Antidiabetic Activity of Spondias mombin Extract in NIDDM Rats

Abstract

The antidiabetic activity of the methanol leaf extract of Spondias mombin Linn (Anacardiaceae), a medicinal plant used in the treatment of diabetes and other diseases, was evaluated in oral glucose tolerance test and alloxan induced diabetic rats using the O-toluidine and glucose-oxidase methods. A dose of the methanol extract and its chloroform fraction was administered to experimental diabetic rats and their blood glucose levels evaluated over 8 h. The extract exhibited significant blood glucose lowering effect in the oral glucose tolerance test and the alloxan induced diabetic rats when compared with the control. The chloroform fraction of Spondias mombin showed a peak hypoglycaemic effect of 60.6% at 4 h. This study, therefore, justifies the use of the plant in the treatment of diabetes mellitus.

Keywords::

• Spondias mombin
• alloxan-induced
• OGTT
• antidiabetic activity

Introduction

Diabetes mellitus a serious and chronic metabolic disorder characterised by hyperglycaemia in postprandial and/or fasting state is a threat to human health. Epidemiological studies and clinical trials indicate that hyperglycaemia is the main cause of complication related with coronary artery disease, renal failure, blindness, limb amputation, neurological complications and pre-mature death (Lopez-Candales, 2001).

The increase in prevalence of diabetes is expected to be 42% in developed countries and 70% in developing countries by 2025 (WHO, 2006; King et al., 1998). The management of diabetes without side effect is still a challenge, hence, a growing interest in the evaluation of plant and herbal remedies which are considered less toxic and with minimal side effects.

Spondias mombin Linn (Anarcadiaceae) is known commonly as ‘Hog plum’, ‘Iyeye’ and ‘Olosan’ (Yoruba, South West Nigeria) (Gbile, 1984). It is a deciduous tree, with large panicles of small white flowers and yellow plum-like fruits, common in farmland and villages, especially in the forest region as well as the savannah. In traditional folklore medicine, S. mombin is used in treating intestinal disorders, as an emetic, for treating gonorrhoea, diabetes, psychiatric disorders, for the expulsion of placenta in goats and women. The plant is also useful as an antidiarrhoeal agent (Iwu, 1993), as an antimicrobial agent, for the treatment of wounds, and as an astringent (Oliver-Bever, 1960; Kokwaro 1976; Abo et al, 1999) and as an oxytocic (Ayensu, 1978). Spondias mombin has been reported to contain pelandjuaic acid, ellagitannins, caffeoyl esters and anacardic acid (Corthout et al., 1991, 1992, 1994; Coates et al, 1994). The current study investigates the antidiabetic activity of Spondias mombin leaves using the oral glucose tolerance test and alloxan-induced diabetic rats.

Materials and Methods

Plant material

Spondias mombin leaves were collected in August 2001 from Sagamu, Ogun State Nigeria. The plant material was authenticated by Mr T.K Odewo at the herbarium of the Forestry Research Institute of Nigeria Ibadan. A voucher specimen (FHI 106132) has been deposited in the herbarium.

Preparation of methanol extract

Finely powdered, dried leaves of S. mombin (100 g) were macerated with 80% methanol for four days at room temperature. The brown extract obtained was filtered and concentrated in vacuo in a rotary evaporator at 40°C (10.7% w/w dry weight basis). The methanol extract was suspended in distilled water for animal studies.

Preparation of n-hexane and chloroform fractions of Spondias mombin leaf

The dried methanol extract of Spondias mombin leaf was suspended in MeOH-H₂O (1:9), partitioned sequentially with n-hexane, chloroform to yield the n-hexane and chloroform fractions.

Animals

Healthy Wistar albino rats weighing between 80–250 g of either sex were used for the study. The animals were housed in polypropylene cages and maintained under standard conditions (12 h light and dark cycle at 25°C). They were fed with standard rat pellet diet (Ladokun feeds, Ibadan) and water ad libitum. All experimental protocol were conducted in accordance with internationally accepted standard guidelines for care and use of laboratory animals.

Biochemical studies

For the biochemical studies, rats divided into four groups (n = 5) were administered methanol extract, glibenclamide and water for 10 days. Group 1 were normal non- diabetic rats treated with water only (2 ml/kg). Groups 2 and 3 were diabetic rats treated with S. mombin methanol leaf extract (1 g/kg) and glibenclamide (5 mg/kg), respectively, while Group 4 was the diabetic untreated control rats administered water (2 ml/kg).

