Antidiabetic activity of extracts and fraction of Zizyphus mauritiana

Abstract

Various extracts, petroleum ether, chloroform, acetone, ethanol, aqueous, and crude aqueous, of fruits of Zizyphus mauritiana Lam. (Rhamnaceae) and the fractions of petroleum ether and aqueous extracts were tested for antihyperglycemic activity in glucose overloaded hyperglycemic rats. The effective antihyperglycemic extracts and fraction were tested for their hypoglycemic activity at two dose levels, 200 and 400 mg/kg, respectively. To confirm their utility in a higher model, the effective extracts and fraction of Z. mauritiana were also subjected to an antidiabetic study in the alloxan-induced diabetic model at two dose levels, 200 and 400 mg/kg. The aqueous extract and the non-polysaccharide fraction of the aqueous extract of Z. mauritiana were found to exhibit significant antihyperglycemic and hypoglycemic activities. The petroleum ether extract was found to exhibit only an antihyperglycemic effect. Treatment of diabetic rats with petroleum ether extract, aqueous extract, and non-polysaccharide fraction of this plant restored the elevated biochemical parameters, glucose, urea, creatinine, serum cholesterol, serum triglyceride, HDL, LDL, hemoglobin, and glycosylated hemoglobin significantly to the near normal level. Comparatively, the non-polysaccharide fraction of the aqueous extract was found to be more effective, followed by the aqueous extract, and the petroleum ether extract. The activity of the non-polysaccharide fraction was comparable to that of the standard drug glibenclamide.

Keywords::

• Alloxan
• antidiabetic activity
• biochemical parameters
• hypoglycemic activity
• lipid profile
• Zizyphus mauritiana

Introduction

Diabetes is a disease associated with elevated blood glucose levels, leading to major complications such as diabetic neuropathy, nephropathy, retinopathy and cardiovascular diseases (Merlin et al., 2005). More than 100 million of the world’s population has already reached the diabetic mark and the disease now kills more people than AIDS (Sunday Times India, 2006). Decreased physical activity, increasing obesity, stress, and changes in food consumption have been implicated in this increasing prevalence over the past two decades (Shastri, 1980). Overt diabetes affects 2 to 3% of the total world population. In conventional therapy, Type I diabetes is treated with exogenous insulin and Type 2 with oral hypoglycemic agents (sulphonylureas, biguanides, etc.) (Pepato et al., 2005). Though different types of oral hypoglycemic agents are available along with insulin for the treatment of diabetes, there is an increased demand by patients to use natural products with antidiabetic activity (Venkatesh et al., 2003). One such plant being used by the traditional practitioners in Tamil Nadu, India to treat diabetes is Zizyphus mauritiana Lam. (Rhamnaceae). Traditionally, it is also used in diarrhea, wounds, abscesses, liver diseases, asthma, fever (Dahiru et al., 2005), sleeplessness (Oh et al., 2004), constipation, urinary diseases, gastrointestinal disorders, constipation, and abdominal pains (Warrier et al., 2005; Nadkarni, 1954). The constituents reported in this plant are benzaldehyde, benzenoids, aliphatic carboxylic acids, aldehydes, hydrocarbons, oxygenated monoterpenes (Ruy et al., 2005), mucilages, polysaccharides (Clifford et al., 2002), flavonoids (Bhargava et al., 2005), alkaloids (Akino et al., 1996), and triterpenes (Shoei et al., 1996).

Only the aqueous extract of fruits of this plant is being used in traditional herbal preparations. Moreover, researchers focus mainly on ethanol and aqueous extracts for diabetes, but a considerable number of studies state that the petroleum ether, benzene and chloroform extracts were also found active against diabetes (Nagarajan et al., 2005; Nalamolu & Srinivasu, 2006; Phuong et al., 2004). Therefore, knowing the most effective solvent extract and isolating the active fraction from the most effective extract would be useful in the development of new drugs from plants. The standard fraction of an active extract may prove better therapeutically, less toxic and inexpensive compared to pure isolated compound drugs. Keeping these facts in mind, the present study was undertaken to identify the active antidiabetic extract of Zizyphus mauritiana in diabetes-associated complications and to identify the active antidiabetic fraction of the active extract.

