Antidiabetic and Antihyperlipidemic Activities of Methanolic Extract of Leaves of Coccinia grandis in Diabetic Rats

Coccinia grandis is a one of the edible plant and its antidiabetic and antihyperlipidemic activities are not well explored. Hence, the present is planned to study the antidiabetic and antihyperlipidemic activities of methanolic extract of Coccinia grandis leaves in streptozotocin-induced diabetic rats. Coccinia grandis leaves was dried under the shade and extracted with methanol. Pre-liminary phytochemical and pharmacological analysis were conducted using methanolic extract of Coccinia grandis (MECG) leaves. Female Sprague dawley (SD) was used for acute toxicity studies. Female SD was induced with diabetes by administering streptozotocin (55 mg/kg, i.p.). Glibenclamide (20 mg/kg, p.o.) or MECG leaves (125, 250 and 500 mg/kg, p.o.) used to treat diabetic rats for 28 days. Blood samples were collected at regular intervals to check the antidiabetic effect of MECG. On 28 th day, blood sample was collected from the rats to analyse biochemical and lipid pro(cid:976)ile. MECG did not show any toxic symptoms up to the dose 2000 mg/kg/ BW. MECG at 125, 250 and 500 mg/kg marked signi(cid:976)icant antidiabetic and antihyperlipidemic activities. Coccinia grandis reduced streptozotocin-induced weight loss and signif-icantly recovered lipid levels. At the end of the study, MECG exhibited signi(cid:976)i-cant antidiabetic and antihyperlipidemic activities in streptozotocin-induced diabetes in rats.


INTRODUCTION
Diabetes is known as a type of heterogeneous disease linked with glucose metabolism due to abnormalities in insulin secretion and action. In 2019, approx. 463 million peoples living with diabetes and this will rise to 700 million in 2045. It was classiied as one of the top 10 causes of death in adults, and approximately 4.2 million deaths were recorded globally in 2019. Besides this, global health expenditure on diabetes was reported to be USD 760 billion (IDF Diabetes Atlas ninth edition, 2019). It is described as hyperglycemia, various microvas-cular and macrovascular diseases, including glucosuria (Gong et al., 2017). Illness in diabetes is due to hyperglycemia-induced oxidative stress which reduces anti-oxidant activity by scavenging free radicals (Ayepola et al., 2014). Impairment in lipid metabolisms such as hyperlipidemia and hypercholesterolemia found in late stages and as risk factors in the development of atherosclerosis (Krishnakumar et al., 1999). Chances of liver damage caused by diabetes are high due to excess ketogenesis and gluconeogenesis (Agius et al., 1986). No effective treatment available for diabetes and some of the drugs and insulin preparations which currently being used in controlling the disease are causing various unexpected side effect (Chaudhury et al., 2017). Expensive and unexpected side effects of anti-diabetic drugs tiggers inding of plants with hypoglycemic properties and their role in the management of diabetes (Ajiboye et al., 2014;Calixto, 2000).
Medicinal plants have a signi icant role with more signi icant support for alternative medicine and new drug development. Since the suggestions made by World Health Organisation (WHO) on diabetes mellitus, the application of medicinal plant to treat diabetes mellitus gained demand in the past few years. This encourages the inding for effective and safer anti-diabetic compounds obtained from medicinal plants (El-Abhar and Schaalan, 2014). Because of the increasing prevalence of diabetes, investigation on the bene its from traditional medicines might help the indings of possible therapeutic methods to manage and prevent diabetes mellitus and its illness (Patel et al., 2012). Plants showed hypoglycemic properties in managing diabetes due to its contents such as plant polysaccharides, glycosides, alkaloids terpenoids, lavonoids and other bioactive compounds.
Cucurbitaceae belongs to a plant family and contains around 125 genera and 960 species. The number of species such as Citrullus colocynthis (L), Schrad, Coccinia indica Wight et Arn., sativus L., Cucumis Bryonia alba L., Momordica cymbalaria Hook.,Momordica charantia L., Tricosanthes dioica Roxb., Momordica foetida Schumach. Coccinia indica Wright and Arn., and Coccinia cordifolia (L.) Cogn., Cephalandra indica, Naud., and Bryonia cordifolia (L.) Voigt. are the other names of Coccinia grandis (Nagare et al., 2015) . Scienti ic investigations have supported the potency of Coccinia grandis leaves extracts in curing skin diseases, urinary tract infections, bronchitis, itchy skin eruptions and ulcers (Krishnakumari et al., 2011). Furthermore, the leaves also act as antioxidative, anti-in lammatory agent and showed ability to treat antimicrobial infections (Yadav et al., 2010). However, limited scienti ic information available on ef iciency of glucose tolerance and biochemical assessment. Therefore, aim of current study is to analyse the antidiabetic and antihyperlipidemic activities of methanolic extract of Coccinia grandis leaves in streptozotocin-induced diabetic rats.

