EVALUATION OF BLACK TEA BIOACTIVE COMPOUNDS USING IN SILICO AND IN VITRO METHODS AS ACETYLCHOLINESTERASE INHIBITOR

• Main Author: NUR AZIZAH , ROSI
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/64748
• Institution: Institut Teknologi Bandung

Alzheimer's (AD) is a neurodegenerative disease characterized by progressive failure of thinking, lost of memory and language. AD is more specifically characterized by the formation of neuritic plaque accumulations of ?-amyloid peptide (A?) and neurofibrillary tangles (NFT) caused by hyperphosphorylation tau protein. AD often attacks the elderly. Percentage of AD sufferers every year has increased. Problems associated with AD are very complex, including expensive to treat, etiology is unclear and available treatments have side effects to patient. Types of AD treatment approved by FDA are N-methyl-D-aspartate (NMDA) such as memantine and acetylcholinesterase inhibitors such as donepezil, rivastagmine and galantamine. Acetylcholinesterase inhibitor is widely used to treat AD patients. The mechanism of action from the drug is inhibition acetylcholinesterase enzyme. Acetylcholinesterase catalyzes hydrolysis of acetylcholine to choline and acetate. Candidate inhibitors are expected to be drugs that can provide therapeutic benefits for patients and are more environmentally friendly than commercial drugs. Back to nature treatment is becoming a trend, including in overcoming AD. Herbal medicines are widely explored to minimize the side effects that occur in patients. Tea is an herbal drink that is widely consumed and favored by world community. In addition, tea is rich in bioactive compounds that are beneficial for human health. Black tea is a fermented product from tea leaves. Black tea undergoes enzymatic oxidation which gives it a distinctive color and aroma. This study has an urgency to explore natural black tea bioactive compounds as acetylcholinesterase inhibitors with efficient, low cost and low risk. Tea as an acetylcholinesterase inhibitor has been studied before. However, studies regarding black tea as acetylcholinesterase inhibitors have not been conducted. The aims of this study were (1) to obtain candidates for acetylcholinesterase inhibitors from black tea and examine their interactions using in silico method, (2) to obtain bioactive compounds from Indonesia black tea which have inhibitory activity against acetylcholinesterase using in vitro method and (3) to determine the inhibitory power of black tea bioactive compound from Indonesia and donepezil as standard inhibitor against acetylcholinesterase. The stages of the research carried out are as follows. (1) Identification inhibition profile of black tea bioactive compounds using molecular docking method, (2) Extraction of black tea by maceration method using 70% ethanol, (3) Separation of black tea bioactive compounds using Qualitative Thin Layer Chromatography, (4) Separation of black tea bioactive compounds by Preparative Thin Layer Chromatography, (5) Inhibitory activity assay of black tea bioactive compounds against acetylcholinesterase and (6) Structural elucidation of black tea bioactive compounds using LC-MS/MS instruments. In this study, the black teas used for in vitro studies are Wonosari Black Tea and Bogor Black Tea. Both teas come from East Java and West Java, Indonesia. The molecular docking results showed that black tea bioactive compounds that had the best inhibitory ability against acetylcholinesterase were theaflagallin, epicatechin gallate, epigallocatechin gallate, neotheaflavin and epicatechin. The predicted Ki obtained from each compound sequentially is 2.06????10?10; 1.08????10?9; 1.30????10?9; 6.83????10?9 ???????????? 1.19????10?8 . Theaflagallin and neotheaflavin are two compounds that are spesifically present in black tea. The interaction formed in general of those two compounds with acetylcholinesterase are hydrogen bonds and hydrophobic interactions. Therefore, the type of inhibition that occurs is reversible. Probability activity (Pa) analysis shows that black tea bioactive compounds have not been explored as acetylcholinesterase inhibitors. Therefore, black tea has a chance and can be used as a new breakthrough for further exploration as acetylcholinesterase inhibitors. The profiles of those five black tea bioactive compounds if viewed from Ro5 can be used as non-oral drug candidates. The results of the in silico test were then confirmed by in vitro study. The in vitro study gave the results that the thick extracts obtained for Wonosari Black Tea and Bogor Black Tea were 5.00 g and 5.99 g, respectively. The best eluent that can separate bioactive compounds from each viscous extract is ethyl acetate:formic acid:methanol:water 40:1:5:4 (v/v/v/v). The separation results obtained from Wonosari Black Tea and Bogor Black Tea were 15 spots and 14 spots, respectively. The compounds in these spots were tested for inhibition. Black tea bioactive compounds have the ability to reduce the activity of acetylcholinesterase. The best inhibitory power was obtained from ITB_TW_2, ITB_TW_6, ITB_TW_8, ITB_TW_15, ITB_TB_1, ITB_TB_7, ITB_TB_8 and ITB_TB_14. At the enzyme concentration of 1 mU/mL, all of these samples gave better inhibitory values than donepezil as a commercial Alzheimer's drug. The LC- MS/MS results showed that each sample was a mixture. The bioactive compounds contained in black tea have the ability to inhibit acetylcholinesterase and can be used as potential drug candidates to replace donepezil.