A STUDY OF ANTIDYSLIPIDEMIA ACTIVITY ON EXTRACTS OF LERAK FRUIT, MANGROVE CRAB SHELL, BINTANGUR LEAF, CASHEW FRUIT PEEL, TENGKAWANG FRUIT, AND A MECHANISM STUDY OF EXTRACTS, FRACTIONS, AND SUBFRACTIONS OF SELECTED SAMPLES

• Main Author: Fajriaty, Inarah
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/78085
• Institution: Institut Teknologi Bandung

Dyslipidemia is a condition characterized by disorder in lipoprotein metabolism, which may present with an increase in the concentration of total cholesterol, Low Density Lipoprotein (LDL), triglycerides, or a decrease in High Density Lipoprotein (HDL) cholesterol in the blood. Furthermore, it stands as one of the main risk factors for coronary heart disease. The mortality rate due to coronary heart disease continues to increase from year to year, indicating the persistently high prevalence of dyslipidemia and its yearly escalating trend. The therapy for this condition is still under development, given that coronary heart disease remains the leading cause of death. Several biological resources, including lerak fruit (Sapindus rarak DC.), mangrove crab shell (Scylla serrata), bintangur leaf (Calophyllum soulattri, Burm), cashew fruit peel (Anacardium occidentale L.), and tengkawang fruit (Shorea stenoptera Burck), have been traditionally employed as dyslipidemia therapy. The antidyslipidemia activity of these resources was reported by several studies. However, there are limited investigations on their mechanism of action. This study aimed to evaluate the effect extracts of lerak fruit, mangrove crab shell, bintangur leaf, cashew fruit peel, and tengkawang fruit, and mechanism study on selected extracts, fractions and subfractions. The study encompassed various approaches, including in vitro, in vivo, in histology, and in silico. The in vitro investigations were conducted to test the activity of selected extracts, fractions and subfractions through inhibition of HMG CoA reductase enzyme and antioxidant tests with DPPH (2,2-diphenyl-1-picryl-hydrazyl) and FRAP (Ferric Reducing Antioxidant Power) methods as free radical sources. Prior to evaluating the antidyslipidemia activity, research was done to find the best animal models of dyslipidemia. Activity of selected extracts and fractions were performed in vivo to assess lipoprotein repair, and histology studies were conducted to assess normal cells, hydropic degeneration, fatty degeneration and necrosis in rat liver cells. Rats were induced with a high-cholesterol formula for 30 days, followed by oral administration of test materials. Furthermore, the lipoprotein and liver cell histology profiles were then observed. The in vitro studies, extracts and active subfractions were selected based on their viability. The expression and population viii of LDLR in HepG2 cells were examined. To complement the experimental results, in silico analyses were conducted, which involved molecular docking and ADMET tests. These analyses aimed to verify the inhibition of active compounds from active subfractions against the HMG CoA reductase enzyme when compared to the comparator simvastatin. Additionally, preliminary in silico tests were performed on various compounds, such as anacardic acid, beta-sitosterol, caloxanthon B, hederagenin, chitin, resveratrol, and stigmasterol. These compounds were identified in the literature study as having an affinity to bind to the active sites of the HMG CoA reductase receptor, HMG synthase, LDL receptor, PPAR-Alpha (excluding hederagenin), and Hydroxycarboxylic Acid Receptor 2 (HCAR 2). Phytochemical screening conducted on lerak fruit extract showed groups such as alkaloid, phenol, flavonoid, saponin and steroid/triterpenoid. Bintangur leaf extract detected phenol, tannin, flavonoid, saponin, and steroid/triterpenoid groups. Meanwhile, cashew fruit peel extract identified alkaloids, phenols, quinones and steroids/triterpenoids; Tengkawang