INTRODUCTION:

Diabetes mellitus is one of the common metabolic disorders with micro and macro vascular complications that results in significant morbidity and mortality[1]. It is considered as one of the five leading causes of death in the world [4, 5]. Diabetes mellitus is a group of syndrome characterized by hyperglycemia, altered metabolism of lipids, carbohydrates and proteins. It is often called ‘The silent killer’ resulting in serious damage to eyes, kidneys, nerves, heart and blood vessels [2, 3]. The common signs and symptoms are excessive thirst and urination, weight loss or gain and fatigue. Early diabetes symptoms can be very mild and often even unnoticeable. Diabetes mellitus is seen when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces. Most patients can be classified clinically as having either Type 1 diabetes mellitus (IDDM),

An autoimmune disorder where there is destruction of beta cells and cell loss resulting in T-cell mediated autoimmune attack [6].Type 2 diabetes mellitus (NIDDM) is due to insulin resistance or reduced insulin sensitivity, combined with reduced insulin secretion. Variants in 11 genes significantly associated with the risk of Type 2 diabetes of these 8 genes are responsible for impaired beta-cell function[7]. The potential role of medicinal plants as hypoglycemic agents has been reported in many studies supported by ethnobotanical surveys and the use of traditional medicines in numerous cultures. Euphorbia nivulia Buch has been used for treating diabetes.

Therefore, the purpose of this study was to investigate the hypoglycemic effect using petroleum ether, methanolic and aqueous extracts of Euphorbia nivulia in rats and also to determine biochemical data.

MATERIALS AND METHOD:

Plant material:

The leaves of Euphorbia nivulia Buch were collected from the Ramnagar, Karnataka, identified and authenticated by Dr. M. D. Rajanna Professor (Curator) Botanical Garden University of Agricultural Sciences GKVK, Bangalore, Karnataka, India.

Prepration of extracts:

Petroleum ether (PEEN), methanolic (MEEN) and aqueous extracts (AEEN) of Euphorbia nivulia Buch were obtained from Green Chem, Anakel road Bangalore-107.

Animals:

Albino Wistar rats weighing 150-220g were procured from Biogen, Bangalore. They were maintained in the animal house of Gautham College of Pharmacy. Animals were maintained under controlled condition of temperature at 27^o ± 2^oC and 12-h light-dark cycles. They were housed in polypropylene cages and had a free access to standard pellets (Amruth) and water ad libitum. All the studies conducted were approved by the Institutional Animal Ethical Committee (IAEC) of Gautham College of pharmacy, Bangalore (REF-IAEC/003/5/2010) according to prescribed guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (REF-IAEC/02/06/2012-13), Govt. of India.

Chemicals used:

Glibenclamide and Alloxan Monohydrate was purchased from Sigma Swemed Diagnostics Pvt, Ltd, Bangalore. All other chemicals and reagents used were of analytical grade.

Phytochemical screening:

Preliminary phytochemical analysis was carried out to detect the secondary metabolites present in PEEN, MEEN and AEEN extracts – alkaloids, flavonoids, saponins, sterols tannins and terpenes. (Trease 1983)

Acute Toxicity studies [11]:

According to the OECD guideline no. 425, the acute oral toxicity study was performed. Female Albino rats weighing 150-220g were used for the study. They were nulliparous and non-pregnant. These were acclimatized to laboratory condition for one week prior to start of dosing. All the three extracts were studied to determine the safety and dose.

Experimental Design [10, 12]:

A. Single dose study in normal rats:

Albino Wistar rats weighing 150-200 g were divided into five groups of six in each group. Animals were fasted overnight for 16 h prior to the experiment. The blood glucose levels were measured just prior to 0, 1, 2, 4, 8 and 12 h after drug administration. The blood glucose levels were measured from the tail vein by using Sugar check glucometer manufactured by Wockhardt.

Group-I: Vehicle control.

Group-II: Animals received a dose of 5 mg/kg b.w. of Glibenclamide p.o. and served as standard

Group-III: Animals received a single dose of 100 mg/kg b.w. of PEEN p.o.

Group-IV: Animals received a single dose of 100 mg/kg b.w. of MEEN p.o.

Group-V: Animals received a single dose of 100 mg/kg b.w. of AEEN p.o.

