Abstract: Background and Objective: Diabetes has become a global health burden and its management without side effects is still a challenge which leads to an increase in search for herbal anti-hyperglycaemic agents. Traditional medicines are used in Nigeria to treat diabetes due to their acceptability and availability. The plants are used in crude form or as extract, individually or as a mixture. The objective of this study was to investigate the effects of different solvents extracts of Persea americana leaf in alloxan induced-diabetic rats. Materials and Method: Phytochemical analysis of the fractions was carried out using colour reaction method. Hyperglycaemia was induced by intraperitoneal administration of alloxan monohydrate to albino rats. Different solvents extract of P. americana leaf were administrated to group of experimental rats. Blood samples were collected by tail tipping at the end of the experiment and analyzed using glucometer. Results: Ethyl acetate fraction was found to have the highest amount of total extractable compounds with chloroform having the least amount. Phytochemical analysis of P. americana leaf revealed the presence of saponins, flavonoids, tannins, alkaloids, cardiac glycosides, triterpenoids and catechins with significant levels of total flavonoids and phenolics found in ethyl acetate fraction. The results of hypoglycemic study showed significant reduction in fasting blood glucose with concomitant reversal of body weight lost, polydipsia and polyphagia in alloxan induced-diabetic rats administered with ethyl acetate extract. The order of effectiveness was ethyl acetate>chloroform>hexane>butanol fraction. Conclusion: The hypoglycemic potential of P. americana leaf observed in this study may be associated with total flavonoids and phenolics with antioxidant activities via insulin modulation and thus, could served as a potential candidate in developing drugs to manage diabetes mellitus and its complications.
INTRODUCTION
Medicinal plants have continued to attract attention in the global search for effective methods of using plant parts (e.g. seeds, stems, leaves, roots and bark etc.) for the treatment of many diseases affecting humans1. Many important drugs used in medicine today are directly or indirectly derived from plants. The most important of these bioactive constituents of plants are alkaloids such as (nicotine, reserpine, actinidine etc), steroids, tannins, flavonoids, saponins etc.1. The avocado (Persea americana) is a tree native to Pueblo, Mexico2 classified in the flowering plant family Lauraceae along with cinnamon, camphor and bay laurel. Diabetes Mellitus (DM) defined as a syndrome characterized by persistent hyperglycaemia associated with disturbance in carbohydrate, protein and lipid metabolism as a result of absolute or relative deficiency in insulin secretion or action as well as increased cellular resistance to that hormone with dysfunction in organ systems. Diabetes is divided into 2 types, Type 1 diabetes, also term as juvenile diabetes which occurs when the body failed to produce enough insulin, the primary hormone in the body responsible for regulating the level of sugar within the blood stream. This condition can usually be treated effectively with insulin injections3,4. Type 1 diabetes is a relatively rare immunological disorder. On the other side 95% of diabetes cases are Type 2 diabetes which results mostly due to a combination of insulin resistance and an inadequate compensatory insulin secretory response. This results in severe microvascular and macrovascular problems, including neuropathy, nephropathy and retinopathy, cardiovascular and peripheral vascular disease3. Hyperglycaemia generates Reactive Oxygen Species (ROS), which in turn cause damage to the cells in many ways. Damage to the cells ultimately results in secondary complications in diabetes mellitus3. Modern medicines like biguanides, sulphonylureas and thiazolidinediones are the ordinary dugs for the treatment of Type 2 diabetes5,6. The undesirable side effect of these drugs pulled the medicinal researchers to obtain new treating materials characterized by high rate of effectiveness, little adverse effects, easily used and less expensive for all these things plants have become representing the main target for alternative medicine and plant origin drugs become the drugs of choice7,8. The treatment of diabetes and related diseases with medicinal plants has increased in recent years4. Reasons for the increased popularity of these herbal medicines may include their relatively low cost compared to orthodox medicines, availability (since they are almost derived from available plants in the local region) and efficacy3,9. Therefore, the aim of this present study was to evaluate the effect of different solvents fractions of Persea americana in the management of hyperglycaemia in alloxan-induced hyperglycemic rats.
MATERIALS AND METHODS
Experimental animals: Albino wistar rats (70-100 g b.wt.) were purchased from Physiology Department, Bayero University, Kano (B.U.K). They were kept in animal cages at the animal room of same Physiology Department. They were acclimatized for 3 days and had free access to feed and water ad libitu prior to the studies.
Sample collection, preparation and identification: The leaf of Persea americana was collected from Jos, Plateau state, Nigeria. It was authenticated by a Botanist at Plant Science Department, Bayero University, Kano with accession number BUKHAN 0326. The sample was chopped into small pieces and then shades dried and ground into powdered form.
