Introduction

Diabetes is becoming something of a pandemic, and despite the recent surge in new drugs to treat and prevent the condition, its prevalence continues to soar. Perhaps the most worrying aspect of all is that the rise is even reflected in children. Besides hyperglycemia, several other factors, including hyperlipidemia or hyperlipidemia, are involved in the development of micro and macrovascular complications of diabetes which are the major cause of morbidity and death (Thiruvenkatasubramaniam & Jayakar, 2010).

Although several drugs targeted for carbohydrate hydrolyzing enzymes (psuedosaccharides), the release of insulin from pancreatic β-cells (sulphonyl ureas), glucose utilization (biguanides), insulin sensitizers, PPARg agonists (glitazones) are in clinical practice, the growing diabetes market observes several changes. Some of these drugs are linked to liver toxicity (troglitazone), including a few deaths from hepatic failure and raising the symptoms and risk factors of heart disease leading to heart failure (rosiglitazone).

Therefore, as the long term of risk and effect on the complications of diabetes-related with these drugs are not at clear. On the other hand, traditional medicinal plants with various active principles and properties have been used since ancient times by physicians and laymen to treat a great variety of human diseases such as diabetes, coronary heart disease and cancer.

The beneficial multiple activities like manipulating carbohydrate mechanism by various mechanism, preventing and restoring integrity and function of β-cells, insulin-releasing activity, improving glucose uptake and utilization and the antioxidant properties present in medicinal plants offer exiciting opportunity to develop them into novel therapeutics. The multifactorial pathogenicity of diabetes demands a multimodal therapeutic approach. Thus, future therapeutic strategies require a combination of various types of multiple agents. Thus, plant-based herbal drugs and botanicals with free radical scavenging activity are emerging as the primary components of holistic approaches to diabetes management (Büyükbalci & El, 2008; Sabu & Kuttan, 2002).

Wound healing is the process of repair that follows injury to the skin and other soft tissues. Following injury, an inflammatory response occurs, and the cells below the dermis (the deepest skin layer) begin to increase collagen (connective tissue) production. Later, the epithelial tissue (the outer skin layer) is regenerated (Nayak & Pereira, 2006). There are three stages to the process of wound healing: inflammation, proliferation, and remodeling. The proliferative phase is characterized by angiogenesis, collagen deposition, epithelialisation and wound contraction. Current methods used to treat chronic wounds include debridement, irrigation, antibiotics, tissue grafts and proteolytic enzymes, which possess major drawbacks and unwanted side effects (Nayak, Sandiford, & Maxwell, 2009).

Benincasa hispida (Thunb) Cogn. (Family: Cucurbitaceae) is commonly known as white pumpkin or wax gourd or ash gourd. The fruit B.hipsida is an important ingredient of Kusmanda lehyam (Ayurvedic medicine), which is widely used in nervous disorders. The fruits and seeds of B.hipsida possess a number of pharmacological properties and uses laxative, tonic, diuretic, aphrodisiac, antiperiodic, haemoptysis, other internal hemorrhages in insanity, epilepsy and other nervous disorders (Chopra, 1956). In this backdrop, the present study was conducted to evaluate the antidiabetic and wound healing activity of methanol and petroleum ether extracts of B. hispida leaves on streptozocin induced diabetes and excision wound models.

Materials and Methods

Plant extraction

Fresh leaves of B. hispida were collected locally and dried under shade. They were identified and authenticated by Dr. Madhavachetty, Head of Department, Botany, Sri Venkateshwara University, Tirupathi, Andhra Pradesh. The dried leaves were pulverized and then the coarse powder was subjected to methanol and ethylacetate extraction using cold maceration. Then the resultant product is filtered, dried in a desiccator and stored until further use.

Animals

Healthy adult male Wistar rats weighing 250–300 g were used in this study. They were maintained on a standard laboratory pellet chow diet, provided water ad libitum and were kept under standard conditions at 23-25° C, 35 to 60% humidity, 12hr light /dark cycle. The mice were acclimatized to the laboratory conditions a week prior to the experiment. The experimental protocol was duly approved by an institutional animal ethics committee (IAEC). The animal experimentation was carried out under CPCSEA registration.

Acute toxicity studies

The acute toxicity studies for methanolic (MEBH) and petroleum ether (PEMD) of B. hispida were done according to the OECD guidelines No. 423. The extract did not produce any signs of toxicity when given in doses up to 2000 mg/kg by an oral route. Hence, for further studies, 100 & 200 mg/kg dose of the extract were selected.

Streptozocin induced diabetes

A freshly prepared solution of STZ (60 mg/kg in 0.1 M citrate buffer, pH 4.5) was injected intraperitonially to overnight-fasted rats exhibited hyperglycemia within 48 h of STZ administration (Vogel, 1051). The rats having fasting blood glucose (FBG) values of 250 mg/dl or above were considered for the study.

Study design

The experiment was carried out in groups of six rats each:

Group I- Normal control rats received saline.

Group II- Diabetic control rats received streptozocin (60mg/kg)

Group III- Diabetic rats treated with standard drug, Metformin (50 mg/kg).