Oral glucose tolerance test (OGTT)

The oral glucose tolerance test was performed in overnight fasted (18 h) normal animals. Rats divided into four groups (n = 5) were administered methanol extract (1 g/kg), glibenclamide (5 mg/kg), glucose solution (2 g/kg) and water (2 ml/kg), respectively. Glucose (2 g/kg) was fed just before administering the extract. Blood was withdrawn from the tail vein of the animals at 0, 1, 2, 3, 4, 5, 6, and 7 h after extract administration. The fasting blood glucose levels were estimated by using the O-toluidine methods (Dubowski, 1962; Frings et al., 1970).

Induction of non-insulin dependent diabetes mellitus

NIDDM was induced (Abdel-Barry et al., 1997) in overnight fasted animals by a single intraperitoneal injection of 60 mg/kg alloxan monohydrate (Sigma Aldrich, UK). Hyperglycaemia was confirmed by the elevated glucose level in the blood, determined at 72 h after injection. Rats found with NIDDM were used for the antidiabetic study.

Experimental design

The diabetic animals, divided into seven groups (n = 5), were administered methanol extract (1 g/kg), n-hexane fraction (1 g/kg), chloroform fraction (1 g/kg), aqueous fraction (1 g/kg), glibenclamide (5 mg/kg), and water (2 ml/kg) respectively. The fasting blood glucose was determined at 0, 2, 4, 6, and 8 h after extract administration using the glucose-oxidase method (Life scan, Johnson and Johnson Inc., California). The blood cholesterol, protein and creatinine level of the diabetic rats were estimated enzymatically by using specific colorimetric kits (Randox, UK).

Statistical analysis

All values were expressed as mean ± S.E.M. The significance of the differences between the means of the test and control animals were established by the Student’s t-test.

Results

The study showed the non toxic effect of the methanol extract of S. mombin leaf. There was no lethality nor toxic reactions observed throughout and up to the end of the study. The effect of the extract on cholesterol, creatinine and protein levels of the diabetic rats after continuous administration for ten days are indicated in Table 1.The oral glucose tolerance test revealed the antidiabetic activity exhibited by the methanol extract of S. mombin leaf (Table 2). In the alloxan-induced diabetic rats, the extract significantly lowered the blood glucose of the animals when compared with the untreated control group (Table 3). The chloroform fraction of the methanol extract also exhibited a pronounced hypoglycaemic effect (p < 0.05), with decreased blood glucose levels at 2, 4, 6, and 8 h. The effect of the methanol extract and its chloroform fraction are, however, comparable to that of glibenclamide (Table 3).

Table 1.  Mean blood cholesterol, protein and creatinine levels after 10 days.

	Mean values after 10 days
	Cholesterol (mg/dl)	Protein (g/dl)	Creatinine (mg/dl)
S. mombin leaf methanol extract (1 g/kg)	35.6 ± 1.15	1.10 ± 0.115	1.32 ± 0.034
Glibenclamide (5 mg/kg)	87.6 ± 1.274	1.40 ± 0.122	1.18 ± 0.033
Diabetic untreated (water only)	71.0 ± 0.096	1.10 ± 0.077	1.55 ± 0.012
Normal non-diabetic (water only)	35.6 ± 1.58	0.78 ± 0.036	0.91 ± 0.011

Table 2.  Effect of Spondias mombin extract on blood of glucose loaded rats.

Time (h)	Mean blood glucose level (mg/dl )
	0	1	2	3	4	5	6	7
Cage 1 S. mombin	97.8 ± 0.353	270.7 ± 1.661	191.3 ± 0.283	165.2 ± 0.141 (7.4%)	154.3 ± 0.495 (1.8%)	117.4 ± 0.565 (13.7%)*	98.8 ± 1.360 (17.7%)*	85.3 ± 1.369 (26.3%)*
Cage 2 Glibenclamide (5 mg/kg)	96.0 ± 0.707	150.2 ± 0.282 (36.0%)*	120.1 ± 0.100 (40.8)*	100.0 ± 0.283 (43.9 %)*	68.2 ± 0.601 (56.6 %)*	67.1 ± 0.077 (50.9 %)*	66.0 ± 0.028 (45.1 %)*	52.2 ± 0.113 (54.9%)*
Cage 3 untreated Control (2 ml/kg water)	100.2 ± 0.7071	234.8 ± 0.283	186.1 ± 0.990	178.3 ± 0.283	157.2 ± 0.6011	136.1 ± 0.422	120.1 ± 0.345	115.7 ± 0.074
Cage 4 Normal (2 ml/kg water)	88.0 ± 0.211	91.3 ± 0.062	92.2 ± 1.554	87.0 ± 0.198	89.1 ± 1.020	90.4 ± 0.331	89.6 ± 0.119	89.0 ± 0.120