Ethical clearance: The study was approved by the College of Health Sciences Animal Ethical Committee of Bayero University, Kano. Authors declared that animal NIH 1999 rules and regulations regarding the use experimental animals were observed. The ethical clearance was assigned a reference number 651.
Quantitative analysis
Estimation of total phenolic content: The Total Phenolic Content (TPC) was determined by spectrophotometric method. Briefly, 1 mL of sample (1 mg mL–1) was mixed with 1 mL of Folin-Ciocalteu’s phenol reagent. After 5 min, 10 mL of 7% Na2CO3 solution was added to the mixture followed by the addition of 13 mL of deionized distilled water and mixed thoroughly. The mixture was kept in the dark for 90 min at 37°C, after which the absorbance was read at 750 nm. The TPC was determined from extrapolation of calibration curve which was made by preparing gallic acid solution. The estimation of the phenolic compounds was carried out in triplicate. The TPC was expressed as milligrams of Gallic Acid Equivalents (GAE) per gram of dried sample10.
Estimation of total flavonoid content: The extract (0.3 mL) was placed in 10 mL test tube, 3.4 mL of 30% methanol, 0.15 mL of NaNO2 (0.5 M) and 0.15 mL of AlCl.6H2O (0.3 M) were mixed. After 5 min, 1 mL of NaOH (1 M) was added. The solution was mixed well and the absorbance was measured against the reagent blank at 506 nm. The standard curve for total flavonoids was constructed from the data generated using rutin standard solution (0-100 mg L–1) under the same procedure as earlier described. The total flavonoids were expressed as milligrams of rutin equivalents per gram of dried fraction11,12.
Fasting blood glucose determination: Fasting Blood Glucose (FBG) levels were determined with the aid of ACCU-CHEK active glucometer and strips. The plasma glucose estimation was done by glucose oxidase/peroxidase (GOD/POD) method using a standard kit13. The test strip was inserted into the glucometer; blood sample was collected from the tail of the rat by tail tipping using a surgical blade. The blood was then applied to the test area on the strip and the glucometer reads and display the FBG concentration (mg dL–1).
RESULTS
The extraction yield (%) of n-Hexane, chloroform, ethyl acetate and n-Butanol fractions of P. americana leaf was presented in Table 1. Ethyl acetate had the highest (%) of extractable compounds with chloroform having the least quantity. The qualitative phytochemical analysis (Table 2) of solvents fraction showed the presence of some phytochemicals with terpenoids and steroid present in ethyl acetate fraction while anthraquinones was found to be absent in all the fractions. Total phenolics and flavonoids content (Table 3) of the fractions was determined, the results showed appreciable amount of these compounds with significant amount (p<0.05) present in ethyl acetate fraction. There was decrease in fasting blood glucose level in alloxan-induced diabetic rats administered with the various fractions with significant effect (p<0.05) exhibited by ethyl acetate fraction (Fig. 1). Significant decrease in body weight (Fig. 2) was observed in groups administered with hexane, chloroform and butanol with significant increase in groups administered with ethyl acetate and glucophage (p<0.05). Figure 3 and 4 presented mean feed and water intake of alloxan induced diabetic rats treated with the fractions, the results revealed significant decrease in feed and water intake in group administered with ethyl acetate fraction.
| Fig. 1: | Reduction (%) in FBG of alloxan-induced diabetic rats administered with n-Hexane, chloroform, ethyl acetate, n-Butanol fractions of P. americana leaf (400 mg kg–1 b.wt.) and Glucophage (84 mg kg–1 b.wt.) for 7 days |
| Results are presented as Mean±SD (n = 5 #Significant difference (p<0.05) with reference to diabetic control group | |
| Fig. 2: | Initial and final body weight of rats administered with n-Hexane, chloroform, ethyl acetate, n-Butanol fractions of P. americana leaf (400 mg kg–1 b.wt.) and Glucophage (84 mg kg–1 b.wt.) for 7 days |
| Results are presented as Mean±SD (n = 5), values with the different superscripts in the same cluster are significantly different (p<0.05), NC: Normal control, DC: Diabetic control, HF: Hexane fraction, CF: Chloroform fraction, EF: Ethyl acetate fraction, BF: Butanol fraction, GP: Glucophage | |
| Fig. 3: | Mean feed intake (g) of rats administered with n-Hexane, chloroform, ethyl acetate, butanol fractions of P. americana leaf (400 mg kg–1 b.wt.) and Glucophage (84 mg kg–1 b.wt.) for 7 days |
| Fig. 4: | Mean water intake (mL) of rats administered with n-Hexane, chloroform, ethyl acetate, butanol fractions of P. americana leaf (400 mg kg–1 b.wt.) and glucophage (84 mg kg–1 b.wt.) for 7 days |
| Table 1: | Extraction yield of n-Hexane, chloroform, ethyl acetate and n-Butanol fractions of P. americana leaf |
| Table 2: | Phytochemical analysis of n-Hexane, chloroform, ethyl acetate and n-Butanol fractions of P. americana leaf |
| +ve: Present, -ve: Absent | |
| Table 3: | Total flavonoids and phenolics content of n-Hexane, chloroform, ethyl acetate and n-Butanol fractions of P. americana leaf |