B. Oral glucose tolerance test in normal rats (OGTT):

The oral glucose tolerance test was performed in rats weighing 150-200g. The animals were fasted for 16 h before the experiment but allowed free access to water. These rats were divided into five groups, six in each group. Rats of all groups were loaded with glucose 2g/kg p.o 30 min after drug administration. Blood samples were collected from the tail vein prior to drug administration 0, 30, 60, 90 and 120 min of glucose administration.

Group-I: Vehicle control.

Group-II: Animals received a dose of 5 mg/kg b.w. of Glibenclamide p.o. and served as standard

Group-III: Animals received a single dose of 100 mg/kg b.w. of PEEN p.o.

Group-IV: Animals received a single dose of 100 mg/kg b.w. of MEEN p.o.

Group-V: Animals received a single dose of 100 mg/kg b.w. of AEEN p.o.

C. Antidiabetic activity (Alloxan induced diabetes model in rats):

i. Preparation of alloxan (ALX) solution:

Alloxan monohydrate dissolved on 0.9% sodium chloride solution.

ii. Experimentally Induced Diabetes Mellitus:

Female Wistar rats weighing 150-220g were used for this study. The animals were overnight fasted for 16h before the induction of diabetes. Diabetes was induced by a single dose of 120 mg/kg body weight of alloxan by intraperitoneal route. After a period of 2 days blood glucose levels were checked by snipping the tail of alloxan treated fasted rats. Rats showing the blood glucose levels more than 300 mg/dL were selected for the study.

Diabetic rats were divided into five groups (Group II – VI).

Ø Group-I: Vehicle control (Non diabetic rats/ normal rats)

Ø Group-II: Diabetic rats.

Ø Group-III: Diabetic rats received Glibenclamide (5 mg/kg.b.w.,p.o.) for 21 days and served as standard.

Ø Group-IV: Diabetic rats received PEEN (100 mg/kg.b.w.,p.o.) for 21 days.

Ø Group-V: Diabetic rats received MEEN (100 mg/kg.b.w.,p.o.) for 21 days.

Ø Group-VI: Diabetic rats received AEEN (100 mg/kg.b.w.,p.o.) for 21 days.

Fasting blood glucose levels were measured before the administration of PEEN, MEEN and AEEN. It was recorded as day 0. The blood glucose levels were checked on day 7, 14, and 21 of the treatment period. Blood was collected from snipping of the rat tail. Blood glucose levels were measured by using Sugarchek glucometer manufactured by Wockhardt.

At the end of the experimental period, all the animals were sacrificed and different organs heart, pancreas, liver, kidney and spleen was weighed. Blood was collected and serum was used for the estimation of various biochemical parameters like albumin, urea, creatinine, total protein, HDL, VLDL, TG and TC. LDL was also calculated.

Statistical Analysis:

The values are expressed as Mean ± SEM. The data was analysed by using one way ANOVA followed by Dunnett’s test using Graph pad prism software (version 6.01). Statistical significance was set at P ≤ 0.05.

RESULTS AND DISCUSSION:

Preliminary phytochemical constituents [13]:

All the different extracts revealed the presence of alkaloids, carbohydrates, flavonoids, glycosides, phytosterols/ terpenes, proteins and saponins.

Acute oral toxicity studies:

Single dose administration of PEEN, MEEN and AEEN at 2000mg/kg b.w. showed no mortality in any of the animals. Hence, 1/20^th of the dose (100 mg/kg b.w.) was selected for the present antidiabetic study.

Antidiabetic activity:

The effect of oral administration of petroleum ether, methanolic and aqueous extract of the leaves of Euphorbia nivulia (PEEM, MEEN and AEEN) showed an significant changes in single dose study in normal rats and oral glucouse tolerance test in normal rats was showed in table - 1. The effect of repeated oral administration of methanolic, petroleum ether and aqueous extract of the leaves of Euphorbia nivulia (PEEN, MEEN and AEEN) showed an significant changes in blood glucose levels and various organ weight in alloxan-diabetic rats is presented in table- 2 , and the effect on body weight and different serum parameters levels is presented in table- 3. PEEN and MEEN (100mg/kg.,b.w.) in alloxan-treated diabetic rats caused significant dose related and duration dependent reduction of blood glucose levels when compared to AEEN(100mg/kg.,b.w.). Maximum reduction was observed on day 21in ALX induced diabetic rats. Gradual increase in body weight was also observed. PEEN and MEEN (100mg/kg.,b.w.) exhibited maximum glucose lowering effect in diabetic rats and also significant changes in the serum parameters level when compared with the AEEN (100mg/kg.,b.w.). Glibenclamide exhibited significant reduction in blood glucose levels at the end of the study when compared to diabetic control.

