In vitro antiviral activity of plant extracts from Asteraceae medicinal plants

Abstract

Background: Due to the high prevalence of viral infections having no specific treatment and the constant appearance of resistant viral strains, the development of novel antiviral agents is essential. The aim of this study was to evaluate the antiviral activity against bovine viral diarrhea virus, herpes simplex virus type 1 (HSV-1), poliovirus type 2 (PV-2) and vesicular stomatitis virus of organic (OE) and aqueous extracts (AE) from: Baccharis gaudichaudiana, B. spicata, Bidens subalternans, Pluchea sagittalis, Tagetes minuta and Tessaria absinthioides. A characterization of the antiviral activity of B. gaudichaudiana OE and AE and the bioassay-guided fractionation of the former and isolation of one active compound is also reported.

Methods: The antiviral activity of the OE and AE of the selected plants was evaluated by reduction of the viral cytopathic effect. Active extracts were then assessed by plaque reduction assays. The antiviral activity of the most active extracts was characterized by evaluating their effect on the pretreatment, the virucidal activity and the effect on the adsorption or post-adsorption period of the viral cycle. The bioassay-guided fractionation of B. gaudichaudiana OE was carried out by column chromatography followed by semipreparative high performance liquid chromatography fractionation of the most active fraction and isolation of an active compound. The antiviral activity of this compound was also evaluated by plaque assay.

Results: B. gaudichaudiana and B. spicata OE were active against PV-2 and VSV. T. absinthioides OE was only active against PV-2. The corresponding three AE were active against HSV-1. B. gaudichaudiana extracts (OE and AE) were the most selective ones with selectivity index (SI) values of 10.9 (PV-2) and > 117 (HSV-1). For this reason, both extracts of B. gaudichaudiana were selected to characterize their antiviral effects. Further bioassay-guided fractionation of B. gaudichaudiana OE led to an active fraction, FC (EC50 = 3.1 μg/ml; SI = 37.9), which showed antiviral activity during the first 4 h of the viral replication cycle of PV-2 and from which the flavonoid apigenin (EC50 = 12.2 ± 3.3 μM) was isolated as a major compound.

Conclusions: The results showed that, among the species studied, B. gaudichaudiana seemed to be the most promising species as a source of antiviral agents.

Figure 1

Virucidal activity and the effect of pretreatment with B. gaudichaudiana OE and AE. The virucidal activity and the pretreatment of 10xEC₉₀ (OE= 300 μg/ml AE = 350 μg/ml) and 1xEC₉₀ (OE= 30 μg/ml, AE = 35 μg/ml) were evaluated against PV-2 and HSV-1, respectively. Data represent % of virus inhibition compared to untreated controls as mean ± SD (n = 3), each time in quadruplicate.

Figure 2

Effect of B. gaudichaudiana OE and AE in the adsorption and post-adsorption steps of PV-2. A. Scheme of addition of OE or AE. Open and black arrows indicate the absence and presence of extract, respectively. B.- Percentage of viral inhibition under different experimental conditions. 1xEC₉₀ was used for the experiments. Data represented % of virus inhibition compared to untreated control as mean ± SD (n = 3), each time by quadruplicate.

Figure 3

Effect of F_C on PV-2 replication cycle. A. Kinetics of PV-2 extracellular production during one replication cycle Vero cell monolayers were infected with PV-2 ( m.o.i. = 10). Viral titers were determined at different hours by plaque assay. B. Effect of addition of F_C on the PV-2 production during a one step replication cycle At different h p.i. after the adsorption period, F_C (22 μg/ml) was added and the extracellular viral production was determined at 10 h p.i. of incubation at 37°C, by the plaque assay. Data represent % of virus production respect to untreated control. The viral production at 10 h p.i. in the kinetic curve of control virus was considered 100%. * p < 0.05 vs 0 and vs 2 h (one-way ANOVA with Bonferroni a posteriori test).

Figure 4

Antipoliovirus (PV-2) activity of subfractions derived from F_C. The antiviral activity of each subfraction was determined by the reduction of plaque assay. Results are shown as mean ± SD (n = 3), each concentration in quadruplicate.

Figure 5

HPLC profile of F_C3. HPLC: RP-18 column, using a water (A)-methanol (B) gradient: 0–2 50% A; 2–15 min: 50 → 98% A, 15–25 min: isocratic 98% A, 26–30 min: 98 → 50% A, flow rate=1 ml/min, monitored at 336 nm. The insert shows the UV adsorption spectra of the major peak detected.

Figure 6

Chemical structure of apigenin: 5, 7-dihydroxy-2-(4-hydroxylphenyl)-4H –chromen-4-one, C₁₅H₁₀O₅, MW: 270.24.