Antioxidants and Antidiabetic Potency of Aqueous and Ethanolic Extracts of Bawang Dayak Bulbs (Eleutherine palmifolia) in vitro and in vivo.

Abstract

Eleutherine palmifolia is an indigenous plant from Kalimantan and is widely used by the Dayak tribes to cure several diseases including diabetes mellitus. The purpose of this research is to scientifically prove antioxidant and antidiabetic properties of bawang dayak bulbs and to evaluate its safety that can be used as a source of functional food. Bawang dayak bulbs (BDB) were extracted with water and ethanol solvents. The solutions which contain the aqueous and the ethanolic extracts then freeze dryed. Phytochemical screening that was conducted upon aqueous and ethanolic extracts showed that both of extracts contained alkaloids, tannins, phenolics, flavonoids, triterpenoids, and steroids. Prominent types of phytochemical compounds in aqueous extracts are alkaloids phenolic and triterpenoids, whereas in the ethanolic extract are alkaloid, tannin, phenolic, flavonoids and triterpenoids. Both types of extracts showed antioxidant activity and alpha glucosidase inhibition activity. The antioxidant activity of the ethanolic extract is very significantly higher (P<0.01) than that of the aqueous extract, with IC50 112 ppm for ethanolic extract and 526 ppm for aqueous extract. Alpha glucosidase inhibition activity of ethanolic extract was also very significantly greater (P<0.01) than that of the aqueous extract, with IC50 241 for ethanolic extract and 505 ppm for aqueous extract. The result of NMR (Nuclear Magnetic Spectroscopy) analysis showed that there were eleutherinoside A, eleutherol, and eleuthoside B in ethanolic extract of BDB, but there was not enough sinyal in NMR spectra of aqueous extract to conduct NMR-J resolved data analysis. According to Ieyama et al. (2011) those compounds showed alpha glucosidase inhibitory activity, and eleutherinoside has the biggest activity with IC50 0.5 mM. So it is suggested that eleutherinoside A, eleutherol and eleuthoside B are the active compounds of alpha glucosidase inhibitory activity in BDB ethanolic extract. It is also suggested that alphaglucosidase inhibitory capacity in BDB aqueous extract was presented by the activities of phenolic, alkaloid and triterpenes compounds. Oral glucose tolerance test (OGTT) was done to decide the dose of bawang dayak extracts for in vivo analysis in diabetic experimental rats. The chosen dose was 100 mg/kg BW to diabetic rats, whereas a higher dose was chosen for nondiabetic rats (500 mg/kg BW). The reason of higher dose implementation to non diabetic rats is to evaluate the safety of aqueous and ethanolic extracts to the function and morphology of liver and kidney organs. Sprague-Dawley (SD) male rats age ± 8 weeks old and weighs ± 180 grams were used in this experiment. Conditions of diabetes mellitus (DM) is obtained by induction of 110 mg/kg BW of alloxan monohydrate to SD rats intraperitoneally. BDB aqueous and ethanolic extracts were administered orally by gavage to diabetic and non-diabetic rats for 28 days. Termination was done by using ketamine on day 29 and followed by blood, liver, kidneys and pancreas collections. Observations and analysis were conducted on the body weight, blood glucose levels, serum insulin levels, blood lipid profile (total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides), serum catalase and superoxide dismutase (SOD) enzymes activities, liver malonaldehyde levels, serum creatinine levels, serum albumin levels, Serum Glutamic-Piruvic Transaminase (SGPT) and Serum Glutamic-Oxaloacetic Transaminase (SGOT) levels, Langerhans islets numbers, pancreatic beta cells numbers, the profile of Cu,Zn-SOD enzyme in kidney, and morphology of liver and pancreatic tissues. The results showed that administration of 100 mg/kg BW of BDB aqueous and ethanolic extracts for 28 days could maintained the body weight and lowered the blood glucose level of diabetic rats. The serum levels of insulin, SOD activity and catalase activity in BDB treated diabetic rats were very significantly higher (P<0.01) than that of diabetic untreated rats. The levels of liver MDA, LDLcholesterol and total cholesterol in BDB treated diabetic rats were very significantly lower (P<0.01) than that of diabetic untreated rats. But there were no significantly difference in the levels of serum HDL, triglycerides, Glutamic- Piruvic Transaminase (GPT) and Glutamic-Oxaloacetic Transaminase (GOT) of BDB treated diabetic rats and untreated ones. Administration of 500 mg/kg BWof BDB extracts gave no significantly different in body weight change and blood glucose levels of non diabetic rats compare to untreated ones. They were also no significantly different in the level of liver MDA between BDB treated non diabetic rats and untreated ones. The levels of serum SOD activity, catalase activity, LDL, HDL, total cholesterol, triglycerides, creatinin, albumin, GPT and GOT of BDB treated non diabetic rats also showed no significantly different with untreated ones. Cu,Zn-SOD enzymes in the kidney of BDB aqueous and ethanolic extract treated diabetic rats were more plenty than that of diabetic untreated ones. So did in non diabetic rats, administration of BDB aqueous and ethanolic extract gave more plenty of Cu,Zn-SOD enzymes compare to untreated ones. The effect of BDB extracts administration also present in Langerhans islet and pancreatic beta cells. Diabetic rats treated with BDB extracts had very significantly higher Langerhans islet numbers than that of untreated ones. But BDB extracts had no effect to the number of Langerhans islet in pancreas tissue of non diabetic rats. BDB extracts administration to diabetic rats also gave significantly higher in the number of pancreatic beta cells compare to untreated ones. But it gave no effect to non diabetic rats. Histological observation showed that the administration of 100 mg/kg BW of bawang dayak ethanolic extracts for 28 days could inhibit the inflammation of renal tissue and necrosis and degeneration of tubulus renalis cells in diabetic kidney. The administration of 100 mg/kg BW bawang dayak aqueous and ethanolic extracts could prevent the inflammation of liver tissue and inhibit necrosis and hidropic degeneration of hepatocytes. Morphologic observation of kidney and liver tissues also showed that administration of 500 mg/kg BW bawang dayak aqueous and ethanolic extracts for 28 days did not give adverse effect to non diabetic rats. The result of this study suggested that antidiabetic mechanisms of bawang dayak aqueous and ethanolic extracts is through the inhibition of alphaglucosidase enzyme activity, and through the inhibition of the damage rate of pancreatic beta cells. The alpha-glucosidase is a carbohydrate-digesting enzyme that works in the gut. As a result of this inhibitor, the uptake of glucose from the intestine into the blood to be down and can suppress postpandrial glucose levels. The inhibition of the damage rate of pancreatic beta cells suggested due to the antioxidants activity of the extracts. Bioactive compounds such as polyphenols and flavonoid donored their hydrogen ions to scavenge free radical that lead on stopping of lipoperoxidation process which could damage the membrane cells and causing the death of cells. As conclusions, the results of this study successfully prove the antioxidant and antidiabetic properties of bawang dayak aqueous and ethanolic extracts. Based on its efficacy and safety profile obtained in this study, bawang dayak aqueous and ethanolic extracts can be used as functional food with antioxidant and antidiabetic properties.