IN VIVO ANTIHYPERLIPIDEMIC, ANTIOXIDATIVE EFFECTS OF COCCINIA GRANDIS (L.) VOIGT (CUCURBITACEAE) LEAF EXTRACT: AN APPROACH TO SCRUTINIZE THE THERAPEUTIC BENEFITS OF TRADITIONAL SRI LANKAN MEDICINES AGAINST DIABETIC COMPLICATIONSHTML Full Text
IN VIVO ANTIHYPERLIPIDEMIC, ANTIOXIDATIVE EFFECTS OF COCCINIA GRANDIS (L.) VOIGT (CUCURBITACEAE) LEAF EXTRACT: AN APPROACH TO SCRUTINIZE THE THERAPEUTIC BENEFITS OF TRADITIONAL SRI LANKAN MEDICINES AGAINST DIABETIC COMPLICATIONS
- A. P. Attanayake*1, K.A.P.W. Jayatilaka 1, L.K.B. Mudduwa 2 and C. Pathirana 1
Department of Biochemistry 1, Department of Pathology 2, Faculty of Medicine, University of Ruhuna, Karapitiya, Sri Lanka.
ABSTRACT: Coccinia grandis (Linn.) Voigt (Cucurbitaceae) has been widely used for the management of diabetes mellitus in Sri Lankan traditional medicine. The aim of the present study was to investigate antihyperlipidemic, antioxidative effects of the leaf extract of C. grandis in sterptozotocin induced diabetic rats and to standardize the leaf extract of C. grandis by standard analytical methods. The results showed that the concentration of serum total cholesterol, triglycerides and low density lipoprotein cholesterol were significantly lower in C. grandis (0.75 g/kg) treated rats than in diabetic control rats (P < 0.05). In addition, an improvement in the antioxidant potential is also showed in C. grandis treated diabetic rats. P < 0.05). The polyphenolic compounds, alkaloids flavonoids, saponins and sterols/tritepenoids were present in the C. grandis extract. The results revealed that the aqueous leaf extract of C. grandis (0.75 g/kg) exerted antihyperlipidemic and antioxidative effects in diabetic rats, corroborating the therapeutic use of C. grandis leaf extract in the management of diabetic complications in traditional medicine.
Antihyperlipidemic effect; Antioxidative effects; Coccinia grandis; Streptozotocin induced diabetic rats
INTRODUCTION: Diabetes mellitus has been considered as one of the emerging health problems worldwide because of its high prevalence, adverse clinical outcomes, marked reduction in the quality of life of patients and high healthcare costs 1, 2, 3. Accordingly, it has become an adverse public health crisis in most of the South Asian countries including Sri Lanka with a prevalence of 8.5% in the general population.
The total number of deaths caused by diabetes mellitus and its associated complications reached to 16,318 of Sri Lankan population in 2015 4.
Over the past few decades, there has been a rapid discovery of diverse therapeutic agents for the management of diabetes mellitus. Present pharmacological agents as insulin and oral hypoglycemic drugs aim at correcting/overcoming hyperglycemia and disturbances in the metabolism of carbohydrate, fat and protein resulting from absolute or relative lack of secretion of insulin 5. Despite the use of different types of oral hypoglycemic agents as insulin releasers, insulin sensitizers and glucosidase inhibitors etc. for the treatment of diabetes mellitus, studies have consistently demonstrated that patients’ adherence to present therapeutic regimes have not been satisfactory. Regime complexity, occurrence of hypoglycemia, associated side effects, lack of confidence in immediate or future benefits and education/beliefs of people are among the common reasons limiting compliance 6. Although, the present drugs are effective in reducing hyperglycemia, inadequacies in current pharmaceutical agents have resulted in developing new antidiabetic drugs with increased efficacy, lack of toxicity and side effects for the treatment of diabetes and its complications. Thus, despite some encouraging findings on the discovery of novel antidiabetic agents from medicinal plant extracts, extensive research is required for the discovery of antidiabetic drugs/lead compounds from medicinal plant extracts which act against hyperglycemia, dyslipidemia and oxidative stress 7, 8.
Medicinal plants in particular, have formed the basis of sophisticated traditional medicinal systems. Ethnobotanical studies have identified more than 1,200 species of plants with antidiabetic activity 9. Accordingly, a number of medicinal plant extracts in the form of decoctions have been used by Sri Lankan Ayurvedic practitioners for the management of diabetes mellitus and associated conditions of dyslipidemia and oxidative stress induced microvascular complications 10. However, this traditional knowledge derived empirically has to be further supported by scientific testing.