PEEN – Petroleum ether extract of Euphorbia nivulia.

MEEN – Methanolic extract of Euphorbia nivulia.

AEEN – Aqueous extract of Euphorbia nivulia.

Table 1 : Single dose and Oral Glucouse Tolerance Test

Effect of extracts on blood glucose levels in normal rats.
Blood glucose levels(mg/dL)
Time in hours		Groups
		Group-I Vehicle control		Group-II Glibenclamide (5mg/kg, b.w.)		Group-III PEEN (100mg/kg, b.w.)	Group-IV MEEN (100mg/kg, b.w.)	Group-V AEEN (100mg/kg, b.w.)
Fasting		96.50 ± 3.16		93.17 ± 2.63		94.17 ± 1.95	93.33 ± 2.18	95.83 ± 2.33
1^sth		94.00 ± 2.53		82.33 ± 2.07^**		87.83 ± 1.62^ns	87.17 ± 1.62^ns	92.17 ± 1.79^ns
2^nd h		95.33 ± 2.72		75.67 ± 1.80^***		85.83 ± 2.12^*	81.83 ± 1.86^***	88.17 ± 1.72^ns
4^th h		92.00 ± 2.32		67.33 ± 1.38^***		83.67 ± 2.20^*	76.67 ± 1.45^***	85.00 ± 1.29^*
8^thh		91.17 ± 2.16		80.83 ±2.75^***		91.17 ± 2.04^ns	90.33 ± 1.33^ns	93.17 ± 2.30^ns
12^th h		93.67 ± 3.90		90.67 ± 2.48^ns		93.83 ± 2.12^ns	93.50 ± 1.80^ns	94.33 ± 1.33^ns
Oral glucose tolerance test in normal rats.
Blood glucose levels(mg/dL)
Time in minutes	Groups
	Group-I Vehicle control		Group-II Glibenclamide (5mg/kg, b.w.)		Group-III PEEN (100mg/kg, b.w.)		Group-IV MEEN (100mg/kg, b.w.)	Group-V AEEN (100mg/kg, b.w.)
Fasting	96.17 ± 5.93		94.67 ± 6.02		94.50 ± 3.61		90.83 ± 4.86	98.17 ± 2.63
30 min	161.8 ± 3.19		87.00 ± 0.93^***		138.2 ± 6.77^**		134.7 ± 5.37^**	144.7 ± 4.52^**
60 min	147.8 ± 4.61		74.33 ± 5.54^***		125.5 ± 3.88^**		119.8 ± 2.28^***	126.6 ± 5.97^**
90 min	130.5 ± 6.26		68.17 ± 4.93^***		113.7 ± 2.66^*		108.8 ± 2.77^**	114.0 ± 4.56^*
120 min	123.2 ± 4.87		54.83 ± 2.70^***		102.0 ± 3.93^**		99.17 ± 3.26^***	101.5 ±3.65^**

Values are expressed as mean ± SEM (n=06)

Data were analyzed by one way ANOVA followed by Dunnett’s t test. *** P<0.001, ** P<0.01 and *P<0.05.

^a compared with Vehicle control, ^bcompared with the alloxan treated group

Table 2: ALX Model Sheet Blood Glucouse level and Various Organs Weight.

