The Hypoglycaemic, Antihyperlipidemic and Antioxidative Effects of Anacardium Occidentale Methanolic Nut Extract in Streptozotocin-Induced Diabetic Male Wistar Rats

Background: This research work investigated the antidiabetic, anti-hyperlipidemic, and antioxidative effects of Anacardium occidentale methanolic nut extract in Streptozotocin (STZ)-induced diabetic Wistar rats. Methods: Forty (40) Wistar rats weighing 250±30g were randomly divided into ve groups of 8 rats each. Group 1 served as the control; Group 2-5 were induced with diabetes with a single dose of 50mg/kg bw of streptozotocin intraperitoneally. After diabetes induction, Group 2 served as the STZ-only group, Groups 3 and 4 were administered 100mg/kg bw and 200mg/kg bw p.o Anacardium occidentale nut extract, respectively, while Group 5 was administered 2mg/kg bw of glimepiride as a reference drug for a period of 4 weeks. Food and water intake were monitored daily, body weight, and blood glucose levels weekly throughout the experiment. On day 29, the animals were sacriced, and blood samples were collected through cardiac puncture for biochemical studies. Results: Administration of the nut extract signicantly (p<0.05) increased the food intake and body weight of diabetic treated rats, fasting blood glucose level and oral glucose tolerance test (OGTT) decreased signicantly (p<0.05) in treated rats. On lipid prole, administration of nut extract signicantly decreased (p<0.05) triglyceride, low-density lipoprotein, total cholesterol, and very-low-density lipoprotein concentrations while it signicantly increases (p<0.05) the high-density lipoprotein. Anacardium occidentale nut extract caused signicantly (p<0.05) increases in SOD, GPx, GSH, and CAT levels with a decrease MDA level in diabetic treated rats. Markers of liver and kidney functions were also improved in diabetic treated rats. Conclusion: Treatment with Anacardium occidentale methanolic nut extract has hypoglycemic, hypolipidemic, and hepato-protective effects in diabetic rats. It also

The uptake of glucose by cells and metabolic utilization is disrupted, and the conversion of excess glucose to either glycogen in the liver or as fat for storage is usually decreased compared to non-diabetic persons. Hyperlipidemia is common in diabetes mellitus [9] especially elevated triglyceride and cholesterol levels. Hypercholesterolemia is one of the risk factors responsible for the onset of the development of atherosclerosis during the course of diabetes mellitus [10].
Oral hypoglycemic drugs used in management of these diseases have side effects such as gastrointestinal discomfort, weight gain, and hepatic dysfunction [11]. Therefore, there is an increasing need to nd more safe and e cient therapies for prevent diabetes mellitus and its related complica tions. Plants are known to possess a wide variety of pharmacological effects and extraordinary therapeutic possibilities.
The Cashew tree, also known as Anacardium occidentale (Latin name), is a member of the Anacardiaceae family. Anacardium occidentale is grown widely in tropical countries like Malaysia, India, Brazil, Nigeria, and occurs widely in Senegal and is known as Darkassou [12].
The stem bark, fruits, and leaf extract, have pharmaceutical properties and extensively used as antiin ammatory, antioxidant, antibacterial, and antidiarrheal [13][14][15][16]. The anti-diabetic and anti-in ammatory properties have also been reported [17]. Phytochemical study of the methanolic leave extract revealed the presence of phenolic, avonoids, steroids and triterpenes [18]. The hypoglycemic effect of the administration of methanol plant extract at a single dose of 800.0 mg/kg bw was found to be more pronounced than that of the aqueous extract in both the normal and streptozotocin-diabetic rats [19]. Thus, the current study was aimed at investigating the antidiabetic, anti-hyperlipidemic, and antioxidative effects of Anacardium occidentale methanolic nut extract in Streptozotocin (STZ)-induced diabetic rats.

Animals
Healthy male Wistar rats (180g ±20g) were used. The animals were received from the Animal House of The nut of Anacardium occidentale plant was sun-dried at room temperature in the laboratory, powdered and stored in airtight container. The powdered nut of Anacardium occidentale was extracted with 95% ethanol in the Soxhlet apparatus [21].