fruit extract detected alkaloids, phenols, tannins, flavonoids and steroids/triterpenoids while mangrove crab shell extract contains alkaloids. Further tests, such as Spektrofotometer Fourier Transform Infra Red (FTIR), showed the presence of chitin compounds. The IC50 test results of HMG CoA reductase enzyme inhibitory activity of lerak fruit extract, mangrove crab shell, bintangur leaf, cashew fruit peel, tengkawang fruit, and pravastatin as a comparison are 75.95 ± 1.13 µg/mL; 36.65 ± 0.78 µg/mL; 63.91 ± 0.12 µg/mL; 66.82 ± 0.16 µg/mL; 25.33 ± 0.54 µg/mL, 6.95 ± 0.19 µg/mL, respectively. The results of the antioxidant activity test with IC50 2,2- diphenyl-1-picryl-hydrazyl (DPPH) and antioxidant activity index (AAI) of lerak fruit extract, mangrove crab shell, bintangur leaf, cashew fruit peel, tengkawang fruit, and ascorbic acid as a comparison were 6.63 ± 0.01 µg/mL; 5.94 ± 0.07, 24.38 ± 0.03 µg/mL; 1.62 ± 0.06, 6.11 ± 0.03 µg/mL; 6.44 ± 0.06, 2.12 ± 0.05 µg/mL; 18.58 ± 0.22, 0.28 ± 0.02 µg/mL; 140.71 ± 5.03, dan 0.60 ± 0.03 µg/mL; 65.67 ± 1.06, 6.63 ± 0.01 µg/mL; 5.94 ± 0.07, 24.38 ± 0.03 µg/mL; 1.62 ± 0.06, 6.11 ± 0.03 µg/mL; 6.44 ± 0.06, 2.12 ± 0.05 µg/mL; 18.58 ± 0.22, 0.28 ± 0.02 µg/mL; 140.71 ± 5.03, and 0.60 ± 0.03 µg/mL; 65.67 ± 1.06, respectively.The results of the antioxidant activity test with IC50 Ferric Reducing Antioxidant Power (FRAP) and AAI of lerak fruit extract, mangrove crab shell, bintangur leaf, cashew fruit peel, tengkawang fruit, and ascorbic acid as a comparison were 418.82 ± 7.06 µg/mL; 0.75 ± 0.09, 430.00 ± 10.07 µg/mL; 0.73 ± 0.07, 8.63 ± 0.05 µg/mL; 3.83 ± 0.15, 10.47 ± 0.06 µg/mL; 29.88 ± 0.13, 2.70 ± 0.09 µg/mL; 115.64 ± 5.66, and 5.35 ± 0.02 µg/mL; 58.36 ± 0.93, respectively. Tengkawang fruit extract had the highest HMG CoA reductase inhibitory activity. The in vitro analysis showed that tengkawang fruit extract had the highest antioxidant activity, therefore, it was classified as a very strong antioxidant. Lerak fruit, bintangur leaf, cashew fruit peel, and tengkawang fruit were fractionated by liquid-liquid extraction (ECC) to obtain n-hexane, ethyl acetate and water fractions. Phytochemical screening was conducted on lerak fruit fractions. ix According to the results, water fraction yielded phenol, tannin, flavonoid and saponin groups. The ethyl acetate fraction detected phenol groups, while n-hexane contains alkaloid, saponin and steroid/triterpenoid groups. Based on the phytochemical screening conducted on bintangur leaf, the water and ethyl acetate fractions detected phenols, tannins, flavonoids, saponins, and steroids/triterpenoids, while the n-hexane identified quinones, saponins and steroids/triterpenoids. For cashew fruit peel, the aqueous fraction detected phenols and flavonoids, while the ethyl acetate fraction detected phenols, quinones and steroids/triterpenoids. n-Hexane fraction yielded alkaloids, phenols, flavonoids, quinones, saponins and steroids/triterpenoids. According to the screening on tengkawang fruit, water and ethyl acetate fractions detected phenols, tannins, flavonoids, while the n-hexane fraction detected alkaloids, quinones and steroids/triterpenoids. The n-hexane fraction of tengkawang fruit had the highest HMG CoA reductase enzyme inhibitory activity with IC50 of 10.26 ± 0.31 µg/mL, while pravastatin as a comparator was 6.25 ± 0.21 µg/mL. Antioxidant activity of n-hexane, ethyl acetate, and water fractions of cashew fruit peel and tengkawang fruit did not differ significantly compared to ascorbic acid. Therefore, these fractions were classified as having very strong antioxidant properties. The ethyl acetate fraction of tengkawang fruit demonstrated the highest antioxidant activity according to the DPPH method, with an IC50 value of 0.43 ± 0.12 µg/mL and an AAI (antioxidant activity index) of 92.70 ± 1.95. On the other hand, the ethyl acetate fraction of cashew