Plant material
Coccinia grandis leaves were collected from Semeling, Sungai. Petani, Kedah and botanist recognized it at the herbarium section, voucher code (USM 11768/09/2018). The leaves were cleaned in running water; dried under the shade and powdered using a grinding machine. The powder was stored in an airtight container for further use.

Chemicals
Streptozotocin (STZ) and all other chemicals used in this experiment were purchased from Sigma, Malaysia and Merck or SD ine Chemicals respectively and were of analytical grade.

Extraction
Coccinia grandis powder were weighed and subjected to maceration with methanol solvent in a conical lask and kept aside for 7 days at the room temperature with frequent agitation. After the completion of maceration process, the extract was then iltered through muslin cloth and the extract was concentrated to a solid mass by evaporation under reduced pressure using rotary evaporator (Rotavapor ® R-210, BUCHI Corporation). The MECG was stored at room temperature until use.

Phytochemical screening
The methanolic extract of Coccinia grandis (MECG) leaves tested for the presence of secondary metabolites like phenolic compounds, alkaloids, lavonoids, carbohydrate, steroids, saponins, tannins by using standard procedures. Total phenolic content of methanol was quanti ied by spectrophotometric method. Sample (0.5ml) was mixed with 0.75% sodium carbonate solution (2.5 ml) followed by 1% Folin-Ciocalteu reagent (2.5 ml). The mixture was incubated for 15 minutes at a temperature of 45 • C and absorbance was measured at 765 nm. The standard calibration curve was plotted using gallic acid concentration. The total phenolic content was calculated from the calibration curve, and the results were expressed as gallic acid equivalent in mg/g (Kumari et al., 2016).
Total lavonoid content of methanol was quanti ied using aluminium chloride colorimetric assay. The sample (1ml) mixed with standard quercetin solution (1ml), distilled water (4ml) and 5 % sodium nitrite solution (0.3ml) followed by 1M sodium hydroxide (2 ml). The absorbance was measured at 510 nm. The standard calibration curve was plotted using standard quercetin. The total lavonoid content was calculated from the calibration curve, and the results were expressed as quercetin equivalent in mg/g (Kumari et al., 2016).

Experimental animals
Adult female Sprague Dawley (SD) rats rats free from diseases weighing 180-200 g were purchased from (Universiti Sains Malaysia, Penang, Malaysia). The rats were kept at 23 ± 2 • C in 50 ± 5% humidity and 12 hours of light-dark cycles. Standard rat pellet diet and tap water was given and acclimatized for seven days before the rats are used in the experiment. All the protocols are approved by the Committee on the Care of Laboratory Animal Resources, AIMST University (AUHAEC/FAS/2017/01) and conducted regarding Guide for the Care and Use of Laboratory Animals.

Acute toxicity
Adult female Sprague Dawley (SD) rats free from diseases were selected for this study. The ixeddose procedure was performed to test acute toxicity. Coccinia grandis with dose levels of 250, 500, 1000 and 2000 mg/kg body weight (n = 6 per dose) were given orally to the rats after overnight fasting. Behavioural, neurological and autonomic proiles of the rats were observed continuously for 24 hours followed by 14 days observation for mortality accordance with the current guidelines of Organization for Economic Co-operation and Development (OECD), revised draft guidelines 423 ( Organization for Economic Cooperation and Development (OECD), 2001).

