Antidiabetic Effect of Anacardium occidentale. Stem-Bark in Fructose-Diabetic Rats

Abstract

The antidiabetic activity of the cashew plant (Anacardium occidentale. Linn. [Anacardiaceae]) stem-bark methanol extract was investigated in fructose-fed (diabetic) and normal rats. Animals were randomly divided into group A (control), group B (treated with 200.0 mg/kg body weight of the extract; orally), group C (fed with enriched fructose diet only, 25%; w/w) and group D (fed with enriched fructose diet along with 200.0 mg/kg body weight of the extract; orally). Animals were treated with extract and/or enriched fructose diet for 21 days. The enriched fructose diet resulted in significant increases in plasma glucose, total cholesterol, triglyceride, total cholesterol/HDL-cholesterol ratio, malonyldialdehyde, total protein, urea, and creatinine. Administration of the extract significantly prevented changes in plasma glucose, triglyceride, total cholesterol/HDL-cholesterol ratio, malonyldialdehyde, urea, and creatinine induced by enriched fructose diet. On the other hand, treatment with enriched fructose diet and/or extract did not have any significant effect on plasma alkaline phosphatase level. These results show that chronic oral administration of methanol extract of Anacardium occidental. stem-bark at a dose of 200.0 mg/kg body weight may be a safe alternative antihyperglycemic agent that has beneficial effect by improving plasma glucose and lipids in fructose-induced diabetic rats, which is associated with a reduced lipid peroxidation.

Keywords:

• Anacardium occidentale
• antihyperglycemic
• antioxidant
• diabetes mellitus
• fructose
• plasma lipids

Introduction

Diabetes mellitus is characterized by loss of glucose homeostasis, altered metabolism of lipids and proteins, and increased risk of atherosclerosis, coronary heart disease, renal failure, nerve damage, and blindness (Sobrevia & Mann, 1997). Type II diabetes mellitus accounts for approximately 85% of all cases of diabetes mellitus and is an important risk factor for cardiovascular morbidity and mortality (Jarret, 1989; Abbott et al., 1990). Treatment with blood lipid-lowering agents has been shown to reduce micro- and macro vascular complications of the coronary artery and cerebral vascular diseases in type 2 diabetic patients in particular (UKPDSG, 1998).

Fructose is an important dietary source of carbohydrates. Fructose is a common monosaccharide that is found naturally in its free form in honey, fruits, and other plants, and in a combined form as half of the disaccharide sucrose. When ingested by mouth, fructose is initially absorbed by the small intestine and flows through the liver (Scriver et al., 1995). However, fructose has been reported to promote adverse metabolic changes, including glucose intolerance, insulin resistance, and hyperlipidemia (Thorburn et al., 1989; Lee et al., 1994). Reactive oxygen species such as superoxide ions, hydroxyl radical, hydrogen peroxide, and lipid peroxide have been reported to contribute to the genesis of atherosclerosis, heart diseases, hypertension, and diabetes (Ross, 1993; Manzella et al., 2001). Thus, prevention of oxidative stress–induced damage is an area of growing interest. As a consequence of a number of serious adverse effects of already known oral hypoglycemic drugs, it is reasonable to search for hypoglycemic plants.

Acute administration of aqueous or methanol stem-bark extracts of cashew plant (Anacardium occidentale. Linn. [Anacardiaceae]) has been shown to have a hypoglycemic effect in fasted streptozotocin-diabetic rats (Ojewole, 2003). The hypoglycemic effect of the administration of methanol plant extract at a single dose of 800.0 mg/kg body weight was found to be more pronounced than that of the aqueous extract in both the normal and streptozotocin-diabetic rats (Ojewole, 2003). The current study was therefore undertaken to validate the effect of chronic administration of an oral daily dose of methanol extract of occidentale stem-bark (200.0 mg/kg body weight) on fasting blood glucose, lipid profile, and lipid peroxidation in fructose-fed and normal rats. In addition, the effect of the extract on some biochemical markers of tissue damage was investigated.