Values are Mean ± SEM; P < 0.05; N = 5; Figures in parenthesis = % decrease; * P < 0.05

Table 3.  Effect of Spondias mombin leaf methanol extract, n-hexane, chloroform and aqueous fractions on alloxan-induced diabetic rats.

Time (h)	Mean blood glucose level (mg/dl)
	0	2	4	6	8
Group 1 Methanol extract of S. mombin leaf (1 g/kg)	180.0 ± 0.040	154.3 ± 0.123 (42.8%)*	144.5 ± 0.127 (49.7%)*	141.5 ± 0.310 (56.6%)*	146.6 ± 0.249 (51.1%)*
Group 2 n-hexane fraction of S. mombin leaf (1 g/kg)	220.1 ± 0.127	317.6 ± 0.566	400.9 ± 1.598	479.4 ± 1.797	476.1 ± 1.073
Group 3 Chloroform fraction of S. mombin leaf (1 g/kg)	208.7 ± 0.042	169.6 ± 0.057 (37.1%)*	113.0 ± 0.368 (60.6%)*	176.8 ± 0.099 (45.8%)*	186.9 ± 0.014 (37.7%)*
Group 4 Aqueous fraction of S. mombin leaf (1 g/kg)	187.0 ± 0.127	365.2 ± 1.626	413.0 ± 1.440	504.3 ± 0.251	514.8 ± 0.240
Group 5 Glibenclamide (5 mg/kg)	175.2 ± 0.092	141.1 ± 0.180 (47.7%)*	140.3 ± 0.209 (51.1%)*	136.9 ± 0.215 (58.0%)*	134.6 ± 0.010 (55.1%)*
Group 6 Untreated diabetic control	258.7 ± 0.731	269.6 ± 0.508	287.0 ± 1.361	326.1 ± 1.483	300.0 ± 1.164
Group 7 Normal (non-diabetic)	92.4 ± 0.495	91.4 ± 0.080	93.4 ± 0.781	95.6 ± 0.052	96.6 ± 0.012

Values are Mean ± SEM; P < 0.05; n = 5; Figures in parenthesis = % decrease; * Represents statistical significance vs. control P < 0.05

Discussion

Goldner and Gomon (1943) reported that alloxan causes a massive reduction in insulin release by destroying the β-cells of the islets of Langerhans and thereby inducing hyperglycaemia. In the oral glucose tolerance test, the methanol extract of S. mombin leaf showed a significant (P < 0.05) reduction in blood glucose level of the rats 5 h after extract administration and normalised the blood glucose levels of the rats at 6 and 7 h when compared with the glucose loaded untreated control rats. The methanol extract and chloroform fraction of Spondias mombin significantly reduced the blood glucose levels of the alloxan-induced diabetic rats compared with the control (untreated alloxan induced diabetic rats). There was, however, no significant difference in the antidiabetic activity of the methanol extract, chloroform fraction and glibenclamide.

In addition, the methanol extract normalised the blood cholesterol level of the diabetic rats by the tenth day of continuous extract administration. This effect is important in preventing complications such as coronary heart disease associated with diabetes mellitus.

The exact mechanism of action of the methanol extract and chloroform fraction of S. mombin leaf is unknown. It is, however, possible that the hypoglycaemic effect observed is due to a pancreatic and/or extra pancreatic mechanism of action. This could be by possible stimulation of the residual β-cells to release more insulin or an increased peripheral utilization of glucose.

Plant sterols have been shown to exert antidiabetic activity and inhibit the absorption of dietary cholesterol (Grundy et al., 1969; Lees et al., 1977) therefore, the presence of sterols in the leaf of S. mombin may be responsible for the antidiabetic and cholesterol lowering effects exhibited in the rats. A further study is in progress to identify the compound(s) responsible for the cholesterol lowering and hypoglycaemic effect in S. mombin leaf. This study justifies the use of Spondias mombin in folklore medicine as treatment for diabetes mellitus.

Acknowledgements

Declaration of interest: The authors report no conflicts of interest.