Antidiabetic Effect of Ethanolic Extract of Carica papaya Leaves in Alloxan-Induced Diabetic Rats

The growing number of diabetes coupled with the harsh side effects of some synthetic drugs has led to the increasing search for alternatives which are relatively cheap with minimal side effects. This study sought to investigate the antidiabetic effect of ethanolic extract of Carica papaya leaves in alloxan-induced diabetic rats. Thirty adult male albino rats were induced intraperitoneally with alloxan. The rats were grouped into six groups of five animals per group: Group A was not induced with alloxan, Group B animals were induced but not treated, Group C animals were treated with glibenclaimide, Group D, E and F animals were treated with 200, 400 and 600 mg/kg body weight of ethanolic leaf extract of C. papaya respectively. The extracts were administered to the animals orally for 14 days. The animal’s blood sugar levels were assayed using glucometer. The lipid profiles parameters were assayed using standard methods. The animals administered with 200, 400 and 600 mg/Kg B.W of extract showed significant decrease (P<0.05) in blood sugar level compared to the untreated animals. The decrease in the blood glucose level of the animals following the administration of the plant extract suggested that the plant extract possesses antidiabetic effects. The extract of C. papaya produces hypolipidaemic effect and this is evident as there are significant decrease in plasma total cholesterol, triglycerides, low density lipoprotein-cholesterol and an increase in plasma high density lipoprotein-cholesterol in the treated groups compared to the untreated group. This implies that C. papaya leaves could replace glibenclamide in the treatment of diabetes as it has no side effect. The leaves could also be exceedingly helpful in the control of obesity and hypertension.


Introduction
Carica papaya belongs to the family of Caricaceae, and several species of Caricaceae have been used as remedy against a variety of diseases [1,2]. Originally derived from the southern part of Mexico, C. papaya is a perennial plant, and it is presently distributed over the whole tropical area. In particular, C. papaya fruit circulates widely, and it is accepted as food or as a quasi-drug. Many scientific investigations have been conducted to evaluate the biological activities of various parts of C. papaya, including fruits, shoots, leaves, rinds, seeds, roots or latex. The leaves of C. papaya have been shown to contain many active components that can increase the total antioxidant power in blood and reduce lipid peroxidation level, such as papain, chymopapain, cystatin, à-tocopherol, ascorbic acid, flavonoids, cyanogenic glucosides and glucosinolates [3] (Figure 1a).
Fruit and seed extracts have pronounced bactericidal activities [5]. Leaves have been poulticed into nervous pains, elephantoid growths and it has been smoked for asthma relief amongst tropical tribal communities. The antiplasmodial potency of ethanolic leaf extract of C. papaya against Plasmodium berghei in infected Swiss albino mice has also been reported [4]. Moreover, C. papaya leaf juice is consumed for its purported anti-cancer activity by people living on the Gold Coast of Australia, with some anecdotes of successful cases being reported in various publications. C. papaya leaf extracts have also been used for a long time as an aboriginal remedy for various disorders, including cancer and infectious diseases. C. papaya contains two important biologically active compounds viz., increase in the rate of mortality [9]. Among the multiple risk factors underlining the incidence and progression of diabetes, diet is the main modifiable factor. Both experimental and epidemiological evidences have shown that consumption of vegetables rich in phenolic compounds and possess high antioxidant capacity may have inverse relationship with the incidence and prevalence of diabetes [10]. Dietary control remains one of the most desirable avenues for the prevention and management of chronic degenerative diseases such as diabetes and cardiovascular diseases. Although various plants have been employed in traditional medicine in Nigeria to treat diabetes, a lot remains to be done scientifically to confirm the efficacy of these herbal drugs. This present study sought to investigate the antidiabetic effect of ethanolic leaf extract of C. papaya leaves in alloxan-induced diabetic rats.

Collection and Extraction of Plant Material
Fresh and health leaves of C. papaya free from disease were harvested from the Institute of Agricultural Research and Training, Moor Plantation, Ibadan and were identified by a botanist.
They were washed in running water to remove contaminants. They were air dried at room temperature in an open laboratory space for 14 days and milled into powder using an electronic blender (Moulinex). The extraction was done using soxhlet apparatus and ethanol as the solvent according to the method described [11,12].