Coccinia grandis (Linn.) Voigt (Cucurbitaceae) is an edible perennial climber distributed in tropical Asia, commonly found in Sri Lanka, India and Pakistan. Every part of this plant is valuable in medicine and various decoctions have been prepared in traditional Sri Lankan medicine for the treatment of various skin diseases, diabetes mellitus, urinary tract infections, bronchitis, itchy skin eruptions and ulcers 11. Further, leaf extract of C. grandis is widely used by traditional physicians for the management of hyperglycemia and related microvascular complications associated with diabetes mellitus 10. Thus, extensive research on bioactivity studies of C. grnadis has been carried out by our research group based on the highly demanding therapeutic use of C. grandis in traditional medicine
Dose response experiments of hot water leaf extract of C. grandis in streptozotocin induced (STZ) diabetic rats revealed that the optimum therapeutic dose of the extract was 0.75 g/kg 12. Accordingly, this particular dose was used in the present investigation as well. In addition, extensive research carried out by our research group proved that the extract was able to induce β-cell regeneration as one of the antidiabetic mechanisms in vivo 13. Even though the leaf extract of C. grandis has been used in the management of dyslipidemia and oxidative stress associated diabetic complications in traditional medicine, the in vivo antihyperlipidemic and antioxidative effects of the aqueous leaf extract of C. grandis at its optimum therapeutic dose have not beenreported to date.
The aim of the present study was to investigate antihyperlipidemic, antioxidative effects of the leaf extract C. grandis (0.75 g/kg) in sterptozotocin induced diabetic rats and to standardize the C. grandis by standard analytical methods.
Chemicals and instruments: D-glucose, glibenclamide and streptozotocin were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). A UV visible spectrophotometer (Gallenkamp PLC, UK) and Olympus CX 21(Japan) microscope were used for spectrophotometric measurements and for the assessment of histopathology of the liver tissue respectively. A rotatory evaporator (Buchi, B-480) will be used in the standardization of C. grandis extract.
Plant material: Leaves of C. grandis were collected during May-June 2013 from the Southern region of Sri Lanka. Botanical identity was determined by the descriptions given by Jayaweera 11 and confirmed by comparing authentic samples at the National Herbarium, Royal Botanical Gardens, Peradeniya, Sri Lanka. A voucher specimen was preserved at the Department of Biochemistry, Faculty of Medicine, University of Ruhuna, Sri Lanka (Attanayake/2011/ 01).
Preparation of the plant extract: The leaves of C. grandis were cut into small pieces, dried at 40°C until a constant weight. Powdered plant material (50.00 g) was dissolved in 400.0 mL of distilled water and refluxed for 4 h. The mixture was strained and the final volume was adjusted to 50.0 mL. The optimum effective therapeutic dose (0.75 g/kg) was prepared for in vivo experiments.
Animals: Healthy adult male rats of Wistar strain (200 ± 25 g body weight) were purchased from the Medical Research Institute (MRI), Sri Lanka and were used to carry out the experiments. They were housed in standard environmental conditions at the animal house of Faculty of Medicine, University of Ruhuna, Sri Lanka (Tem 25 ± 2°C, relative humidity 55- 65% and 12 ± 1 h light/ dark cycle). Rats were fed with a standard diet (MRI rat formulae, Sri Lanka) with free access to water before and during the experiment. The rats were randomized into various groups and allowed to acclimatize for a period of seven days under standard environmental conditions before the commencement of the experiments. The animals described as fasting were deprived of food and water for 12 h ad libitum.
All protocols used in this study were approved by the Ethical Review Committee of Faculty of Medicine, University of Ruhuna, Sri Lanka guided by the Council for International Organization of Medical Sciences (CIOMS) international guiding principles of biomedical research involving animals.
Development of diabetes mellitus in Wistar rats: Streptozotocin dissolved in citrate buffer (0.1M, pH 4.4) at a dose of 65 mg/kg was administered intraperitonially to rats fasted for 8 h. Thereafter, rats were maintained on 5% D-glucose solution for the next 24 h. Rats were allowed to stabilize for three days and on the 4th day, blood samples were drawn from tail vein to determine the blood glucose concentration to confirm the development of diabetes mellitus. Rats with fasting blood glucose concentration of 12.0mmol/L or above were considered as hyperglycemic and on the 4th day onwards the hyperglycemic rats were used in the experiments 14.
Experimental group design: Group one and two served as the healthy and diabetic control rats and received distilled water. Group three and four were diabetic rats, received the optimum effective therapeutic dose of the extract C. garndis (0.75 g/kg) and glibenclamide (0.50 mg/kg) daily for 30 days respectively. At the end of the study (on the 30th day), blood was collected by cardiac puncture and the liver was excised from sacrificed rats. Serum was separated from blood of all rats for the estimation of biochemical parameters. Liver tissue was excised for the assessment of histopathology.