fruit peel displayed the highest antioxidant activity using the FRAP method, with an IC50 value of 1.54 ± 0.17 µg/mL and an AAI of 202.75 ± 3.99. For comparison, ascorbic acid had IC50 and AAI of 0.68 ± 0.03 µg/mL and 57.94 ± 1.88 for DPPH as well as 5.10 ± 0.18 µg/mL and 61.22 ± 1.67 for FRAP. Animal models of dyslipidemia was induced to exhibit an increase in total cholesterol, triglyceride, LDL, and a decrease in HDL levels by 1.63, 1.56, 2.53, and -1.08 times, respectively. The obtained levels of hepatic cell damage include normal cells, hydropic degeneration, fatty degeneration, and necrosis. These models are formed through the induction method, achieved by administering high cholesterol formula for 30 days. Extracts and fractions of tengkawang fruit as the best, were selected to perform in vivo testing. The results showed the ethanol extract at a dose of 300 mg/kg bw. Furthermore, the n-hexane fraction of tengkawang fruit 2 at a dose of 255.9 mg/kg bw was the best selected extracts and fractions in improving lipoprotein profiles in the form of decreased cholesterol levels, triglycerides and LDL as well as an increase in HDL levels of Wistar rats. These improvements were not significantly different from the simvastatin group, which was administered at a dose of 3.6 mg/kg bw. There was an enhancement in hepatic cells in the form of repair of hydropic degeneration, fatty degeneration, and necrosis, leading to repair of cells into normal its normal state. These positive effects were observed after administering the extracts and n-hexane fractions of tengkawang fruit for a duration of 30 days in the Wistar rats. x The n-hexane fraction of tengkawang fruit was selected to proceed to the next stage of subfraction studies. Its subfractionation was conducted using vacuum liquid chromatography (VLC) with a silica gel 60 H stationary phase, employing gradient elution with n-hexane-ethyl acetate. This process yielded 16 distinct subfractions which were monitored by thin layer chromatography (TLC) with sulfuric acid, FeCl3, citroborate, Liebermann-Burchard, DPPH, and 2,4,6-tripyridyl-S-triazin (TPTZ) being the spotting agents. Based on monitoring the 16 subfractions, those with the same chromatogram pattern were combined to produce 3, namely combined subfraction 1 ( 1) - 3 (SGF 3). The 3 of tengkawang fruit had the highest HMG CoA reductase enzyme inhibitory activity with IC50 of 2.398 ± 0.106 µg/mL, while the comparator pravastatin was 6.184 ± 0.126 µg/mL. Furthermore, both 2 and 3 of tengkawang fruit exhibited noteworthy antioxidant activity, as determined by the IC50 DPPH (1,1-diphenyl-2- picrylhydrazyl) assay. Their performance was notably superior to that of the comparator substances, including linoleic acid, oleic acid, beta-sitosterol, and ascorbic acid. In particular, the 3 had the highest antioxidant activity using the DPPH method, with an IC50 DPPH and AAI (antioxidant activity index) of 0.34 ± 0.074 µg/mL and 115.88 ± 0.74 µg/mL, respectively. Based on the in vitro viability test results conducted on HepG2 liver cells using the Microtetrazolium (3-(4,5-dimetiltiazol-2-il)-2,5-difenil tetrazolium bromide (MTT) method, data was obtained to determine the number of healthy cells in the sample. of tengkawang fruit extract. The 3 of tengkawang fruit and atorvastatin exhibited the largest percentage of cell viability. The results of cytotoxic tests on HepG2 cells in vitro show that lerak fruit, mangrove crab shell, cashew fruit peel, and tengkawang fruit extracts had moderately active cytotoxic potential. Meanwhile, bintangur leaf extract and 3 exhibited weak potential. The mechanism of lowering cholesterol, specifically LDL, by tengkawang fruit extract and 3 involved increasing the expression and population of LDLR in HepG2 cells. This mechanism was not significantly different from the atorvastatin group, which served as a comparison. After