Materials and methods

Plant material

Zizyphus mauritiana was collected in March 2006 from Tamil Nadu, India. The taxonomical identification of the plant was done by H. S. Chatree, Botanist, Government Arts and Science College, Mandsaur, India. The voucher specimen (BRNCP/Z/001/2006) was deposited in the Herbarium of the Department of Pharmacognosy, B. R. Nahata College of Pharmacy, Mandsaur.

Preparation of extracts

Dried and powdered plant material (500 g) was successively Soxhlet extracted with petroleum ether (60°–80°C), chloroform, acetone, ethanol, and water for 72 h each. Crude aqueous extract of this plant was prepared separately by boiling the plant material (25 g) with 200 mL of water for 15 min. The obtained extracts were evaporated in vacuum to give residues.

Fractionation of aqueous and petroleum ether extracts

The aqueous extract (15 g) obtained through sequential extraction of Z. mauritiana was dissolved in water (250 mL) and excess of ethanol was added to completely precipitate polysaccharides (Li et al., 2006; Chandrika et al., 2006). Precipitates (polysaccharide fraction, AqPF) were filtered and dried. The remaining non- polysaccharide (AqNPF) fraction was also dried.

Fractionation of petroleum ether extract of Z. mauritiana fruits was done by column chromatography. The solvent system for column chromatography, petroleum ether (60°–80°C), was selected on the basis of separation achieved by thin-layer chromatography (TLC). The column was packed using a wet packing technique with hexane as the solvent and silica gel (60–120 mesh) as the adsorbent. Extract (10 g) was loaded and the column was eluted using the chosen solvent until 90% of the extract loaded eluted out. Fifteen fractions (100–150 mL) were collected on the basis of the color bands appearing and according to the total volume of solvent eluted. Fractions showing similar TLC patterns were pooled together and finally 4 fractions were obtained (Petroleum ether F₁- Petroleum ether F₄). Percentage yield of fractions was determined with respect to the total weight of the extracts.

Phytochemical screening

In order to determine the presence of alkaloids, glycosides, flavones, tannins, terpenes, sterols, saponins, fats, and sugars, a preliminary phytochemical study (color reactions) with various plant extracts and fractions was performed (Kokate et al., 2001).

Animals and treatment

Healthy Wistar rats of either sex (150–180 g) with no prior drug treatment were used for the present studies. The animals were fed with commercial pellet diet (Hindustan Lever, Bangalore, India) and water ad libitum. The animals were acclimatized to laboratory hygienic conditions for 10 days before starting the experiment. The animal study was performed in the Division of Pharmacology, B.R. Nahata College of Pharmacy, Mandsaur, with due permission from the Institutional Animal Ethics Committee (registration number 918/ac/05/CPCSEA). The extracts and fractions that were not soluble in water were suspended in 1% Tween 80 just before administration to rats.

Acute toxicity studies

The acute toxicity test of the extracts and fractions was determined according to OECD guideline No. 420 (Organization for Economic Co-operation and Development). Female Wistar rats (150–180 g) were used for this study. After the sighting study, a starting dose of 2000 mg/kg per os (p.o.) of the test samples were given to various groups containing 5 animals in each groups. The treated animals were monitored for 14 days for mortality and general behavior. No deaths were observed by the end of the study. The test samples were found to be safe up to the dose of 2,000 mg/kg and from these results a 400 mg/kg dose was chosen for further experimentation as the maximum dose.