Blood/serum glycemic parameters: Oral glucose tolerance test was performed in all groups on the 1st, 7th, 14th, 21st, 28th and 30th day. Blood glucose concentration was determined by using a spectrophotometric enzyme assay kit 15. The effect was evaluated over a four hour period using area under the oral glucose tolerance curve 16.
Serum lipid parameters in diabetic rats: The concentrations of serum total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), triglyceride (TG) were estimated in all rats using spectrophotometric enzyme assay kits 17, 18, 19. The concentrations of serum low density lipoprotein cholesterol (LDL-C), very low density lipoprotein cholesterol (VLDL-C) were calculated using the Friedewald formulae 20.
Antioxidant markers in diabetic rats: Fasting serum activities of liver enzymes; alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were estimated using spectrophotometric enzyme assay kits 21, 22. The estimation of reduced glutathione (GSH), activities of glutathione reductase (GR, EC 188.8.131.52), glutathione peroxidase (GPx, EC 184.108.40.206) and glutathione S-transferase (GST, EC220.127.116.11) in the liver homogenates were done using reported protocols 23, 24, 25. Further the extent of lipid peroxidation and total protein were estimated in liver homogenates by the formation of malondialdehyde (MDA) using thiobarbituric acid and Lowry methods respectively 26, 27.
Qualitative and quantitative analysis of chemical constituents:
Phytochemical screening: Preliminary qualitative phytochemical screening for the presence of alkaloids, cardenolide glycosides, phenols, flavonoids, phytosterols, saponins, tannins, reducing sugars, and proteins was carried out by the reported protocol 28.
Total polyphenol content: Total polyphenol content was measured using Folin-Ciocalteu colorimetric method 29. Plant extract (1. 0 mL of 0.05 g/mL) was mixed with 1.0 mL of 95% ethanol, 5.0 mL of distilled water and 0.5 mL of 50% Folin-Ciocalteu reagent. The mixture was allowed to react for 5 min and 1.0 mL of 5% sodium carbonate was added. Thereafter, it was thoroughly mixed and placed in dark at room temperature (27°C) for one hour and the absorbance was measured spectrophotometrically at 725 nm. Quantification was done with respect to the standard curve of gallic acid (0-50 μg/mL). The results were expressed in gallic acid equivalents mgGAE/g of the dry weight.
Total flavonoid content: The total flavonoid content of plant extract was determined using aluminum chloride colorimetric method of Koksal and Gulcin 30 based on the method of Chang et al. 31. The plant extract (0.50 mL) was mixed with ethanol 95% (1.5 mL) followed by aluminium chloride 10% (0.10 mL), potassium acetate 1M (0.10 mL) and distilled water (2.8 mL). The resultant mixture was incubated at 27°C for 30 minutes. The absorbance of the reaction mixture was measured spectrophotometrically at 415 nm. The flavonoid content was calculated using standard calibration of quercetin solution in range of 0-50 µg. The results are expressed as micrograms of quercetin equivalent (QE)/g of the dry weight. cyclohexane in a ratio of 0.1:1:1 (v/v/v). Spots were observed under UV (both 254 nm and 366 nm) light and they were visualized using vanillin sulphuric acid.
Statistical analysis: The replicates of each sample were used for statistical analysis and the values were expressed as mean ± standard deviation in the in vitro study. The data were analyzed using analysis of variance (ANOVA) and the mean values for each group were compared by Dunnett’s multiple comparison tests in the in vivo study. The level of significance was set at P < 0.05.
Blood/serum glycemic parameters: Effect of the extract of C. grandis (0.75 g/kg) on fasting blood glucose concentration in diabetic rats is shown in Fig.1. The fasting blood glucose concentration of C. grandis treated diabetic rats was reduced significantly on the 14th day on wards for the period of 30 days (P < 0.05). The reduction in fasting blood glucose concentration with the administration of C. grandis and glibenclamide was 48% and 61% in streptozotocin induced diabetic rats on the 30th day respectively (P < 0.05). The healthy animals were normoglycemic throughout the experimental period. The total area under the curve values of plant extract treated diabetic rats showed a statistically significant improvement of 54% on the 30th day (P < 0.05, Fig.2).
FIG. 1: EFFECT OF C. GRANDIS EXTRACT ON FASTING BLOOD GLUCOSE CONCENTRATION IN DIABETIC RATS AT SPECIFIC INTERVALS FOR 30 DAYS Each line represents mean ± SEM (n=6/group) FBG: Fasting blood glucose concentration.
FIG. 2: TOTAL AREA UNDER THE ORAL GLUCOSE TOLERANCE CURVE VALUES OF C. GRANDIS EXTRACT TREATED DIABETIC RATS AT SPECIFIC INTERVALS FOR 30 DAYS
Each column represents mean ± SEM (n=6/group). TAUC: Total area under the curve from the 21st day onwards, total area under the curve value of C. grandis (0.75 g/kg) treated rats is significantly different from the total area under the curve values of diabetic untreated rats (P < 0.05).
