In vitro Antiviral Activity of Rubia cordifolia Aerial Part Extract against Rotavirus

Abstract

The root of Rubia cordifolia has been used traditionally as a hemostatic agent, while the aerial part of the plant consisting of leaf and stem is known to exhibit anti-diarrheal properties and has been widely used as a remedy in many parts of China. As rotavirus is one of the most commonly associated diarrhea-causing pathogen, this study aims to investigate the anti-rotaviral effect of R. cordifolia aerial part (RCAP). The cytotoxicity of RCAP toward MA-104 cells was evaluated using the WST-8 assay. Colloidal gold method and real time polymerase chain reaction (qPCR) assay were used to confirm the findings of the antiviral assay. Then, 4',6-diamidino-2-phenylindole (DAPI) staining method was subsequently used to investigate the mode of death among the cells. And the representative components of aqueous extract were isolated and identified. It was shown that both the viability of MA-104 cells and the viral load were reduced with increasing concentration of the extract. DAPI staining showed that virus-induced apoptosis was the cause of the low cell viability and viral load, an effect which was accelerated with incubation in the aqueous herbal extract. The major compounds postulated to exhibit this activity were isolated from the aqueous herbal extract and identified to be compounds Xanthopurpurin and Vanillic Acid. This study showed that RCAP extract effectively inhibited rotavirus multiplication by promoting virus-induced apoptosis in MA-104 cells.

Keywords: Rubia cordifolia; aerial part; antiviral activity; apoptosis; diarrhea; rotavirus.

FIGURE 1

Cytotoxicity of RCAP extract in MA-104 cells. The non-toxic groups were determined by comparing the cell viability. Cell viability was calculated as OD_ex / OD_con × 100%, and OD_con = 1.74 ± 0.06. Data are represented as mean ± S.D. n = 11. ^∗p < 0.05 vs mock; ^∗∗p < 0.01 vs mock by one-way ANOVA test, post hoc Tukey test.

FIGURE 2

Morphological observation of MA-104 cells in antiviral assay. (A) Non-infected cells incubated with RCAP extract; (B) Rotavirus-infected cells treated with RCAP extract. Morphological changes were characterized by rounding and enlargement of the cells and by detachment of the cells from the substrate.

FIGURE 3

Effect of RCAP on cell viability and viral load in antiviral assay. (A) Cell viability of each group in mixed and post treatment assay. Cell viability was calculated as OD_rv / OD_con × 100%, OD_con of mixed treatment assay was 1.11 ± 0.03, OD_con of post treatment assay was 1.18 ± 0.03; (B) Detection of rotavirus in each group using the colloidal gold method. Control line indicates valid testing; test line indicates presence of rotaviruses.

FIGURE 4

Quantification of rotavirus by real time qPCR assay (A) Viral load in each group of mixed treatment assay; (B) Viral load in each group of post treatment assay. 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.

FIGURE 5

Characteristic nuclear fluorescence of MA-104 cells with DAPI staining. (A) Non-infected cells being treated with different concentrations of RCAP extract; (B) Rotavirus-infected cells being treated with different concentrations of RCAP extract. Red arrows correspond to nuclear fragmentation of apoptotic cells.

FIGURE 6

Structures of components isolated and identified from the ethyl acetate fraction of RCAP aqueous extraction. (A) 1,3-dihydroxy-9, 10-anthracenedione (xanthopurpurin); (B) 4-hydroxy-3-methoxybenzoic acid (vanillic acid); (C) 1-(3,4-dihydroxyphenyl)-1,2,3,4-tetrahydro-7-hydroxy-6-methoxy-2, 3-naphthalenedimethanol (isotaxiresinol).