Materials and Methods

Animals

A total of 24 male albino rats weighing between 150 and 200 g obtained from the Animal Breeding Unit of the Department of Biochemistry, University of Ilorin (Ilorin, Nigeria) were used for this study. All animals were maintained 6 per cage in standard environmental condition and were allowed free access to normal and/or high-fructose rat diet and tap water.

Plant extracts

The fresh samples of the stem-barks of A. occidentale. were collected from trees within Ilorin and authenticated by Prof. F.A. Oladele of the Department of Botany, University of Ilorin. Collected materials (200 g) were thoroughly washed in distilled water, chopped, air dried, and pulverized in an electric grinder. The sample obtained was percolated in 500 ml of methanol. The percolated mixture was filtered and evaporated at room temperature to dryness as described by Ojewole (2003).

Experimental design

Diabetes was induced in rats by feeding the animals with 25% (w/w) fructose mixed with normal rat chow for 21 days as in past studies (Lee et al., 1994; Olatunji-Bello et al., 2001). The rats were randomly divided into four groups of 6 rats each; group I received distilled water by gavage (normal rats), group II received 200.0 mg/kg body weight of methanol extract of A. occidentale. stem-bark by gavage, group III received distilled water by gavage and was fed with a 25% fructose diet, and group IV received 200.0 mg/kg body weight of methanol extract of A. occidentale. stem-bark by gavage and was fed with a 25% fructose diet.

Blood collection

All animals fasted overnight and were sacrificed by cervical dislocation at the end of the experimental period. The jugular vein was exposed and cut with a sterile scalpel blade, and the rats were bled into lithium-heparinized specimen bottles. Plasma was obtained from a portion of the blood sample by centrifugation at 3000 rpm for 10 min. All plasma samples were stored in a refrigerator at 4°C before analysis.

Biochemical parameters

Plasma total cholesterol (TC; Allain et al., 1974), high-density lipoprotein cholesterol (HDL-C; Warnick et al., 1982), and triglyceride (TG; Carr et al., 1993) levels were determined by standard enzymatic colometric methods using respective assay kits supplied by Randox Laboratory Ltd. (Co. Antrim, UK). Plasma levels of low-density lipoprotein-choloesterol (LDL-C) were determined using Friedwald's formula (Friedwald et al., 1972). Fasting plasma glucose levels were determined using the glucose oxidative method (Manzella et al., 2001). Plasma lipid peroxidative activity was evaluated by plasma malonyldialdehyde (MDA) content as described by Esterbauer and Cheeseman (Esterbauer & Cheeseman, 1990). Total plasma protein (TPP) was estimated by the microbiuret method, and plasma levels of creatinine and urea were determined as previously published (Odigie et al., 2003) using an assay kit supplied by Randox Laboratory Ltd. Alkaline phosphatase (E.C. 3.1.3.1) (ALP) activities were done by spectrophotometry using an assay kit obtained from Quinica Clinical (Applicada, Spain) (Babson et al., 1966).

Statistical analysis

All results are presented as mean ± SEM. Data were analyzed by ANOVA followed by Student-Neuman-Keuls as the post hoc. test. Results were considered statistically significant at p < 0.05.

Results

The effect of treatment with A. occidentale. on fasting blood glucose, lipid variables, and lipid peroxidation index (MDA) in normal and fructose-fed rats is summarized in Table 1. Fasting plasma glucose levels of fructose-fed rats were significantly higher (p < 0.05) than those in normal rats. Treatment with extract of A. occidentale. significantly abolished the increase in fasting plasma glucose levels induced by high-fructose diet (6.3 ± 0.3 mmol/l versus 4.0 ± 0.4 mmol/l; p < 0.05). The extract did not produce any significant effect on fasting blood glucose in normal rats. The plasma TC, TG, TC/HDL-C ratio (atherogenic index) and MDA levels were significantly higher in fructose-fed rats compared with those in normal rats, whereas plasma HDL-C and LDL-C levels in fructose-fed rats were not significantly different from those in the controls. Treatment with A. occidentale. in the fructose-fed rats led to a significant reduction in plasma TG, LDL-C, TC/HDL-C ratio, and MDA content. On the other hand, administration of the extract significantly enhanced plasma HDL-C levels in both fructose-fed and normal rats. Treatment with A. occidentale. produced significant reduction in MDA and TC/HDL-C ratio in normal rats, whereas the levels of plasma TC, TG, and LDL-C were not significantly affected in normal rat treated with A. occidentale..