Acute toxicity test
Acute toxicity test was carried out according to the modi ed Lorke's method [24] using a total of 12 rats. At the initial phase, the rats were assigned randomly into three groups of 3 rats each. The rats in each group were administered an intraperitoneal injection of extract at 10, 100, and 1000mg/kg. Their body weight was observed for signs of toxicity and death in the rst 24hours. In the second phase, another set of rats were randomly assigned into four groups of one rat each and administered the Anacardium occidentale methanolic nut extract intraperitoneally, at 1600, 2900, and 5000mg/kg based on the result of the rst phase. The LD 50 was then calculated as the square root of the product of the maximum dose for all surviving and minimum dose for all mortality using the formula; LD 50 = (D X D )

Experimental induction of diabetes
Diabetes was induced through a single intraperitoneal injection of (50 mg/ kg b.w.) freshly prepared streptozotocin (STZ) in 0.1 M citrate buffer (pH = 4.5) to overnight fasted rats [25]. To prevent the initial drug induced hypoglycemic death, diabetic rats were permitted to drink a 20% glucose solution overnight. The blood glucose level was measured after three days, and rats with glucose levels >200 mg/dL were considered as diabetic. Control rats however injected with 0.2 mL of the vehicle (0.1 M citrate buffer, pH 4.5) alone.

Experimental Design
A total of 40 experimental rats were used to assess the effect of the nut extracts on the experimental rats: 32 STZ induced diabetic rats plus 8 normal control rats. Animals were divided into ve (5) major groups and housed under controlled environmental conditions. Rats were divided into the following groups: The body weight measurement and fasting plasma glucose levels were assessed before and during the administration of the extracts weekly, till the end of the study. The glucose level in plasma was determined by glucose oxidase/peroxidase method as described by [26] using a digital glucometer and test strips (Accu-Chek Advantage, Roche Diagnostic, Germany)

Biochemical Parameters
At the end of the experimental treatment, after 12h fasting, the animals were anaesthetized with ketamine-75mg/kg and xylazine-20mg/kg intraperitoneal injection. The unconscious animals were sacri ced by cervical dislocation and the hearts were exposed by thoracotomy. The fasting blood was collected via cardiac punctured into heparinized tubes, centrifuged at 13000 rpm for 5 mins, and the plasma was then retrieved. The plasma determination of plasma total cholesterol (TC), triglycerides (TG) and HDL-cholesterol, was done using a commercial Diagnostic Kit (Genzyme Diagnostics, MA. USA).
Antioxidant enzyme activities in the plasma were assayed using commercial kits: Serum GSH was measured based on the method described by [27]. Serum MDA, SOD, and GPx levels were measured by enzyme-linked immunosorbent assay (ELISA) methods using Rat MDA, SOD, and GPx Elisa Kit (Elabscince, China).
Markers of kidney function (blood urea nitrogen (BUN), plasma creatinine, and uric acid) were determined by using the commercially kits from Siemens Health Care Diagnostics.The liver biomarkers such as Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), Total protein (TP), and Albumin were assayed in plasma spectrophotometrically by standard automated techniques according to the procedures described by the manufacturers.

Statistical analysis
Data statistical analysis was carried out using SPSS, Version 21 software. The results were expressed as mean ± SEM, and the statistical difference was evaluated using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. Data were considered statistically signi cant at p less than 0.05 (p<0.05)

3.2
Effects of Anacardium occidetale nut extract on water intake in streptozotocin-induced diabetic rats Figure 1 shows the water intake of control and treated rats after administration of Anacardium occidentale nut extract. There was a signi cant increase (p<0.05) in the water intake of diabetic rats when compared with the control. Administration of 100mg/kg body weight and 200mg/kg body weight of Anacardium occidentale nut extract and glimepiride signi cantly decrease water intake in groups 3, 4 and 5 compared to the diabetic group.

3.3
Effects of Anacardium occidetalenut extract on food intake in streptozotocin-induced diabetic rats Figure 2 shows the food intake of control and treated rats after administration of Anacardium occidentalenut extract. There was a signi cant decrease (p<0.05) in the food intake of diabetic rats when compared with the control. Administration of 100mg/kg body weight and 200mg/kg body weight of Anacardium occidentale nut extract and glimepiride signi cantly increase food intake in groups 3, 4 and 5 compared to the diabetic group.

