ANTI-HYPERGLYCEMIC AND ANTI-OXIDANT ACTIVITIES OF METHANOL EXTRACT OF GONGRONEMA LATIFOLIUM

Objective: Gongronema latifolium (Asclepiadiaceae) is used as bitter spice or flavouring agent in many traditional Nigerian dishes. It is well known for medicinal and nutritional purposes like anti-hyperglycemic, anti-oxidant, antitussive and widely distributed in the southeastern states of Nigeria. Objective of present study was to evaluate the anti-hyperglycemic and anti-oxidant effect of the methanol extract of Gongronema latifolium leaves. Methods: The methanol extract of G. latifolium at doses of 250 and 500 mg/kg were studied for anti-hyperglycemic effect in alloxan-induced hyperglycemic rats. The variation in blood glucose level in normal and experimental rats on 0, 7, 14 and 21 days of treatment has also been recorded. The effect of different treatment on body weight of rats was also determined. The antioxidant properties of the extracts as determined by the DPPH free radical scavenging assay. Results: Treatment with G. latifolium extract showed signs of recovery as comparable with the standard drug glibenclamide (0.25 mg/kg). The result showed that G. latifolium extract caused a concentration dependent percentage increase of antioxidant activity. Conclusion: Study concludes that methanol leaf extract of G. latifolium possesses significant anti-hyperglycemic and anti-oxidant activities.


INTRODUCTION
Plants are extremely an important source of medicinal agents. Since ancient times, plants have been used due to their impactful pharmacological properties. WHO esti mates that 80% of the population living in rural areas is dependent on herbal medicine for their health needs. Crude plant extracts in the form of infusion, decoction, tincture or herbal extracts have been traditionally used by the population for the treatment of various diseases 1 . Plant used in treatment of diseases is as old as civilization and traditional medicines are still a major part of habitual treatments of different maladies 2 . Diabetes mellitus is the commonest endocrine disorder that affects more than 171 million people worldwide. It is a condition in which a person has a high blood sugar level. The reason may be either the body doesn't produce enough insulin, or because body cells don't properly respond to the insulin that is produced 3 . When the blood glucose increases for example, after eating food material, insulin is released from the pancreas to normalize the glucose level. If the body cells do not absorb glucose, the glucose accumulates in the blood, leading to vascular, neuron, and other complications 4 . Our body is exposed to a variety of oxidizing agents. Oxidation is a chemical reaction that produces free radicals, leading to chain reactions that may damage cells 5 . Body is equally inbuilt with antioxidants to cater for the free radicals generated from the oxidants, so a balance between the production of free radicals and neutralization by antioxidants is remains maintained. Antioxidants are substances known to protect the body from damage caused by reactive oxygen species induced oxidative stress 6 . Oxidative stress results because of imbalance between formation and neutralization of free radicals by antioxidants, it results to oxidative stress. Oxidative stress has been implicated in the etiology of diseases such as cardiovascular diseases and lung cancer. Antioxidants from natural sources have a higher bioavailability and therefore higher protective efficacy comparison of synthetic antioxidants 7 . Gongronema latifolium (Asclepiadiaceae) is used for medicinal and nutritional purposes widely distributed in the southeastern states of Nigeria 8 . Apart from being used as bitter spice or flavouring agent in many traditional Nigerian dishes the plant leaves has been found very efficacious as an antidiarrhoeal and antitussive. Aerial parts are taken to treat cough, intestinal worms, dysentery, dyspepsia and malaria 9 . It is also taken as a tonic to treat loss of appetite. A decoction of leaves or leafy stems is commonly taken to treat diabetes and high blood pressure 10 . The objective of the present study was to evaluate the antihyperglycemic and anti-inflammatory properties of methanol leaf extract of Gongronema latifolium.

MATERIALS AND METHODS Collection and preparation of leaf samples
Fresh leaves and roots of G. latifolium were collected from Niger Delta and authenticated.

Extraction of plant materials
Fresh leaves of G. latifolium were washed and allowed to dry air at room temperature (28 to 30 o C) for two weeks, to avoid the escape of volatile components by oven-drying. The dried leaves were milled into fine powder using the Christy-Norris hammer mill and passed through a1mm sieve to obtain a fine powder. 2 kg of the sample was percolated in methanol for 48 hrs; this was then filtered through a Whatman filter paper No.1. The filtrate was evaporated to dryness on a hot plate at an initial temperature of 100 0 C and the dry powder obtained was suspended in 10ml distilled water, stirred and refiltered 11 .

