In Vitro Evaluation of the Anti-Chikungunya Virus Activity of an Active Fraction Obtained from Euphorbia grandicornis Latex

Abstract

Chikungunya virus (CHIKV) is classified as a pathogen with the potential to cause a pandemic. This situation becomes more alarming since no approved drug exists to combat the virus. The present research aims to demonstrate the anti-CHIKV activity of molecules present in the latex of Euphorbia grandicornis. Therefore, a biodirected assay was carried out to find the molecules with anti-CHIKV activity. Extractions with hexane, dichloromethane, and methanol and subsequent purification by column chromatography were carried out to later evaluate cytotoxic activity by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and antiviral activity by plaque assay. Our findings show that unlike the others, methanolic extract has a low cytotoxic effect and a good anti-CHIKV effect (EC₅₀ = 26.41 µg/mL), which increases when obtaining the purified active fraction (pAFeg1) (EC₅₀ = 0.4835 µg/mL). Time-of-addition suggests that the possible mechanism of action of pAFeg1 could be inhibiting any of the non-structural proteins of CHIKV. In addition, both the cytotoxic and anti-CHIKV activity of pAFeg1 demonstrate selectivity since it killed cancer cells and could not inhibit DENV2.

Keywords: Euphorbia grandicornis; antiviral; cancer cells; chikungunya virus; cytotoxic; latex; oleanolic acid; roburic acid; selectivity.

Figure 1

Flowchart of the extraction, fractionation, and obtention of an active fraction pAFeg 1 process carried out from Euphorbia grandicornis latex.

Figure 2

Dose–response curve of the cytotoxic activity of the methanol extract. * Represents a p-value < 0.05 with respect to the vehicle control estimated by one-way ANOVA followed by Dunnett’s test.

Figure 3

Evaluation of the cytotoxic activity of fractions obtained from ME. (a) Evaluation of the six fractions obtained; a represents a value of p < 0.05 concerning the control in viability, and b represents p < 0.05 in% PFU/mL. (b) Evaluation of AFeg in different cell lines; 1, 2, and 3 represent a value of p < 0.05 from the indicated concentration concerning the control in A549, HeLa, and HaCat, respectively. p values were estimated by one-way ANOVA followed by Dunnett’s test.

Figure 4

Evaluation of the cytotoxic activity of pAFeg1. * Represents a p-value < 0.05 with respect to the vehicle control estimated by one-way ANOVA followed by Dunnett’s test.

Figure 5

Evaluation of anti-CHIKV activity of ME in VERO cells. * Represents a p-value < 0.05 with respect to the vehicle control estimated by one-way ANOVA followed by Dunnett’s test.

Figure 6

Evaluation of anti-CHIKV activity of pAFeg1 in HaCat cells. * Represents a p-value < 0.05 with respect to the vehicle control estimated by one-way ANOVA followed by Dunnett’s test.

Figure 7

Evaluation of selectivity of pAFeg1. No decrease in the number of PFUs was observed at the concentrations in which CHIKV was inhibited, even at a higher concentration.

Figure 8

Confirmation of the anti-CHIKV activity of pAFeg1 by RT-qPCR. (a) PFU standard curve, (b) inhibition of viral RNA replication. * Represents a p-value < 0.05 with respect to the vehicle control estimated by one-way ANOVA followed by Dunnett’s test.

Figure 8

Confirmation of the anti-CHIKV activity of pAFeg1 by RT-qPCR. (a) PFU standard curve, (b) inhibition of viral RNA replication. * Represents a p-value < 0.05 with respect to the vehicle control estimated by one-way ANOVA followed by Dunnett’s test.

Figure 9

AFeg1 time-of-addition assay.