Experimental Animals
A total of 30 male albino rats with body weight ranging from 160 to 180 g were obtained from the Federal College of Animal Health and Production Technology, Moor Plantation, Ibadan, Nigeria. They were acclimatized for seven days to Laboratory condition.
They were kept in plastic cages and fed with commercial rat chow and supplied with water ad libitum. The rats were used in accordance with NIH Guide for the care and use of laboratory animals; NIPRD Standard Operation Procedures (SOPs). After the acclimatization period, the rats were injected with alloxan monohydrate dissolved in sterile normal saline in a dose of 150 mg/kg body weight intraperitoneally [13]. After 72 hours of the injection, rats with fasting blood glucose (FBG) at or above 126 mg/dL. (7.0 mmol/L) were considered diabetic.

Grouping of Animals
The animals were randomly divided into six groups of five animals each. They were grouped as follows: Group A: Normal control (non-diabetic rats)

Determination of Fasting Blood Sugar
Fasting blood sugar was determined three times in the course of this study. Firstly, before the induction of diabetes by alloxan secondly, after the induction of diabetes by alloxan administration and lastly, after fourteen (14) days treatment. The blood glucose level were taken by sterilizing the tails of the animals with 10% alcohol, and cutting the tails using scissors then allowing the blood to touch the test strip which was inserted into a calibrated glucose meter (One touch Glucometer, Acon Laboratory INC. San Diego, USA) according to the methods described [14]. This gave direct reading after 5 seconds in mg/dL.

Determination of Lipids
Lipids were extracted and determined according to previously described methods [15,16].

Statistical Analysis
Data were subjected to analysis of variance using Graph Pad Prism. Results were presented as Mean ± Standard Error of the Mean (SEM). One-way analysis of variance (ANOVA) was used for comparison of the means. Differences between means were significant at p<0.05.

Results and Discussion
Diabetes is a major health challenge globally and it is on the increase as carbohydrate is the main food consumed by an average human. Up till now, no synthetic drug has been able to provide permanent cure to diabetes. Various in vivo models (e.g., diazoxide, alloxan, or streptozotocin-induced diabetic rats) are used in evaluating medicinal plants with suspected hypoglycemic potentials [17].
In this study, diabetes mellitus was induced using intraperitoneal injection of alloxan at a dose of 140 mg/kg of body weight. This dose induced diabetes mellitus in the treated rats 72 hours after administration. Alloxan induces diabetes mellitus by selectively destroying the pancreatic beta cells which are involved in the synthesis, storage, and release of insulin which is a peptide hormone that regulate carbohydrate, protein, and lipid metabolism [17].  In this study, no significant difference was observed in the fasting blood sugar of all the animals before induction of diabetes (Fig-ure 1b). After the induction of diabetes by alloxan administration, a significant increase was observed in the fasting blood sugar of all the treatment groups when compared with the normal control group at p< 0.05 ( Figure 2). In the normal control group, the beta cells of the pancreas which produce insulin are intact. But alloxan might have destroyed these beta cells in animals in groups B to F.
Alloxan-induced diabetic rats, therefore, no longer have functional pancreatic beta cells and have lost the capacity to secrete insulin which is required for glucose absorption [18]. profile of albino rats. It also corresponds with another report [19] who studied the effect of methanolic extract of Corchorus olitorius leaves on hypoglycemic and hypolipidaemic activities in albino rats. Several other plants and extracts have also been reported to have an antihyperglycemic and an insulin-stimulatory effect [20][21][22][23].
Most of the plants with hypoglycemic properties have been found to contain metabolites such as glycosides, alkaloid and flavonoids [24][25][26]. One interesting finding of this study is that 600 mg/kg body  Apart from the regulation of carbohydrate metabolism, insulin plays an important role in lipid metabolism. Insulin insufficiency, as in diabetes mellitus, is associated with hypercholesterolemia and hypertriglyceridemia, which have been reported to occur in experimental diabetic rats [31][32][33]. Hypercholesterolemia could result in a relative molecular ordering of the residual phospholipids, resulting in a decrease in membrane fluidity [34]. Accumulation of triglycerides is one of the leading risk factors in coronary heart dis-