3.4
Effects of Anacardium occidentale nut extract on fasting blood glucose in streptozotocin-induced diabetic rats Figure 3 shows the fasting blood glucose of control and treated rats after administration of Anacardium occidentale nut extract. There was a signi cant increase (p<0.05) in the fasting blood glucose of diabetic rats when compared with the control. Administration of 100mg/kg body weight and 200mg/kg body weight of Anarcadium occidentale nut extract and glimepiride ameliorates the increase in fasting blood glucose in groups 3, 4 and 5 compared to the diabetic group.

3.5
Effects of anarcardium occidetale nut extract on body weight in streptozotocin-induced diabetic rats Figure 4 shows the body weight of control and treated rats after administration of Anacardium occidentale nut extract. There was a signi cant decrease (p<0.05) in the body weight of diabetic rats when compared with the control. Administration of 100mg/kg body weight and 200mg/kg body weight of Anacardium occidentale nut extract and glimepiride attenuates the decreased body weight in groups 3, 4 and 5 compared to the diabetic group.
3.6 Assessment of weekly blood glucose level in streptozotocin-induced diabetic rats In gure 5, blood glucose level of control group was 86.5±4.5mg/dl while that of the diabetic groups increased signi cantly when compared with control induction of diabetes (Day 1) Blood glucose in the diabetic rats treated with 100mg/kg and 200mg/kg of Anacardium occidentale nut extract and 2mg/kg Glimepiride (GMP) respectively decreased when compared with diabetic non-treated rats on Days 7, 14, 21 and 28 of the experiment.

Effect of Anacardium occidentale nut extract on Antioxidant Enzymes and
Oxidative Stress Parameters in streptozotocin-induced male Diabetic rats. 3.8 Effect of Anacardium occidentale nut extract on Lipid pro le in streptozotocin-induced Diabetic rats.
The lipid pro le result ( Figure 6) showed signi cant increases in total cholesterol, triglyceride, low density lipoprotein and very low density lipoprotein concentrations in the STZ group when compared to control. The high density lipoprotein concentration however decreased signi cantly. After treatment of diabetic rats with both doses of the extract, the high density lipoprotein concentration increased signi cantly while the total cholesterol, triglyceride, low density lipoprotein and very low density lipoprotein concentrations were signi cantly reduced comparable to the GMP group.

3.9
Effects of Anacardium occidentale nut extract on Markers of Liver Function in Diabetic Wistar Rats There were signi cant (p<0.01) increases in aspartate aminotransferase (AST), alkaline phosphatase (ALP) and alanine transaminase (ALT) in the STZ group compared to control (Table 3). However, in administration of Anacardium occidentale nut extracts to STZ-induced diabetic rats, apartate aminotransferase, alkaline phosphatase and alanine transaminase activitiesreduced signi cantly (p<0.05) comparable to the GMP group.

Effects of Anacardium occidentale nut extract on Some Markers of Kidney Function in Streptozotocin-Induced Diabetic Rats
The markers of kidney function assessed in this study were presented in Table 4. The urea, uric acid and creatinine levels in the STZ group increased signi cantly (p<0.05) compared to control. However, there were signi cant (p<0.01) reductions in levels of urea, uric acid, and creatinine in the extract and GMP groups compared to STZ group after treatment.