Table 1.. Effect of administration of methanolic extract of A. occidentale. stem-bark (200.0 mg/kg body weight) on fasting plasma glucose, lipid profile and malonyldialdehyde in fructose-fed and normal rats

Variables	Normal	Treated-normal	Untreated-fructose	Treated-fructose
FPG (mmol/l)	3.4 ± 0.3^a.	3.0 ± 0.5^a.	6.3 ± 0.3^a.	4.0 ± 0.4^a.
TC (mmol/l)	2.6 ± 0.1^a.	2.4 ± 0.1^a.	3.5 ± 0.2^b.	3.4 ± 0.3^b.
LDL-C (mmol/l)	0.89 ± 0.10^a.	1.0 ± 0.13^a.	1.1 ± 0.14^a.	0.60 ± 0.07^b.
HDL-C (mmol/l)	1.27 ± 0.01^a.	1.42 ± 0.04^b.	1.35 ± 0.05^a.	2.30 ± 0.12^c.
TC/HDL-C	1.94 ± 0.06^a.	1.69 ± 01.0^b.	2.65 ± 0.12^c.	1.52 ± 0.16^b.
TG (mmol/l)	0.90 ± 0.50^a.	0.80 ± 0.10^a.	1.61 ± 0.02^b.	0.90 ± 0.02^a.
MDA (mmol/l)	1.50 ± 0.03^a.	1.34 ± 0.04^b.	1.81 ± 0.06^c.	1.60 ± 0.03^d.

FPG, fasting plasma glucose; TC, total cholesterol; LDL-C, low-density lipoprotein-cholesterol; HDL-C, high-density lipoprotein-cholesterol; TC/HDL-C, atherogenic index; TG, triglyceride; MDA, malonyldialdehyde.

Values across each row with different superscript letters differ significantly (p < 0.05).

*Data are presented as means in ± SEM of 6 rats by group.

Plasma alkaline phosphatase (ALP) activity, urea, creatinine, and total plasma (TPP) levels in the four groups of rats are shown in Table 2. Plasma levels of urea, creatinine, and TPP levels in fructose-fed rats were significantly higher (p < 0.05) than those in control rats. However, administration of A. occidentale. prevented the increase in plasma levels of urea and creatinine induced by high-fructose diet. In contrast, the extract administration led to increases in TPP levels in both normal and fructose-fed rats.

Table 2.. Effect of administration of methanolic extract of A. occidentale. stem-bark (200.0 mg/kg) on total plasma proteins (TPP), urea, creatinine, and alkaline phosphatase activity (ALP) in fructose-fed and normal rats

Variables	Normal	Treated-normal	Untreated-fructose	Treated-fructose
TPP (g/l)	59.3 ± 0.6^a.	68.0 ± 1.0^b.	64.3 ± 0.4^b.	66.0 ± 1.8^b.
Urea (mmol/l)	3.60 ± 0.23^a.	4.00 ± 0.20^a.	5.20 ± 0.14^b.	3.50 ± 0.35^a.
Creatinine (nmol/l)	46.0 ± 1.4^a.	46.2 ± 1.6^a.	53.3 ± 2.0^b.	45.4 ± 1.5^a.
ALP (IU/l)	26.3 ± 1.9^a.	25.6 ± 1.4^a.	28.7 ± 1.0^a.	25.6 ± 1.5^a.

Values across each row with different superscript letters differ significantly (p < 0.05).

*Data are presented as means ± SEM of 6 rats by group.