Induction of Diabetes
Adult female SD rats free from diseases were selected for this study. After acclimatized for one week, diabetes mellitus was induced in rats by intraperitoneally injecting STZ (55 mg/kg body weight) dissolved in 0.05M citrate buffer and pH maintained to 4.0 to 4.5 before injecting. Twentyfour hours later upon induction of diabetes mellitus, 5% w/v of glucose solution (2 mL/kg body weight) were given to the rats to avoid hypoglycemic mortality. Citrate buffer alone was intraperitoneally injected to the control rats. Two days after STZ treatment, the blood sample was collected from the tail vein to record glucose level using a glucometer (ACCU-CHEK ® Active, Roche Diagnostics, Mannheim, Germany). Non-fasting rats with glucose levels above 11 mmol/L were considered as diabetic rats and introduced in the study.

Experimental Design
The total of 30 diabetic induced rats and six normal control rats were used in this study to analyze the effect of the MECG. The rats were divided into six groups (n = 6) as follows: Group 1-Normal control The rats in group 1 and 2 were administered with 0.5% w/v carboxymethyl cellulose (CMC). Glibenclamide (20mg/kg body weight) was administered to rats in group 3. Coccinia grandis dose levels of 125, 250, 500 mg/kg body weight were administered to the rats in group 4 to group 6. The doses of Coccinia grandis were selected from the acute toxicity study indings. The rats were treated with respective assigned treatment for 28 days. During the study, changes in body weight and blood glucose levels were measured at regular intervals. At the end of the study, the blood sample was collected through the tail vein and used for biochemical and lipid analysis.

Blood glucose levels measurement
To evaluate the glucose levels, few drops of blood samples were retrieved from the tail vein, and glucose levels were then measured using a blood glucometer by using test strips to analyze glucose oxidoreductase mediated dye reaction, as per manufacturer's guide. Blood glucose levels were recorded on day 0, 7, 14, 21 and 28 th day of the experiment.

Statistical analysis
The mean ± standard error of the mean (SEM) values was calculated for all the groups. One-way Values are Mean ± SEM.,n = 6 per group. ***P<0.001 compare with control. Values are Mean ± SEM.,n = 6 per group. ***P<0.001compare with control. ### P<0.05, ## P<0.01and # P<0.001 compare with diabetic control. ANOVA, followed by Tukey's post-hoc test, was used to calculate statistical differences among the groups. P < 0.05 was considered to be signi icant.

