In Vitro Antiviral Activity of Potential Medicinal Plant Extracts Against Dengue and Chikungunya Viruses

Abstract

Dengue and chikungunya are two important mosquito-borne infections which are known to occur extensively in tropical and subtropical areas. Presently, there is no treatment for these viral diseases. In vitro antiviral screening of 25 extracts prepared from the plants of Vitex negundo, Plumeria alba, Ancistrocladus heyneanus, Bacopa monnieri, Anacardium occidentale, Cucurbita maxima, Simarouba glauca, and Embelia ribes using different solvents and four purified compounds (anacardic acid, chloroquinone, glaucarubinone, and methyl gallate) were carried out for their anti-dengue virus (DENV) and anti-chikungunya virus (CHIKV) activities. Maximum nontoxic concentrations of the chloroform, methanol, ethyl acetate, petroleum ether, dichloromethane, and hydroalcoholic extracts of eight plants were used. The antiviral activity was assessed by focus-forming unit assay, quantitative real-time RT-PCR, and immunofluorescence assays. Extracts from Plumeria alba, Ancistrocladus heyneanus, Bacopa monnieri, and Cucurbita maxima showed both anti-DENV and CHIKV activity while extract from Vitex negundo showed only anti-DENV activity. Among the purified compounds, anacardic acid, chloroquinone and methyl gallate showed anti-dengue activity while only methyl gallate had anti-chikungunya activity. The present study had identified the plant extracts with anti-dengue and anti-chikungunya activities, and these extracts can be further characterized for finding effective phytopharmaceutical drugs against dengue and chikungunya.

Keywords: antivirals; chikungunya virus; dengue virus; phytopharmaceuticals; plant extracts.

Figure 1

Antiviral screening of different plant extracts at maximal nontoxic concentration against DENV (A) and CHIKV (B) under posttreatment condition. Vero CCL-81 cells were treated with highest maximum nontoxic dose of extracts 24 h postinfection and incubated for 96 h for DENV (1A) and 24 h in the case of CHIKV (1B), and after the incubation, the plates were frozen and the culture filtrates were used for the different assays. The experiments were performed at two independent time points in triplicates, and the results are expressed as mean log₁₀ focus-forming unit/ml ± standard error. All the treatment conditions were compared with the virus control. ^**** p < 0.0001.

Figure 2

Antiviral effect of Plumeria alba bark extract prepared using chloroform (M2C) and leaf-based extract prepared using methanol (M8M) against DENV under different treatment conditions. Vero CCL-81 cells were pre-, co-, and posttreated with different concentrations of extracts, and 120 h incubation after infection, the plates were frozen and the culture filtrates were used for the FFU assay (A, M2C; C, M8M) and real-time PCR (B, M2C; D, M8M). The experiments were performed at two independent time points in triplicates, and the results are expressed as either mean log₁₀ focus-forming unit/ml ± standard error (A, M2C; C, M8M) or mean log₁₀ viral RNA copies/ml ± standard error (B, M2C; D, M8M). All the treatment conditions were compared with the virus control. ^**** p < 0.0001; ^*** p < 0.001.

Figure 3

Antiviral effect of bark and leaves of Plumeria alba methanol extract (M8M) against CHIKV under different treatment conditions. Vero CCL-81 cells were pre-, co-, and posttreated with different concentrations of extracts, and 48 h incubation after infection, the plates were frozen and the culture filtrates were used for the FFU assay (A) and real-time PCR (B). The experiments were performed at two independent time points in triplicates, and the results are expressed as either mean log₁₀ focus-forming unit/ml ± standard error (A) or mean log₁₀ viral RNA copies/ml ± standard error (B). All the treatment conditions were compared with the virus control. ^**** p < 0.0001.

Figure 4

Antiviral effect of leaves of Vitex negundo chloroform extract (M4C) against DENV under different treatment conditions. Vero CCL-81 cells were pre-, co-, and posttreated with different concentrations of extracts, and 120 h incubation after infection, the plates were frozen and the culture filtrates were used for the FFU assay (A) and real-time PCR (B). The experiments were performed at two independent time points in triplicates, and the results are expressed as mean log₁₀ focus-forming unit/ml ± standard error as well as mean log₁₀ viral RNA copies/ml ± standard error. All the treatment conditions were compared with the virus control. ^**** p < 0.0001; ^*** p < 0.001; ^** p < 0.005.

Figure 5

Antiviral effect of bark of Ancitrocladus heyneanus chloroform extract (M5C) against DENV under different treatment conditions. Vero CCL-81 cells were pre-, co-, and posttreated with different concentrations of extracts, and 120 h incubation after infection, the plates were frozen and the culture filtrates were used for the FFU assay (A) and real-time PCR (B). The experiments were performed at two independent time points in triplicates, and the results are expressed as either mean log₁₀ focus-forming unit/ml ± standard error (A) or mean log₁₀ viral RNA copies/ml ± standard error (B). All the treatment conditions were compared with the virus control. ^**** p < 0.0001.

