RCAP was collected from Hanzhong city of Shaanxi Province. All the plant materials were air dried, powdered and deposited at the Department of Pharmacy, Tongji Hospital. Hot water extract of the plant was prepared according to the procedures as described. The plant (1 kg) was boiled in 1 L of distilled water for 1 h. The aqueous solution was collected and the residual was extracted again with another 1 L of distilled water. This step was repeated, until the extract became transparent. All of the aqueous extracts were combined, filtered through gauze, concentrated under low pressure and lyophilized. The lyophilized powder was dissolved in sterile distilled water.

The rhesus monkey kidney cell line MA-104 and human rotavirus G9 were obtained from Dr. Jihong Yang. The MA-104 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS; Hyclone Laboratories Inc., USA) and 1% v/v of 10,000 unit/mL penicillin and 10,000 μg/mL streptomycin. The cell cultures were maintained at 37°C in a humidified 5% CO₂ atmosphere under aseptic condition. Rotavirus was activated with 15 μg/mL trypsin for 30 min at 37°C and propagated in MA-104 cells monolayer in the presence of 5 μg/mL trypsin. The virus titer was estimated from cytopathogenicity by limit-dilution method and expressed as 50% tissue culture infectious dose (TCID₅₀/mL) by the Reed and Muench (1938) statistical method.

The cytotoxicity of the tested extract was evaluated using WST-8 assay (Tominaga et al., 1999). MA-104 cells (5000 cells/well) were grown in 96-well plate for 24 h. Different concentrations of aqueous extract were applied to culture wells in quadruplicate (1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 mg/mL). After incubation at 37°C, 5% CO₂ for 48 h, 10 μL of WST-8 solution was added to each well. Plates were then incubated at 37°C for 2 h. A microplate reader was used to test the absorbance of each well at 450 nm. The control for this assay was monolayer MA-104 cells incubated only with DMEM. Results were estimated by regression analysis. Cell viability was calculated as OD_ex / OD_con × 100%. OD_ex indicates the absorbance of extract treated group. OD_con indicates the absorbance of the control cells without the extract. The highest concentration of the extract which showed no significant statistical difference from the controls in cell viability was deemed as the maximum non-toxic concentration (MNTC).

Antiviral assays used in this study have been described by Barnard et al. (1997). In the mixed treatment assay: Extract diluted with DMEM containing 5 μg/mL trypsin was mixed with rotavirus at various concentrations (0.12, 0.24, 0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5 mg/mL), which were then incubated at 37°C for 2 h. The mixtures were inoculated in quadruplicate onto near confluent MA-104 cells monolayer in two 96-well plates, each well containing 100TCID50 of the rotavirus. The cells were incubated at 37°C with 5% CO₂ atmosphere for 48 h and observed with an inverted microscope. Then, WST-8 assay was done in one 96-well plate as previously described and the supernatant of each group in the other plate was gathered and tested with the rotavirus detection kit (Wantai BioPharm, Beijing, China) at 4°C. Cell viability was calculated as OD_rv / OD_con × 100%. OD_rv indicated the absorbance of rotavirus infected group with or without RCAP extract. OD_con indicates the absorbance of non-infected cells without RCAP extract (control).

In the post treatment assay: 100TCID₅₀ of the rotavirus was inoculated onto the near confluent MA-104 cells monolayer for 1 h. Extract of various concentrations (0.12, 0.24, 0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5 mg/mL) diluted with DMEM containing 5 μg/mL trypsin were added in quadruplicate. The cell viability was tested and the content of each group was gathered as described before.

The contents gathered from the antiviral assay were used in the extraction of the rotavirus RNA. RNAs were extracted by a TRI Reagent method (Kim et al., 2014). For TRI Reagent extraction, 500 μL of each sample was incubated with 500 μL TRI reagent (Sigma-Aldrich Chemical Company) for 10 min at room temperature, followed by supplementation with 100 μL chloroform. After vigorous mixing for 10 s and centrifugation at 13000 rpm for 15 min at 4°C, the upper layer was transferred into a fresh tube. An aliquot of 400 μL iso-propanol was added and the resulting mixture incubated for 10 min at room temperature, followed by centrifugation at 13000 rpm for 10 min. After centrifugation the supernatant was discarded, and the remaining was washed with 1 mL 75% ethanol and air-dried for 10 min. RNA was dissolved in 20 μL RNase-free water.

