DEVELOPMENT OF EXTRACTS AND ACTIVE COMPOUNDS FROM SIRIH BUMI HERB (PEPEROMIA PELLUCIDA (L.) KUNTH) AS ANTIOSTEOPOROSIS AGENT

• Main Author: Gusti Agung Ayu Kartika, I
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/53762
• Institution: Institut Teknologi Bandung

Osteoporosis is one of diseases that greatly affects the quality of human life, especially in the elderly. However, to this day, the available conventional treatments are still limited and unsafe in long-term use. Therefore, an alternative treatment that can overcome these problems is needed. One of the promising candidates is sirih bumi (Peperomia pellucida (L.) Kunth). Some previous studies have proved that extracts from this plant have antiosteoporosis effect both through in vitro and in vivo study. Nevertheless, further tests are still needed in order to determine the most active type of extract, fraction, or compound, the dominant active compounds play an important role in this activity as well as know the mechanism of its action. This study aims to determine potential compounds as anti-osteoporotic agents from sirih bumi herb and study its mechanism of action. This research included an anti-osteoporosis activity test which was supported by an anti-inflammatory and estrogenic activity test on extracts, fractions and some isolated compounds from sirih bumi. The activities and mechanism of actions of the plant were examined through in vivo tests, which was supported by data from in vitro and in silico studies. This research began with the extraction process with sequential maceration from the simplicia of sirih bumi which produced three types of extracts, namely nhexane, ethyl acetate, and ethanol extracts. The water extract was obtained from a separate maceration process. Five chemical compounds were isolated from ethyl acetate extract by monitoring the results of estrogenic tests and chromatographic profiles. The five compounds are a derivative of dillapiol (6- allyl-5-methoxy-1,3-benzodioxol-4-ol), pachypostaudin B, pellucidin A, dillapiol and apiol. 6-allyl-5-methoxy-1,3-benzodioxol-4-ol and pachypostaudin B were compound that had been isolated for the first time from sirih bumi and their pharmacological activity had never been reported. In addition, there are four chemical compounds that have been identified in sirih bumi, namely stigmasterol in n-hexane and ethyl acetate extract, apigenin and apigetrin in water extract, and quercetin in all extract. The presence of apigetrin in the sirih bumi herb has never been reported in scientific journals. In preliminary research, the crude ethanol extract of the sirih bumi (ethanol extract A) had better anti-osteoporotic effect than its juice through in vivo experiments. Moreover, through in vitro antioxidants tests (DPPH, ABTS, CUPRAC) against four extracts, ethyl acetate extract and ethanol extract were found had the best antioxidant effect. In the in vitro anti-inflammatory test using cell membrane permeability inhibitory models, ethyl acetate extract showed the highest anti-inflammatory activity. This extract also had a thin layer chromatographic pattern that was most similar to the ethanol extract A with the result that the antiosteoporotic activity test was only carried out on ethyl acetate extract and ethanol extract. The in vivo antiosteoporotic activity test proved that the two extracts were comparable in accelerating the bone healing process based on qualitative observations of bone microarchitecture parameters and bone cells histology. The only significant difference was found in the increase in the value of the trabecular thickness parameter shown by the ethanol extract. Both extracts were significantly able to increase the number of fibroblasts (203.32% ethyl acetate extract and ethanol extract of 206.30%, p <0.01) and decreased the number of polymorphonuclear cells (PMN) (35.35% ethyl acetate extract, p <0.05 and ethanol extract 40.49%, p<0.01). Ethyl acetate extract had the potential for better antiosteoporotic activity based on liquid chromatography-mass spectroscopy testing and had a better flavonoid content. Estrogenic activity test which was carried out on n-hexane extract, ethyl acetate extract, ethanol extract, water extract, and two fractions, nine subfractions, and five chemical compounds isolated from ethyl acetate extract, revealed that the five compounds (derivate of dillapiol 22.28%; pachypostaudin B 36.54%; pellucidin A 13.46%; dillapiol 12.73%; and apiol 33.08%) had lower estrogenic activity than methanol fraction (114.37%). Estrogenic activity of all of the compounds except pachypostaudin Beven lower than ethyl acetate extract. The type of samples with the highest activity were water extract and methanol fraction from ethyl acetate extract. The higher the estrogenic activity, the greater the possibility of antiosteoporotic activity. The experiment also revealed that estrogenic activity was mediated by the binding to estrogen receptors. Water extract consistently demonstrated its best activity in an in vitro antiosteoporosis test by demonstrating an excellent inhibition toward osteoclast differentiation (inhibitory activity was 92.45 ± 1.82% at a concentration of 10 µg/mL). This activity might be supported by the ability of water extract to induce polarization of M0 macrophages into M1 macrophages that played a role in the inhibition of osteoclastogenesis. Apigenin is thought to be one of the active compounds in water extract. The compound had been reported to have osteoporotic activity through the inhibition of formation and function of osteoclasts as well as the increase of osteoclasts apoptosis. A series of in silico tests carried out in this study included molecular docking at alpha and beta estrogen receptors, Receptor Activator of Nuclear Factor-?B Ligand (RANKL), osteoprotegerin (OPG), cathepsin K, and Matrix Metallopeptidase-9 (MMP-9). In silico analysis to predict the profile of absorption, distribution, metabolism, excretion, and the toxicity (ADMET) of chemical compounds, and predict the possible biological activities were also carried out. The results of in silico estrogenic activity test supported the in vitro test results The in silico test results also predicted that apigetrin, 6-allyl-5-methoxy-1,3- benzodioxol-4-ol, pachypostaudin B, dillapiol and apiol can bind to cathepsin K while apigenin and apigetrin can bind to MMP-9 indicated by their high binding energy and the bond similarity in certain amino acid residues to natural ligands. 6-allyl-5-methoxy-1,3-benzodioxol-4-ol, dillapiol, and apiol were predicted to be involved in the anti-inflammatory activity of ethyl acetate extract by inhibiting cell membrane permeability. 6-allyl-5-methoxy-1,3-benzodioxol-4-ol, pellucidin A, dillapiol, and apiol were predicted to be harmful if swallowed indicated by LD50 values and toxicity class levels. Special attention needs to be paid to the possible carcinogenic effects and immunotoxicity caused by 6-allyl-5-methoxy-1,3- bensodioksol-4-ol, dillapiol, and apiol.