Group IV- Diabetic rats treated with MEBH (50 mg/kg in 0.5% CMC).

Group V- Diabetic rats treated with MEBH (100 mg/kg in 0.5% CMC).

Group VI- Diabetic rats treated with MEBH (200 mg/kg 0.5% CMC).

Group VII-Diabetic rats treated with PEBH (50 mg/kg 0.5% CMC).

Group VIII-Diabetic rats treated with PEBH (100 mg/kg 0.5% CMC).

Group IX- Diabetic rats treated with PEBH (200 mg/kg in 0.5% CMC).

Serum glucose was estimated on day 1, 7, 14 and 21 by the Glucose oxidase method and the absorbance was measured at 505 nm by UV-Spectrophotometer (ELICO-Sl-159). On the 22^nd day, the animals were sacrificed, and the liver was removed and homogenized for the estimation of antioxidants parameters.

Preparation of liver homogenate

The liver was quickly removed and perfused immediately with ice-cold saline (0.9% NaCl). A portion of the liver was homogenized in chilled Tris–HCl buffer (0.025 M, pH 7.4) using a homogenizer. The homogenate obtained was centrifuged at 5000 rpm for 10 min, the supernatant was collected and used for the estimation of antioxidants Estimation of antioxidants.

The hepatic levels of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA), an index of lipid peroxidation was estimated by standard kits.

Estimation of lipid profiles

The serum levels of total cholesterol, LDL, HDL. VLDL and triglycerides were estimated by standard biochemical kits.

Wound healing activity

Animals were under light ether anaesthesia through the surgical procedure. An impression of 2.5 cm diameter (500 sq mm) as described by Morton and Malone was made after leaving at least 5 mm complete space from the ears. The skin of the impressed area was excised carefully to the complete thickness, and a wound of 500 sq mm was formed. Homeostasis was achieved by the application of a normal saline solution. The rats were kept individually in separate cages. The physical attributes of wound healing viz wound closure (contraction) and epithelization were recorded. The wound contraction was studied by tracing the raw wound area on transparent paper on 0,3^rd, 6th, 9th, 12th, 15th and 18th day. (Morton & Malone, 1972) The criterion for complete epithelization was fixed as the formation of a scar with an absence of raw wound area. The animals were grouped as follows,

Group I: Control, applied topically (0.5 g), simple ointment.

Group II: Standard, applied topically (0.5g), 5% w/w Povidone ointment.

Group III: Excision Wound.

Group IV: Treated with MEBH 2% w/w ointment (0.5g), topically.

Group V: Treated with MEBH 5% w/w ointment (0.5g), topically.

Group VI: Treated with MEBH 10% w/w ointment (0.5g), topically.

Group VII: Treated with PEBH 2% w/w ointment (0.5g), topically.

Group VIII: Treated with PEBH 5% w/w ointment (0.5g), topically.

Group IX: Treated with PEBH 10% w/w ointment (0.5g), topically

Statistical analysis

The data were expressed as mean ± SEM. The results were analyzed by SPSS version 19 using a one-way analysis of variance. The differences between mean values were considered significant at P < 0.05.

Results and Discussion

Acute toxicity studies

Acute oral toxicity studies revealed the non-toxic nature of the MEBH and PEBH. There was no lethality observed and any profound toxic reactions found at a dose of 2000mg/kg b.wt. p.o. which indirectly pronounces the safety profile of the plant extract.

Antidiabetic activity of B.hispida on STZ induced diabetes

In this study, a single intraperitoneal injection of streptozocin (60mg/kg; b.wt) had displayed noxious biochemical changes. Whilst treatment with MEBH and PEBH significantly attenuated the toxic manifestation and thus inhibited the state of diabetes. In the present study, STZ intoxicated rats displayed significant (p<0.001) elevation of blood glucose level on the 1^st, 7^th, 14^th and 21^st day as that of the control rats. Meanwhile, MEBH and PEBH at the doses of 5 100mg/kg had not display any significant alteration in the blood glucose level as that of the diabetic control. However, treatment with MEBH and PEBH at the dose of 200 mg/kg showed a significant (p<0.001) reduction in the blood glucose level on the 7th, 14th and 21st day as compared to the diabetic control (Table 1).

The values are expressed as Mean ± SEM, n=6. * p<0.05, ** p<0.01, *** p<0.001, extract-treated groups were compared with diabetic control group. ^NS non-significant One-way ANOVA followed by Dunnett’s t-test. ^*** p<0.001, the diabetic control group was compared with the normal group.

Effect of B.hispida on antioxidant enzymes and lipid peroxidation in STZ induced diabetic rats

In the present study, STZ intoxicated rats displayed a significant (p<0.001) reduction in the level of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) and an increase in the MDA in the liver tissue of diabetic rats. However, treatment with MEBH and PEBH at the dose of 100mg/kg had not displayed effects, but at the dose of 200mg/kg, there was a significant increase in the level of antioxidants and a decrease in lipid peroxidation (Table 2).