Serum lipid parameters: The concentrations of serum TC, HDL-C, LDL-C, VLDL-C, TG in streptozotocin induced diabetic rats followed by the plant treatment is shown in Fig. 3. The streptozotocin induced diabetic control rats had a significant elevation in the concentration of serum TC (57%), LDL-C (93%), VLDL-C (95%), TG (94%) and a reduction in HDL-C (12%) as compared with the untreated healthy control rats. As shown in Fig.3, the extract of C. grandis treated streptozotocin induced diabetic rats showed a significant reduction in the concentration of serum TC (25%), LDL-C (33%), VLDL-C (21%), TG (21%) and an elevation in HDL-C (4%) on the 30th day of the study (P < 0.05). The concentration of serum TC, LDL-C, VLDL-C, TG were reduced by 32%, 38%, 49% and 48% in glibenclamide treated diabetic rats. In contrast, there was no significant change in the concentration of serum HDL-C with the glibenclamide treatment in diabetic rats (P > 0.05).
FIG. 3: EFFECT OF C. GRANDIS EXTRACT ON SERUM LIPID PARAMETERS IN DIABETIC RATS AFTER 30 DAYS OF TREATMENT
Each column represents mean ± SEM (n=6/group). TC: Total cholesterol, HDL-C: High density lipoprotein cholesterol, LDL-C: Low density lipoprotein cholesterol, VLDL-C: Very low density lipoprotein cholesterol, TG: Triglyceride. The concentrations of TC, HDL-C, LDL-C, VLDL-C and TG of C. grandis (0.75 g/kg) treated rats is significantly different from the total area under the curve values of diabetic untreated rats (P < 0.05).
Serum hepatic enzymes and oxidative stress markers: The effect of the C. grandis extract on liver enzymes and hepatic oxidative stress markers in STZ-diabetic rats is shown in Table 1 and 2 respectively. There was an elevation in the activities of ALT, AST, ALP and the concentration of MDA in streptozotocin induced diabetic rats when compared to the untreated healthy rats. On the other hand, there was a reduction in the concentration of GSH, GR, GPx and GST in the same treatment groups. Treatment of diabetic rats with the extract and glibenclamide decreased the activities of ALT (by 18%, 16%), AST (by 16%, 17%) and ALP (by 40%, 5%) (P<0.05). The protective effect of the extract on lipid peroxidation was also demonstrated; a significant reduction in the concentration of MDA by 14% when compared to untreated diabetic rats (P<0.05). The administration of the plant extract restored the concentration of GSH, activities of GR, GPx, GST to near normalcy (by 41%, 49%, 40%, 57%) and it was more effective than the attainment of above biochemical parameters by glibenclamide (by 35%, 20%, 38%, 12%).
As shown in Fig. 4, liver histology was normal in untreated healthy rats. In contrast, untreated diabetic rats showed very early microvesicular fatty change in centrilobular areas of the liver, mild congestion, moderate lymphocytic infiltrates mostly around the portal tract, increased fibrosis with paranchymal infiltrates and focal necrosis. The light microscopic appearance of the liver tissue in C. grandis treated rats are in line with biochemical results, with a reduction in microvesicular fatty change, mild lymphocytic infiltrates and no necrosis. Further, reduction in microvesicular fatty change, moderate lymphocytic infiltrates were also observed in glibenclamide treated diabetic rats.
TABLE 1: EFFECT OF C. GRANDIS EXTRACT ON SERUM HEPATIC ENZYMES IN DIABETIC RATS AFTER 30 DAYS OF TREATMENT
|Healthy control rats||12.4±0.1||44.2±1.7||61.4±1.0|
|Diabetic control rats||36.6± 2.2||90.1±0.1||165.6±2.5|
|C. grandis (0.75 g/kg)
Glibenclamide (0.50 mg/kg)
The values are expressed as mean ± SEM (n=6/group). *Statistically significant from streptozotocin induced diabetic control rats at P < 0.05 (ANOVA followed by Dunnett’s test). ALT: Alanine aminotransferase, AST: Aspartate aminotransferase, ALP: Alkaline phosphatase.