Antihyperglycemic activity in glucose overloaded hyperglycemic rats

Antihyperglycemic activity was studied in glucose overloaded hyperglycemic rats (Babu et al., 2002). Animals were divided in to various treatment groups (n = 5) as mentioned in Tables 1 and 2. Glibenclamide (5 mg/kg) was used as the reference standard and the negative control group animals received only vehicle. Remaining groups were treated with 400 mg/kg of various extracts and fractions of plant suspended in 1% Tween 80. Zero hour blood sugar level was determined from overnight fasted animals. After 30 min of the drug treatment, animals were fed with glucose (4 g/kg) and blood glucose was determined after 1/2, 1, 2, and 3 h of the glucose load. Blood glucose concentration was estimated by the glucose oxidase enzymatic method using a commercial glucometer and test-strips (Accu-chek Active^™ test meter).

Table 1.  Effect of various extracts of Z. mauritiana in glucose loaded hyperglycemic rats.

Treatment	Dose mg/kg	Blood glucose concentration (mg/dL)
		0 h	1/2 h	1 h	2 h	3 h
Glucose control	–	89.80 ± 3.02	144.40 ± 4.85	150.60 ± 4.01	124.40 ± 3.32	105.20 ± 4.77
Glibenclamide	5	90.20 ± 3.59	105.20 ± 3.49**	92.20 ± 4.60**	78.40 ± 4.20**	66.20 ± 3.68**
Petroleum ether	400	85.40 ± 5.40	110.20 ± 5.30**	95.40 ± 3.38**	83.60 ± 5.36**	82.20 ± 3.02**
Chloroform	400	86.40 ± 4.41	142.40 ± 6.80	140.20 ± 5.20	120.80 ± 3.86	96.20 ± 3.83
Acetone	400	90.80 ± 5.18	130.60 ± 10.20	131.80 ± 4.22	108.20 ± 6.32	98.40 ± 3.22
Ethanol	400	88.80 ± 3.35	144.40 ± 5.85	142.40 ± 7.23	122.20 ± 5.77	95.20 ± 3.83
Aqueous	400	88.60 ± 7.60	118.20 ± 3.24**	96.20 ± 4.20**	80.20 ± 3.88**	70.40 ± 4.20**
Crude aqueous	400	85.00 ± 3.22	128.20 ± 6.20	122.40 ± 5.20*	100.20 ± 4.88*	90.80 ± 3.84

Each value represents the mean ± SEM of five observations. *P < 0.05; **P < 0.01 versus control (ANOVA followed by Dunnett’s test).

Table 2.  Effect of fractions of petroleum ether and aqueous extracts of Z. mauritiana in glucose loaded hyperglycemic rats.

Treatment	Dose mg/kg	Blood glucose concentration (mg/dL)
		0 h	1/2 h	1 h	2 h	3 h
Glucose control	–	85.80 ± 3.20	131.00 ± 4.46	110.00 ± 4.22	85.40 ± 2.20	83.80 ± 4.20
Glibenclamide	5	82.40 ± 2.15	85.40 ± 1.93**	71.80 ± 2.49**	63.80 ± 1.85**	58.80 ± 1.85**
Petroleum ether	400	85.40 ± 5.40	97.80 ± 3.30**	92.40 ± 3.38**	83.80 ± 3.02	83.20 ± 5.36
Petroleum ether-F1	400	82.20 ± 2.10	118.00 ± 5.12	98.45 ± 3.65	85.00 ± 2.20	82.20 ± 3.02
Petroleum ether-F2	400	80.00 ± 1.94	128.20 ± 4.02	110.40 ± 4.12	90.00 ± 3.44	85.40 ± 2.10
Petroleum ether-F3	400	83.40 ± 2.40	130.00 ± 3.60	105.60 ± 2.80	96.40 ± 2.84	82.00 ± 3.01
Petroleum ether-F4	400	84.60 ± 2.12	121.40 ± 3.20	107.80 ± 3.54	95.40 ± 3.12	83.60 ± 2.80
Aqueous	400	83.00 ± 2.29	98.20 ± 3.98**	95.80 ± 2.72*	75.40 ± 2.04*	71.80 ± 2.10**
Polysac. fraction	400	80.00 ± 1.20	118.80 ± 3.70	102.40 ± 3.20	90.40 ± 2.88	85.40 ± 3.66
Non Polysac. fraction	400	81.20 ± 2.25	90.60 ± 1.69**	86.40 ± 1.50**	71.80 ± 3.20**	63.80 ± 2.22**

Each value represents the mean ± SEM of five observations. *P < 0.05; **P < 0.01 versus control (ANOVA followed by Dunnett’s test).

