In vitro antiviral activity of plant extracts from Asteraceae medicinal plants

Six Argentinean Asteraceaes were selected for this study. Chemical, ethnopharmacological data and previously reported antiviral studies are shown in Table 1. The antiviral activity of plant extracts against BVDV, HSV-1, PV-2 and VSV was assessed in vitro by the viral CPE reduction assay. Results obtained from this screening (Table 2) showed that B. gaudichaudiana and B. spicata OE were active against PV-2 and VSV, the AE of both species and the AE of T. absinthioides were active against HSV-1 and T. absinthioides OE was active only against PV-2. None of the twelve extracts was active against BVDV.

To confirm the inhibitory effect detected in the screening, we evaluated the antiviral activity of the positive extracts by the plaque reduction assay and the SIs were determined (Table 3). Regarding the active extracts, the thin layer chromatographic (TLC) profiles of the OE of the two Baccharis species were very similar. Bands corresponding to flavonoid aglycones and terpenoids were observed after spraying with NPR and anisaldehyde/H₂SO₄. On the other hand, Tessaria absinthioides OE showed strong bands corresponding to flavonoid glycosides and only weak bands corresponding to terpenoids in the OE and AE (Additional file 1).

In the pretreatment assay, none of the extracts protected Vero cells against PV-2 or HSV-1 infection after 7 h of incubation at the evaluated concentrations (Figure 1).

When the virucidal activity was assessed, the OE did not prove to have this effect against PV-2 since 10 and 20% of reduction of viral infectivity was obtained at r.t. and 37°C, respectively. In contrast, higher values were obtained for the AE against HSV-1 with values of 85% and 97%, at r.t. and 37°C, respectively (Figure 1).

With the aim to determine whether the inhibitory effect of the extracts occurs during the adsorption or post-adsorption steps of the viral cycle, different experimental conditions were evaluated with 1xEC₉₀ of OE and AE (Figure 2A). The results obtained demonstrated that B. gaudichaudiana OE (30 μg/ml) reduced the formation of PV-2 plaques when it was added after the adsorption period. This reduction in the number of plaques was similar to that obtained when the OE was present during all the experimental time (Throughout) (Figure 2B).

In contrast, B. gaudichaudiana AE (35 μg/ml) interfered in the adsorption step of HSV-1 to Vero cells and caused an inhibition degree similar to that obtained when it was present throughout the experimental time. The reduction of 25% observed in the post-adsorption condition could be due to the inhibitory effect of this extract on the adsorption steps on the subsequent HSV-1 replication cycles occurring during the 48 h incubation period carried out at 37°C (Figure 2B).

B. gaudichaudiana OE was fractionated by a silicagel column chromatography. Eight final fractions were obtained according with their TLC profiles. The results obtained in the evaluation of the anti-PV-2 activity demonstrated that F_C was the most active fraction with a SI = 56.4 and EC₅₀ = 2.1 ± 0.1 μg/ml followed by F_D SI = 44.1 and EC₅₀ = 2.5 ± 0.3 μg/ml (Table 4). The HPLC profile obtained for F_D was similar to that of F_C but based on the SI value and the yield, F_C was selected for further characterization (Additional file 2).

To define the post-adsorption steps of the viral cycle that could be targeted by F_C, the effect of the addition of F_C (10xEC₉₀ = 22 μg/ml) at different times of infection on PV-2 production at 10 h p.i. was evaluated (Figure 3). The results obtained demonstrated that the maximum inhibition level was exerted when F_C was present before 4 h of infection.

A semipreparative HPLC of F_C was then performed and four subfractions (F_C1- F_C4) were collected. The antiviral activity against PV-2 was detected in F_C2 (EC₅₀ = 3.3 ± 0.3 μg/ml) and F_C3 (EC₅₀ = 1.8 ± 0.1 μg/ml) (Figure 4). The CC₅₀ value of F_C3 was higher than 100 μg/ml and the SI was > 55.6. A major pure compound was isolated from F_C3 by semipreparative HPLC (Figure 5) and identified as apigenin (Figure 6) by comparison of its spectral data (Additional file 3 and Additional file 4) with literature values [20] and by HPLC comparison with a reference standard (Additional file 5). The antiviral activity of apigenin was determined by plaque assay with EC₅₀ = 12.2 ± 3.3 μM. Its CC₅₀ value was 230.7 ± 4.4 μM; in consequence the SI was 18.9. The apigenin standard exhibited similar values of EC₅₀ and CC₅₀ (data not shown).

