Anti-influenza virus activity of extracts from the stems of Jatropha multifida Linn. collected in Myanmar

Abstract

Background: To contribute to the development of novel anti-influenza drugs, we investigated the anti-influenza activity of crude extracts from 118 medicinal plants collected in Myanmar. We discovered that extract from the stems of Jatropha multifida Linn. showed anti-influenza activity. J. multifida has been used in traditional medicine for the treatment of various diseases, and the stem has been reported to possess antimicrobial, antimalarial, and antitumor activities. However, the anti-influenza activity of this extract has not yet been investigated.

Methods: We prepared water (H₂O), ethyl acetate (EtOAc), n-hexane (Hex), and chloroform (CHCl₃) extracts from the stems of J. multifida collected in Myanmar, and examined the survival of Madin-Darby canine kidney (MDCK) cells infected with the influenza A (H1N1) virus, and the inhibitory effects of these crude extracts on influenza A viral infection and growth in MDCK cells.

Results: The H₂O extracts from the stems of J. multifida promoted the survival of MDCK cells infected with the influenza A H1N1 virus. The EtOAc and CHCl₃ extracts resulted in similar, but weaker, effects. The H₂O, EtOAc, and CHCl₃ extracts from the stems of J. multifida inhibited influenza A virus H1N1 infection; the H₂O extract possessed the strongest inhibitory effect on influenza infection in MDCK cells. The EtOAc, Hex, and CHCl₃ extracts all inhibited the growth of influenza A H1N1 virus, and the CHCl₃ extract demonstrated the strongest activity in MDCK cells.

Conclusion: The H₂O or CHCl₃ extracts from the stems of J. multifida collected in Myanmar demonstrated the strongest inhibition of influenza A H1N1 viral infection or growth in MDCK cells, respectively. These results indicated that the stems of J. multifida could be regarded as an anti-influenza herbal medicine as well as a potential crude drug source for the development of anti-influenza compounds.

Keywords: Anti-influenza; Anti-virus; Herbal medicine; Jatropha multifida; Stem.

Fig. 1

Effect of extracts from the stems of J. multifida on the viability of MDCK cells infected with influenza A H1N1 virus. H₂O, EtOAc, Hex, and CHCl₃ extracts from the stems of J. multifida (0.8-25 μg/mL in DMSO) were mixed with or without (virus-) influenza A H1N1 virus (A/PR/8/34) (MOI; 10), and added to MDCK cells. DMSO (0.008-0.5%) and (+)-(S)-bakuchiol (bakuchiol; 0.8-25 μM in DMSO) were used as negative and positive controls, respectively, for the inhibition of influenza A viral infection. After incubation for 4 days, cell viability was determined by naphthol blue black staining. Data are representative of three independent experiments, and the results were found to be reproducible

Fig. 2

Toxicity of extracts from the stems of J. multifida to MDCK cells. H₂O, EtOAc, Hex, and CHCl₃ extracts from the stems of J. multifida (12.5-100 μg/mL) in DMSO (concentrations of DMSO: 12.5 μg/mL, 0.125%; 25 μg/mL, 0.25%; 50 μg/mL, 0.5%; 100 μg/mL, 1%) were added to the MDCK cells. Cell viabilities were determined via MTT assay after 24 h (n = 6 each) (a), 72 h (n = 6 each) (b), and 96 h (n = 6 each) (c). Data are the mean ± SEM representative of two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 in comparison with DMSO

Fig. 3

Extracts from the stems of J. multifida inhibited influenza A H1N1 viral infection. H₂O, EtOAc, Hex, or CHCl₃ extracts from the stems of J. multifida (3.1-25 μg/mL) (n = 9 each) or (+)-(S)-bakuchiol (bakuchiol; 3.1-25 μM) (n = 9) were mixed with influenza A virus (A/PR/8/34) at a MOI of 0.1 and added to MDCK cells. DMSO (0.031-0.25%) (n = 9) was used as the negative control. After 24 h, the cells were fixed and permeabilized. To visualize influenza A virus-infected cells, we performed immunofluorescent staining of influenza A viral NP (green) and cell nuclei (blue), using the nuclear-staining compound, DAPI. Cells were subsequently photographed under a fluorescence microscope (a), and the percentage of influenza A viral NP-positive cells per DAPI-positive cells was calculated based on influenza A viral NP-positive and DAPI-positive cell numbers (b). The white scale bar in each image represents 100 μm. Data are presented as means ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 in comparison with DMSO

Fig. 4

Extracts from the stems of J. multifida inhibited influenza A H1N1 viral growth. MDCK cells were infected with A/PR/8/34 (MOI; 0.001) for 1 h, and then the infected cells were washed. H₂O, EtOAc, Hex, or CHCl₃ extracts from the stems of J. multifida (25 μg/mL in 0.5% DMSO) (n = 7 each) (a) were added to the cells in the infection medium supplemented with 3 μg/mL TPCK-treated trypsin. DMSO (0.5%) (n = 7) or ribavirin (50 μM in 0.5% DMSO) (n = 7) were the negative and positive controls, respectively, for the inhibition of influenza A viral growth. In addition, the same experiment was performed with DMSO (0.5%), ribavirin (50 μM in 0.5% DMSO), and 12.5 μg/mL CHCl₃extract from the stems of J. multifida (in 0.5% DMSO) (n = 12 each) (b). The conditioned culture medium was collected at the indicated time points, added to MDCK cells, and the treated cells were immunostained with an antibody to influenza A viral NP. The viral titers (PFU/mL) were calculated from the number of stained cells. The data are the mean ± SEM representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 in comparison with DMSO