IN VITRO, IN VIVO AND IN SILICO ANTIDISLIPIDEMIC ACTIVITY OF BLACK GRASS JELLY (MESONA PALUSTRIS BLUME) LEAF ETHANOL EXTRACT

• Main Author: Andri Deswati, Dytha
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/87835
• Institution: Institut Teknologi Bandung

Ischemic heart disease is the largest cause of death in the world, with the death rate reaching 8.9 million in 2019. In Indonesia, the prevalence of heart disease continues to increase, with dyslipidemia as one of the main risk factors. According to Riskesdas 2018, around 34.82% of the Indonesian population has problematic total cholesterol levels, while the prevalence of low High Density Lipoprotein (HDL) and high triglycerides respectively reach 24.3% and 13.8%. Dyslipidemia is characterized by an imbalance of plasma lipids often caused by a diet high in fat and cholesterol. Conventional treatment using drugs such as HMG-CoA reductase (HMGR) inhibitors, fibrates, and others, although effective, often cause side effects such as myopathy, gastrointestinal disorders, and increased risk of metabolic disease. To reduce the risk of side effects, the use of safe and effective natural ingredients is a promising alternative. One potential plant is black grass jelly (Mesona palustris Blume), which has traditionally been used by the Indonesian people and has potential as an antidislipidemia agent. The purpose of this research is to test the antidislipidemia activity of the ethanol extract from black grass jelly leaves (EECH). The research stages began with determination, extraction and fractionation, phytochemical screening, characterization, compound identification and determination of the total flavonoid content of black grass jelly leaves. The research continued with in vitro activity testing of antioxidant activity with ABTS, DPPH, FRAP method; HMGR and lipase enzyme activity, in vivo testing of ethanol extract from black grass jelly leaves against rats induced dyslipidemia by observing lipid profiles (total cholesterol, LDL triglycerides, HDL), oxidative stress biomarkers (MDA, SOD, catalase, glutathione peroxidase) and anti-inflammatory (TNF-?, IL-6). In the final stage of the research, in silico study was carried out, including molecular docking and molecular dynamic approaches. Determination result showed that the plant used was Mesona palustris Blume. Extraction was done using the maceration method with 96% ethanol solvent while fractionation used n-hexane, ethyl acetate and water solvent. The yield of ethanol extract, water fraction, ethyl acetate fraction dan n-hexane were 8.28%, 32.6%, 8.0% and 15.6% respetively. Phytochemical screening of simplisia, ethanol extract and water fractions showed the presence of alkaloids, flavonoids, saponins, tannins, polyphenols and steroids/triterpenoids. The ethyl acetate fraction does not contain alkaloids, while the n-hexane fraction contains polyphenol and steroid/triterpenoid compounds. Characterization results show that the extract has a moisture content of 16.19 ± 0.19%, total ash content of 20.54 ± 0.49%, water soluble content of 58.03 ± 0.165%, then ethanol soluble content of 32.89 ± 0.24 %. Extract identification results with TLC indicate the presence of flavonoid, polyphenol and steroid/terpenoid groups. From the results of the HPLC, it is suspected that the compounds caffeic acid, quercetin 3-O-galactoside, isoquercetin, astragalin, and rosmarinic acid were identified. Identification with UPLC-MS compounds caffeic acid, 4'-methylnaringenin, epicatechin, quercetin, betulinic acid, quercetin 3-O-galactoside, then myricetin 3-(6-acetylgalactoside). The total flavonoid content of the ethanol extract is 4.9 ± 0,18 mgQE/g. In in vitro testing, antioxidant activity using the ABTS method showed that the ethyl acetate fraction had the strongest activity compared to the ethanol extract, water fraction and the n-hexane fraction with an IC50 value of 2.52 ± 0.02 µg/mL; 3.02 ± 0.11 µg/mL; 3.21 ± 0.03 µg/mL then 3.62 ± 0.09 µg/mL, respectively. Testing with the DPPH method showed that ethyl acetate fraction had the