(i) Complementary DNA (cDNA) synthesis – the cDNA synthesis was conducted in a 25 μL reaction container using M-MLV Reverse Transcriptase. Firstly, 4 μL of extracted viral RNA, 1 μL of each primer and 11 μL RNase-free water, were incubated at 70°C for 10 min and then quickly chilled on ice for 5 min. After this, 5 × reaction buffer was added, 1 μL of each deoxynucleotide (dNTP), 1 μL RNasin Ribonuclease inhibitor and 1 μL M-MLV Reverse Transcriptase. The reaction was incubated for 60 min at 37°C, 5 min at 70°C. The cDNAs were maintained at 80°C until required. (ii) Primers – the primers used for rotavirus protein (VP6) region amplification were: Rota A (forward primer): 5′-GACGGVGCRACTACATGGT-3′ (V = A/C R = A/G) and Rota A (reverse primer): 5′-GTCCAATTCATNCCTGGTGG-3′ (N = A/G), which amplify a 379-bp product (Kang et al., 2004). They were synthesized commercially (Takara Bio Inc., China). (iii) The qPCR assay was performed on a CFX96 Touch^TM Real-Time PCR Detection System (Bio-Rad, USA) using SYBR premix Ex Taq. The assay was carried out in a total volume of 25 μL reaction mixture containing 12.5 μL SYBR premix Ex Taq master mix, 2 μL of cDNA, 1 μL of each primer and 8.5 μL RNase-free water. The optimized cycling conditions were as follow: initial denaturation at 95°C for 10 s, followed by 40 cycles of denaturation at 95°C for 10 s, primer annealing and extension at 56°C for 30 s. Fluorescence signals were recorded at the end of each cycle. A melt curve analysis was measured following amplification to confirm the specificity of the amplified products. Melting curve analysis consisted of 65°C for 5 s, and followed by increase in temperature to 95°C for 5 s with continuous fluorescence reading. RNase-free water, 5 × reaction buffer, dNTP, RNasin Ribonuclease inhibitor, SYBR premix Ex Taq used in qPCR were all obtained from Takara (Takara Bio Inc., China).

Cell nuclear morphology was evaluated by fluorescence microscopy following DAPI (Roche Diagnostics GmbH., Germany) staining. After gathering the contents from the antiviral assay, the monolayer consisting of MA-104 cells was washed once with DAPI-methanol (working solution, 1 μg/mL). Then, the cells were covered with DAPI-methanol and incubated for 15 min at 37°C. The staining solution was discarded after 15 min of incubation and the sample was washed once with methanol. The 96-well plates were examined under a fluorescence microscope (Nikon Eclipse Ti, Nikon Instruments Inc., USA). Different concentrations of aqueous extract were added to MA-104 cells for 48 h in another 96-well plate without incubation with rotavirus. The cells were observed under an inverted microscope. Then, DAPI staining was performed after discarding the supernatant to observe the effects of the extract on the cells, before and after treatment with RCAP extract.

The aqueous extract of RCAP was partitioned with ethyl acetate, CHCl₃ and n-butanol, consecutively. Comparing the volumes of each fraction, ethyl acetate fraction was chromatographed on silica gel column (Qing-dao Marine Chemical Inc., China). The column was washed and eluted, then the eluates were separated and confirmed by thin-layer chromatography (Yantai Chemical Industry Research Institute, China). For further purification of active components, semi-preparative HPLC was carried out on a Dionex quaternary system with a diode array detector at a flow rate of 2.5 mL/min using a reversed-phased C18 column (5 μm, 10 × 250 mm, YMC-pack ODS-A). The representative active compounds isolated were identified by analyzing them on a Bruker AM-400 spectrometer (Bruker Corporation, USA).

Study results were analyzed using SPSS software (ver. 18.0; SPSS, Inc., USA). Results were presented as mean ± standard deviation (SD). One-Way ANOVA test was used to calculate differences. Statistical significance was indicated at p < 0.05. Curves of real-time qPCR were analyzed using CFX Manager Software (ver. 3.0; Bio-Rad, Inc., USA).

