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

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.


Background
In 1918, the Spanish influenza A (H1N1) virus pandemic caused 50 million deaths worldwide [1,2]. In 2009, influenza A virus originating in swine (H1N1) caused a pandemic, and the avian H5N1 and H7N9 influenza A viruses in China are highly pathogenic to humans [1][2][3][4]. Currently, the application of three antiviral medicines known as neuraminidase (NA) inhibitors, oral oseltamivir, zanamivir, and peramivir, is recommended for the treatment of influenza. However, oseltamivir resistance has been detected in some of the 2009-derived H1N1 viruses and the seasonal H1N1 viruses between 2007 and 2009, but little in H3N2 viruses [5]. In the future, zanamivir-and peramivir-resistant strains, similar to oseltamivir-resistant strain, will emerge. Therefore, the development of novel anti-influenza drugs to prevent and control future influenza epidemics and pandemics is desired.
Traditional medicinal plants have been recognized as a rich source of candidate compounds for the development of pharmaceuticals [6,7]. A large number of natural products and extracts from medicinal plants have been reported to possess anti-influenza virus activity [8][9][10]. Therefore, many studies have focused on traditional medicinal plants as an important source of candidate compounds for the discovery of novel anti-influenza drugs.
The abundance of medicinal plants in Myanmar has enabled the population to use traditional medicines to maintain their own health and treat various diseases. Thus, to discover sources for novel anti-influenza drugs, we screened extracts from 118 medicinal plants collected in Myanmar to analyze the cell viability of influenza A H1N1 virus (A/PR/8/34)-infected MDCK cells using naphthol blue black staining. We identified six medicinal plants that promoted the survival of influenza A virus-infected cells selected by the criteria described at the Methods section. Of these six plants, the activity of extract from the stems of Jatropha multifida Linn (J. multifida) was strongly pronounced. J. multifida, a member of the family Euphorbiaceae, is a tree of 2-3 m in height, and widely distributed in sub-tropical and tropical areas throughout Asia and Africa [11]. Popularly known as "Say-ma-khan", it is commonly used as a folk medicine in Myanmar and has been used as a purgative, and against fever, indigestion, colic, wounds, and skin infection [11]. The seed oil, latex, and leaves are effective purgatives and abortifacients, have been used as wound dressings, and for the treatment of neurodermatitis, eczema, and itches [11]. The roots and stems have antimicrobial, antimalarial, antitumor, antileishmanial, and antiulcer activities [11,12]. In addition, previous phytochemical studies on J. multifida reported the presence of cyclic peptides, diterpenoids, and phenolic compounds [11]. However, pharmacological and phytochemical investigations of J. multifida stems originating from Myanmar have not yet been conducted, which attracted us to investigate whether extracts from the stems of J. multifida, obtained using various solvents, possessed anti-influenza virus activity.

Plant material
The stems of J. multifida were purchased from Sandhi Brothers Trading Co. Ltd (Yangon, Myanmar) in November 2015. A voucher specimen (TMPW 28729) was deposited at the Museum of Materia Medica, Analytical Research Center for Ethnomedicines, Institute of Natural Medicine, University of Toyama, Japan.

Plant extraction
We performed the plant extraction as described previously [13,14]. In brief, dried stems of J. multifida were chopped into small pieces (3.0 kg), which were macerated four times with 70% aqueous EtOH (7 L) in an ultrasonic bath for 90 min at 25°C. After filtration of the suspension, the resulting solution was evaporated under reduced pressure to yield a crude extract (180 g). The crude extract was suspended in water and partitioned into n-hexane (Hex), chloroform (CHCl 3 ), and ethyl acetate (EtOAc) fractions, to yield Hex-soluble (12.2 g), CHCl 3 -soluble (11.0 g), and EtOAc-soluble portions (11.0 g), respectively. Finally, the residual aqueous layer was evaporated under reduced pressure to yield a water (H 2 O)-soluble portion (140.8 g). The extracts were stored at a concentration of 10 mg/mL in dimethyl sulfoxide (DMSO).

Viral strain
This study used the Puerto Rico 8/34 (A/PR/8/34; H1N1) strain of the influenza A virus. Viral titers were determined by immunostaining influenza A viral nucleoprotein (NP) as previously described [16].

Analysis of cell viability of influenza A virus-infected MDCK cells using naphthol blue black staining
MDCK cells were seeded in a 96-well plate (1 × 10 4 cells/well). H 2 O, EtOAc, Hex, or CHCl 3 extracts from the stems of J. multifida (0.8-25 μg/mL in DMSO) were mixed with influenza A virus in 10% FBS-supplemented growth medium at a multiplicity of infection (MOI) of 10, and then incubated for 30 min at 37°C in the presence of 5% CO 2 . DMSO (0.008-0.5%) and (+)-(S)-bakuchiol (0.8-25 μM in DMSO) were used as negative and positive controls, respectively, for the inhibition of influenza A viral infection [15]. The mixture was added to the cells, which were then incubated for 4 days at 37°C in the presence of 5% CO 2 . After incubation, the cells were stained using naphthol blue black as previously described [15,17]. The viable cells in each well were stained blue, while dead cells remained unstained. The selection criteria for the six plants are more than 50% cell survival at 96 h after the viral infection with the concentration of 50 μg/mL.

