GC-MS, LC-MS/MS, Docking and Molecular Dynamics Approaches to Identify Potential SARS-CoV-2 3-Chymotrypsin-Like Protease Inhibitors from Zingiber officinale Roscoe.

This study aims to identify and isolate the secondary metabolites of Zingiber officinale using GC-MS, preparative TLC, and LC-MS/MS methods, to evaluate the inhibitory potency on SARS-CoV-2 3 chymotrypsin-like protease enzyme, as well as to study the molecular interaction and stability by using docking and molecular dynamics simulations. GC-MS analysis suggested for the isolation of terpenoids compounds as major compounds on methanol extract of pseudostems and rhizomes. Isolation and LC-MS/MS analysis identified 5-hydro-7, 8, 2'-trimethoxyflavanone (9), (E)-hexadecyl-ferulate (1), isocyperol (2), N-isobutyl-(2E,4E)-octadecadienamide (3), and nootkatone (4) from the rhizome extract, as well as from the leaves extract with the absence of 9. Three known steroid compounds, i.e., spinasterone (7), spinasterol (8), and 24-methylcholesta-7-en-3ß-on (6), were further identified from the pseudostem extract. Molecular docking showed that steroids compounds 7, 8, and 6 have lower predictive binding energies (MMGBSA) than other metabolites with binding energy of -87.91, -78.11, and -68.80 kcal/mole, respectively. Further characterization on the single isolated compound by NMR showed that 6 was identified and possessed 75% inhibitory activity on SARS-CoV-2 3CL protease enzyme that was slightly different with the positive control GC376 (77%). MD simulations showed the complex stability with compound 6 during 100 ns simulation time.

Antioxidant and antiviral potency of Begonia medicinalis fractions.

OBJECTIVES: This study aims to evaluate the antioxidant and antiviral potency of n-hexane, ethyl acetate and, water fractions of Begonia medicinalis Ardi & D.C.Thomas as well as to identify the chemical constituents. METHODS: Assays for antioxidant and antiviral activity (HIV-1) were carried out on MT-4 cells infected with HIV using the DPPH method and the determination of the cytopathic effect. Meanwhile, GC-MS was used to identify the chemical compounds. RESULTS: The determination of antioxidants showed that all fractions possessed potent activity with the IC50 ranging from 2.61 to 8.26 µg/mL. From the antiviral activity of MT-4 cells infected by HIV, the n-hexane fraction of B. medicinalis showed the most potency with the IC50 of 0.04 ± 0.05 µg/mL. It has less cytotoxicity (11.08 ± 4.60 µg/mL) affording the high selectivity index of 238.80. Furthermore, GC-MS analysis of n-hexane fraction found the major compound of carboxylic acid derivate with the area percentage of 76.4% and the presence of phenolic compounds (8.38%). Meanwhile, in water fraction, terpenoids were found in a higher concentration (10.05%) than others. CONCLUSIONS: Therefore, this study supports the application of B. medicinalis as a herbal medicine for antioxidant and antiviral.

Antiviral screening on Alpinia eremochlamys, Etlingera flexuosa, and Etlingera acanthoides extracts against HIV-infected MT-4 cells.

Alpinia eremochlamys K. Schum, Etlingera flexuosa A.D. Poulsen, and Etlingera acanthoides A.D. Poulsen are endemic Zingiberaceae plants from Central Sulawesi, Indonesia. This study is the first report on screening the potential antiviral activity of ethanol extracts of the leaves, pseudostems, and rhizomes parts on HIV-infected MT-4 cells and identifying chemical constituents by GC-MS. The plants were extracted by the maceration method using 96% ethanol as a solvent. The antiviral activity was measured using Viral-ToxGlo colorimetric method and using the extracts at concentrations ranging from 7.8 to 1000 µg/mL. GC-MS was used to identify the secondary metabolites of potential extracts. The results showed that ethanol extract of E. acanthoides rhizome was the most potent antiviral activity (IC50 of 1.74 ± 2.46 µg/mL) and less toxic on lymphocyte (MT-4) cells (CC50 of 204.90 ± 106.35 µg/mL), affording the highest value of selectivity index (SI) of 117.76. A. eremochlamys rhizomes also showed promising antiviral activity with IC50 of 64.18 ± 2.58 µg/mL and no toxicity on MT-4 cells affording a high SI value 19.05. Preliminary GC-MS identification showed the presence of terpenoids and fatty acids as major compounds. Zerumbone, ar-turmerone, caryophyllene, and caryophyllene oxide were also detected. Chemical constituents identified by GC-MS might be responsible for the antiviral activity of extracts, suggesting further isolation and antiviral testing of the purified compounds.

Docking Study on Anti-HIV-1 Activity of Secondary Metabolites from Zingiberaceae Plants.