TABLE 2: EFFECT OF C. GRANDIS EXTRACT ON HEPATIC ANTIOXIDATIVE STRESS MARKERS IN DIABETIC RATS AFTER 30 DAYS OF TREATMENT
|(nmol/mg protein )||(µg/g liver)||(nmol/min/mg of||(nmol/min/mg of||(nmol/min/|
|Healthy control rats||12.1±0.3||719.8±7.4||9.1±1.1||10.5±0.9||10.9±1.4|
|Diabetic control rats||36.3±1.1||419.6±8.7||5.3±0.5||4.6±1.0||4.7±0.1|
|C. grandis (0.75 g/kg)||31.1±0.3*||590.0±22.6*||7.9±0.7*||7.7±0.2*||7.4±0.6*|
|Glibenclamide (0.50 mg/kg)||22.1±0.2*||564.7±19.8*||6.3±0.9*||6.3±0.6*||7.3±0.2*|
The values are expressed as mean ± SEM (n=6/group). *Statistically significant from streptozotocin induced diabetic control rats at P < 0.05 (ANOVA followed by Dunnett’s test). MDA: Malondialdehyde, GSH: Reduced glutathione, GR: Glutathione reductase, GPx: Glutathione peroxidase, GST: Glutathione S-transferase
FIG. 4(A-D): PHOTOMICROGRAPHS OF HISTOPATHOLOGY OF THE LIVER TISSUES, STAINED WITH HEMATOXYLIN AND EOSIN (X 400)
(A) Healthy control rats, liver tissue is within normal histological limits
(B) Diabetic control rats, focal necrosis associated with inflammation
(C) Coccinia grandis (0.75 g/kg) treated diabetic rats, restoration of hepatocytes
(D) Glibenclamide treated (0.50 mg/kg) diabetic rats, restoration of hepatocytes
DISCUSSION: In the present study, the antihyperlipidemic and antioxidant activities of the leaf extract of C. grandis were evaluated in streptozotocin induced diabetic rat model. Streptozotocin is a pancreatic toxin that induces diabetes mellitus by destroying pancreatic β- cells resulting insulin deficiency. The cytotoxic effects
of streptozotocin are dependent on DNA alkylation by site-specific action with DNA bases and by excessive formation of free radicals 32. The area under the oral glucose tolerance curve of diabetic rats showed a highly significant elevation at fasting state 1h, 2h, 3h and 4h after glucose loading as compared to healthy (normoglycemic) animals. However, the C. grandis extract appeared more effective at all tested points after oral glucose loading throughout the study period. In addition, from the observations made in Fig. 1, it is further confirmed that the leaf extract of C. grandis possesses significant antihyperglycemic activity as it could produce a fall of 48% in fasting blood glucose in diabetic rats after the plant treatment for 30 days.
Lipid abnormalities are postulated to be major causes for complications associated with diabetes mellitus. Indeed, diabetes is associated with profound alterations in the serum concentration of TG, lipoprotein profile and with an increased risk of coronary heart disease 33. The hyperlipidemia associated with diabetes mellitus may result from an accelerated hepatic biosynthesis of triglycerides and a release of VLDL without an increase in its rate of clearance from the blood by lipoprotein lipase, which is dependent on the insulin/glucagon ratio. Furthermore, the alterations in lipid profile may be a consequence of increased breakdown of lipids and mobilization of free fatty acids from peripheral deposits 34. Accordingly, an increase in the concentration of serum TC, TG and a decrease in the concentration of HDL-C were demonstrated in diabetic control rats in the present study. This is similarly mentioned by other authors who also observed alterations in the serum lipid parameters in diabetic rats 35, 36. High serum concentration of TC and importantly, LDL-C are the predictors of atherosclerosis 37. Therefore, the ideal treatment for diabetes mellitus should have favorable effects on serum lipid parameters in addition to the glycemic control.
Treatment of diabetic rats with C. grandis extract reversed dyslipidemia, as evidenced by a significant decrease in the serum concentration of TC, LDL-C and TG coupled to an increase in the serum concentration of HDL-C (P < 0.05). The increase in the concentration of HDL- C by the plant treatment is of therapeutic advantage as it improves the glycemic control as well as reduces the cardiovascular risk associated with diabetes mellitus. The strong antihyperlipidemic effect by the administration of C. grandis in diabetic rats could be through its’ control on hyperglycemia or/and direct actions on the absorption of intestinal cholesterol and biosynthesis of cholesterol. Several authors have reported the antihyperlipidemic activities of medicinal plants in experimental diabetic animals which are similar to our results 38, 39.
Preliminary phytochemical screening revealed the presence of polyphenolic compounds, alkaloids flavonoids, saponins and sterols/tritepenoids in the extract of C. garndis. Saponins possess antihyperlipidemic activity and this has been reported to increase the lipoprotein lipase activity, which helps to remove free fatty acids from circulation, causing decrease in the concentration of serum cholesterol 40. Flavonoids and other polyphenols may also contribute to the antihyperlipidemic activity by increasing the cholesterol metabolism and by modulating the enzymes involved in cholesterol metabolism, such as HMG CoA reductase, cholesterol 7α-hydroxylase and acyl-CoA:cholesterol acyltransferase 41, 42.
Liver is the vital organ of metabolism, detoxification, storage and excretion of xenobiotics and their metabolites 43.