(Polysac., Polysaccharide)

Hypoglycemic activity

Animals were divided into 8 groups (n = 5). Group 1 was kept as control, and received a single dose of 0.5 mL/100 g of the vehicle, group 2 was treated with glibenclamide (5 mg/kg) as a hypoglycemic reference drug. Groups 3 to 8 were treated with petroleum ether extract, aqueous extract, and non-polysaccharide fraction of aqueous extract at two dose levels (200 and 400 mg/kg), as mentioned in Table 3. Blood samples were collected from the tail tip at 0 (before oral administration), ½, 1, 2, and 3 h after vehicle, samples, and drug administration (Ekrem et al., 2005). The blood sugar level was measured as mentioned above.

Table 3.  Effect of active antihyperglycaemic extracts and fraction of Z. mauritiana on blood glucose levels in normal rats.

Treatment	Dose mg/kg	Blood glucose concentration (mg/dL)
		0 h	1/2 h	1 h	2 h	3 h
Normal control	–	84.40 ± 2.32	83.00 ± 1.80	81.80 ± 2.42	82.20 ± 1.70	80.20 ± 2.38
Glibenclamide	5	82.00 ± 2.15	45.20 ± 3.86**	36.60 ± 1.43**	33.20 ± 1.39**	35.00 ± 3.16**
Petroleum ether	200	80.40 ± 2.19	81.00 ± 3.22	82.20 ± 1.88	79.60 ± 2.87	80.40 ± 1.90
	400	82.00 ± 1.89	80.00 ± 2.40	81.20 ± 2.84	80.00 ± 3.10	81.40 ± 2.42
Aqueous	200	83.00 ± 2.76	76.40 ± 2.80	66.20 ± 2.15**	62.60 ± 2.06**	58.80 ± 1.82**
	400	83.20 ± 2.70	68.80 ± 2.03*	61.20 ± 2.26**	55.00 ± 4.12**	44.60 ± 1.40**
Non polysac. fraction	200	81.40 ± 2.20	71.80 ± 2.80*	63.40 ± 2.10**	56.80 ± 2.02**	53.00 ± 1.32**
	400	82.20 ± 2.89	63.20 ± 2.01**	53.00 ± 3.12**	42.80 ± 2.20**	38.20 ± 2.12**

Each value represents the mean ± SEM of five observations. *P < 0.05; **P < 0.01 versus control (ANOVA followed by Dunnett’s test).

Antidiabetic activity in alloxan-induced diabetes model

Diabetes was induced in rats by injecting 120 mg/kg of alloxan monohydrate intraperitoneally in 0.9% w/v NaCl to overnight-fasted rats. The rats were then kept for the next 24 h on 10% glucose solution bottles, in their cages to prevent hypoglycemia. After 72 h of injection, fasting blood glucose level was measured. Animals which did not develop more than 300 mg/dL glucose levels, were rejected (Jamal et al., 1997; Sabu & Subburaju, 2002). The selected diabetic animals were divided in to 8 groups (n = 5) and one more group of normal non-alloxanized animals was also added in the study. Group 1 was kept as normal control (non-alloxanized rats), and received a single dose of 0.5 mL/100 g of the vehicle, group 2 was kept as negative control, alloxan-induced and received a single dose of 0.5 mL/100 g of the vehicle, group 3, diabetic-induced was treated with glibenclamide (5 mg/kg) as reference drug. Groups 4 to 9, diabetic-induced were treated with petroleum ether extract, aqueous extract, and non- polysaccharide fraction of aqueous extract at two dose levels, (200 and 400 mg/kg) as in Table 4. Treatment was continued for 7 consecutive days (p.o.). At the end of day 7, the rats were fasted for 16 h and blood parameters were determined.