strongest activity compared to ethanol extract, water fraction and n-hexane fraction with IC50 values of 17.58 ± 0.53 µg/mL; 22.69 ± 0.58 µg/mL; 23.92 ± 0.16 µg/mL then 24.97 ± 0.64 µg/mL, respectively. By FRAP method, the highest antioxidant capacity was also found in the ethyl acetate fraction, followed by ethanol extract and aqueous fraction, while the n-hexane fraction showed the lowest activity with antioxidant activity values of 20.34 ± 0.23 mgAAE/g; 11.50 ± 0.46 mgAAE/g; 9.20 ± 0.21 mgAAE/g then 5.79 ± 0.16 mgAAE/g. In the HMGR enzyme inhibition test, the percentage of inhibition obtained by pravastatin, ethanol extract, water fraction, ethyl acetate fraction, and fraction n-hexane, respectively, was 75.96 ± 5.07%; 74.18 ± 1.05%; 74.87 ± 7.6%; and 63.84 ± 8.5%. Meanwhile, the inhibition test against lipase enzyme showed the percentage inhibition by orlistat, ethanol extract, aqueous fraction, ethyl acetate fraction, then n-hexane fraction were 83.68 ± 1.65%; 93.67 ± 1.05%; 97.49 ± 0.67%; 81.74 ± 5.68% and 108.72 ± 2.68%, respectively. In in vivo, the administration of EECH at doses of 200, 400, and 600 mg/kg bw significantly decreased total cholesterol (18.67%, 25.50%, 29.53%), triglycerides (9.46%, 24, 57%, 28.77%), LDL (35.65%, 53.75%, 53.65%), and increase HDL (1.50%, 2.42%, 4.22%) at doses of 200, 400, and 600 mg/kg bb. Measurement of the level of MDA in the group given simvastatin showed a value of 2.13 mmol/mL, while the group with an extract doses of 200, 400 and 600 mg/kg bw produced value of 2.69; 2.57 and 2.31 mmol/mL. Measurement of SOD activity shows that the simvastatin group and the extract group doses 200, 400 and 600 mg/kg body weight produce an activity of 132.97; 83.85; 86.59 then 127.86 U/mL. The result of measuring catalase activity separately in the simvastatin group, extract dose 200, 400 and 600 mg/kg bw was 19.13; 16.20; 20.24 and24.19 U/mL. The result of measurement of glutathione peroxidase levels in the group given simvastatin and extract doses 200, 400, and 600 mg/kg bw, respectively 906.56 ng/L; dose group 200, 400 and 600mg/kg bw was 881.86; 893.75 and 988.75 ng/L. Measurement of the level of IL-6 in the simvastatin-treated group, the 200, 400 and 600 mg/kg body weight dose extract groups showed values of 5.99; 7.09; 6.66 and 6.53 ng/L. Measurement of TNF-?levels in the same four groups, consecutively, showed values of 115.94; 176.22; 168.85 and 151.01 ng/L. In in silico testing, molecular docking results against HMGR obtained inhibition constant values of simvastatin, betulinic acid and myricetin 3-(6-acetylgalactoside) of 55.20 nm; 3.86 µM and 11.81 µM. On PPAR-?target, phenofibric acid, betulinic acid and myricetin 3-(6-acetylgalactoside), showed inhibition constant 1.40 µM; 62.68 µM and 2.29 µM. While on the NPC1L1 target, ezetimib, betulinic acid and myricetin 3-(6-acetylgalactoside) respectively, showed inhibition constants of 528.75nM; 633 nM and 363.81 nM. The result of molecular dynamic (MD) testing on HMGR, simvastatin as a control showed the most negative ?G total (-37.125 kcal/mol), high structural stability, and stable hydrogen interaction. While betulinic acid has a total ?G of -28.0973 kcal/mol with better stability in a hydrophobic environment than myricetin 3-(6-acetylgalactoside) (-12.169 kcal/mol). In PPAR ?, phenofibric acid showed the most negative ?G total (-15,432 kcal/mol) with high structural fluctuation, while betulinic acid had a total ?G of -13,028 kcal/mol and the highest stability in MD simulation, with low RMSD and RMSF. Myricetin 3-(6- acetylgalactoside) has a ?G total of -12,492 kcal/mol with slightly larger fluctuation but remains stable. In NPC1L1, betulinic acid showed the best binding affinity (?G total -59.2625 kcal/mol) followed by the control, ezetimib (-44.434 kcal/mol), and myricetin 3-(6-acetylgalactoside) (-28.354 kcal/mol). MD analysis showed that myricetin 3-(6-acetylgalactoside) had better structural stability (lowest average RMSD), while betulinic acid showed the most stable position in the active site (lowest ligand RMSD).