Assessment of hypoglycaemic activity
The approval of the Institutional Animal Ethics Committee was obtained before starting the study. An international protocol for conducting experiments on animals were followed. Healthy wistar rats of either sex having weight 150-200 g were selected for this activity 12 . They were housed in standard condition of temperature (25±2 0 C) with 12 h light per day cycle. Before induction of diabetes weigh and normal glucose levels of rats were determined and recorded as Day 0.
The acclimatized animals were fasted for 24 h with water ad libitum 13 . Hyperglycemia was induced using a single intraperitoneal injection of alloxan monohydrate (160 mg/kg). All animals were returned to their cages and given free access to food and water. Blood glucose levels were monitored by using a Glucometer after 72 h of injection and recorded as 1 st day. Rats with fasting blood glucose >7.8 mmol/l or 140 mg/dl were considered hyperglycemic and were selected for the study. Diabetic rats were randomly assigned to 5 groups, each group contains six animals. The animals were grouped as follow  Group I: Normal control  Group II: Diabetic control  Group III: Diabetic rats treated with G. latifolium extract (250 mg/kg)  Group IV: Diabetic rats treated with G. latifolium extract (500 mg/kg)  Group V: Diabetic rats treated with glibenclamide (0.25 mg/kg). Blood samples were obtained from the cut tail tip of the conscious rat and glucose test strip soaked with blood and then inserted to be read by the glucometer. Blood glucose levels were examined after 2, 12, 24, 72 hrs of orally administration of test drugs 14 .

Anti oxidant study DPPH antioxidant assay
The free radical scavenging activity of G. latifolium extract was analyzed by, 1, 1-diphenyl-2-hydrazyl (DPPH) photometric assay. 2 ml of test extract at concentrations ranging from 10 to 400 µg/ ml was each mixed with 1 ml of 0.5mM DPPH in methanol 14 . Absorbance at 520 nm was taken after 30 min incubation in the dark at room temperature using a spectrophotometer 15 . The experiment was done in triplicates and the percentage antioxidant activity was calculated as follows: Where; AbsC=Absorbance of control, AbsB= Absorbance of blank. One ml methanol and 2ml extract were used as blank, while 1 ml 0.5 mM DPPH solution and 2 ml methanol was used as control. Ascorbic acid was used as reference standard.

RESULTS AND DISCUSSION
The anti-hyperglycemic effect of methanol extract of G. latifolium leaves was evaluated for 21 days. Alloxan-induced hyperglycemia is due to selective toxicity of alloxan on the pancreatic beta cells, generation of superoxide radicals and cytotoxic action mediated by generation of reactive oxygen species. The cytotoxic action of alloxan is mediated by reactive oxygen species, with a simultaneous massive increase in cytosolic calcium concentration, leading to a rapid destruction of beta cells 16 . The effect of administration of extracts of G. latifolium on blood glucose level of ISSN: 2456-8058 diabetic rats is shown in Table 1. Treatment of extract of G. latifolium showed a significant reduction in the blood glucose level. It also shows that 0.25 mg/kg glibenclamide is lowering glucose level significantly compared to normal control. Leaf extract at a dose of 500 mg/kg was more effective in reducing the blood glucose level than a dose of 250 mg/kg. The variation in blood glucose level in normal and experimental rats on 0, 7, 14 and 21 days of treatment has also been recorded. Treatment with G. latifolium extract showed signs of recovery as comparable with the standard drug glibenclamide. There was significant loss in body weight of diabetic rats compared to normal rats. In diabetics, glucose is not available therefore the cells use alternatively proteins for energy; consequently due to excessive breakdown of tissue protein a loss in body weight occurs. Body weight slightly increased in the normal control rats compared to initial body weight, whereas in diabetic control rats there was a significant decrease in body weight. Groups that were treated with glibenclamide and G. latifolium extract (250 and 500 mg/kg) showed significant reduction in body weights. The final body weights of treated groups were significantly lower than the final weights of normal control group. Hence, present study showed a good antidiabetic response of leaf extract against the experimental animals. The antioxidant activity was done in vitro using 1, 1-diphenyl-2-hydrazyl (DPPH) spectrophotometric assays. Figure 1 shows the antioxidant activity of G. latifolium and ascorbic acid standard. The result showed that G. latifolium extract caused a concentration dependent percentage increase of antioxidant activity. DPPH is a stable free radical, it accepts an electron or hydrogen radical to become a stable diamagnetic molecule which is widely used to investigate radical scavenging activity. The degree of discoloration indicates the radicalscavenging potential of the antioxidant. Result shows that DPPH scavenging was increased in a concentration dependent manner compared to ascorbic acid 16 .

AUTHOR'S CONTRIBUTION
The manuscript was carried out, written, and approved in collaboration with all authors.