A STUDY OF HIBISCUS SURATTENSIS L. ANTIDIABETIC ACTIVITY MECHANISM AND IDENTIFICATION OF ITS ACTIVE COMPOUND

• Main Author: Yuliet
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/49125
• Institution: Institut Teknologi Bandung

Diabetes mellitus (DM) is a disease or chronic metabolic disorder with multiple etiologies characterized by high blood sugar levels, accompanied by carbohydrate, lipid, and protein metabolism disorders. This might be caused by disruption or deficiency of insulin production by beta Langerhans cells in the pancreas gland or caused by insensitive insulin receptors. Uncontrolled disease will cause various metabolic complications, macrovascular and microvascular disorders that cause loss of quality and life expectancy of the patient. As an agricultural country, Indonesia has the potential for the development of herbal plants to treat DM. One that has been used empirically is Hibiscus surattensis L. leaves. However, scientific studies are yet to be done to confirm its activity as an antidiabetic agent. This study aims to examine the antidiabetic activity of H. surattensis L. leaves to understand the mechanism of action the extract and active fractions of H. surattensis L. leaves and to identify active compounds in H. surattensis L. leaves. In addition, an acute toxicity test was performed to determine the safety profile of H. surattensis L. leaves. In vitro test of ?-glucosidase and dipeptidyl peptidase-4 (DPP-4) inhibition and in vivo oral glucose tolerance test, by induction with glucose at 3 g/kg body weight (bw), were performed initial screening to determine the antidiabetic activity of ethanol extract, n-hexane fraction (FNH), ethyl acetate fraction (FEA), and the water fraction (FA) of H. surattensis L. leaves. Glucose tolerance test was carried out in male Swiss Webster mice which were randomly divided into eleven groups of five, as follows: normal; control; glibenclamide (0.65 mg/kg bw, as the standard group); ethanol extract (50 mg/kg bw); and FNH, FEA, and FA (each given at 25 and 50 mg/kg) Activity test on Streptozotocin (100 mg/kg bw) induced diabetic was carried out in male Swiss Webster mice, divided into 8 groups of six, namely: normal, diabetic animal, glibenclamide (0.65 mg/kg), ethanol extract (50 mg/kg), FEA and FA (each given at 25 and 50 mg/kg). The treatments were given for 28 days. The parameters observed were blood glucose level, insulin level, ex vivo plasma antioxidant capacity using DPPH and reducing power methods, pancreatic histopathology with Gomori staining, and hepatic histopathology using Hematoxylin-Eosin staining. Activity test on animal model of DM induced by high fat-diet and fructose 1.8 g/kg bw was performed in male Wistar rats, divided into 8 groups of five, namely: normal, diabetic animal, metformin (100 mg/kg), ethanol extract (50 mg/kg), FEA and FA (each given at 25 and 50 mg/kg). The treatments were given for 21 days. The parameters observed were blood glucose, insulin, HbA1c, and AGE levels, hepatic histopathology using Hematoxylin-Eosin staining, and gastrocnemius muscle immunohistochemistry for GLUT-4 translocation analysis. The identification of the active compound was carried out using the classical column chromatography method. Isolates were characterized using mass spectroscopy, Magnetic Resonance Spectroscopy (RMI)- 1 H, RMI- 13 C, RMI- Heteronuclear Single Quantum Correlation (HSQC,) and RMI-Heteronuclear Multiple Bond Correlation (HMBC) spectroscopy. The isolates were tested for ?- glucosidase and DPP-4 inhibitor enzymes. ?-glucosidase enzyme inhibitory activity test showed that ethanol extract, FNH, and FA had IC50values of > 5 mg/mL, while FEA had IC50value of 3.89 ± 0.13 mg/mL. DPP-4 enzyme inhibitory activity test showed that ethanol extract, FNH, FEA, and FA had IC50 values of 236.02 ± 13.95; > 1000; 17.95 ± 2.80, and 847.90 ± 31.42 µg/mL, respectively. Glucose tolerance test showed that ethanol extract, FEA, and FA significantly reduced the increase in blood glucose levels (KGD) from minute 60 to 150 and increased glucose tolerance with respective hypoglicemic effect percentages for ethanol extract, FNH 25, FNH 50, FEA 25, FEA 50 FA 25 and FA 50 of 29,76; 18,57; 23,04; 32,84; 41,41; 19,92, and 24,77%. The results of antidiabetic activity tests in animal model induced streptozotocin showed that ethanol extract, FEA (25 and 50) and FA (25 and 50) reduce blood glucose levels with potential hypoglycemic effect of 29.84; 27.47; 31.26;, 18.69, and 19.62%, respectively; increased insulin levels to 8.36, 7.49, 8.04, 6.86, and 7.16 mIU/L, respectively; increased plasma antioxidant capacity; repaired damage to pancreatic beta cells with HOMA ?values of 23.06; 19.96; 25.66; 14.10; and 13.66%, respectively; and hepatocyte damage score of 29.67; 28.00; 24.67; 29.67; and 32.00, respectively, which were significantly different compared to diabetic control group (p <0.05). Tests in animal model of type 2 DM further showed that ethanol extract, FEA, and FA reduced blood glucose levels with potential hypoglycemic level of 26.43; 30.60; 36.84; 33.33, and 27.98%, respectively; reduced total cholesterol levels by 42.41; 43.80; 52.32; 40.12, and 42.20%, respectively; increased insulin sensitivity by 88.47; 89.29; 91.66; 79.86, and 75.06%, respectively; reduced HbA1c level to 64.91, 59.10, 55.65, 59.41 and 65.71 ng/mL, respectively and AGEs level to 424.33, 312.98, 278.33, 417.14, and 379.00 ng/mL, respectively; and significantly increased GLUT-4 translocation compared to diabetic control group (p <0.05). Structural characterization and elucidation results showed isolates were kaempferol compounds (C15H10O6) with levels of 0.02% (g / 100 g fraction). The content of isolate in this study was 0.02% (g/100 g of fraction). The inhibitory activities against ?-glucosidase and DPP-4 were shown at IC50 values of 27.78 ± 0.86 and 7.37 ± 0.06 ?g/mL, while the values for the standard acarbose and sitagliptin were 17.80 ± 0.27 and 25.56 ± 0.43 ?g/mL, respectively. The ethanol extract of H. surattensis L. leaves at a dose of 5,000 mg/kg bw neither show any signs of toxicity or death that were no significantly different compared to the normal controls. These results indicate that ethanol extract of H. surattensis L. leaves is safe and practically non-toxic. Based on the findings of the study, it can be concluded that the extract and active fraction of H. surattensis L. leaves have antidiabetic activity with increasing insulin sensitivity by regenerating pancreatic ?cells, increasing the expression of GLUT 4, and inhibiting the activity of the DPP-4 enzyme (DPP-4 inhibitor) as the main mechanisms of action. Results of this study further indicate that FEA is the most potential fraction.