Antiviral effect of compounds derived from the seeds of Mammea americana and Tabernaemontana cymosa on Dengue and Chikungunya virus infections

Abstract

Background: The transmission of Dengue virus (DENV) and Chikungunya virus (CHIKV) has increased worldwide, due in part to the lack of a specific antiviral treatment. For this reason, the search for compounds with antiviral potential, either as licensed drugs or in natural products, is a research priority. The objective of this study was to identify some of the compounds that are present in Mammea americana (M. americana) and Tabernaemontana cymosa (T. cymosa) plants and, subsequently, to evaluate their cytotoxicity in VERO cells and their potential antiviral effects on DENV and CHIKV infections in those same cells.

Methods: Dry ethanolic extracts of M. americana and T. cymosa seeds were subjected to open column chromatographic fractionation, leading to the identification of four compounds: two coumarins, derived from M. americana; and lupeol acetate and voacangine derived from T. cymosa.. The cytotoxicity of each compound was subsequently assessed by the MTT method (at concentrations from 400 to 6.25 μg/mL). Pre- and post-treatment antiviral assays were performed at non-toxic concentrations; the resulting DENV inhibition was evaluated by Real-Time PCR, and the CHIKV inhibition was tested by the plating method. The results were analyzed by means of statistical analysis.

Results: The compounds showed low toxicity at concentrations ≤ 200 μg/mL. The compounds coumarin A and coumarin B, which are derived from the M. americana plant, significantly inhibited infection with both viruses during the implementation of the two experimental strategies employed here (post-treatment with inhibition percentages greater than 50%, p < 0.01; and pre-treatment with percentages of inhibition greater than 40%, p < 0.01). However, the lupeol acetate and voacangine compounds, which were derived from the T. cymosa plant, only significantly inhibited the DENV infection during the post-treatment strategy (at inhibition percentages greater than 70%, p < 0.01).

Conclusion: In vitro, the coumarins are capable of inhibiting infection by DENV and CHIKV (with inhibition percentages above 50% in different experimental strategies), which could indicate that these two compounds are potential antivirals for treating Dengue and Chikungunya fever. Additionally, lupeol acetate and voacangine efficiently inhibit infection with DENV, also turning them into promising antivirals for Dengue fever.

Keywords: Antiviral; Chikungunya Virus; Dengue Virus; Mammea americana; Tabernaemontana cymosa.

Fig. 1

Structures of the compounds that were identified from the seeds of M. americana and T. cymosa. They were identified by using Nuclear Magnetic Resonance in one and two dimensions (1D and 2D NMR) and mass spectrometry (MS) and were compared with data reported in the literature. a and b Coumarins derived from M. americana. c Voacangine derived from T. cymosa. d Lupeol acetate derived from T. cymosa

Fig. 2

Evaluation of compound cytotoxicity in VERO cells. Each compound was evaluated by MTT at concentrations from 6.25 μg/mL to 400 μg/mL and compared with the untreated controls. a Compounds extracted from the seeds of M. americana. b Compounds extracted from the seeds of T. cymosa. *Only coumarin B significantly decreased the cellular viability at a concentration of 400 μg/mL (ANOVA-LSD, p < 0.05)

Fig. 3

Antiviral effects on viral production (DENV-2/NG or CHIKV/ACol) using the pre-treatment strategy. The cells were treated with each compound at a concentration of 200 μg/mL and were subsequently infected. a  Effects of the compounds extracted from the M. Americana seeds. b Effects of the compounds extracted from the T. cymosa seeds. Statistically significant inhibitions are observed only in the cultures treated with coumarin A or coumarin B (Student’s t-test, p < 0.05)

Fig. 4

Antiviral effect on the viral genome replication of DENV-2/NG during the post-treatment strategy. The cells were infected and subsequently treated with the compounds at concentrations from 0.8 to 200 μg/mL and compared with the untreated controls. a Effects of the compounds extracted from the M. Americana seeds. b Effects of the compounds extracted from the T. cymosa seeds. Statistically significant inhibitions are observed in the cultures treated with coumarin A (concentrations greater than 3.1 μg/mL) or treated with coumarin B, lupeol acetate, or voacangine (all of the concentrations evaluated here) (ANOVA-LSD, p < 0.05)

Fig. 5

Antiviral effect on the production of infectious viral CHIKV/ACol particles during the post-treatment strategy. The cells were infected and subsequently treated with compounds at concentrations from 0.8 to 200 μg/mL and compared with the untreated controls. a Effect of the compounds extracted from the M Americana seeds. b Effect of the compounds extracted from the T. cymosa seeds. Statistically significant inhibitions are observed in the cultures treated with coumarin A (concentrations greater than 12.5 μg/mL) or coumarin B (all of the evaluated concentrations) (ANOVA-LSD, p < 0.05)