Thus far, ALT, AST and ALP are reliable markers of liver function. Therefore, an increase in the activities of above liver enzymes in the serum of diabetic rats might be mainly due to the leakage of these enzymes from the liver cytosol into the blood stream which gives an indication of the hepatotoxic effect of streptozotocin 44. Treatment of the diabetic rats with the leaf extract of C. grandis caused a reduction in the activity of these enzymes in serum compared to the diabetic untreated group. Further, the liver histopathology matches with biochemical results. These results are in agreement with those obtained by Kasetti et al. 45 and Eliza et al. 46 in diabetic rats.
The experimental data from the present study indicated that the concentration of malonaldehyde, an end-product of lipid peroxidation was elevated in the liver homogenates of diabetic rats. In contrast, the activities of hepatic antioxidant enzymes as GR, GPx and GST were decreased in diabetic rats. This may be due to the rise in generation of reactive oxygen species results in hyperglycemia and/or excessive formation of superoxide and hydroxyl radicals by the induction of streptozotocin 47. Indeed, the free radicals may inactivate the activities of these enzymes 48. This may also be responsible for the insufficiency of antioxidant defences in mitigating reactive oxygen species mediated damage 48. However, the administration of leaf extract of C. grandis was able to improve the activities of these antioxidant enzymes, increase the concentration of reduced glutathione and decrease the concentration of malonaldehyde in diabetic rats. The in vivo antioxidative activity of C. grandis may be due to the high content of polyphenolic compounds. Indeed, natural polyphenols are capable of scavenging free radicals, chelate metal catalysts, activate antioxidant enzymes, reduce α-tocopherol radicals and inhibit oxidases 48.
CONCLUSIONS: The results revealed that the leaf extract of C. grandis exert antihyperglycemic and antihyperlipidemic effects in streptozotocin induced diabetic rats, corroborating the therapeutic use of C. grandis leaf extract in the management of diabetic complications in traditional Sri Lankan medicine. Furthermore, the data obtained from the study, confirmed the safe consumption of C. grandis as a dietary adjunct and in the development of pharmaceutical products and neutarceuticals for the management of diabetes mellitus and its’ associated complications.
ACKNOWLEDGEMENT: The financial assistance given by University Grants Commission (UGC/ICD/CRF 2009/2/5) in Sri Lanka is greatly appreciated. The authors wish to thank Dr D.A.B.N. Gunarathne of the Department of Crop Science, Faculty of Agriculture, University of Ruhuna, Sri Lanka for the guidance given in statistical data analysis, Mrs. B.M.S. Malkanthie, Mr G.H.J.M. Priyashantha and Mr D.G.P. Pathmabandu, Mrs G.G.D.D. Gunawardane, Faculty of Medicine, University of Ruhuna, Sri Lanka for technical assistance.
- Chen L, Magliano DJ and Zimmet PZ: The worldwide epidemiology of type 2 diabetes mellitus present and future perspectives. Nature Reviews Endocrinology. 2012; 8: 228-236.
- Guariguata L, Whiting DR, Hambleton I, Beagley J, Linnenkamp U and Shaw JE: Global estimates of diabetes prevalence for 2013 and projections for 2035. Diabetes Research and Clinical Practice 2014; 103: 137-149.
- Ramachandran A, Snehalatha C and Ma RC: Diabetes in South-East Asia: An update. Diabetes Research and Clinical Practice 2014; 103: 231-237.
- International diabetes federation atlas. [online] Available at: http://www.idf.org/diabetesatlas/5e/the-global-burden 2015.
- Tahrani AA, Bailey CJ, Del Prato D and Barnett AH: Management of type 2 diabetes: New and future developments in treatment. Lancet 2011; 378: 182-197.
- Bastaki S: Diabetes mellitus and its treatment. International Journal of Diabetes and Metabolism 2005; 13: 111-134.
- Hung HY, Qian K, Morris-Natschke SL, Hsu CS and Lee KH: Recent discovery of plant-derived anti-diabetic natural products. Natural Product Reports 2012; 29: 580-606.
- Prakash O, Kumar R, Srivastava R, Tripathi P, Mishra S and Ajeet: Plants explored with anti-diabetic properties: A review. American Journal of Pharmacological Sciences 2015; 3: 55-66.
- Sharma SB, Gupta S, Ac R, Singh UR, Rajpoot R and Shukla SK: Antidiabetic action of Morus rubra leaf extract in streptozotocin- induced diabetic rats. Journal of Pharmacy and Pharmacology 2010; 62: 247-255.
- Ediriweera ERHSS and Ratnasooriya WD: A review on herbs used in treatment of diabetes mellitus by Sri Lankan Ayurvedic and traditional physicians Ayu 2009; 30: 373-391.
- Jayaweera DMA: Medicinal Plants (Indigenous and Exotic) Used in Ceylon, National Science Foundation in Sri Lanka, Colombo, Sri Lanka 1982.
