ANTIDYSLIPIDEMIC ACTIVITY OF ETHANOL EXTRACT OF PANDAN (PANDANUS AMARYLLIFOLIUS ROXB.) LEAVES USING IN VITRO, IN VIVO, AND IN SILICO METHODS, AND ITS SAFETY ASSESSMENT.

• Main Author: Parotua Lumban Raja, Martohap
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/84630
• Institution: Institut Teknologi Bandung

Dyslipidemia is a lipid metabolism disorder, that is characterized by lipid fraction abnormalities in the form of increased total cholesterol levels, LDL levels, triglyceride levels, and decreased HDL levels. Dyslipidemia is a risk factor for coronary heart disease and stroke. Pandan (Pandanus amaryllifolius Roxb) is an annual plant that grows in tropical areas, including Indonesia, traditionally used as a dye, food fragrance, and medicine. Pandan plants have been studied to have pharmacological activities such as antihyperglycemic, antibacterial, antioxidant, antiviral, anticancer, and hepatoprotective. However, research into pandan plants' potential antidyslipidemia activity and safety is still limited. The plant was determined as Pandanus amaryllifolius Roxb. The plant extract was then standarized using specific and non-specific parameters. Pandan leaf extract was prepared using the maceration method with 96% ethanol as the solvent. The characteristics and identity of the metabolite content of pandan leaf ethanol extract (EEDP) were determined using phytochemical screening procedures and then analyzed using liquid chromatography high-resolution mass spectrometry (LC- HRMS). The yield of pandan leaf ethanol extract was 15.4%. The results of standardization of crude drug and pandan leaf ethanol extract showed that the water- and ethanol soluble extracts of the crude drugs were 15.758 ± 0.282% and 6.581 ± 0.193%, respectively, with loss on drying of 6.327 ± 0.018%. EEDP water content was 9.967 ± 0.015%. Phytochemical screening results showed that EEDP contained flavonoids, alkaloids, saponins, steroids, terpenoids, glycosides, and tannins. Characterization results using LC-HRMS showed that EEDP contained the alkaloid pandamarilactonine A or B. Quantitative determination of total phenols using the folin-ciocalteu method is expressed as gallic acid equivalent (GAE) per gram of extract, total flavonoid content using the AlCl3 method is expressed as quercetin equivalent (QE). Determination of total alkaloids using the UV-vis spectrophotometric method using quinine as the standard. The results of measurement of total phenol, flavonoid and alkaloid levels of pandan leaf ethanol extract were indicated to be 80.910 ± 0.190 mg GEA/g extract and 31.762 ± 0.271 mg QE/g extract, 0.028±0.002% respectively. In vitro test results show that the IC50 of EEDP for the inhibitory activity of the HMG-CoA reductase enzyme was 3.159 µg/mL, while the IC50 of pravastatin is 0.072 µg/mL. The results of the in vivo anti-dyslipidemia activity test showed that induction of dyslipidemia by feed high in fat, cholesterol, cholic acid, and propylthiouracil for 8 weeks significantly increased total cholesterol levels. Administration of EEDP at 200, 300, and 600 mg/kg bw caused decreases in totalAdministration’s cholesterol (15.92%; 14.46%; 34.73%), LDL (24.33%; 40.15%; 47.13%), and triglycerides (39, 67%; 40.47%; 56.62%), with EEDP at 600 mg/kg bw as the most potent extract. The results of measurement of pro-inflammatory cytokines, namely IL-6, TNF-?, and NF-?B p65 showed that the levels of IL-6 and TNF-?in the treatment group (doses of 200, 300, and 600 mg/kg bw) was significantly lower than the vehicle group. The levels of the NF-?B p65 were significantly lower compared to control with the 300 and 600 mg/kg bw treatment groups. The safety assessment of EEDP was carried out by oral acute toxicity test and a 28- day short oral subchronic toxicity test. Tests rats were divided into 3 groups: the control group, the treatment group (dose of 300 mg/kg bw, 600 mg/kg bw, and 1000 mg/kg bw), and the satellite group (control satellite group and satellite group dose of 1000 mg/kg bw). Toxicity parameter testing includes behavioral parameters, organ indices, urin analysis, hematology examination, clinical biochemistry, and macropathology observations. The results of acute toxicity testing on female Wistar rats showed that there were no deaths or toxic symptoms at a dose of 5000 mg/kg bw. The results of the 28-day short oral subchronic toxicity test, observing signs oftoxicity, body weight, relative organ weights, urin, hematology, and clinical biochemistry, shwed there was no significant difference after administration of EEDP in all dose groups compared to the control group. The results of microscopic observations of organs (liver, kidney, lung, heart, and spleen) in the 28-day oral subchronic toxicity test of EEDP after doses of 300, 600, and 1000 mg/kg bw showed no significant differences compared to control. Molecular docking and molecular dynamics were used for the first part of the research, which aimed to find the possible secondary metabolite content of pandan leaves that have antidyslipidemic activity. Testing the activity of pandan plant metabolite content as anti-dyslipidemia in silico was based on the interaction of pandan leaf metabolite compounds with several receptors that play a role in lipid metabolism, namely the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase receptor with the PDB code (PDB ID: 1HW9), peroxisome proliferator- activated receptor (PPAR) alpha (PDB ID: 6LX4), and Niemann-Pick C1-like 1 (NPC1L1) (PDB ID: 7DFZ). Overall, the molecular docking results showed that the alkaloid compounds contained in pandan leaves (pandamarilactonine A, pandamarilactonine B, pandanusine B, and pandanamine) may have potential antidyslipidemic activity through the mechanisms of HMG-CoA reductase receptor inhibition, PPAR?receptor agonism, and NPC1L1 receptor inhibition. The stability of the ligand-receptor interactions of pandan leaf metabolite compounds (pandamarilactonine A, pandamarilactonine B, pandanusine B, and pandanamine) as antidyslipidemia was obtained from validated molecular docking results followed by dynamic molecular tests with parameters analyzed including root mean square deviation (RMSD), root mean square fluctuation (RMSF), and solvent accessible surface area (SASA). Based on the results of testing the molecular dynamics simulation parameters (RMSD, RMSF, and SASA), the compounds pandamarilactonine A, pandamarilactonine B, pandanusine B, and have stable interactions with the 1HW9, 6LX4, and 7DFZ receptors so that they have the potential to be used as antidyslipidemia drug candidates.