Alloxan induced diabetes model in rats.
Physical Parameters
Parameters	Groups
Parameters	Group-I Vehicle control		Group-II ALX (120mg/kg, b.w.) + Saline	Group-III ALX (120mg/kg, b.w.) + Glibenclamide (5mg/kg, b.w.)	Group-IV ALX (120mg/kg, b.w.) + PEEN(100mg/kg, b.w.)	Group-V ALX (120mg/kg, b.w.) + MEEN (100mg/kg, b.w.)	Group-VI ALX (120mg/kg, b.w.) + AEEN(100mg/kg, b.w.)
Body weight(g)	Day 0	169.00 ± 1.12	165.00 ± 1.82	172.50 ± 1.52	166.50 ± 0.71	163.70 ± 1.11	174.70 ± 1.82
	Day 7	174.80 ± 1.01	153.5 ± 1.38^a***	174.0 ± 1.84^b***	162.0±0.77^b***	160.8±0.90^b**	171.0 ± 1.88^b***
	Day 14	183.7 ± 1.02	138.7 ± 2.18^a***	176.7 ± 1.28^b***	164.3 ± 1.25^b***	164.5 ± 0.99^b***	172.3 ± 2.37^b***
	Day 21	195.5 ± 2.52	129.2 ± 0.94^a***	180.3 ± 1.70^b***	168.7 ± 1.52^b***	167.8 ± 1.35^b***	173.8 ± 2.49^b***
Organ weight(g)	Pancreas	0.82 ± 0.02	0.40 ± 0.02^a***	0.70 ± 0.06^b***	0.57 ± 0.01^b**	0.61 ± 0.09^b***	0.56 ± 0.02^b*
	Liver	6.83 ± 0.40	4.20 ± 0.30^a***	6.23 ± 0.18^b***	5.32 ± 0.25^b*	5.73 ± 0.17^b**	5.33 ± 0.16^b*
	Heart	0.81 ± 0.03	0.46 ± 0.03^a***	0.73 ± 0.04^b***	0.59 ± 0.01^b*	0.62 ± 0.01^b***	0.59 ± 0.03^b*
	Kidney	1.70 ± 0.05	0.97 ± 0.06^a***	1.59 ± 0.04^b***	1.33 ± 0.03^b***	1.38 ± 0.03^b***	1.33 ± 0.04^b***
	Spleen	0.82 ± 0.02	0.44 ± 0.03^a***	0.77 ± 0.04^b***	0.64 ± 0.04^b*	0.69 ± 0.05^b**	0.62 ± 0.03^b*

Values are expressed as mean ± SEM (n=06)

Data were analyzed by one way ANOVA followed by Dunnett’s t test. *** P<0.001, ** P<0.01 and *P<0.05.

^a compared with Vehicle control, ^bcompared with the alloxan treated group

Table 3: ALX Model Sheet of Body Weight and Various Organs weight.