Table 4.  Biochemical parameters of normal and experimental animals on 7^th day post treatment.

Parameters	Experimental groups
	Normal control	Diabetic control	Petroleum ether		Aqueous		Nonpolysac. fraction		Glibenclamide 5mg/kg
			200 mg/kg	400 mg/kg	200 mg/kg	400 mg/kg	200 mg/kg	400 mg/kg
Blood glucose	81.40 ± 3.22**	512.00 ± 15.29	285.00 ± 16.19**	197.40 ± 21.11**	198.00 ± 16.40**	174.40 ± 10.80**	152.40 ± 9.80**	122.00 ± 8.72**	124.40 ± 7.84**
S. urea	30.20 ± 1.77**	279.00 ± 14.01	285.20 ± 10.20	253.00 ± 10.24	71.20 ± 4.09**	46.40 ± 2.60**	38.20 ± 2.12**	33.40 ± 3.22**	32.80 ± 1.39**
S. creatinine	0.45 ± 0.03**	1.85 ± 0.36	1.20 ± 0.15	0.88 ± 0.04*	0.75 ± 0.04**	0.40 ± 0.24**	0.43 ± 0.14**	0.46 ± 0.18**	0.44 ± 0.03**
S. cholestrol	34.00 ± 1.74**	84.00 ± 4.94	89.20 ± 3.80	75.60 ± 2.63*	64.20 ± 3.50**	45.00 ± 1.83**	39.00 ± 2.03**	37.00 ± 2.33**	32.20 ± 2.49**
S. triglyceride	33.40 ± 3.45**	123.00 ± 6.63	111.20 ± 5.60	98.00 ± 4.80*	86.20 ± 2.51**	39.00 ± 2.80**	41.00 ± 1.92**	34.20 ± 1.80**	38.20 ± 1.90**
HDL	24.60 ± 1.47**	10.20 ± 1.12	14.00 ± 0.55**	14.80 ± 1.40**	16.00 ± 2.22**	18.80 ± 1.10**	22.20 ± 1.22**	24.60 ± 1.02**	25.80 ± 0.98**
LDL	22.00 ± 2.10**	58.80 ± 3.22	38.20 ± 2.22**	39.00 ± 1.61**	34.00 ± 1.42**	28.40 ± 2.68**	25.20 ± 2.44**	24.20 ± 2.80**	23.60 ± 1.90**
Hemoglobin	11.20 ± 0.33**	6.90 ± 0.41	8.40 ± 0.65*	8.80 ± 0.88*	8.00 ± 0.60*	10.60 ± 0.53**	11.60 ± 0.74**	11.40 ± 0.94**	10.90 ± 0.47**
Gly. hemoglobin	1.92 ± 0.17**	5.70 ± 0.37	4.60 ± 0.24*	3.80 ± 0.44**	3.20 ± 0.26**	2.80 ± 0.15**	2.00 ± 0.25**	1.80 ± 0.35**	2.00 ± 0.15**

Each value represents the mean ± SEM of five observations. *P < 0.05; **P < 0.01 versus diabetic control (ANOVA followed by Dunnett’s test).

(S. urea, Serum urea; S. creatinine, Serum creatinine; S. cholestrol, Serum cholestrol; S. triglyceride, Serum triglyceride; HDL, High Density Lipoprotein; LDL, Low Density Lipoprotein; Gly. Hemoglobin, Glycosylated hemoglobin)

Collection of blood and estimation of biochemical parameters

The blood sugar level was measured using Accu-chek Active^™ test strips in an Accu-chek Active^™ test meter by collecting the blood from rat tail vein. For other plasma profiles, blood was collected from the retro-orbital plexus of the rats under light ether anesthesia using capillary tubes into Eppendorf tubes containing heparin. The plasma was separated by centrifugation (5 min, 5,000 rpm) and was analyzed for lipid profiles (serum cholesterol, serum triglyceride, HDL cholesterol, LDL cholesterol), serum creatinine, serum urea, hemoglobin, and glycosylated hemoglobin. The plasma profiles were measured by standard enzymatic methods with an automatic analyzer (Phuong et al., 2004) and glycosylated hemoglobin by colorimetric method.