Results are presented as Mean±SD, values bearing different superscripts in the same row are significantly different (p<0.05) |
|
DISCUSSION
Although different types of oral hypoglycaemic agents are available along with insulin for the treatment of diabetes mellitus, demand for the use of natural products with antidiabetic activity is on the increase. Insulin cannot be used orally and the continuous use of synthetic drugs has its risk of side effects and toxicity14,15. The yield (%) of ethyl acetate fraction of P. americana leaf was found to exceed that of n-hexane, chloroform and butanol fractions. However, differs from the yields reported previously by using methanolic extract16. The difference in yield might be due to the extraction solvents, procedures and duration used. Previous studies concerning the phytochemical constituents of medicinal plants, revealed the presence of secondary metabolites that were shown to have hypoglycemic and hypolipidemic effects such as Saponins17, alkaloids18, flavonoids19,20, tannin21, phenol derivatives22 and terpenoids23. Results from this study shows that total phenolics and flavonoids composition of EPAL was found to exceed that of n-hexane, chloroform and n-butanol fractions significantly, which was consistent with the observed antihyperglycaemic activity. The 5 h effect of a single dose (400 mg kg–1) of these fractions on blood glucose level in alloxan-induced diabetic rats was evaluated. The results indicated that all fractions tested displayed antihyperglycaemic effects under acute conditions. However, the effect of ethyl acetate leaf fraction of P. americana (EPAL) was more consistent at acute level and thus was considered the fraction with the highest antihyperglycaemic effect. The profound action of all the tested fractions was also observed in the 7 day antihyperglycaemic study which reveals significant reduction in blood glucose in ethyl acetate administered group. However, this observation disagrees with a previous study which indicated that n-butanol fraction had the highest activity24, a similar study indicated that the ethyl acetate fraction was the most effective in lowering glucose compared to the n-hexane and n-butanol fractions25. A number of plants were found to possess hypoglycemic effects and the possible mechanism suggested for such hypoglycemic actions could occurs at the pancrease level by protecting, regenerating, repairing pancreatic beta cells, increasing the size and number of cells in the islets of Langerhans26, stimulation of insulin secretion from surviving beta cells of islets or/and inhibition of insulin degradative processes27. While other possible mechanism could be exerted by inhibition in renal glucose re-absorption28 stimulate reduction in insulin resistance29 stimulation of glycogenesis and hepatic glycolysis25. The antidiabetic action of several medicinal plants has been attributed to the phenolic and flavonoids contents30,31. The high composition of phenols and flavonoids and other phytoconstituents present in EPAL may also be responsible for the observed glucose lowering action. The decrease in blood glucose level observed in this study is in line with the works of some authors which supported to the use of P. americana leaf preparations as an anti-diabetic agent32-34. One or more of the other chemical constituents of the plant especially flavonoid31 may have played a role in the anti-hyperglycemic action of plant extract31 in diabetic and normal mammal35.
In the anti-hyperglycaemic study, EPAL significantly reversed the excess feed and water intake as well as body weight loss in hyperglycaemic treated rats. These observations may be due to reversal of hyperglycaemia and the effect of the plant extract on lipolysis, thus, countering the basic symptoms of DM through polyphagia and polydipsia. Decreased body weight seen in diabetic groups was improved following oral administration of both EPAL and glucophage. Diabetes mellitus causes a drastic change in body weight36-39, which may develop due to an excessive breakdown of tissue proteins and lipids caused by insulin insufficiency. The improvement in body weight seen in hyperglycaemic rats treated with EPAL might be underpinned by an improved metabolic activity, making the body system more capable of maintaining blood glucose homeostasis40,41.
CONCLUSION
The result of the study showed reduction in fasting blood glucose (54.6%), with concomitant reversal of body weight lost, polydipsia and polyphagia in alloxan induced-diabetic rats administered with 400 mg kg–1 of ethyl acetate extract compared to chloroform, n-hexane and n-butanol fractions. The hypoglycaemic potential of P. americana leaf observed in this study may be associated with total flavonoids and phenolics with antioxidant activities via insulin modulation and thus, could served as a potential candidate in developing drugs to manage diabetes mellitus and its complications.
ACKNOWLEDGMENT
The authors wish to acknowledge the efforts of Dr Aminu Ibrahim and Dr Aminu Idi of Biochemistry Department, Faculty of Basic Medical Sciences, Bayero University, Kano in ensuring the success of this work. This work was partially supported by Department of Biochemistry, Faculty of Basic Medical Sciences, Bayero University Kano.