Alloxan induced diabetes model in rats.
Biochemical Parameters
Parameters		Groups
Parameters		Group-I Vehicle control	Group-II ALX (120mg/kg, b.w.) + Saline	Group-III ALX (120mg/kg, b.w.) + Glibenclamide (5mg/kg, b.w.)	Group-IV ALX (120mg/kg, b.w.) + PEEN(100mg/kg, b.w.)	Group-V ALX (120mg/kg, b.w.) + MEEN(100mg/kg, b.w.)	Group-VI ALX (120mg/kg, b.w.) + AEEN(100mg/kg, b.w.)
Blood glucose levels (mg/dL)	Day 0	110.0 ± 3.152	465.2 ± 10.15	410.7 ± 8.50	422.3 ± 7.68	410.7 ± 8.31	431.2 ± 6.47
	Day 7	111.3 ± 2.84	437.5 ± 15.15^a***	208.7 ± 4.70^b***	396.0 ± 5.16^b**	378.3 ± 12.52^b***	400.2± 2.63^b*
	Day 14	99.50± 1.92	374.0 ± 5.54^a***	172.5 ± 6.94^b***	321.8 ± 12.45^b**	312.5 ± 12.80^b***	322.8± 14.41^b**
	Day 21	97.67 ±1.33	333.7 ± 10.60^a***	101.8 ± 2.88^b***	166.5 ± 7.92^b***	143.5 ± 7.99^b***	152.2± 6.48^b***
Serum albumin (g/dL)		5.23 ± 0.11	2.82± 0.26^a***	4.91 ± 0.13^b***	3.41 ± 0.12^b*	3.60 ± 0.10^b**	3.48 ± 0.09^b*
Serum urea (mg/dL)		30.95 ± 2.33	100.8 ± 23.33^a***	40.68 ± 0.15^b***	58.15 ± 0.29^b***	54.15 ± 0.61^b***	57.16 ± 0.25^b***
Serum total protein (mg/dL)		9.26 ± 0.13	4.53± 0.13^a***	8.29 ± 0.13^b***	5.84 ± 0.49^b*	6.10 ± 0.12^b**	5.95± 0.54^b*
Serum creatinine (mg/dL)		0.959 ± 0.02	1.673 ± 0.06^a***	1.031 ± 0.008^b***	1.170 ± 0.02^b***	1.126 ± 0.03^b***	1.153± 0.01^b***
Hemoglobin (mg/dL)		15.38 ± 0.85	9.16± 0.27^a***	14.10 ± 0.79^b***	11.76 ± 0.27^b**	12.25 ± 0.23^b***	11.20 ± 0.07^b*
Lipid profile (mg/dL)	TG	71.01 ± 2.15	134.1 ± 2.07^a***	94.93 ± 2.61^b***	115.2 ± 1.86^b***	108.0 ± 2.61^b***	115.9 ± 2.15^b***
	TC	89.44 ± 2.84	133.1 ± 3.45^a***	91.39 ± 1.45^b***	104.7 ± 2.08^b***	100.6 ± 2.90^b***	106.1± 2.42^b***
	HDL-C	28.33 ± 0.61	12.50 ± 0.91^a***	24.38 ± 1.10^b***	18.13 ± 0.53^b***	19.79 ± 0.75^b***	18.96 ± 1.26^b***
	LDL-C	46.91 ± 2.99	93.41 ± 3.33^a***	51.06 ± 0.99^b***	63.55 ± 2.19^b***	59.17 ± .88^b***	63.97 ± 2.38^b***
	VLDL-C	14.20 ± 0.42	26.81 ± 0.41^a***	18.98± 0.52^b***	23.04 ± 0.37^b***	21.59 ± .52^b***	23.19 ± 0.42^b***
SOD U/mg protein		15.71 ± 0.19	7.91± 0.12^a***	13.38 ± 0.18^b***	9.57 ± 0.12^b***	10.26 ± .10^b***	10.04 ± 0.04^b***
TBARS (nmoles of MDA/ 100 mg of tissue)		1.13± 0.01	3.91± 0.06^a***	1.67± 0.03^b***	2.35 ± 0.03^b***	2.08 ± 0.01^b***	2.22 ± 0.01^b***
GSH (mM/ 100 mg of tissue)		45.54 ± 0.32	26.09 ± 0.31^a***	40.11 ± 0.07^b***	33.89 ± 0.04^b***	36.12 ± .12^b***	34.24 ± 0.09^b***

Values are expressed as mean ± SEM (n=06)

Data were analyzed by one way ANOVA followed by Dunnett’st test. *** P<0.001, ** P<0.01 and *P<0.05.

^a compared with Vehicle control, ^bcompared with the alloxan treated group

CONCLUSION:

The present study indicates that petroleum, methanolic and aqueous leaves extract of Euphorbia nivulia Buch at the doses 100mg/kg, b.w. p.o. possesess anti diabetic activity studied from dexamethasone induced insulin resistant and ALX induced diabetes model in rats. The acute toxicity study indicated that the PEEN, MEEN and AEEN was devoid of major toxic effects. The effect of PEEN, MEEN and AEEN in normal rats and glucose loaded rats also indicated that the PEEN, MEEN and AEEN exhibited better glycemic control compared with the normal control animals. The entire drug treated diabetic rats showed a significant reduction in blood glucose levels and the other serum biomarker levels and also increased the haemoglobin levels. PEEN, MEEN and AEEN also exhibited antioxidant activity in diabetic rats. The reports of histopathology study concluded there is a recovery to the damage of β-cells in the pancreatic islets.

These observations concluded that the leaves extract of the plant Euphorbia nivulia Buch possesses hypoglycemic, insulin resistance and antioxidant activity. Overall observed significant activity may be due to presence of various phytoactive chemical constituents present in PEEN, MEEN and AEEN.

Further, the work could be extended to evaluate the effectiveness of the marker compounds for the treatment of diabetes at its cellular level to elucidate its exact mechanism for the traditional claim.

ACKNOWLEDGMENT:

The author sincerely thanks to guide Saraswathi C.D, Uday Mohan Reddy P, Archana Swamy P. and Varshitha C. for rendering their suggestions and helping them in each and every step of completing this research paper successfully.

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Received on 29.10.2013 Modified on 18.11.2013

Res. J. Pharmacology & P’dynamics. 6(1): Jan.-Mar. 2014; Page 08-12