ease (CHD). Lipid and lipoprotein abnormalities have been shown
to play a major role in the pathogenesis and progression of several disease conditions [35].
In this study, total cholesterol and triglycerides concentrations were observed to decrease significantly when diabetic animals  [42]. Hypertriglyceridaemia has been reported in diabetic animals [43]. This was reported to be due to increased absorption and formation of triglycerides in the form of chylomicrons following exogenous consumption of diet rich in fat or through increased endogenous production of triglyceride-enriched hepatic VLDL-cholesterol and decreased triglyceride uptake in peripheral tissues [43]. The effect of C. papaya leaves observed in this study might suggest that C. papaya leaves can inhibit the absorption and formation of triglycerides in the form of chylomicrons. Hypercholesterolemia has also been reported in diabetic animals [43]. This was attributed to the increased dietary cholesterol absorption from the small intestine following the intake of high fat diet in a diabetic condition [44]. Hypertriglyceridaemia has also been reported to be a predictor of hypertension [45]. In the while simultaneously decreasing HDL-cholesterol, and has generally been considered pro-atherogenic [46]. This probably explains why C. papaya leaves may lead to a reduction in the risk of developing heart diseases since a high HDL-cholesterol/LDL-cholesterol ratio has been shown to be beneficial and is indicative of a lower risk of cardiovascular diseases [47]. HDL-cholesterol and LDL-cholesterol are two of the four main groups of plasma lipoproteins that are involved in lipid metabolism and the exchange of cholesterol, cholesterol ester and triglycerides between tissues [48,49]. Numerous population studies have shown an inverse correlation between plasma HDL-cholesterol levels and risk of cardiovascular disease, implying that factors associated with HDL-cholesterol protects against atherosclerosis. Some of these factors appear to have antioxidant and anti-inflammatory effects which may obviate processes that initiate atherogenesis [50,51].
Epidemiological studies have also shown that elevated concentrations of total cholesterol and/or LDL-cholesterol in the blood are powerful risk factors for coronary heart disease [52]. Most extra-hepatic tissues, although having a requirement for cholesterol, have low activity of the cholesterol biosynthetic pathway.
Their cholesterol requirements are supplied by LDL, which is internalized by receptor-mediated endocytosis. A major function of HDL-cholesterol is to enhance reverse cholesterol transport by scavenging excess cholesterol from peripheral tissues followed by esterification through lecithin: cholesterol acyltransferase and delivering it to the liver and steroidogenic organs for subsequent syn-thesis of bile acids and lipoproteins and eventual elimination from the body [53,54]. This role of HDL-cholesterol has been shown to be responsible for its atheroprotective properties. HDL-cholesterol also regulates the exchange of proteins and lipids between various lipoproteins.   In addition, HDL-cholesterol provides the protein components required to activate lipoprotein lipase which releases fatty acids that can be oxidized by the ß-oxidation pathway to release energy [48,49]. Most importantly, HDL-cholesterol can inhibit oxidation of LDL-cholesterol as well as the atherogenic effects of oxidized LDL-cholesterol by virtue of its antioxidant property [54]. LDL is a lipoprotein that transports cholesterol and triglyceride from the liver to peripheral tissues. It enables fat and cholesterol to move within the water-blood solution of the blood stream. LDL is often called bad cholesterol; hence low levels are beneficial [55].  (Figure 8). This strongly supports the notion that dietary supplementation with the extract of C. papaya leaves may lead to a reduction in the risk of developing heart diseases, because a high HDL-cholesterol/LDL-cholesterol ratio has been shown to be beneficial and is indicative of a lower risk of CHD [56]. Although, the activities of enzymes were not investigated in this study, but it is possible that C. papaya leaves decreased the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (the rate-limiting enzyme in cholesterol biosynthesis). This implies that C. papaya leaves are of significant health importance as far as hyperlipidaemia is concern. It could also be exceedingly helpful in the control of obesity (Figure 9).

Conclusion
The present study proved that C. papaya leaves is a potent antidiabetic agent with effect like glibenclamide, the standard diabetes drug. It could also reduce the risk of obesity and hypertension due to its hypolipidemic effect.

Conflict of Interest
Authors declare that no conflict of interest exist in this paper.