Discussion
Diabetes mellitus can be characterized as exposure to hyperglycemia and increase in total lipids, total cholesterol and LDL-cholesterol in diabetic rats which is now recognized as the primary causal factor in the pathogenesis of diabetic complications as well as induce a large number of alterations in vascular tissue that potentially promotes or accelerates atherosclerosis [28]. Hyperglycemia generates reactive oxygen species (ROS), which in turn causes cellular damage in many ways. Damage to the cells ultimately results in secondary complications in diabetes mellitus [29]. The elevations of total lipids are due to the decrease in lipoprotein lipase (LPL) activity secondary to insulin de ciency [30].
In the study of Locke et al. [31], it has been stated that acute oral toxicity studies are performed in animals to evaluate the safety of plant-based products and other formulations for humans [32]. In this study, the non-toxic nature of the methanolic nut extract of Anacardium occidentale was observed by the acute oral toxicity test. The safe doses in animals were extrapolated to human doses, and at the dose level of 100mg/kg and 200mg/kg of the extract, no mortality or any toxic reactions were found at any of the doses; thus they are used as the low (100mg/kg) and high (200mg/kg) doses in this study respectively.
The phytochemical constituents available in the methanolic nut extract of Anacardium occidentale in this research work revealed the presence of tannins, terpenoids, and reducing sugar while saponins, phlobotannins, cardiac glycosides, anthraquinones, alkaloids, avonoids and steroids tested were all absent which correlates with the previous research [33].
In the present investigation, weight loss was one of the symptoms noticed after the induction of diabetes with STZ, which showed a signi cant decrease in body weight, which may be due to increased muscle wasting. STZ, by causing hyperglycemia and hyperinsulinemia causes a decrease in body weight of diabetic rats as reported by [34]. Treatment with Anacardium occidentale nut extract at low (100mg/kg) and high (200mg/kg) doses for 28 days increase their body weight compared to the levels of untreated diabetic rats.
Fasting blood sugar (FBS) is an important and necessary basal parameter among DM patients [35]. This study showed that Anacardium occidentale nut extract decreased FBS levels and reversed its value in STZ-induced diabetic rats. STZ can damage pancreatic β-cells and decrease endogenous insulin secretion, thereby reducing glucose utilization of tissues. Therefore, the possible mechanism for decreased lipid levels could either be an insulin-releasing effect of Anacardium occidentale nut extract or insulin-sensitizing activity, because insulin has been proved to inhibit the activity of the hormone-sensitive lipases in adipose tissue and suppresses the release of lipids. The HDL-cholesterol is involved in transport of cholesterol from peripheral tissues to the liver and thereby, it acts as a protective factor. In the present study, the level of HDL-cholesterol was also found to be reduced in diabetic rats. The level of HDL-cholesterol was increased in STZ-induced diabetic rats when treated with Anacardium occidentale methanolic nut extract. This indicates that Anacardium occidentale methanolic nut extract may help to increase transport of peripheral tissue cholesterol to the liver and thereby decrease blood cholesterol levels [36].
The food intake decreased while water intake increased in STZ-induced diabetic rats compared to normal control. Treatment with Anacardium occidentale methanolic nut extract showed increased food intake and decreased water intake in extract-treated rats compared to diabetic control. This is similar to the ndings of Al-Awwadi et al. [37], who reported that the classical clinical manifestations of diabetes type I, including weight loss, increased food, and water intakes and reduced insulin concentrations in rats administered STZ. This may be due to Anacardium occidentale alleviating the symptoms of type I diabetes mellitus.
The effect of Glimepiride and Anacardium occidentale on FBS was evaluated after the 28 days study period. Both treatments showed (p<0.05) hypoglycemic activity compared to diabetic control. Experimental animals treated with Anacardium occidentale methanolic nut extract showed a decrease in FBS level but not as much as those treated with standard drugs. Improved blood glucose in the treated animals suggests either increased insulin release or improved insulin activity, both of which could be attributed to improvement in the integrity of β . This research work hence showed that Anacardium occidentale methanolic nut extract contains some components, especially antioxidants capable of suppressing oxidative stress.

Conclusion
Administration of Anacardium occidentale methanolic nut extract exerts signi cant antidiabetic, anti-oxidative, and anti-hyperlipidemic effects as well as improvement in liver and kidney functions in STZ-induced diabetic rats. Anacardium occidentale methanolic nut extract can therefore be a therapeutic application for diabetes mellitus. There is a need for further studies to con rm the mechanism of actions and isolate the active ingredients of this extract so as to develop it as a potent antidiabetic formulation.

Declarations
Ethics approval and consent to participate: All procedure were approved by the Animal care committee of the Ladoke Akintola University of Technology and conducted according to the "Principles of Laboratory Animal Care" and speci c national laws where applicable.

Consent for publication: Not applicable
Availability of data and Materials: The datasets used and/ or analysed during this current study are included in this manuscripts.     Effects of Anacardium occidentale nut extract on weekly blood glucose levels in streptozotocin-induced diabetic rats. Values are expressed as mean ± SEM (n=8). *signi cant at p<0.05 compared with control, #signi cant at p<0.05 compared with STZ group. Key: CON-Control group STZ-Streptozotocin-induced untreated group GMP-Glimepiride treated group