The values are expressed as Mean ± SEM, n=6. * p<0.05, ** p<0.01, *** p<0.001, extract-treated groups were compared with diabetic control group. ^NS non-significant One-way ANOVA followed by Dunnett’s t-test. ^*** p<0.001, the diabetic control group was compared with the normal group.

Effect of B.hispida on lipid profiles in STZ induced diabetic rats

In this study, STZ intoxicated rats displayed significant (p<0.001) elevation in the level of total cholesterol, LDL, VLDL triglycerides and a decrease in HDL in serum. Meanwhile, treatment with MEBH and PEBH at the dose, 100 mg/kg, displayed a minimal restoration of lipid profiles, but it was statistically non-significant. However, MEBH and PEBH at the dose of 200mg/kg showed a significant (p<0.001) decrease in the level of total cholesterol, LDL, VLDL and triglycerides with an increase in HDL level and thus restored the lipid profiles to normalcy (Figure 1)

Wound healing activity of B.hispida on excision wound models

On day 0 till day 3 there were no significant wound contractions observed in both control and wound-induced and extracts treated experimental groups. On the 6th day, a significant wound contraction process started in experimental animals treated with 5 and 10% ointment of MEBH and PEBH. On day 12, the wound contraction rate was rapid in 10% ointment of MEBH and PEBH as that of the 2% and 5% ointment of MEBH and PEBH. The complete healing was observed in 10% ointment of MEBH and PEBH on 15^th day, whilst in 5% ointment of MEBH and PEBH, the complete healing was observed only on the 18^th day (Figure 2).

Effect of B.hispida on a period of epithelization on wound-induced rats

In this study, the epithelialization period was significantly (p<0.01; P<0.001) lower in 10% and 5 % ointment of MEBH and PEBH as that wound-induced group (Table 3).

The values are expressed as Mean ± SEM, n=6. ** p<0.01, *** p<0.001, extracts treated groups were compared with a wound-induced group. ^NS non-significant One-way ANOVA followed by Dunnett’s t-test.

Discussion

The present investigation discusses the antidiabetic and wound healing potential of the leaves of the latex of A. megalacantha in STZ-induced diabetic mice. The use of STZ to induce DM in rodent models is widely accepted, and STZ-induced diabetes is reported to resemble human DM (Adisa, Choudhary, & Olorunsogo, 2011), which is characterized by glycosuria, hyperglycemia, polyphagia, polydipsia, and body weight loss when compared with normal rodents (Kumar, Kumar, & Prakash, 2010). Metformin is often used as a standard antidiabetic drug in STZ-induced moderate diabetes to compare the antidiabetic effects of a variety of bioactive compounds (Ramkumar et al., 2011).

The B.hispida and metformin-treated groups showed a reduction of the glucose levels compared to diabetic control. The decrease of the glucose levels may be due to the plasma insulin levels elevation or the enhancement of the blood glucose transportation in the peripheral tissue (Petchi, Vijaya, & Parasuraman, 2014). Glibenclamide could enhance the insulin secretion from the pancreatic beta cells by the closure of K_ATP channels. As a result, the membrane will be depolarized and cause the activation of the voltage-dependent Ca²⁺ channels. The influx of Ca2+ to the cells will initiate the secretion of insulin (Fridlyand, Jacobson, & Philipson, 2013). The production of insulin could lower down the glucose level and reverse back the glycemic control. There was a significant elevation of the glucose levels in the diabetic control and this may due to the damage of the beta cells of the pancreas.

Diabetic rats also showed increased levels of serum cholesterol, TG, LDL, VLDL, and decreased HDL levels. Increased in levels of TC, LDL and VLDL indicate the development of hyperlipidemia in rats, and this may be due to an increase in the activity of hormone-sensitive lipase, which catalyzes the mobilization of fatty acids from triacylglycerols stored in adipocytes (Ahmadian, Duncan, Jaworski, Sarkadi-Nagy, & Sul, 2007).

Wound healing is a process by which damaged tissue is restored as closely as possible to its normal stage and wound contraction is a process of shrinkage of an area of the wound. It depends upon the reparative abilities of the tissue, type and extent of the damage and general state of health of the tissue. The granulation tissue of the wound is generally composed of fibroblasts, collagen, edema and small new blood vessels (Singh et al., 2005). Since B.hispida enhanced wound contraction, it would have either enhanced the contractile property of myofibroblasts or increased the number of myofibroblasts recruited in the wound area. In the excision wound model, B.hispida longifolia fasten the period of epithelization. It appears that B.hispida was able to promote epithelization either by a proliferation of epithelial cells or by increasing the viability of epithelial cells.

Conclusion

The present study indicates the significant anti-diabetic effect of the various extracts of leaves extracts of Benincasa hispida and supports its traditional usage in the control of diabetes. And also concluded that the leaves extracts of B.hispida has a strong effect on wound healing in albino rats. Further studies are required for the detailed studies in isolation of the compounds and pharmacological investigations to explore the exact mechanism of action.

Conflict of Interest

The authors declare that they have no conflict of interest for this study.

Funding Support

The authors declare that they have no funding support for this study.