Quantitative approach in screening the antiviral properties of Kandis Hutan in animal cell culture

This main aim of this study was to determine the concentration of Kandis Hutan leaf extracts that can inhibit the infectivity of pseudorabies virus (PrV) in Vero cells. The leaf extracts were crude and extracted with 3 organic solvents namely hexane, ethyl acetate and ethanol. The cytotoxic effect of extracts on Vero cells was assessed by both MTT assay and cell cytotoxicity scoring method. Two-fold serial dilutions of each extracts were prepared from the highest concentration of 1000µg/ml in 0.1% DMSO. For MTT assay, the highest cytotoxicity was found in the hexane extract (CC50 <1.25 µg/mL), followed by ethyl acetate extract (CC50 = 237.5 µg/mL), whilst minimal cell cytotoxicity was observed in ethanol extracts (CC50 = 555.0 µg/mL). There was a significant correlation between cell scoring system and MTT assay in term of cell cytotoxicity whereby the least toxic was ethanol extracts, followed by ethyl acetate extract and the most toxic hexane extract. Four antiviral assays were conducted for each extracts, namely plaque reduction assay, cytopathic effect (CPE) reduction assay, inhibition assay and virucidal assay. The most promising result was obtained from the inhibition assay, in which ethyl acetate extracts produced 75% viral inhibition at 125 µg/mL concentration. In virucidal assay, both ethyl acetate and ethanol extracts produced 100% viral inhibition at 250 µg/mL. For plaque reduction assay, there was a significant dose dependent inhibition for ethyl acetate extract but not for ethanol extract and hexane extract. In comparison to CPE reduction assay, findings from plaque reduction assay showed better viral inhibition by ethyl acetate extract (47%) at concentration of 300 µg/mL. The estimated selectivity index (ESI) calculated from the inhibition assay showed the highest antiviral response by ethyl acetate extract (2.7) in comparison with ethanol extract (1.8) and hexane extract (0.1). Therefore, the most promising antiviral activity was produced by ethyl acetate extract which showed consistent viral inhibition in all tested antiviral assays. In contrary, hexane extracts showed the least antiviral efficacy among the tested extracts

Quantitative in vitro assays for screening antiviral characteristics of Berembang Bukit leaf extracts

Berembang Bukit or Megawasih, is a native of our tropical rainforest and other ASEAN countries. In a preliminary review, a qualitative screening on the antiviral activities of its leaf extracts revealed various degrees of antiviral potential. Therefore this study was done to evaluate its antiviral characteristics in a quantitative approach. The leaf extracts were prepared using hexane, ethyl acetate and ethanol extractions and dissolved in DMSO. DMSO cytotoxicity was initially evaluated to ensure a safe working concentration. A negligible cytotoxicity was observed at a concentration of ≤ 0.1% DMSO. The cytotoxic effect of extracts on Vero cells was assessed by both MTT assay and cell cytotoxicity scoring method. Two-fold serial dilutions of each extracts were prepared from the highest concentration of 1000µg/mL in 0.1% DMSO. For MTT assay, the highest cytotoxicity was found in the ethyl acetate extract (CC50 = 218µg/mL), whilst minimal cell cytotoxicity was observed in both hexane (CC50 = 833µg/ml) and ethanol (CC50 = >1000µg/mL) extracts. However, there were no correlation between MTT and cell scoring for cytotoxicity in this study. A series of experiments including CPE reduction assay, plaque reduction assay, inhibition assay and virucidal assay were done to evaluate the total antiviral potential of the leaf extracts. The leaf extracts produced a dose-dependent antiviral response. Both ethyl acetate and ethanol extracts showed 100% plaque formation inhibition in plaque reduction assay, inhibition assay and virucidal assay. Hexane extracts showed absence of plaque inhibition in all the tested antiviral assays. In inhibition assay, the estimated selective index (ESI) for ethanol and ethyl acetate extracts were 8.3 and 1.9, respectively. Whilst in CPE reduction assay, the ESI for the respective extracts were 6.7 and 2.9. In conclusion, the ethanol extracts exhibited the highest antiviral efficacy among the tested extracts

Antiviral properties of Berembang Bukit and Kandis Hutan against psedorabies virus in animal cell culture

Tropical rainforest in Malaysia represents and untapped potential source of antiviral compounds. Bioactive compounds in plant species from the same genus as Kandis Hutan such as xanthones, benzophenones, biflavonoids and lupeol had been studied. Eugeniin is an anti-herpesvirus compound which had also been found n Berembang Bukit. This preliminary study was carried out to discover the presence of antiviral properties in Berembang Bukit and Kandis Hutan using different antiviral assays. In this study, MTT cell viability assay was used in addition to microscopic evaluation of pseudorabies virus (PrV)- induced cytopathic effects (CPE) on Vero cells. The cellular toxicity of DMSO was also evaluated. DMSO was less than 10% cytotoxic at concentration of 0.1% to Vero cells and its effect can be negligible. Both plants had demonstrated antiviral properties in thyl acetate and ethanol extracts. From our findings from all three antiviral assays, the ethanol-extracted Kandis Hutan possessed the most promising antiviral properties. Nevertheless, antiviral potential of ethyl acetate and ethanol-extracted Berembang Bukit and ethyl acetate-extracted Kandis Hutan also merit further investigatio