A total of 132 extracts obtained from 21 plant species were subjected to the antiviral activity screening against the chikungunya virus. As six different solvents were used, each extractant yielded 22 extracts. The ability of an extract to protect Vero cells from the cytopathic effect caused by the virus was used as a measurement of antiviral activity for the extract. As an extract is a mixture of many phytochemicals extracted from a particular plant part, it may contain compounds that are toxic to Vero cells. Thus, it is necessary to determine the non-toxic concentrations for use in the cytopathic effect inhibitory assay. As such, a standardized test concentration range of an extract is not feasible.

In order to express and classify the cytopathic effect inhibitory activity of an extract, three scales were established based on percentage of cell viability, these being strong inhibitory activity when the cell viability is ≥70%, intermediate inhibitory activity when the cell viability is 31–69%, and weak inhibitory activity when the cell viability is ≤30%. The inhibitory activity for each extract is shown in Table 2. Forty-two extracts (31.8%) were found to have strong inhibitory activity in the concurrent mode compared to only three extracts (2.3%) in the non-concurrent mode. These extracts were derived from 12 medicinal plants, i.e., Azadirachta indica, Clinacanthus nutans, Diodella sarmentosa, Diplazium esculentum, Ficus deltoidea, Gynura bicolor, Hydrocotyle sibthorpioides, Homalocladium platycladum, Ocimum americanum, Petroselinum crispum, Sechium edule, and Strobilanthes crispus. The results indicate that the cytopathic effect inhibitory activity was dependent on plant species and sample introduction mode. The results also suggest that phytochemicals in the extracts could exert an inhibitory effect against the virus in the concurrent mode but lost their activity in the non-concurrent mode. The exposure of Vero cells to the extracts for 24 h before the addition of virus inoculum could result in the metabolism of active phytochemicals into metabolites devoid of inhibitory activity. An exception was noted for the three extracts with strong inhibitory activity in the non-concurrent mode. They were ethyl acetate, ethanol, and methanol extracts of F. deltoidea. The corresponding cell viabilities in the concurrent mode were 66.8% ± 4.2% at 10 µg/mL, 71.9% ± 5.3% at 40 µg/mL, and 1.5% ± 2.9% at 40 µg/mL, respectively, and increased to 76.5% ± 4.1% (p = 0.046), 90.3% ± 0.8% (p = 0.024), and 79.8% ± 6.7% (p < 0.001), respectively, in the non-concurrent mode, suggesting that the metabolites produced (in the non-concurrent mode) may have stronger activity than their parent compounds.

The data in Figure 1 indicate that extractants such as chloroform, ethyl acetate, ethanol, and methanol resulted in higher activity compared to hexane and water, both in the concurrent mode and non-concurrent mode. The type of solvent used to extract phytochemicals from plants is an important contributing factor to the results of the bioassay. Phytochemicals of a plant part are solubilized in an extractant based on their polarity [30].

Among the 45 extracts which exhibited strong inhibitory activity, 20 extracts of seven plants from the concurrent mode and one extract of a plant from the non-concurrent mode were able to protect ≥90% of Vero cells from the cytopathic effect caused by the virus, as shown in Figure 2. These 21 extracts are of great potential for further drug developments. A wide half-maximal effective concentration (EC₅₀) range was observed for these extracts, ranging from 1.33 µg/mL for the ethanol extract of O. americanum to 394.0 µg/mL for the water extract of H. sibthorpioides (Table 3). Consequently, the selectivity indices for these extracts ranged from 2.62 to 170.2. The indices for the ethanol extract of S. edule and the water extract of H. sibthorpioides could not be calculated as no significant cytotoxicity (p > 0.05) was recorded.

In order to yield some indications of the antiviral mechanisms of the 21 extracts, quantification of the viral copy number in the experiments was performed using a real-time RT-PCR. The results are shown in Table 3. All extracts of C. nutans and S. edule, ethanol extract of H. sibthorpioides, and ethanol and methanol extracts of O. americanum produced a viral copy number similar to the virus control (p > 0.05), suggesting the virus was successfully replicated in the Vero cells but the release of the viral progeny was inhibited by these extracts, and this prevented the occurrence of cytopathic effect in the cells. In contrast, the viral copy numbers for the infected cells treated with the extracts of F. deltoidea (ethanol), G. bicolor (water), H. sibthorpioides (water), and O. americanum (chloroform and ethyl acetate) were not significantly different (p > 0.05) from the copy number of the viral inoculum. The results suggested that these extracts may work as a fusion inhibitor and block the entry of the virus into the cells. The virus was not able to replicate in the experiments and the copy number remained similar to that of the viral inoculum throughout the 72-h incubation period. The viral copy numbers for five extracts, i.e., ethyl acetate extract of D. sarmentosa, chloroform, ethyl acetate, and ethanol extracts of D. esculentum, and ethanol extract of G. bicolor, were significantly lower (p < 0.05) than that of the virus inoculum. These extracts caused 5.41-log to 6.63-log reductions of viral load compared to the virus control (Table 3) as quantified by real-time RT-PCR, suggesting the active phytochemicals in the extracts possessed a virucidal effect on the chikungunya virus. The viral copy number indicates that phytochemicals may have different modes of action against the virus, as illustrated by the extracts of H. sibthorpioides and O. americanum whereby these extracts could prevent the release of viral progeny and the entry of the virus into cells. Similarly, the extracts of G. bicolor could kill the virus, as well as block the virus from entry into the cells.