Statistical analysis

The values are expressed as mean ± SEM. The results were analyzed for statistical significance using one-way ANOVA followed by Dunnet’s test. P < 0.05 was considered significant.

Results

Preliminary phytochemical screening

The petroleum ether extract (yield 2.42% w/w) contained fats and triterpenoids, while the chloroform extract (yield 0.34% w/w) contained only steroids. Acetone extract (yield 6.07% w/w) contained tannins and steroids. Ethanol extract (yield 6.54% w/w) contained carbohydrates, flavonoids, tannins and saponins. Aqueous extract (yield 8.8% w/w) contained carbohydrates, flavonoids, alkaloids and saponins. Crude aqueous extract (yield 12.42% w/w) contained carbohydrates, flavonoids, tannins, alkaloids and saponins. The non-polysaccharide fraction (yield 58% w/w) contained alkaloids, flavonoids, and saponins, while the polysaccharide fraction (yield 36.25% w/w) contained only carbohydrates. Fractions F₁ (yield 21% w/w) and F₂ (yield 33.08% w/w) of petroleum ether extract contained fats and triterpenoids, while F₃ (yield 14% w/w) and F₄ (yield 20.2% w/w) contained only steroids.

Effect of extracts and fractions in glucose loaded hyperglycemic animals

Tables 1 and 2 show the antihyperglycemic effect of plant extracts and fractions at a dose of 400 mg/kg in glucose loaded hyperglycemic rats. There was a significant rise in the blood glucose level of control animals after ½ h of the glucose load, which declined at the end of 2 h. The petroleum ether and aqueous extracts exhibited the antihyperglycemic effect (P < 0.01) at ½, 1, 2 and 3 h after the glucose load compared to control (Table 1). Crude aqueous extract was found to exhibit the effect only at 1 h and 2 h (Table 1). When the most active petroleum ether and aqueous extracts were fractionated and subjected to antihyperglycemic studies, only the non-polysaccharide fraction of aqueous extract (Table 2) exhibited significant antihyperglycemic activity. The aqueous extract and the non-polysaccharide fraction of aqueous extract were found to produce hypoglycemia at the end of 2 h (P < 0.05) (P < 0.01) (Table 2). Comparatively, the non-polysaccharide fraction of aqueous extract of Z. mauritiana was found to be more active (Table 2).

Effect of extracts and fraction in fasted normal rats

The extracts and fraction that produced antihyperglycemic activity were further subjected to hypoglycemic studies at two dose levels (200 and 400 mg/kg) and the results are given in Table 3. The aqueous extract and non-polysaccharide fraction exhibited significant (P < 0.05) (P < 0.01) hypoglycemic activity and the activity was found to be dose-dependent.

Effect of extract and fraction in alloxan-induced diabetic rats

The basal blood glucose levels of all the groups were statistically not different from each other and three days after alloxan administration, blood glucose values were 5-fold higher in all the groups. After 7 days, values of blood glucose decreased in all the treatment groups and the diabetic control rats showed a slight increase in blood glucose level. The administration of plant extracts, fraction and glibenclamide to diabetic rats restored the level of blood glucose significantly (P < 0.01) (Table 4).

The level of total hemoglobin, glycosylated hemoglobin, serum urea, serum creatinine and lipid profiles of different experimental groups are also represented in Table 4. The diabetic rats showed a significant decrease in the level of total hemoglobin and significant increase in the level of glycosylated hemoglobin. The administration of petroleum ether extract, aqueous extract, fraction and glibenclamide to diabetic rats restored the changes in the level of total hemoglobin and glycosylated hemoglobin to near normal levels (P < 0.05) (P < 0.01).

