In vitro antiviral activity of twenty-seven medicinal plant extracts from Southwest Nigeria against three serotypes of echoviruses

Twenty-seven different morphological parts from 26 plants (Table 1), selected based on their ethnobotanical use in the treatment of infectious diseases, were collected from various locations in Ibadan, South-west Nigeria, identified and authenticated at Forestry Herbarium Ibadan (FHI). Plant parts used were air-dried, pulverized and extracted by maceration in methanol for 72 h at room temperature (26–33 °C). The resulting extracts were filtered and concentrated in vacuo using the rotary evaporator.

The most active crude extract (Macaranga barteri) was subjected to bioassay guided fractionation using liquid-liquid partitioning and vacuum liquid chromatography (VLC). Briefly, 150 g of the crude methanol extract of M. barteri was dissolved in 250 mL of 70% aqueous methanol and the mixture was partitioned successively in n-hexane (400 mL × 4), dichloromethane (DCM; 400 mL × 4), ethylacetate (EtOAc; 400 mL × 4) and aqueous methanol (400 mL × 4). As the DCM fraction displayed the highest antiviral activity, it was subjected to vacuum liquid chromatography (VLC) on silica gel using a gradient elution solvent system that comprised of n-hexane/EtOAc (10:1 to 5:5, each 250 mL). Based on the similarity of their analytical TLC profiles, the fractions obtained were pooled into six sub-fractions (DCMA – DCMF). The VLC fractions were sprayed with chromogenic spray reagents including 1% vanillin/sulphuric acid and aluminium chloride (AlCl₃) to detect the classes of possible compounds present.

Three serotypes of echovirus (E7, E13, and E19) used in this study were obtained from stool isolate at the WHO Polio Laboratory, Department of Virology, University of Ibadan, Nigeria. All viruses were stored at − 70 °C until use. The human rhabdomyosarcoma (RD) cells were obtained by Centre for Disease Control, Atlanta, Georgia. The cells were grown in Eagle’s minimum essential medium (MEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, 100 μg/mL of streptomycin, 2 mM L-glutamine, 0.07% NaHCO₃, 1% non-essential amino acids and vitamin solution. The cells were cultured in 37 °C in 5% CO₂ humidified incubator for 72 h. The test medium used for cytotoxic assays and antiviral assays contained only 2% of fetal bovine serum (maintenance medium).

Crude extracts and fractions, 10 mg each were dissolved in 1 mL dimethlysulfoxide (DMSO) and filtered through a sterile syringe filter (0.2 μm pore diameter) to obtain a stock solution (SS; 10 mg/mL). The SS was diluted with the maintenance medium (culture medium that contained only 2% FBS) to obtain a final concentration of 1000 μg/mL (0.1% DMSO).

Virus titres were determined by virus-induced cytopathic effect in RD cell culture and were expressed as 50% tissue culture infective concentration (TCID₅₀) per mL using Sperman-Karber’s method. Briefly, RD cells were seeded into T25 cell culture flask (Corning®, UK), and incubated at 37 °C/5% CO₂ environment for 24 h. Thereafter, 100 μL of E7 were incorporated into the T25 flask containing the cultured RD cells and incubated for 72 h, leading to an increment in the virus stock quantity as a result of the 100% cytopathic effect. One hundred microliters RD cell suspension (1 × 10⁶ cells/mL) was seeded into three 96-well microtitre plates and incubated for 24 h to form monolayer. Ten-fold serial dilutions of the virus stock was made and 100 μL of each dilution was inoculated into the wells. A well that contained only RD cells without any virus served as the cell control. The microtitre plate was incubated at 37 °C, and daily CPE scoring was done till the control wells started dying. The TCID₅₀ values was determined using Spearman-Karber’s method and 100 TCID₅₀ was used for the assay. The procedure was done in triplicate and repeated for the E13 and E19.

The MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay, which is known to be a reliable measure of cell viability, was used to determine the cytotoxicity of the plant extracts and fractions. The principle of this assay involves the reduction of yellow MTT dye by mitochondrial succinate dehydrogenase to an insoluble, coloured (dark purple) formazan product. The purple formazan-containing cells are then solubilized with an organic solvent such as DMSO to release the solubilized formazan reagent which is measured by spectrophotometry. The assay was carried out based on the protocol described in an earlier literature [13]. In brief, ten-fold serial dilutions (1000 to 0.01 μg/mL) of the extracts / fractions were made with the maintenance. Monolayers of RD cells were seeded into each well of a 96-well microplate and treated with various concentrations of each extract/fraction. Plates were incubated at 37 °C in 5% CO₂ humidified incubator for 72 h. Plates were then observed under the microscope for cell death/cytopathic effect (CPE). The minimum dilution of extract with no toxic effect on the cells was referred to as the maximum non-toxic concentration (MNTC). After the incubation period, the old medium was removed and 25 μL of MTT solution (2 mg/mL) was added to each well and incubated for 2 h at 37 °C. thereafter, the MTT solution was removed from the wells and DMSO (75 μL) was added to dissolve formazan crystal. Optical density was measured spectrophotometrically (Multiscan 347, MTX lab) at 490 nm. The experiment was conducted in triplicate and the 50% cytotoxic concentration (CC₅₀) was calculated.