Chloroquine, which was used as a positive control, is reported to interfere with the protonation of the endocytic vesicles thereby raising the endosomal pH and preventing the fusion of chikungunya virus to the host cell [53]. The EC₅₀ value of chloroquine obtained for the non-concurrent mode (9.05 µg/mL or 17.5 µM) was significantly higher (p < 0.001) than the concurrent mode (1.92 µg/mL or 3.72 µM) (Table 3), suggesting that the metabolism of chloroquine in the Vero cells may have reduced its efficacy by producing non-active metabolites or metabolites with reduced efficacy against the chikungunya virus. The EC₅₀ value (3.72 µM) obtained in this study was generally lower than the values (5.0–11 µM) reported in the literature, probably resulting from the types of cells and the virus strains used [54,55,56].

To the best of our knowledge, this study constitutes the first report of the antiviral properties of the medicinal plants D. sarmentosa, D. esculentum, F. deltoidea, G. bicolor, H. platycladum, and S. edule. Azadirachta indica, popularly known as neem, has been extensively used in the Unani, Ayurveda, and Chinese traditional systems of medicine [35]. Raghavendhar et al. studied the water extract of the bark of A. indica against chikungunya virus and reported that the extract did not reduce the plaque formation in Vero cells [57]. In contrast, the current study shows that the chloroform, ethyl acetate, ethanol, and methanol extracts of the leaves of A. indica had strong cytopathic effect inhibitory activity against the virus (Table 2). For C. nutans, H. sibthorpioides, O. americanum, P. crispum, and S. crispus, the results of this study further strengthen the case for these plants as potential sources of antiviral compounds. The antiviral activity of C. nutans has well been documented against herpes simplex virus types 1 and 2 [58,59], human papillomavirus [60], and dengue virus [61]. The methanol extract and the asiaticoside isolated from H. sibthorpioides possess anti-dengue virus activity [62] and anti-hepatitis B virus activity [63], respectively. The dichloromethane and methanol extracts of O. americanum and the methanol extract of S. crispus displayed anti-herpes simplex virus activities [64,65]. The methanol extract of P. crispum has been reported to have inhibitory activity against the Sindbis virus, which like the chikungunya virus is an alphavirus [66]. Further studies need to be carried out to elucidate the identity of antiviral compounds in the active extracts, the inhibitory or virucidal potential for the isolated pure compounds, and the possible synergistic effects among the isolated compounds in targeting different mechanisms of the life cycle of chikungunya virus.

3. Conclusions

For this research 132 extracts from 21 medicinal plant species were evaluated for cytopathic effect inhibitory activity against the chikungunya virus using concurrent and non-concurrent sample introduction modes. The inhibitory effect of the extracts was dependent on plant species, extract concentration, type of extractant, and sample introduction mode. More extracts were found to have strong inhibitory activity in the concurrent mode than in the non-concurrent mode. Analysis of selected 21 extracts from eight plants with a strong inhibitory activity using real-time RT-PCR indicates that the active extracts targeted the chikungunya virus life cycle at different stages, including inhibition of virus entry into Vero cells, blocking the release of viral progeny from the cells, and virucidal effect on the virus. Some of the medicinal plants such as G. bicolor, H. sibthorpioides, and O. americanum even possessed multiple antiviral mechanisms. The bioactive compounds in the plant extracts could be isolated and characterized as lead compounds for potential pharmaceutical developments into anti-chikungunya virus drugs. The plant extracts could also be evaluated against other viruses such as dengue virus, which causes another endemic mosquito-borne disease in Malaysia. The results of this study reiterated the fact that medicinal plant extracts contain many phytochemicals with biological activities. Medicinal plants could be explored as an accessible and sustainable source of chemotherapeutic agents for the treatment of emerging or re-emerging viral diseases. More collaborative efforts are needed to pursue the exploration of medicinal plants for human health benefits.