analyzing the tengkawang fruit extract using gas chromatography-mass spectroscopy (GC-MS), the 3 of the n-hexane fraction showed 13 compounds. The composition of fatty acid content in the 3 was rich in unsaturated fatty acids such as oleic and linoleic acids, beta-sitosterol, and stigmast-4-en-3-one compounds. However, saturated fatty acids like palmitic, stearic, arachidic, and myristic acids were also present in the 3. The in silico test of molecular tethering and molecular dynamics was conducted using beta-sitosterol and stigmast-4-en-3-one, the predicted active compounds found and contributed in the 3 of tengkawang fruit. The docking results revealed favorable affinity values of -7.4 kcal/mol and 7.0 kcal/mol for HMG CoA reductase receptors, surpassing simvastatin, a known reference. During the 100 ns molecular dynamics simulation, simvastatin, beta-sitosterol, and stigmast-4-en-3-one demonstrated remarkable stability within the receptor binding pocket, effectively binding to the amino acid residues on the receptor site. xi The results of in silico molecular docking and ADMET tests demonstrated the potential of certain compounds as antidyslipidemia drugs, specifically anacardic acid, beta-sitosterol, caloxanthon B, hederagenin, chitin, resveratrol, and stigmasterol, all of which were mentioned in the literature studies. These tests revealed a strong affinity of these compounds for various receptors involved in dyslipidemia regulation, such as HMG CoA reductase receptors, HMG synthase, LDLR, PPAR-Alpha (with the exception of hederagenin), and HCAR 2. Stigmasterol exhibited the most promising results among the tested compounds in terms of binding affinity. It demonstrated an exceptional affinity value of -8.4 kcal/mol for the HMG CoA reductase receptor, surpassing the positive control, atorvastatin. Moreover, stigmasterol displayed the highest affinity value of -8.6 kcal/mol for the HMG synthase receptor, outperforming simvastatin as a positive control. Caloxanthon B also stood out with a significant affinity value of -4.7 kcal/mol for LDLR, exceeding the affinity of simvastatin used as a positive control. Furthermore, caloxanthon B showed a substantial affinity value of -5.1 kcal/mol for the HCAR 2 receptor, surpassing the positive control, nicotinic acid. All the tested compounds, including anacardic acid, beta-sitosterol, caloxanthon B, hederagenin, chitin, resveratrol, and stigmasterol, adhered to Lipinski's five rules, exhibiting good water solubility. Additionally, these compounds showed indeterminate blood-brain barrier penetration and weak intestinal absorption. Notably, they exhibited 90% plasma protein binding, had no discernible impact on cytochrome P450, and displayed very low hepatotoxicity. The large Log P values of these compounds indicated adequate bioavailability. The results of the Pearson correlation test showed that there were a correlation between the parameters of IC50 DPPH, AAI DPPH, IC50 FRAP, AAI FRAP, HMG CoA reductase, cholesterol, triglycerides, HDL, LDL, normal cells, hydropic degeneration, fatty degeneration, necrosis, MTT, and LDLR. Based on the study results, the tengkawang fruit extract and the n-hexane fraction exhibited several beneficial effects, including HMG CoA reductase inhibition, antioxidant activity, improvement of lipid profile, amelioration of liver cell damage, and increased expression and population of LDLR. Similarly, 3 of tengkawang fruit demonstrated comparable effects, such as HMG CoA reductase inhibition, antioxidant activity, and increased expression and population of LDLR. Further investigation revealed that the active subfraction contained beta-sitosterol and stigmast-4-en-3-one as the active compounds. Computational molecular docking and molecular dynamics analysis showed that beta-sitosterol and stigmast-4-en-3- one exhibited greater inhibition of HMG CoA reductase compared to simvastatin.