- Attanayake AP, Jayatilaka KAPW, Pathirana C and Mudduwa LKB: Acute hypoglycemic and antihyperglycemic effects of ten Sri Lankan medicinal plant extracts in healthy and streptozotocin induced diabetic rats. International Journal of Diabetes in Developing Countries 2015; 35: 177-183.
- Attanayake AP, Jayatilaka KAPW, Pathirana C and Mudduwa LKB: Antihyperglycemic activity of Coccinia grandis (L.) Voigt (Cucurbitaceae) in streptozotocin induced diabetic rats. Indian Journal of Traditional Knowledge 2015; 14: 376-381.
- Vasconcelos CF, Maranhao HM, Batista TM, Carneiro EM, Ferreira F, Costa J, Soares LA, Sá MD, Souza TP and Wanderley AG: Hypoglycemic activity and molecular mechanisms of Caesalpinia ferrea Martius bark extract on streptozotocin- induced diabetes in Wistar rats. Journal of Ethnopharmacology 2011; 137: 1533-1541.
- Trinder P: Determination of blood glucose using an oxidase peroxidase system with a non- carcinogenic chromogen. Journal of Clinical Pathology 1969; 22: 158-161.
- Purves RD: Optimum numerical integration methods for estimation of area-under the curve (AUC) and area under the moment curve (AUMC). Journal of Pharmacokinetics and Pharmacodynamics 1992; 20: 211-226.
- Roeschlau P, Bernt E and Gruber W: Enzymatic determination of total cholesterol in serum. Zeitschriftfürklinische Chemie und klinische Biochemie 1974; 12: 226.
- Assmann G, Schriewer H, Schmitz G and Hagele EO: Quantification of high-density- lipoprotein cholesterol by precipitation with phosphotungstic acid/MgCl2. Clinical Chemistry 1983; 29: 2026-2030.
- Wahlefeld AW: Method of Enzymatic Analysis Academic Press, New York, NY, USA 1974.
- Friedewald WT, Levy RI and Fredrickson DS: Estimation of the concentration of low density lipoprotein cholesterol in plasma without the use of preparative concentrating. Clinical Chemistry 1972; 18: 499-502.
- Bergmeyer HU, Scheibe P and Wahlefeld AW: Optimization of methods for asparatate aminotransferase and alanine aminotransferase. Clinical Chemistry 1978; 24: 58-73.
- Bowers Jr GN and Mc Comb RB: A continuous spectrophotometric method for measuring the activity of serum alkaline phosphatase. Clinical Chemistry 1966; 12:70-89.
- Sedlak J and Lindsay RH: Estimation of total, protein bound and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Analytical Biochemistry 1968; 25:192-205.
- Jodynis-Liebert J, Murias M and Bloszyk E: Effect of sesquiterpene lactones on antioxidant enzymes and some drug metabolizing enzymes in rat liver and kidney. Planta Medica 2000; 66: 199-205.
- Habig WH, Pabst MJ and Jakoby WB: Glutathione S-transferase, the first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry 1974; 249: 7130-7139.
- Ohkawa H. Ohishi N and Yagi K: Assay for lipid peroxides in animal tissues by the thiobarbituric acid reaction. Analytical Biochemistry 1979; 95: 351-358.
- Lowry OH, Rosebrough NJ, Farr AL and Randall RJ: Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 1951; 193: 265-275.
- Trease GE and Evans WC: Pharmacology London: Bailliere Tindall Ltd 1978.
- Singleton VL, Ortofer R and Lamuda-Raventos RM: Analysis of total polyphenol and other oxidation substances and antioxidants by means of Folin-Ciocalteu regent. Methods Enzymology 1999; 29:152-178.
- Koksal E and Gulcin L: Antioxidant Activity of Cauliflower (Brassica oleracea) Turkish Journal of Agriculture and Forestry 2008; 32: 65-78.
- Chang C, Yang M, Wen H and Chern J: Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis 2002; 10: 178-182.
- Nukatsuka M, Yoshimura Y, Nishida M and Kawada M: Importance of the concentration of ATP in rat pancreatic beta cells in the mechanism of streptozotocin- induced cytotoxicity. Journal of Endocrinology 1990; 127: 161-116.
- Maghrani M, Lemhadri A and Zeggwagh NA: Effects of an aqueous extract of Triticum repens on lipid metabolism in normal and recent-onset diabetic rats. Journal of Ethnopharmacology 2004; 90: 331-337.
- Juarez-Rojop I E, Diaz-Zagoya JC, Ble-Castillo JL, Miranda-Osorio PH, Castell- Rodríguez AE, Tovilla-Zárate CA, Rodríguez-Hernández A, Aguilar-Mariscal H, Ramón-Frías T and Bermúdez-Ocaña DY: Hypoglycemic effect of Carica papaya leaves in streptozotocin induced diabetic rats. BMC Complementary and Alternative Medicine 2012; 12: e236.