Alloxan-induced diabetic rats showed significant hypercholesterolemia and hypertriglyceridemia as compared with control. Treatment with plant extracts and fraction showed a significant decrease in cholesterol and triglycerides levels (P < 0.05) (P < 0.01), simultaneously increased the HDL-c. The lower dose of petroleum ether extract didn’t show any preventive effect on hypercholesterolemia and hypertriglyceridemia. A significant increase in creatinine and urea levels of diabetic control rats was observed. Treatment with aqueous extract and non-polysaccharide fraction of aqueous extract of Z. mauritiana significantly decreased these values (P < 0.01). Treatment with petroleum ether extract was found to be inactive in restoring the levels of urea and only 400 mg/kg was active in case of creatinine. The aqueous extract and the non-polysaccharide fraction were found effective in alleviating diabetes and diabetes related complications. Comparatively, the activity of non-polysaccharide fraction was better than that of the activity exhibited by aqueous extract and the activity was comparable with that of the standard drug glibenclamide (Table 4).

Discussion

The present study was undertaken to examine the antidiabetic activity of Z. mauritiana and also to find the active antihyperglycemic fraction of the active extract of this plant. Diabetes is a major health problem affecting major populations worldwide. Epidemiological studies and clinical trials strongly support the notion that hyperglycemia is the principal cause of complications. Thus, sustained reduction in hyperglycemia will decrease the risk of developing microvascular complications and most likely reduce the risk of macrovascular complications (Muniappan et al., 2004). On the basis of this statement we have selected the glucose-induced hyperglycemic model to screen the antihyperglycemic activity of the plant extracts and fractions.

In the glucose-loaded hyperglycemic model, the plant exhibited significant antihyperglycemic activity at a dose level of 400 mg/kg. An excessive amount of glucose in the blood induces the insulin secretion. This secreted insulin will stimulate peripheral glucose consumption and control the production of glucose through different mechanisms (Andrew, 2000). However, from the study (glucose control) it was clear that the secreted insulin requires 2–3 h to bring the glucose level back to normal. In the case of the petroleum ether extract, aqueous extract, crude aqueous extract, non-polysaccharide fraction of aqueous extract, and drug-treated groups, the glucose levels did not increase like the control group, giving an indication regarding the supportive action of the extracts, fraction, and drug in the glucose utilization. The effect of glibenclamide, a standard drug used in this study on glucose tolerance, has been attributed to enhanced activity of beta cells of the pancreas resulting in secretion of larger amounts of insulin. So, the mechanism behind this antihyperglycemic activity of plant extracts and fraction involves an insulin-like effect, probably through peripheral glucose consumption or enhancing the sensitivity of beta cells to glucose, resulting in increased insulin release (Muniappan et al., 2004).

Among the active antihyperglycemic extracts, the aqueous extract and the non-polysaccharide fraction exhibited hypoglycemic activity. The hypoglycemic effect produced by the extract and fraction may be due to increased insulin release resembling the mechanism of action of sulphonyl ureas (Okine et al., 2005; Miura et al., 2001). But the petroleum ether extract did not show any hypoglycemic effect in normal rats and showed antihyperglycemic effect in the glucose-loaded model, suggesting its mechanism is similar to biguanides. Biguanides do not increase insulin secretions, they promote tissue glucose uptake, reduce hepatic glucose output, thereby producing an antihyperglycemic effect and not hypoglycemic effect (Andrew, 2000).

