress of my work without which successful completion of this work would not have been possible. It is my pleasure to express my sincere thanks to Mr. G. Ravinder Reddy, secretary, Teja Educational Society, for providing laboratory facilities and chemicals. I am also grateful to my friends Mr. J. Sunil and Mr. P. Randheer Reddy for their kind help from time to time at each and every step of my project work.

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Received on 02.04.2014 Modified on 06.05.2014

Asian J. Research Chem. 7(6): June 2014; Page 580-585

Group	Treatment	Blood glucose level in mg/dl
Group	Treatment	0 min	30 min	60 min	90 min	120 min
Group I	Normal control(Saline)	101.58 ± 4.53	163.84 ± 7.33	129.03 ± 5.28	110.13 ± 3.71	104.62 ± 3.81
Group II	Diabetic Control	367.97 ± 35.72	562.68 ± 27.47	528.72 ± 15.37	483.78 ± 16.76	454.71 ± 21.11
Group III	Glibenclamide	300.67 ± 27.42	479.22 ± 27.36	440.72 ± 16.54	422.78 ± 12.86	372.89 ± 19.70***
Group IV	Test extract 1 (200mg/kg)	342.77 ± 16.44	507.92 ± 33.28	461.28 ± 28.10	421.40 ± 24.88	382.77 ± 29.53*
Group V	Test extract 2 (400mg/kg)	308.77 ± 18.50	446.93 ± 22.20	429.66 ± 24.99	396.68 ± 14.76	379.52 ± 14.76**