- Ravi K, Rajasekaran S and Subramanian S: Antihyperlipidemic effect of Eugenia jambolana seed kernel on streptozotocin-induced diabetes in rats. Food and Chemical Toxicology 2005; 43: 1433-1439.
- Tchamadeu MC, Dzeufiet PD, Nouga CC, Azebaze AG , Allard J , Girolami JP , Tack I , Kamtchouing P and Dimo T: Hypoglycemic effects of Mammea africana (Guttiferae) in diabetic rats. Journal of Ethnopharmacology 2010; 127: 368-372.
- Temme EH, Van Hoydonck PG, Schouten EG and Kesteloot H: Effects of a plant sterol-enriched spread on serum lipids and lipoproteins in mildly hypercholesterolemic subjects. Acta Cardiologica 2002; 57: 111-115.
- Gupta S, Sharma SB, Bansal SK and Prabhu KM: Antihyperglycemic and hypolipidemic activity of aqueous extract of Cassia auriculata leaves in experimental diabetes. Journal of Ethnopharmacology 2009; 123: 499-503.
- Kasetti RB, Rajasekhar MD, Kondeti VK, Fatima SS and Kumar EGT: Antihyperglycemic and antihyperlipidemic activities of methanol:water (4:1) fraction isolated from aqueous extract of Syzygium alternifolium seeds in streptozotocin induced diabetic rats. Food and Chemical Toxicology 2010; 48: 1078-1084.
- Verma PR, Deshpande SA, Kamtham YN and Vaidya LB: Hypolipidemic and antihyperlipidemic effects from an aqueous extract of Pachyptera hymenaea (DC.) leaves in rats. Food Chemistry 2012; 132: 1251-1257.
- Chen W, Nishimura N, Oda H and Yokogashi H: Effect of taurine on cholesterol degradation and bile acid pool in rats fed a high-cholesterol diet. Advances in Experimental Medicine and Biology 2003; 526: 261-267.
- Eliza J, Daisy P, Ignacimuthu S and Duraipandiyan V: Normo-glycemic and hypolipidemic effect of costunolide isolated from Costus speciosus (Koen ex. Retz.)Sm. in streptozotocin-induced diabetic rats. Chemco Biological Interactions. 2009; 179: 329-334.
- Stephen Irudayaraj S, Sunil C, Duraipandiyan V and Ignacimuthu S: Antidiabetic and antioxidant activities of Toddalia asiatica (L.) Lam. leaves in streptozotocin induced diabetic rats. Journal of Ethnopharmacology 2012; 143: 515-523.
- Kazeem MI, Akanji MA, Yakubu MT and Ashafa AOT: Protective effect of free and bound polyphenol extracts from Ginger (Zingiber officinale Roscoe) on the hepatic antioxidant and some carbohydrate metabolizing enzymes of streptozotocin-induced diabetic rats. Evidence Based Complementary and Alternative Medicine 2013; e935486.
- Pari L and Latha M: Antidiabetic effect of Scoparia dulcis: Effect on lipid peroxidation in streptozotocin diabetes. General Physiology and Biophysics 2005; 24:13-26.
- Bouchenak M and Lamri-Senhadji M: Nutritional quality of legumes and their role in cardiometabolic risk prevention: A review. Journal of Medicinal Food 2013; 16: 1-14.
- Maruthupandian A, Mohan VR and Sampathraj R: Antidiabetic, antihyperlipidaemic and antioxidant activity of Wattakaka volubilis (l. f) Stapf leaves in alloxan induced diabetic rats. International Journal of Pharmaceutical Sciences and Research 2010; 1:83-90.
- Reddy KN, Trimurthulu G and Reddy CHS: Medicinal plants used by people of Medak district Andhra Pradesh. Indian Journal of Traditional Knowledge 2010; 9:184-190
How to cite this article:
Attanayake AP, Jayatilaka KAPW, Mudduwa LKB and Pathirana C: In vivo antihyperlipidemic, antioxidative effects of Coccinia grandis (L.) Voigt (cucurbitaceae) leaf extract: an approach to scrutinize the therapeutic benefits of traditional Sri lankan medicines against diabetic complications. Int J Pharm Sci Res 2016; 7(10): 3949-58.doi: 10.13040/IJPSR.0975-8232.7(10).3949-58.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
A. P. Attanayake*, K.A.P.W. Jayatilaka , L.K.B. Mudduwa and C. Pathirana
Department of Biochemistry, Faculty of Medicine, University of Ruhuna, Karapitiya, Sri Lanka
10 May, 2016
06 September, 2016
23 September, 2016
01 October, 2016