The antiviral activity of the plant extracts and fractions against echoviruses (E7, E13, and E19) was determined using the neutralisation assay, an assay that measures the inhibition of cytopathic effect on cell culture. The method previously described in the literature was modified and used in this assay [14]. Briefly, RD cells were seeded in a 96-well microplate and incubated for 24 h to grow to confluency. After the incubation period, 50 μL of 100 TCID₅₀ virus suspension were added to confluent cell monolayers in a 96-well plate and allowed to stand for 1 h to enable virus adsorption. Thereafter, ten-fold serial dilutions, obtained from the MNTC of each extract were added in triplicate into all the wells with the exception of the negative control wells that contained only RD cells and the virus control that contained an equal virus concentration but lacked the plant extract. The plates were incubated at 37 °C in 5% CO₂ humidified incubator for 72 h after which the cell viability was measured using the MTT assay as described above. The concentration of the plant extract that decreased the CPE by 50%, with respect to the virus control, was estimated from the obtained optical density and defined as the 50% inhibition concentration (IC₅₀). The selectivity index (SI), defined as CC₅₀ over IC_50, for each active extract and fractions were also determined. Since there are no antiviral drugs approved for the treatment of enteroviral infections, no drug control was used in this study. All experiments were carried out in triplicates.

The experiments were conducted in triplicate. The 50% cytotoxic concentration (CC₅₀) and the 50% inhibitory concentration (IC₅₀) for the extracts and fractions were calculated from concentration-effect-curves after linear regression analysis using GraphPad Prism5 and Microsoft Excel. The selectivity index for each active extract and fractions were also determined. Selective index is a comparison of the amount of a test agent that causes the inhibitory effect to the amount that causes toxicity.

The determination of the virus titre (TCID₅₀) for E7, E13, and E19 viruses by Sperman-Karber’s method gave a value of 10^− 6 for the three viruses. Therefore, 100 TCID₅₀ which was used for the study was calculated as 10^− 4.

The plant extracts encountered in this study displayed varying MNTC to RD cells in tissue culture medium. As presented in Table 1, the extract of Macaranga barteri leaves had the lowest MNTC (0.01 μg/mL), while Petiveria alliacea, Mondia whitei, Ageratum conyzoides and Bryophyllum pinnatum extracts had the highest MNTC of 100 μg/mL.

As presented in Table 2, the cytotoxic concentration that inhibited 50% of cell growth (CC₅₀) showed that the crude extract of M. barteri leaves had the highest cytotoxicity with CC₅₀ value of 0.27 μg/mL. This was followed by Crinum jagus (9.88 μg/mL) and Terminalia ivorensis (12.14 μg/mL). The DCM fraction of M. barteri showed the highest cytotoxicity amongst the fractions with CC₅₀ value of 0.18 μg/mL. Other fractions including n-hexane, ethyl acetate and aqueous had CC₅₀ values of 24.9, 2.87 and 3.01 μg/mL, respectively. Amongst the VLC sub-fractions of M. barteri, sub-fraction A (DCMA) displayed the highest cytotoxicity on the RD cells with CC₅₀ value of 0.47 μg/mL (Table 3).

The preliminary antiviral screening showed that only ten out of the 27 crude plant extracts tested were active on E7 and E19, inhibiting the cytopathic effect of the virus in tissue culture (Table 2). On E7, M. barteri displayed 75% CPE inhibition at MNTC (0.01 μg/mL), 50% inhibition at 0.001 μg/mL, while the extract exhibited 75% CPE inhibition at MNTC (0.01 μg/mL) and 50% CPE inhibition at 0.01 ng/mL on E19. None of the extracts inhibited the cytopathic effect caused by E13 serotype. Amongst the active plant extracts, the crude methanol extract of M. barteri leaves had the highest antiviral activity on both E7 and E9 with IC₅₀ values of 0.028 and 0.0017 ng/mL, respectively, followed by the Ageratum conyzoides extracts (0.208 μg/mL, E7; 0.006 μg/mL, E19) and Mondia whitei extracts (0.038 μg/mL, E7; 0.005 μg/mL, E19) (Table 2). Amongst the liquid-liquid partitioned fractions of M. barteri, the DCM was most the active and selective on E7 (IC₅₀ = 0.0075 ng/mL; SI = 19,896.54) and E19 (IC₅₀ = 0.0175 ng/mL; SI = 8581.24) when compared with the hexane fraction which had moderate activity on E7 (IC₅₀ = 0.442 μg/mL; SI = 57.96) and E19 (IC₅₀ = 0.305 μg/mL; SI = 83.85). However, both the ethyl acetate and aqueous fractions of M. barteri were inactive against E7 and E19. In addition, sub-fraction D (DCMD) displayed the highest antiviral activity amongst the sub-fractions on both E7 (IC₅₀ = 0.00477 μg/mL), and E19 (IC₅₀ = 0.00532 μg/mL) (Table 3).

Yellow colouration and mint green spots were observed when sub-fraction D (DCMD) was sprayed with vanillin/sulphuric acid. In addition, yellow spots were observed at short (254 nm) and long (365 nm) wavelengths of UV light when the sub-fraction was sprayed with aluminium chloride.