Figure 6

Antiviral effect of bark of Ancitrocladus heyneanus chloroform extract (M5C) against CHIKV under different treatment conditions. Vero CCL-81 cells were pre-, co-, and posttreated with different concentrations of extracts, and 48 h incubation after infection, the plates were frozen and the culture filtrates were used for the FFU assay (A) and real-time PCR (B). The experiments were performed at two independent time points in triplicates, and the results are expressed as either mean log₁₀ focus-forming unit/ml ± standard error (A) or mean log₁₀ viral RNA copies/ml ± standard error (B). All the treatment conditions were compared with the virus control. ^**** p < 0.0001.

Figure 7

Antiviral effect of whole herb of Bacopa monnieri hydroalcoholic extract (M7M) against DENV under different treatment conditions. Vero CCL-81 cells were pre-, co-, and posttreated with different concentrations of extracts, and 120 h incubation after infection, the plates were frozen and the culture filtrates were used for the FFU assay (A) and real-time PCR (B). The experiments were performed at two independent time points in triplicates, and the results are expressed as either mean log₁₀ focus-forming unit/ml ± standard error (A) or mean log₁₀ viral RNA copies/ml ± standard error (B). All the treatment conditions were compared with the virus control. ^**** p < 0.0001.

Figure 8

Antiviral effect of whole herb of Bacopa monnieri hydroalcoholic extract (M7M) against CHIKV under different treatment conditions. Vero CCL-81 cells were pre-, co-, and posttreated with different concentrations of extracts, and 48 h incubation after infection, the plates were frozen and the culture filtrates were used for the FFU assay (A) and real-time PCR (B). The experiments were performed at two independent time points in triplicates, and the results are expressed as mean log₁₀ focus-forming unit/ml ± standard error as well mean log₁₀ viral RNA copies/ml ± standard error. All the treatment conditions were compared with the virus control. ^**** p < 0.0001.

Figure 9

Antiviral effect of Cucurbita maxima seed methanol extract (M9M) against CHIKV under different treatment conditions. Vero CCL-81 cells were pre-, co-, and posttreated with different concentrations of extracts, and 48 h incubation after infection, the plates were frozen and the culture filtrates were used for the FFU assay (A) and real-time PCR (B). The experiments were performed at two independent time points in triplicates, and the results are expressed as either mean log₁₀ focus-forming unit/ml ± standard error (A) or mean log₁₀ viral RNA copies/ml ± standard error (B). All the treatment conditions were compared with the virus control. ^**** p < 0.0001; ^*** p < 0.001.

Figure 10

Antiviral effect of pure compounds anacardic acid (A-S), chloroquinone (C-S), and methyl gallate (MG-S) against DENV under different treatment conditions. Vero CCL-81 cells were pre-, co-, and posttreated with different concentrations of pure compounds, and 120 h incubation after infection, the plates were frozen and the culture filtrates were used for the FFU assay [(A), A-S; (C), C-S; and (E), MG-S] and real-time PCR [(B), for A-S; (D), C-S; (F), MG-S]. The experiments were performed at two independent time points in triplicates, and the results are expressed as either mean log₁₀ focus-forming unit/ml ± standard error [(A), A-S; (C), C-S; and (E), MG-S] or mean log₁₀ viral RNA copies/ml ± standard error [(B), A-S; (D), C-S; and (F), MG-S]. All the treatment conditions were compared with the virus control. ^**** p < 0.0001; ^*** p < 0.001; ^** p < 0.005; ^* p < 0.05.

Figure 11

Antiviral effect of pure compound methyl gallate (MG-S) against CHIKV under different treatment conditions. Vero CCL-81 cells were pre-, co-, and posttreated with different concentrations of pure extract, and 48 h incubation after infection, the plates were frozen and the culture filtrates were used for the FFU assay (A) and real-time PCR (B). The experiments were performed at two independent time points in triplicates, and the results are expressed as either mean log₁₀ focus-forming unit/ml ± standard error (A) or mean log₁₀ viral RNA copies/ml ± standard error (B). All the treatment conditions were compared with the virus control. ^**** p < 0.0001; ^*** p < 0.001.

Figure 12

Immunofluorescence assay images for DENV after treatment with extracts and compounds. Images represent DENV-2-infected Vero CCL-81 cell lines under posttreatment condition. Virus-infected cells appear green in color (A). Percentage of infected Vero CCL-81 cell line in cultures infected with virus with different concentrations of extracts under posttreatment condition (B). All the treatment conditions were compared with the virus control. ^**** p < 0.0001; ^*** p < 0.001; ^** p < 0.005; ^* p < 0.05. VC, virus control; CC, cell control.

Figure 13

Immunofluorescence assay images for CHIKV after treatment with extracts and compounds. Images represent CHIKV-infected Vero CCL-81 cell lines under posttreatment condition. Virus-infected cells appear green in color (A). Percentage of infected Vero CCL-81 cell line in cultures infected with virus with different concentrations of extracts under posttreatment condition (B). All the treatment conditions were compared with the virus control. ^**** p < 0.0001; ^*** p < 0.001; ^** p < 0.005. VC, virus control; CC, cell control.