INTRODUCTION: Human immunodeficiency virus type-1 (HIV-1) that causes acquired immunodeficiency syndrome (AIDS) has become a worldwide health problem today. There are approximately 30 anti-HIV-1 drugs that have been used in the treatment of AIDS. However, effective anti HIV-1 agents with less side affect and high inhibition potency are still in demand. OBJECTIVE: The objective of this study was to identify the potential compounds from Zingiberaceae plants that might be active as anti-HIV-1 by molecular docking. MATERIALS AND METHODS: Molecular docking simulation was performed by using AutoDock 4.2 on Linux operation system. Docking protocol was validated by using root mean square deviation (RMSD) value using redocking and cross-docking methods. The reported metabolites from Zingiberaceae plants were docked on HIV-1 protease, integrase, and reverse transcriptase protein enzymes. RESULTS: The docking result showed that the genera of Zingiber, Etlingera, Alpinia, Hedychium, and Boesenbergia have potential metabolites that inhibit HIV protease, integrase, and reverse transcriptase enzymes by possessing lower docking energy than native ligand of amprenavir, raltegravir, and nevirapine. Among the metabolites, noralpindenoside B and alpindenoside A from Alpinia densespicata inhibited protease enzymes with the lowest docking energy of -18.02 and -17.90 kcal/mol, respectively. Meanwhile, panduratin E from Boesenbergia pandurata Roxb. and 5α,8α-epidioxyergosta-6,22-dien-3ß-ol from Etlingera elatior showed the lowest docking energy on integrase protein with docking energy of -11.97 and -11.41 kcal/mol, respectively. Pahangensin A from Alpinia pahangensis Ridley showed the lowest docking energy on reverse transcriptase enzyme with docking energy of -13.76 kcal/mol. CONCLUSION: The docking molecular study has identified the possible potential compounds from Zingiberaceae plants that might be used for anti-HIV-1 treatment. So, this study suggested further isolation and purification of the predicted compounds.

ANTIVIRAL ACTIVITY OF Justicia gendarussa Burm.f. LEAVES AGAINST HIV-INFECTED MT-4 CELLS.

BACKGROUNDS: Justicia gendarussa Burm.f. has been known to have anti-HIV activity. This study was conducted to evaluate the effect of incubation time on the antiviral activity of the J. gendarussa leaves extract on HIV-infected MT-4 cells in vitro. Molecular docking test was also conducted to determine the interaction of alkaloids and flavonoids on the J. gendarussa leaves against HIV-1 reverse transcriptase receptor. It is expected that this research will provide scientific information on the development of J. gendarussa leaves as an anti-HIV drug. MATERIALS AND METHODS: In the activity test, the effect of incubation time on the antiviral activity of J. gendarussa leaves on HIV-infected MT-4 cells were evaluated. During the activity test, a parameter of cytolysis effect inhibition on MT-4 cell line was observed after 4 days and 6 days incubation period. The molecular docking test is performed by using Molegro Virtual Docker software to determine the interaction of alkaloid and flavonoid compounds of J. gendarussa leaves with HIV-1 reverse transcriptase receptor. RESULTS: The incubation time influences the CC50 and EC50 value. Fractionated-70% ethanol extract of J. gendarussa leaves showed a higher anti-HIV activity with EC50 = 3.045 x 10-9 µg/mL, SI = 6.309 x 1012 (4 days of incubation) and EC50 = 6.066 µg/mL, SI = 58494.845 (6 days of incubation). From molecular docking test, it was found that flavonoid of J. gendarussa leaves could inhibit the activity of HIV reverse transcriptase enzyme. CONCLUSION: Fractionated-70% ethanol extract of J. gendarussa has potential as an anti-HIV.

EFFECT OF VARYING INCUBATION PERIODS ON CYTOTOXICITY AND VIRUCIDAL ACTIVITIES OF Justicia gendarussa Burm.f. LEAF EXTRACT ON HIV-INFECTED MOLT-4 CELLS.

BACKGROUNDS: Justicia gendarussa Burm.f. has an anti-HIV activity. This study was conducted to evaluate the effects of incubation periods on the cytotoxicity and virucidal activities of the J. gendarussa leaves extract on MOLT-4 cells. MATERIALS AND METHODS: The cytotoxicity assay was evaluated by using the WST-1 test with incubation periods of 3 days and 5 days. The virucidal activity test was determined by measuring the inhibitory activities on the syncytium formation. RESULTS: The cytotoxicity assay showed the value of CC50 on MOLT-4 cell culture with the test material of 70% ethanol extract of J. gendarussa leaves as much as 3928.620 µg /mL and 3176.581 µg /mL (incubation day 3 and day 5, respectively); fractionated-70% ethanol extract = 81782.428 µg /mL and 12175.870 µg/mL; and water extract = 16372.689 µg/mL and 2946.117 µg/mL. The test results of the virucidal activities (inhibit ≥ 90% the formation of syncytium) of 70% ethanol extract of J. gendarussa leaves is at a concentration 250 µg/mL, 500 µg/mL and 1000 µg/mL (3-day incubation) and 250 µg/mL (5-day incubation); and fractionated-70% ethanol extract at a concentration 250 µg /mL, 500 µg/mL and 1000 µg/mL (3-day incubation) and 1000 µg/mL (5-day incubation). CONCLUSION: 70% ethanol extract, fractionated-70% ethanol extract, and water extract of J. gendarussa leaves were relatively nontoxic toward MOLT-4 cells, and fractionated-70% ethanol extract had better potentials in virucidal activities.