Plant samples (aerial parts with flowers) were collected between 2008 and 2010 in their places of origin in Argentina. Voucher specimens are deposited as follows: B. gaudichaudiana (1655): Botany Herbarium at Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina; B. spicata (BAF 711), Bidens subalternans (BAF 704), Pluchea sagittalis (BAF 709) and Tagetes minuta (BAF 714): Herbarium at Museo de Farmacobotánica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires; Tessaria absinthioides (Slanis-Juarez 1041): Herbarium of Fundación Miguel A. Lillo, Universidad de Tucumán. Botanical and vernacular names, popular uses and reported chemical composition are shown in Table 1.

Dried aerial parts of each plant (10 g) were reduced to powder and extracted by soaking in 100 ml of dichloromethane:methanol (1:1) at room temperature (r.t.) for 24 h and then vacuum-filtered. The process was repeated twice and the filtrates were combined and dried under vacuum to obtain the organic extract (OE). The marc of the plant material was further extracted with distilled water under the same conditions. The aqueous extracts (AE) were lyophilized. For the antiviral assays, OE and AE were dissolved in dimethyl-sulfoxide and sterile distilled water, respectively.

Vero cells (ATCC CCL 81) were obtained from Asociación Banco Argentino de Células and cultured in growth medium consisting of Eagle’s Minimal Essential Medium (E-MEM) supplemented with 10% fetal bovine serum (FBS) (PAA), 100 μg/ml streptomycin, 100 IU/ml penicillin, 2 mM L-glutamine, 2.25 g/L sodium bicarbonate and non-essential amino acids (100 μM) (Gibco), at 37°C in a 5% CO₂ incubator. The infection medium (IM), used for the antiviral assays, was the same as the growth medium but 2% FBS was added instead. The plaque medium (PM) was IM supplemented with 1% methylcellulose (Sigma). Madin-Darby Bovine Kidney cells (MDBK) (ATCC CLL 22) were grown in growth medium supplemented with 10% of γ-irradiated FBS. IM for the MDBK cell line was supplemented with 2.5% horse serum (Gibco).

The herpes simplex type 1 (HSV-1) F strain, the poliovirus type 2 (PV-2) Sabin strain and the bovine viral diarrhea virus (BVDV:NADL strain cytopathic biotype were kindly provided by Dr. Albert Epstein, Dr. María Cecilia Freire (ANLIS-Instituto Dr. Carlos G. Malbrán, Argentina) and Dr. Laura Weber (INTA, Castelar, Argentina), respectively. VSV, Indiana strain (ATCC VR-1421), was purchased from ATCC. Virus stocks of HSV-1, PV-2 and VSV were propagated and quantified in Vero cells. BVDV was propagated and quantified in MDBK cells. Virus quantification was performed by plaque assay method as number of plaque forming units per ml (p.f.u./ml). All virus stocks were stored at −70°C until used.

The antiviral activity of each plant extract was screened in 96-well culture plates by measuring the reduction of the viral cytopathic effect (CPE). Confluent Vero and subconfluent MDBK cell monolayers were infected with HSV-1, PV-2 or VSV or with BVDV, respectively, at a multiplicity of infection (m.o.i.) of 0.01 p.f.u./cell in the presence of 25 and 100 μg/ml of each OE/AE. Infected cells in the absence of extract as control virus and mock-infected cells with and without extract as control cells and cytotoxicity control were included. Plates were incubated at 37°C in a humidified atmosphere containing 5% CO₂ until 90% of viral CPE in the CV was reached. The reduction of viral CPE was determined by measuring cell viability by the tetrazolium salt/phenazine methosulfate (MTS/PMS) colorimetric assay (CellTiter 96™ Promega, Madison, WI, USA). The absorbance at 490 nm was measured in a Multi-Mode microplate reader (Synergy™ HT, BioTek). Results of the screening were expressed as positive (+) (reduction in the CPE at both concentrations tested), negative (−) (absence of reduction in the CPE) and (+/−) (reduction in CPE only at 100 μg/ml).

