ISOLATION OF XANTHOANGELOL AND 4- HYDROXYDERRICIN FROM ANGELICA KEISKEI (MIQ.) KOIDZUMI. (ASHITABA) AND PHARMACOLOGY ACTIVITIES: AN IN SILICO AND IN VITRO STUDIES

• Main Author: Lia Aulifa, Diah
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/53629
• Institution: Institut Teknologi Bandung

The Angelica keiskei (Miq.) Koidzumi (Ashitaba) has been well cultivated in Lombok (Rinjani Mountains), Indonesia, and used empirically as a diuretic, controlling blood pressure, diabetes, and preventing obesity. The leaves and stems are parts of A. keiskei often consumed. The stem contains the yellow sap which have the main compounds, i.e. xanthoangelol (XAG) and 4-hydroxyderricin (4-HD). This study aims to isolate and characterize the chalcone XAG and 4-HD, followed by examining the inhibitory activity of the 3-hydroxy-3-methylglutaryl-coA (HMGCoA) reductase, ?-glucosidase enzymes, and dipeptidyl peptidase-IV (DPP-IV) by in silico method, continued examining the inhibitory activity of selected enzymes by in vitro method. The study was initiated by screening the selected plant parts from A. keiskei such as leaves, stems, and yellow sap of stems. Leaves and stems were macerated for 72 hours using 96% ethanol (1: 6.5), then concentrated to obtain yields of 6.48 and 6.53% w/w, respectively. The yellow sap of A. keiskei stems were freeze-dried, and 31.59 g of yellow-orange powder was obtained. The qualitative analysis of the ethanol extract of leaves, stems, and yellow sap was carried out using thin layer chromatography (TLC) F254 to observe the chromatogram of the A. keiskei main compounds. The results of the TLC analysis showed that the main compound A. keiskei was mostly found in the ethanol extract of yellow sap. This study provides preliminary data, which parts of the plant will be used for the isolation process of the main compound. This result was preliminary data used to isolate the main compound of A. keiskei plants. The yellow sap powder of A. keiskei stems (10 g) was isolated by liquid-liquid extraction using three solvents with different polarity (1:1; three replicated) to produce yields consecutively, n-hexane (0.43 g), ethyl acetate (5.86 g), and water (2.90 g). The results of TLC analysis showed that the main compound was in the ethyl acetate fraction. The ethyl acetate fraction (2 g) was further fractionated using a chromatography column (2.0 x 45 cm) packed with 32.33 g Kieselgel 60 and eluted using a mobile phase combination of n-hexane-acetone (100:0 ? 0:100, v/v) to produce six subfractions F1–F6. The results TLC analysis showed that the main compounds were obtained in the subfractions F3 and F5. Furthermore, the separation of subfraction F3 (300 mg) was carried out using column chromatography. The isolate (75.5 mg) was obtained from the subfraction sbf3C, called isolate 1. Isolate 1, a yellowish powder that has a purity of 84.91% at a retention time of 5.36 minutes, was analyzed using liquid chromatography (LC). The further characterization of isolate 1 was conducted using UV spectrophotometry, mass spectroscopy (TOF-MS-ES+), and nuclear magnetic resonance (NMR). The result showed that the UV-wavelengths of maximum absorbance of isolate 1 was 364 nm, while the molecular weight of isolate 1 was [M+H] + m/z = 339.22 with molecular formula C21H22O4. The result of 1 H- 13 C NMR and heteronuclear single?quantum coherence ( 1 H- 13 C HSQC) showed that isolate 1 was confirmed as 4-hydroxyderricin (4-HD). The separation of subfraction F5 (770 mg) was also carried out using column chromatography. The isolate (194.2 mg) was obtained on the subfraction sbf5L, called isolate 2. The melting point of isolate 2 was 114-114.4 o C, while its appearance was a yellow crystal. The UV-wavelengths of maximum absorbance of isolate 2 was 368 nm, while its molecular weight was [M+H] + m/z = 393.20 with molecular formula C25H28O4. The result of 1 H- 13 C NMR, and heteronuclear single? quantum coherence ( 1 H- 13 C HSQC) showed that isolate-2 was confirmed as xanthoangelol (XAG). The interaction of the isolated compound with HMG-CoA reductase, ?-glucosidase, and DPP-IV enzymes was investigated using an in silico study. The proteins used are 1HW9, 3W37, 1X70 obtained from the Protein Data Bank (PDB). Program used for ligands and receptors preparations were Open Babel program, PLANTS1.2, MGLTools1.5.6 shell script. The AutoDock Vina 1.1.2 program was used for the molecular docking process. The Pymol and PoseView programs were used for the calculation of root mean square deviation (RSMD) and visualization of molecular docking results. Simvastatin, acarbose, and sitagliptin are used as ligand drugs (reference). This study provides data on the interaction energy (Ei), inhibition constant (Ki), and the formation of hydrogen bonds between the test ligands (isolate) and the receptor (enzyme). The results of in silico studies between simvastatin and HMG-CoA reductase show that there are five hydrogen bonds with amino acid residues in the enzyme active site, namely Arg590, Ser684, Lys691, Lys735, Asn755, with an interaction energy (Ei) of -6.97 kcal/mol; inhibition constant (Ki) 10.8 ?M. The 4-HD compound shows three hydrogen bonds with Arg590, Ser684, dan Lys735. 