ISOLATION OF ANTIHYPERCHOLESTEROLEMIC AGENT FROM RICE BRAN AND STUDY OF ORYZANOL ANALOG AS HMG-COA REDUCTASE INHIBITOR

• Main Author: Aziz, Syaikhul
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/46949
• Institution: Institut Teknologi Bandung

Rice bran is the outermost layer of rice, a by-product was obtained during rice milling. This layer is rich in macro and micro-nutrients that have health benefits. The oryzanol contained in the rice bran has been known as the major bioactive component, which is responsible for various biological activities, including antioxidant, antimicrobial, antiallergenic, anticarcinogenic, antidiabetic, antiinflammatory, and antihypercholesterolemic. Especially for antihypercholesterolemic activity, oryzanol shows promising results, making it potential to be developed. However, the next challenge is to get it in large quantities, since the rice bran contains a relatively small amount of oryzanol, and the isolation method is not easy. This study has two main objectives. The first objective is to obtain antihypercholesterolemic active compound from rice bran. The second objective is to study the oryzanol analog, ergosteryl ferulate, as HMG-CoA reductase inhibitor through several steps, i.e. in silico study against HMG-CoA reductase, synthesis, and in vitro evaluation of HMG-CoA reductase inhibitor activity. The antihypercholesterolemic active compound was obtained based on antihypercholesterolemic activity study in the rat model. That step was begun with hot solvent extraction by reflux method using single and binary solvents, i.e. nhexane, chloroform, ethyl acetate, dichloromethane, isopropanol, acetone, nhexane - ethyl acetate (1:1, v/v), chloroform - ethyl acetate (1:1, v/v), and n-hexane - isopropanol (1:1, v/v). The selected extract for further fractionation was chosen by the characteristic of extract that has the highest amount of oryzanol. TLC densitometry was used to quantitatively analyze the total oryzanol content in various extracts. The hypercholesterolemic rat models were induced by intragastric administration of 0.5 ml/100 g body weight of cocktail containing 2% of cholesterol, 1% of cholic acid, and 0.6% of propylthiouracil in palm oil which is given for 30 days period. To total cholesterol-lowering fraction, a series of separation and purification methods were then performed by chromatographic techniques. The structure of an isolated compound was determined by onedimensional ( 1 H and 13 C) and two-dimensional (HSQC and HMBC) NMR spectra. The study of oryzanol analog as HMG-CoA reductase inhibitor started with molecular docking simulation using AutoDockTools software to predict the interaction of oryzanol analog in complexes with HMG-CoA reductase. In addition, four major oryzanol compounds i.e. 24-methylene cycloartanyl ferulate, cycloartenyl ferulate, sitosteryl ferulate, and campesteryl ferulate were used for comparison. Ergosteryl ferulate and sitosteryl ferulate were synthesized in four steps, including acetylation of ferulic acid, the formation of acyl chloride, conjugation of ferulate structure with steroid/triterpenoid alcohol, and deprotection of acetyl group. The synthesized product for each step was monitored by thin layer chromatography. The chemical structure of the synthesized product was determined by one-dimensional and two-dimensional NMR spectra. Finally, ergosteryl ferulate and sitosteryl ferulate were evaluated for their HMG-CoA reductase inhibitory activity using the commercial assay kit. The preliminary results showed that all organic solvents could extract oryzanol, in which its content ranged from 27.92 ± 4.45 to 43.59 ± 3.36 mg/100 g rice bran. n- Hexane has been found to be the best solvent to extract oryzanol by reflux method (43.59 ± 3.36 mg/100 g rice bran). Therefore, n-hexane extract was selected for fractionation and evaluation of its antihypercholesterolemic activity. The in vivo results exhibited that the F2 and F5 fractions were able to reduce total cholesterol levels on the 4 th day of administration by 38.15 ± 3.04 and 37.32 ± 4.02 %, respectively, and statistically significant compared to control (p<0.05). In addition, the F2 and F5 fraction also had a higher rate of total cholesterol reduction than the control group. Based on the GC-MS analysis, F2 fraction contained higher unsaturated fatty acid than saturated fatty acid. Compound A has been successfully isolated from the F5 fraction as a white solid with a melting range of 134.3-135.8 ° C. According to one-dimensional and two-dimensional NMR spectra, compound A is ?-sitosterol. The results of molecular docking exhibited that oryzanol and ergosteryl ferulate had binding affinity against HMG-CoA reductase with Gibbs free energy score (?G) and inhibition constant (Ki) of ?G = -4.17 to -3.45 Kcal/mol and Ki = 0.88 to 2.97 mM, respectively. In addition, the study showed that the ergosteryl ferulate (?G = -4.17 Kcal/mol; Ki = 0.88 mM) interacted with important amino acid residue (cis loop) of HMG-CoA reductase - binding pocket, including Ser684, Asp690, and Lys691. Based on ?G score, Ki, and amino acid residue, ergosteryl ferulate was predicted to have greater potential in inhibiting HMG-CoA reductase compared to oryzanol. Ergosteryl ferulate and sitosteryl ferulate have been successfully synthesized and evaluated for their HMG-CoA reductase inhibitory activity. Ergostery ferulate was obtained as a yellowish solid with melting range of 166.4-170.9 ° C, while sitosteryl ferulate as a colorless solid with melting range of 131.2-136.6 ° C. The in vitro assay demonstrated that ergosteryl ferulate and sitosteryl ferulate has the inhibitory activity against HMG-CoA reductase by 16.17 ± 5.84 and 9.57 ± 2.02 %, respectively. Overall, the results of the present study suggest that the composition of phytosterols and unsaturated fatty acids contained in rice bran is responsible for anti hypercholesterolemic activity. In addition, the oryzanol analog (ergosteryl ferulate) has been successfully synthesized and has the potential to improve HMG-CoA reductase inhibitory activity compared to oryzanol. This research was expected to give a positive contribution to the development of antihypercholesterolemic agent from rice bran and drug development, especially for another alternative than oryzanol (ergosteryl ferulate) as HMG-CoA reductase inhibitory activity.