The cytotoxic effect of B. gaudichaudiana, B. spicata and T. absinthioides OE and AE on Vero cells was determined by the MTS/PMS method, as previously described [33]. Briefly, subconfluent monolayers of Vero cells (8×10³ cells/well; 24 h culture) were incubated in quadruplicate in 96-multiwell plates in the presence of two-fold dilutions of the extracts for 72 h at 37°C. Cell viability (%) was calculated for each concentration as Abs _treated/Abs _CC × 100, where Abs_treated and Abs _CC are the absorbance readings for the wells with and without extract, respectively. The CC₅₀ is defined as the concentration that reduced cell viability by 50% with respect to controls without drug. The CC₅₀ value was derived from the corresponding dose–response curves. The maximum non-cytotoxic concentration (MNCC) is defined as the maximum concentration of the extract that leaves 100% of viable cells.

The effective concentration 50 (EC₅₀) is the concentration of extract that reduces the number of viral plaques by 50% with respect to control virus (without extract). This parameter was determined by the plaque reduction assay. Briefly, monolayers of Vero cells grown in a 24-well plate (24 h; 5% CO₂; 37°C) were infected with 100 p.f.u./well of PV-2, VSV or HSV-1 in either the absence or presence of serial two-fold dilutions from the MNCC of B. gaudichaudiana, B. spicata and T. absinthioides extracts (treated). After 45 min incubation at 37°C, the viral inoculum was removed, and the cell monolayers were washed with phosphate buffer saline (PBS) and overlaid with PM supplemented with the corresponding concentrations of each extract. PM without extract was added in CC and CV wells. After 24 h at 37°C for PV-2 and VSV or 48 h for HSV-1, cell monolayers were fixed and stained with 0.75% crystal violet in methanol:water (40:60) and viral plaques were counted. Reduction of plaques (%) was calculated as: [1-(nº plaques _treated/ nº plaques _CV)] × 100. The EC₅₀ values were calculated by regression analysis of the dose–response curves generated with the data.

The selectivity index (SI) was calculated as the CC₅₀/EC₅₀ ratio.

Acyclovir (Filaxis); Guanidine.HCl (Sigma-Aldrich, St. Louis, MO) and Ribavirin (MP Biomedicals, LLC) were tested simultaneously as positive controls for HSV-1, PV-2 and VSV, respectively.

Chromatographic analysis of positive OE were performed by thin layer chromatography (TLC) on silica gel layers (Silica gel 60 F₂₅₄ EMD Chemicals Inc.) using a- ethyl acetate:toluene:formic acid:methanol (2:2:1:1) and Natural Product Reagent (NPR- 2-aminoethildiphenilboric acid -Sigma) as visualization reagents; and b- toluene:ethylacetate (5:5) and sulphuric/anisaldehyde (SAni) as reagent. The positive AE were tested on: a) silica gel layers using ethylacetate:methanol:water (50:6:5) and SAni reagent; and b) Cellulose plate (Polygram^® CEL 300 UV₂₅₄ – Macherey Nagel) using acetic acid 15% and NPR as reagent. In all cases, the TLC plates were visualized under UV light (254 and 366 nm) and visible light.

To assess the effect of the pretreatment with B. gaudichaudiana extracts, Vero cell monolayers seeded in 24-well plates were treated for 7 h at 37°C with two concentrations of the extract 10xEC₉₀ and 1xEC₉₀ (OE: 300 and 30 μg/ml and AE: 350 and 35 μg/ml, respectively). Then, the medium was removed and washed with PBS, and the cell monolayers were infected with 100 p.f.u. of PV-2 or HSV-1/well in the absence of the extracts. Mock-infected cells (CC) and cells pretreated with IM (CV) were included in each assay. After 45 min at 37°C, the viral inoculum was removed and PM without extract was added and further incubated at 37°C for 24 or 48 h. Finally, the number of viral plaques was determined.