RESULTS AND DISCUSSION
MECG contains saponins, steroids, tannins, carbohydrate, phenolic compounds and lavonoids. Folin-Ciocalteu method was used to evaluate total phenolic content in MECG. Total phenolic content of MECG is 69.33 mg GAE/g and recorded 91.17 mg QE/g in total lavonoid analysis. MECG showed no mortality up to 2000mg/kg during acute toxicity study.
Effect of glibenclamide and MEGC on STZ-induced diabetes rats body weights were outlined in Table 1. A decrease in body weight was seen in the untreated diabetic group compared with non-diabetic rats. Bodyweight decreased signi icantly from day 14 (P<0.001) onwards.
Administration of glibenclamide and MECG at oral doses of 125 mg/kg, 250 mg/kg and 500 mg/kg to the rats prevented diabetes-induced weight reduction.
Throughout the study, diabetic rats show a signi icant rise in glucose level when compared with nondiabetic rats. Furthermore, rats treated with glibenclamide and MECG at oral doses of 250 and 500 mg/kg showed a signi icant decline in glucose level on day seven onwards when compared with diabetic control rats. The rats administered with 125 mg/kg MECG showed a signi icant decline in glucose level from day 21 compared with diabetic control rats.. (Table 2).
In biochemical parameter analysis, diabetic rats showed a signi icant increase in AST, ALT, ALP, urea, total bilirubin and creatinine levels (P < 0.001). At the end of the experiment, reduced levels of plasma insulin, albumin, globulin, total protein and A/G (P < 0.001) when compared with normal control. However, the levels of the biochemical parameters are found to be within the normal range in the rats with MECG 500 mg/kg and glibenclamide treatment (Table 3). MECG and glibenclamide altered insulin depletion in diabetic rats and also recovered the plasma insulin level to normal. As exhibited in Table 4, an increase in TC, LDL, VLDL, TG and reduced HDL (P < 0.001) were recorded in diabetic control rats compared with non-diabetic rats. Treatment of MECG to STZ-induced diabetic rats prevented the changes in lipid levels.
Severe body weight loss caused by STZ-induced diabetes mellitus because of a decrease in muscle mass due to lack of carbohydrates (Belayneh et al., 2019). The current study has proven that MECG treatment improved body weights in diabetic rats. Rapid improvement of body weight in glibenclamide treated groups were comparable to non-diabetic rats. This is because of the ability of plant fraction to reduce hyperglycemia and maintain body weight.
ALT and AST play a signi icant role in converting amino acid to ketoacid. Meanwhile, serum ALP can detect both intra-hepatic and extra-hepatic bile obstruction. Furthermore, ALP, ALT and AST are known as enzyme markers for liver function, that released into the serum and increases enzyme activity if liver cells are damaged (Lala et al., 2020). ALP, ALT, AST and total bilirubin levels are increased due to the hepatotoxic effect of STZ. This because, liver cytosol released these enzymes into the bloodstream and caused hepatocellular and posthepatic damages like biliary tract damage and biliary stone formation (Saleem and Naseer, 2014).
As an end product of protein catabolism, urea is excreted in the urine. As an end product of creatine metabolism, creatine will be biosynthesized in the liver and enters into the blood circulation (Mohammed, 2019). Increased levels of urea and creatinine may lead to renal dysfunction. However, treatment with the different doses of MECG and glibenclamide signi icantly decreased ALP, ALT, AST, total bilirubin, urea and creatinine levels in blood indicates ameliorative effect of MECG on renal dysfunction and hepatocytes in the liver after administration of STZ in the diabetic rats (Alshathly, 2019). Restoration of decreased globulin, total protein, albumin/globulin ratio and albumin by plant extract and glibenclamide could be due to inhibition of proteolytic activity due to improved insulin production (Ali et al., 2017).
In diabetic situation, insulin can damage lipid metabolism, and accumulation of lipids in the liver and blood causes abnormal fatty acid metabolism, which occurs after impairment of β-cells function (Erion et al., 2016). Consequently, diabetic dyslipidemia act as signi icant risk factors for nephro-toxicity, myocardial infarction, atherosclerosis and coronary diseases (Xia et al., 2017). Changes in lipid pro ile showed the progress of hyperlipidemia in diabetic rats due to increased hormone-sensitive lipase activity. It catalyzes triacylglycerols stored in adipocytes to fatty acids (Bolsoni-Lopes and Alonso-Vale, 2015). The present study reveals that MECG improved the serum lipid levels by reducing (TC, TG, LDL, and VLDL) and increasing HDL, may be due to high level of lecithin cholesterol acyltransferase activity, that resulted in regulation of blood lipids (Hassan et al., 2015). Moreover, MECG showed favourable results on lipid metabolismrelated hepatic enzymes. Thus, our study indicates MECG able to control dyslipidemia associated with diabetic complications.
The inding of tannins, alkaloid, cardiac glycosides, saponins, glycosides, terpenoids, and reducing sugars in MECG has proven its anti-oxidant and antidiabetic effect. Terpenoids caused insulin-like activity to reduce blood glucose and inhibited glycogenolysis and gluconeogenesis (Grover et al., 2002). Saponin can reduce hyperglycemia related oxidative stress in type 2 diabetes by restoring insulin resistance rather than potentially enhance -cell proliferation (Zheng et al., 2018). Similarly, alkaloids (Wätjen et al., 2005) inhibited α-glucosidase, researchers proven that tannins from plant extract contributed inhibitory activities on α-glucosidase and α-amylase (yi Li et al., 2019), thus reduces intestinal glucose uptake. Flavonoids act as intermediary biosynthetic compound contributed to the inhibition of alpha-amylase, which can restore damaged beta cells in the pancreas. Polyphenolic compounds produced in the plants inhibited glucose transport processes by inhibiting intestinal sodiumglucose co-transporter-1 (S-GLUT-1) (Parasuraman et al., 2019). Referring to the present experimental study, the anti-diabetic effect of the MECG against STZ induced rats might be due to the existence of the polyphenolic compounds, lavonoids, alkaloids, terpenoids, tannins and saponins.

CONCLUSIONS
Methanolic extract of Coccinia grandis leaves has signi icant anti-diabetic activity at the dose levels of 125, 250 and 500 mg/kg body weight in STZ-induced diabetic rats. Furthermore, methanolic extract of Coccinia grandis leaves inhibits STZinduced hyperlipidemia in rats, which may help to prevent hyperlipidemia in diabetes mellitus.

Funding Support
This research was funded by the Ministry of Higher Learning Malaysia under the Fundamental Research