4-HD also forms hydrophobic interactions with Lys692, Leu853, dan Ala856. XAG compound interacts with HMG-CoA reductase through the formation of four hydrogen bonds, namely with Lys691, Lys735, Asn755, and also observed the presence of salt bridges with Arg590 dan Asp690, as well as hydrophobic interactions with Cys561, Lys735, Leu853. Based on the results of the molecular docking of the two prenylated chalcone, both XAG (Ei = -7.31 kcal/mol; Ki = 4.65 ?M) and 4-HD (Ei = -6.7 kcal/mol; Ki = 13.3 ?M) can interact with several important amino acid residues in the cis-loop (active pocket) of HMG-CoA reductase, namely Arg590, Ser684, Asp690, Lys691, Lys692, Lys735, which resembling a simvastatin interaction. Therefore, it can be predicted that the two prenylated chalcone might inhibit HMG-CoA reductase. XAG has better interaction affinity for HMG-CoA reductase than 4-HD. The results of in silico studies between acarbose and ?-glucosidase showed that acarbose interacted to form three hydrogen bonds with the amino acid residues Asn237, Arg552, Asp558. In addition, it was also observed that there were salt bridges with Asp232, Asn237, Trp239, Asp469, Met470 (Ei = -7.81 kcal/mol; Ki = 2.14 ?M). 4-HD compounds interact with ?-glucosidase through the formation of a hydrogen bond and a salt bridge with Asp232, as well as 5 hydrophobic. XAG compounds can interact with ?-glucosidase by forming hydrogen bonds with Ala234, salt bridges with Asp232, and 9 hydrophobic interactions. Based on the results of the molecular docking of the two prenylated chalcone, both XAG (Ei = - 7.81 kcal/mol; Ki = 1.99 ?M) and 4-HD (Ei = -7.4 kcal/mol; Ki 3.99 ?M ) could interact with several important amino acid residues at positions 232-237 on the Nterminal N-loop and (active site) ?-glucosidase, which resembles the interaction of acarbose. Therefore, it can be predicted that the two prenylated chalcone might inhibit ?-glucosidase. XAG has better interaction affinity for ?-glucosidase than 4- HD. The results of in silico studies between sitagliptin dan DPP-IV show that sitagliptin interacts with Glu205 dan Glu206 by forming a salt bridge, then there is an interaction with the amino acid residues Ser209, Arg125, Arg358, Tyr662, and Asn710 through five hydrogen interactions, as well as interactions with the Phe357 residue through interactions ?–?(Ei = -9.24 kcal/mol; Ki = 0.172 M). The 4-HD compound interacts with DPP-IV by means of two hydrogen bonds with Glu206 dan Tyr662 on the 4-HD hydroxyl group, and by the interaction of ?-?with Phe357 in ring A. XAG compounds interact with DPP-IV through two hydrogen bonds with Glu205, Glu206, and ?-?interaction with Phe357 on ring B. The prenylated chalcone compounds, both XAG (Ei = -8.34 kcal/mol; Ki = 0.873 ?M) and 4-HD (Ei = -7.42 kcal/mol; ki = 3.99 ?M) could interact with several important amino acid residues Glu205, Glu206 on the S2 sub-site, and there is an interaction with the amino acid Phe357 residue on the extensive S2 subsite in the active sac of the DPP-IV enzyme, which resembles the interaction of sitagliptin. Therefore it could be predicted that the two prenylated chalcone might inhibit DPP-IV. XAG has a better interaction affinity for DPP-IV than 4-HD. The final stage of this study was to test the inhibitory activity of XAG dan 4-HD compounds against ?-glucosidase dan DPP-IV enzymes in vitro using 96-microwell plates. The first test was carried out on the ?-glucosidase enzyme. The inhibitory activity occurs due to the enzymatic reaction between the ?-glucosidase enzyme and p-nitrophenyl-?-glucopyranose (PNPG) as a substrate. The absorbance of pnitrophenol was analyzed using Multiscanner at a wavelength of 405 nm. Acarbose was used as positive control. The test results showed that the IC50 values of XAG and 4-HD compounds were 14.45 µM and 80.82 µM, respectively, inhibition of the activity of these two compounds was better than acarbose with IC50 = 207 µM.. The inhibitory activity of DPP-IV enzyme occurs due to enzymatic reactions between the DPP-IV enzyme and Gly-Pro-p-Nitroanilide Hydrochloride (GPPN) resulting the release a yellow product p-nitroaniline (p-NA). The absorbance of p- NA was analyzed using Multiscanner at 405 nm. Sitagliptin was used as a reference control. The results of in vitro studies showed that XAG and 4-HD compounds exhibit inhibitory activity against the DPP-IV enzyme with IC50 values of 10.49 µM and 13.95 µM, respectively. While sitagliptin used as reference exhibit inhibitory activity against the DPP-IV enzyme with IC50 values of 0.87 µM. This indicates that the inhibitory activity of XAG and 4-HD compounds were slightly weaker than sitagliptin. The two main compounds of XAG and 4-HD prenylated chalcone are predicted to play a role in the antiT2DM mechanism through inhibition of the ?-glucosidase and DPP-IV enzymes proven in silico and in vitro, furthermore it could be used as markers on A. keiskei plants. The inhibitory activity of the two main compounds against the ?-glucosidase enzyme was better than acarbose. The ethanol extracts from leaves and stems of A. keiskei could be developed into an alternative herbal product in overcoming T2DM. This research could be continued with in vivo antidiabetic activity tests in animals, acute and/or subchronic toxicity tests, and pharmacokinetic tests in animals.