To study the effect of the extracts in the adsorption and post-adsorption events, three different treatments with B. gaudichaudiana OE (1xEC₉₀ = 30 μg/ml) against PV-2 or AE (1xEC₉₀ = 35 μg/ml) against HSV-1 were carried out. B. gaudichaudiana OE and AE were present: (i) only during the adsorption period (Adsorption); (ii) after adsorption and until the end of the experiment (Post-Adsorption), and (iii) during and after the adsorption (Throughout). Briefly, Vero cell monolayers cultured in 24-well plates were precooled for 1 h at 4°C. Cells were then infected with 100 p.f.u. of virus/well in the presence or absence of OE/AE and further incubated at 4°C for 1 h allowing only the adsorption step of the viral particles to the cells (Adsorption). Cell monolayers were washed with PBS, and then PM with or without extract was added. The number of viral plaques was determined after 24 h and 48 h for PV-2 and HSV-1, respectively.

B. gaudichaudiana aerial parts (500 g) were air-dried, ground to powder and extracted with dichloromethane:methanol (1:1) and the extract was taken to dryness. Thirty grams of this OE was fractionated by silica gel 60 (500 g) column chromatography eluted with a step gradient of hexane:ethylacetate (100:0 to 0:100) and ethylacetate:methanol (100:0 to 0:100) to afford 21 fractions of 500 ml each. Eluates were monitored by thin-layer chromatography (TLC) on silica gel 60 F₂₅₄ using toluene-ethyl acetate (1:1) and cellulose layers using acetic acid 40% and combined into eight final fractions (F_A to F_H) according to their TLC profiles.

Fraction F_C was further fractionated by a semipreparative reverse-phase HPLC (Waters 2996 – Photodiode Array Detector–Waters 600 pump) on a RP-18 column (LiChrospher^® 100, 5 μm, LiChroCART 125×4 – Merck). The injection volume was 50 μl. Elution was performed at a flow rate of 1 ml/min. The mobile phase used consisted of water (A) and methanol (B): 0–15 min: isocratic 50% A, 15–25 min: 50 → 98% A, 25–30 min: isocratic 98% A, 30–31 min: 98 → 50% A. Eluates were monitored at 254 nm. Eluates were collected into four subfractions: F_C1 (0–13 min), F_C2 (13–20 min), F_C3 (20–25 min) and F_C4 (25–30 min).

The F_C3 subfraction was subjected to reverse-phase HPLC on RP-18 column (LiChrospher^® 100, 5 μm, LiChroCART 125×4 – Merck), 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 and a flow rate=1 ml/min and a pure compound was isolated. Eluates were monitored at 336 nm.

The anti-PV-2 activity of fractions F_A-F_H and subfractions F_C1-F_C4 and the pure compound was determined by viral plaque reduction assay at concentrations ranging from 100 to 0.1 μg/ml in Vero cells. The cytotoxicity and SI were also evaluated as previously described.

The pure compound obtained from F_C3 was identified by ultraviolet spectroscopy (UV) (Jasco V-630), infrared spectroscopy (IR) (Nicolet 380 FT-IR-Smart Multi Bruce HATR, Zn Se 45°) and HPLC/DAD by comparison with authentic sample (Sigma-Aldrich, St. Louis, MO) and comparison with literature data.

Confluent Vero cell monolayers cultured in a 96-well plate were infected with PV-2 (m.o.i. = 10) for 1 h at 4°C. Following the adsorption period, cells were washed three times, and F_C (22 μg/ml=10xEC₅₀) was added at different hours post-infection (p.i): 0, 2, 4, 6 and 8 h. Cells were further incubated up to 10 h. At this time, supernatants were collected and clarified by centrifugation (3,500 × g at 4°C) and the virus production was determined by viral plaque assays.

Data are presented as means ± standard deviation (SD). A one-way ANOVA with Bonferroni a posteriori test was used to compare differences between groups. A p < 0.05 was considered significant. The EC₅₀ and CC₅₀ values were calculated using GraphPad Prism software v. 5.01.