Discussion

In the current study, a single daily dose of methanol extract of A. occidentale. (200.0 mg/kg) significantly reduced plasma glucose, total cholesterol, triglycerides, atherogenic index (LDL-cholesterol/HDL-cholesterol ratio), urea, and creatinine in fructose-fed rats, a model of type 2 diabetes mellitus. This treatment had no effect on normal rats. These effects are associated with decreased plasma malonyldialdehyde (lipid peroxidation index). To our knowledge, this is the first report showing that chronic antihyperglycemic effect of the extract of A. occidentale. stem-bark is associated with antioxidant activity of the extract. Previous studies demonstrated that acute administration of water or alcoholic extract from the steam-bark of A. occidentale. had an antihyperglycemic effect in streptozotocin-diabetic rats and a hypoglycemic effect in normal (Kamtchouing, 1998; Ojewole, 2003).

Plasma levels of MDA have been shown to be a reliable marker of lipid peroxidation and oxidative stress (Manzella et al., 2001). In the current study, this parameter was increased in fructose-fed rats as compared with normal rats. Increased plasma levels of peroxidized lipids (oxidative stress) has been associated with type 2 diabetes in particular. This, in turn, is linked to cardiovascular diseases (Ross, 1993; Manzella et al., 2001). In this study, long-term treatment with methanol extract of A. occidentale. stem-bark reduced the plasma MDA in fructose-fed rats to levels similar to those of normal rats. This finding suggests that chronic oral treatment with methanol extract of A. occidentale. stem-bark may prevent lipid peroxidation/oxidative stress in the fructose-induced diabetic rats. The reduction in plasma MDA levels in normal rat treated with the extract provides further evidence that the extract possesses antioxidant activity.

Our study shows that high-fructose diet was able to increase the level of fasting blood glucose, triglyceride, total cholesterol, and total cholesterol/HDL-cholesterol ratio, which are commonly associated with diabetes mellitus and are known to be high-risk factors for development of cardiovascular diseases (UKPDS, 1998; Manzella et al., 2001; Olatunji-Bello et al., 2001). The results of the current study showed, in addition to its antihyperglycemic and antioxidant effects, treatment with A. occidentale. also possess lipid-lowering properties in the fructose-induced diabetes model.

Previous studies have demonstrated that patients with diabetes or animal models have impaired cardiovascular functions (Manzella et al., 2001; Olatunji-Bello et al., 2001; Want et al., 2003). Such impaired cardiovascular functions seem to be linked to the degree of oxidative stress (Dhalla et al., 1998; Manzella et al., 2001). These have been shown to be responsible for many cases of sudden death, despite the absence of documented pre-existing cardiovascular disease (Tsuji et al., 1994). Therefore, a therapeutic agent that possesses antioxidant activity may exert beneficial actions.

This study also demonstrated that chronic administration of the extract A. occidentale. causes a significant increase in total plasma protein without significantly altering the plasma levels of urea and creatinine in normal rats. This finding indicates that the extract of A. occidentale. could increase the rate of protein synthesis while renal function is preserved in normal rats. Enhanced protein synthesis seems to be associated with the administration of A. occidentale. and may probably be attributed to improvement in glycemic control or insulin secretion.

In the fructose-fed diabetic rats, the increase blood glucose is accompanied by an increase in the plasma protein, urea, and creatinine levels. The treatment with A. occidentale. led to reductions in plasma urea and creatinine levels toward the control values while the extract failed to affect the total plasma protein levels in fructose-fed rats. These results suggest that the methanol extract of A. occidentale. stem-bark may be able to prevent altered protein metabolism and/or impaired renal function in diabetes mellitus. Alkaline phosphatase is a biochemical “marker” enzyme for plasma membrane integrity (Akanji et al., 1993). The results in all the groups were similar, suggesting that high-fructose diet as well as treatment with the extract at 200.0 mg/kg does not induce any plasma membrane damage.

In conclusion, the results of our current study demonstrate that chronic treatment with methanol extract of A. occidentale. stem-bark at 200.0 mg/kg may be a safe agent that has a protective role against the diabetogenic and atherogenic effects of high-fructose diet by reducing the hyperglycemic, hyperlipidemia, as well as lipid peroxidation. Further studies are needed to elucidate the mechanisms of such antidiabetic effect of A. occidentale..