It was shown that there was no significant difference between the control and the extract treated group in the aspect of cell viability, for RCAP concentrations up to 62.5 mg/mL (Figure 1). Based on this result, the concentrations of extract that exhibited cytotoxicity were excluded from the subsequent antiviral assay and 10 concentrations (0.12, 0.24, 0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5 mg/mL) were used.

In the mixed treatment assay, after various concentrations of extract and rotavirus were mixed and incubated, the mixtures were inoculated onto the confluent monolayer cells. The morphological changes of the virus infected or non-infected RCAP extract treated MA-104 cells were observed with light microscopy, after the cells in the virus control group (VCG, cells were only inoculated with virus) showed viral cytopathic effect (CPE). It was found that these affected cells increased with an increasing concentration of the extract (Figure 2B), while the morphology of non-infected cells treated with RCAP extract remained consistent regardless of the RCAP extract concentration (Figure 2A). This finding was consistent with the cell viability assay (Figure 3A). In the post-treatment assay, a similar situation was observed. As the extract concentration increased, more affected cells can be observed. There was indeed a negative relationship between cell viability and RCAP extract concentration (Figure 3A).

To study the virus quantitatively, a rotavirus detection kit was used on the supernatant isolated from each group. The color of the test line, indicative of the viral load, showed a reduction in intensity with increasing concentration of the extract. The color of the test line became undetectable when the extract reached a concentration of 15.63 mg/mL (Figure 3B). These results showed that the amounts of rotavirus decreased with increasing concentration of extract and could not be detected at higher concentrations of extract.

To study the virus qualitatively, the SYBR Green-based qPCR assay was used. The contents (virus, the extract and cellular debris) collected from 10 different tested groups in mix treatment assay were examined. After cDNAs were prepared, the samples were quantified with ultraviolet spectroscopy. Ct values can be used as a quantitative measurement of the input target number which reflects the viral load in antiviral assay, and there was a negative relationship between Ct value and the viral load. The quantities of rotavirus were compared across treated groups by using their Ct values. It was found the viral load was reduced as the concentration of the extract increased (Figure 4A). A similar trend was observed in post treatment assay as well (Figure 4B).

These results indicated that the extract exerted potential anti-rotavirus effect, not by affecting the rotavirus directly, but by acting on the infected MA-104 cells. The order of adding the rotavirus to the cells did not undermine the anti-viral effect of the extract. The antiviral effect was more pronounced when the virus was added to the cells prior to adding the extract, i.e., the post treatment assay.

In our study, both the cell and viral viability decreased with increasing concentrations of the herbal extract. This may suggest that the death of MA-104 cells was not caused by the viral infection, as the amounts of viruses did not increase significantly with decreasing cell viability (Ward et al., 1984). Programmed cell death, also known as apoptosis, is defined as a physiological cell suicide process alternative to necrosis. Apoptosis can be differentiated from necrosis by their characteristic nuclear fluorescence when excited under fluorescence microscope. In our study, DAPI staining revealed apoptosis in MA-104 cells caused by rotavirus. The apoptosis was shown to be accelerated by the RCAP extract. The morphological changes associated with apoptosis such as chromatin condensation, nuclear fragmentation, and margination of nucleus were evident in MA-104 cells. Moreover, it was shown that the amounts of apoptotic cells increased with increasing concentrations of the extract (Figure 5B). The cells in the viral control group exhibited signs of apoptosis induced by rotavirus, though not as significant compared to the treatment group. However, the extract itself did not result in any apoptosis in the cells (Figure 5A). The results indicated that the elimination of rotavirus may be due to the apoptosis of virus-infected MA-104 cells accelerated by the herbal extract.

There were three compounds isolated and identified from the ethyl acetate fraction of RCAP aqueous extract, which were 1,3-dihydroxy-9,10-anthracenedione (xanthopurpurin; Figure 6A), 4-hydroxy-3-methoxybenzoic acid (vanillic acid; Figure 6B) and 1-(3,4-dihydroxyphenyl)-1,2,3,4-tetrahydro-7-hydroxy-6-methoxy-2,3-naphthalenedi methanol (isotaxiresinol; Figure 6C). The extraction and separation processes, the NMR spectrums of these three compounds were shown in the Supplementary Material. Among them, xanthopurpurin and vanillic acid were considered to be responsible for the anti-viral effect observed with the RCAP aqueous extract.