Chemicals
All of the chemicals that were used in this study were of analytical grade or higher. Ethanol and methanol were obtained from Merck (Darmstadt, Germany). 2,2-Azino-bis(3-ethylbenzothiazo-line-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2-diphenyl-1-picryl hydrazyl (DPPH), Folin & Ciocalteu phenol reagent, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), sodium carbonate, and 2,4,6-tri-pyridyl-s-triazine (TPTZ) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ferric chloride hexahydrate and potassium persulfate were procured from LOBA CHEMIE PVT (Mumbai, India). Gallic acid was procured from Fluka Chemical Co. (Buchs, Switzerland). Dulbecco’s modified Eagle’s medium (DMEM) was procured from Gibco (Massachusetts, USA).
Plant extracts, cells, and viruses
Ethanolic extracts from the Thai medicinal plants C. sappan Linn., G. mangostana Linn., H. cordata, P. frutescens, C. nutans, P. emblica, and T. triandra were purchased from Specialty Natural Product Co. Ltd. (Thailand).
MARC-145 tissue culture cells were grown in Dulbecco’s modified Engle medium (DMEM) containing 10% fetal bovine serum (Gibco) and 1% Penicillin/Streptomycin and incubated at 37°C in a 5% CO2 atmosphere. To produce inoculated cells, PRRSV (VR2332 North American genotype) was propagated in the MARC-145 cells and the virus titer was quantified using the virus titer by immunoperoxidase monolayer assay (IPMA).
Cytotoxicity assay
The cytotoxicity of the seven Thai medicinal plant extracts was determined using the MTT assay. Briefly, MARC-145 cells were plated at a density of 5,000 cells/well in 96-well plates and incubated in a 5% CO2 atmosphere at 37°C for 24 h. When the cells were at least 90% confluent, the medium was removed and replaced with medium containing two-fold serial dilutions of the plant extracts. In addition, medium without plant extract was used as a positive control. Incubation was then continued in a 5% CO2 atmosphere at 37°C for 72 h. After this time, the medium was removed, 20 μl of freshly prepared MTT solution (5 mg/ml) was added to each well, and the plates were incubated at 37°C for 4 h. The medium was then replaced with 150 μl DMSO to dissolve the crystals and the plates were incubated at 37°C for 5 min to dissolve any air bubbles before measuring the MTT signal at an absorbance of 550 nm. The results were reported as 50% cytotoxic concentrations (CC50).
Inhibition of virus infection assay
The inhibition of virus infection test was evaluated as previously described [12]. Briefly, the plant extracts at the concentration that was determined in the cytotoxicity test outlined above, as well as two lower level concentrations in two-fold dilution were mixed with PRRSV at 108 TCID50/ml in the ratio of 1:1 and incubated for 1 h at 37°C. DMSO (1%) containing medium mixed with PRRSV served as the control. Thereafter, the mixture of virus and plant extracts, as well as controls were inoculated into MARC-145 cells as 5,000 cells/well in a 96-well plate and incubated at 37°C for 1 h. Subsequently, the medium was removed and replaced with a fresh medium containing 10% FBS. The plates with MARC-145 cells were cultured under standard conditions for 24 h post-infection (hpi) and then the supernatants were collected to quantify the virus titer.
Inhibition of virus replication assay
The inhibition of virus replication test was performed as previously described [12]. Briefly, MARC-145 cells were plated at a density of 5,000 cells/well in 96-well plates and infected with PRRSV at a multiplicity of infection (MOI) of 1 for 1 h at 37°C. The virus was then removed from each of the wells and replaced with the diluted plant extracts at the concentration that was determined in the cytotoxicity test, as well as two lower levels concentrations in two-fold dilution. Also, 1% DMSO was mixed to medium as a control. The plates were cultured under standard conditions, and the supernatants were collected at 24, 48, and 72 hpi and quantified the virus titer.
Virus Titer
The virus titer was further assessed by Immunoperoxidase Monolayer Assay (IPMA) was adapted from [29]. Briefly, cells were fixed with 100 μl cold 4% formalin for 15 min at room temperature (RT), washed once with 100 μl phosphate-buffered saline (PBS) and twice with 100 μl of 0.5% PBS Tween-20 (PBST), and then blocked with 100 μl of 1% BSA in 0.5% PBST for 30 min at RT. After blocking, the cells were stained with 70 μl anti-PRRSV NC protein monoclonal antibody (Median Diagnostics, Gangwon-do, Korea) diluted 1:400 for 60 min at RT and then washed and incubated with Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch, Pennsylvania, USA) diluted 1:1,200 for 60 min at RT. After washing three times in PBS, the cells were counter-stained with 1,5-diaminopentane (DAP) substrate and examined under a microscope. The virus titer was shown at the viral median tissue culture infectious dose (TCID50) was then determined using the Reed–Muench method.
Phytochemical analysis
The total phenolic contents of the plant extracts were determined using the Folin–Ciocalteu method [30], and their free radical scavenging activities were determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay and 2,2-azino-bis (3-ethylbenzothiazo-line-6-sulfonic acid) diammonium salt (ABTS) scavenging assay, as previously reported [31,32]. The antioxidant activities were determined using the ferric reducing antioxidant power (FRAP) assay, according to the Benzie and Strain method [33].
Statistical analyses
Differences in the antiviral activities among different concentrations of each plant extract were tested using one-way analysis of variance (ANOVA) with Tukey’s post hoc test for a comparison of the means. The CC50 was calculated through regression analysis of the dose-response curves for the MTT assay. All statistical analyzes were performed using the SPSS 23.0 software (SPSS Inc., Chicago, IL, USA) with a significance level of P ≤ 0.05.