Alloxan produces hyperglycemia by a selective cytotoxic effect on pancreatic beta cells. One of the intracellular phenomena for its cytotoxicity is through generation of free radicals demonstrated both in vivo and in vitro (Yadav et al., 2002). In uncontrolled or poorly controlled diabetes, there is an increased glycosylation of a number of proteins including hemoglobin. Glycosylated hemoglobin level was found to be increased in patients with diabetes mellitus to approximately 16%, and the amount of increase was found directly proportional to the fasting blood glucose level. During diabetes, the excess glucose present in blood reacts with hemoglobin; hence the total hemoglobin level is decreased in alloxan diabetic rats (Pari & Amarnath, 2004). Administration of petroleum ether extract, aqueous extract and fraction for 7 days prevented a significant elevation in glycosylated hemoglobin thereby increasing the level of total hemoglobin (P < 0.05) (P < 0.01) in diabetic rats. This could be due to the result of improved glycemic control produced by plant extracts and fraction.

The levels of serum lipids are usually elevated in diabetes mellitus and such an elevation represents a risk factor for coronary heart disease. This abnormal high level of serum lipids is mainly due to the uninhibited actions of lipolytic hormones on the fat depots mainly due to the action of insulin. Under normal circumstances, insulin activates the enzyme lipoprotein lipase, which hydrolyses triglycerides. However, in the diabetic state, lipoprotein lipase is not activated due to insulin deficiency resulting in hypertriglyceridemia (Pushparaj et al., 2007). Also, insulin deficiency is associated with hypercholesterolemia. The mechanisms responsible for the development of hypertriglyceridemia and hypercholesterolemia in uncontrolled diabetes in human beings are due to a number of metabolic abnormalities that occur sequentially (Murali et al., 2002). In our study, the diabetic rats showed hypercholesterolemia and hypertriglyceridemia and the treatment with plant extracts and fraction significantly decreased both cholesterol and triglyceride levels. These findings also support the hypothesis that the activity of plant extracts and fractions may directly impact on improvements in insulin levels (Sharma et al., 2003).

The diabetic hyperglycemia induced by alloxan produces elevation of plasma levels of urea and creatinine, which are considered as significant markers of renal dysfunction (Alarcon et al., 2005). The results showed significant increase in the level of plasma urea and creatinine in the diabetic groups compared to normal control groups. After treatment of alloxan-diabetic rats with aqueous extract and non-polysaccharide fraction, the level of urea and creatinine decreased significantly, when compared to the mean value of diabetic group. This further confirms the utility of this plant in diabetes associated complications. On the other hand, the petroleum ether extract was not found very effective in restoring the values of creatinine and urea. This may be because of the inability of the active constituent present in the petroleum ether extract to revert the damage created by alloxan to kidney.

Though main classes of active constituents are present in the aqueous extract and crude aqueous extract of this plant, the activity was less than that of the fraction and this may be due to the fewer amounts of active constituents present at 400 mg/kg of crude aqueous extract when compared to 400 mg/kg of successively fractionated extract and fraction. Babayi et al. (2004) suggested the same for the better activity of fractions compared to extracts. Synergistic effect of triterpenoids present in the petroleum ether extract may be responsible for the antidiabetic activity, because when these triterpenoids were separated during fractionation the activity was not found in fractions. Many triterpenoids are reported to have antidiabetic effect (Pulok et al., 2006). Alkaloids, flavonoids, and saponins are the main classes of compounds present in the aqueous extract, crude aqueous extract, and non-polysaccharide fraction. Although the ethanol extract contains flavonoids and saponins it was found to be inactive, so we consider the antidiabetic activity exhibited by Z. mauritiana may be due to the alkaloids or the synergistic effect produced by all the three compounds. Most of the alkaloids are reported to have antidiabetic activity (Pulok et al., 2006).

Conclusion

We conclude that the extracts and fraction of the plant tested for antidiabetic activity have shown appreciable results in decreasing the serum glucose level and other complications associated with diabetes. This research supports the inclusion of this plant in traditional antidiabetic preparations, and the formulations made using the identified effective extract and fraction of this plant could serve the purpose better than the existing formulations with crude aqueous extract.

Acknowledgement

The authors are thankful to the Director, BRNCOP for providing all the necessary facilities to carry out this research work.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.