Influenza A viral growth assay
To explore whether the extracts from the stems of J. multifida affected viral growth in pre-infected cells, MDCK cells were seeded in a 24-well plate (1 × 10 5 cells/well). The cells were infected with A/PR/8/34 (MOI; 0.001) in infection medium for 1 h at 37°C in the presence of 5% CO 2 . The infected cells were washed prior to the addition of H 2 O, EtOAc, Hex, or CHCl 3 J. multifida extracts (12.5 or 25 μg/mL in 0.5% DMSO) to the cells in infection medium supplemented with 3 μg/ mL L-tosylamido-2-phenyl ethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich). DMSO (0.5%) and ribavirin (50 μM in 0.5% DMSO) were the negative and positive controls, respectively, for the inhibition on influenza A viral growth [18]. The cells were then incubated for 24, 48, or 72 h at 37°C in the presence of 5% CO 2 . Cell culture media were collected from each well at predetermined time points. Viral titers (plaque forming units per mL [PFU/mL]) were determined as previously described [15].

Statistical analysis
All results were expressed as the mean ± the standard error of the mean (SEM). Differences between more than two groups were analyzed for statistical significance by using one-way analysis of variance (ANOVA). Values of p < 0.05 were considered statistically significant.

Results
Extracts from the stems of J. multifida increased the survival of influenza A viral-infected MDCK cells To evaluate the anti-influenza viral activity of extracts from the stems of J. multifida, we first examined the survival of influenza A virus-infected MDCK cells after treatment with the H 2 O, EtOAc, Hex, or CHCl 3 extracts from the stems of J. multifida. As shown in Fig. 1, cells exposed to DMSO and infected with A/PR/8/34 were not stained. However, cells treated with 3.1-25 μM (+)-(S)-bakuchiol or 3.1-25 μg/mL H 2 O extract and infected with A/PR/8/34 were stained blue. Cells exposed to 25 μg/mL EtOAc or 12.5-25 μg/mL CHCl 3 extract and infected with A/PR/8/34 were also weakly stained blue (Fig. 1).
To evaluate cytotoxicity, we determined the viability of MDCK cells after incubation for 24, 72, or 96 h in infection medium containing BSA using the MTT assay (Fig. 2). The viability of MDCK cells treated with H 2 O, EtOAc, Hex, or CHCl 3 extract from the stems of J. multifida was unaffected after 24 h, compared with cells exposed to DMSO only (Fig. 2a). After 72 or 96 h of incubation, the viability of MDCK cells treated with 100 μg/mL H 2 O or 12.5-100 μg/mL CHCl 3 extracts significantly reduced (Fig. 2b), whereas the viability of cells exposed to ≤ 50 μg/mL H 2 O, ≤ 100 μg/mL EtOAc and Hex, or ≤ 12.5 μg/mL CHCl 3 extracts was unaffected compared with cells exposed to DMSO only (Fig. 2c). Therefore, these data suggested that exposure to ≤ 100 μg/mL H 2 O, EtOAc, Hex, or CHCl 3 extract for 24 h, or ≤ 50 μg/mL H 2 O, ≤ 100 μg/mL EtOAc and Hex, or ≤ 12.5 μg/mL CHCl 3 extracts for 72 or 96 h was not cytotoxic in MDCK cells.
Together, these results proved that the H 2 O extract from the stems of J. multifida promoted the survival of MDCK cells infected with the influenza A H1N1 virus. The EtOAc and CHCl 3 extracts demonstrated similar, but weaker, effects.

The extracts inhibited influenza A viral infection and growth
To investigate whether the extracts inhibited viral infection, we examined viral NP-immunofluorescence staining in MDCK cells treated with a mixture of virus and H 2 O, EtOAc, Hex, or CHCl 3 extract for 24 h. The wells were observed under a microscope and photographed (Fig. 3a). NP-immunostained cells were counted, and the percentage of influenza A NP-positive cells per DAPIpositive cells was calculated (Fig. 3b). The percentage of influenza A NP-positive cells was significantly decreased in a concentration-dependent manner in samples treated with H 2 O, EtOAc, or CHCl 3 extract or (+)-(S)-bakuchiol (positive control), compared with DMSO-treated cells ( Fig. 3a and b). The H 2 O extract produced greater inhibition of influenza A viral infection than the other extracts. These data proved the inhibitory effect of the Next, we investigated the inhibition of viral growth by the H 2 O, EtOAc, Hex, or CHCl 3 extract for 24-72 h in virus-infected MDCK cells. The viral titers in conditioned media from samples treated with CHCl 3 extract between 48 and 72 h, EtOAc or Hex extracts at 72 h, and ribavirin between 24-72 h were significantly decreased compared with those in media conditioned by DMSO-treated cells (Fig. 4a). However, owing to the cytotoxicity observed following a 72-h exposure of MDCK cells to 25 μg/mL CHCl 3 extract in the viral growth experiment (Fig. 2b), we repeated the experiment with 12.5 μg/mL CHCl 3 extract. The viral titers in the conditioned media from cells treated with 12.5 μg/mL CHCl 3 extract at 48 and 72 h significantly decreased compared with those in the media conditioned by DMSO-treated cells (Fig. 4b). These data proved that the EtOAc, Hex, and CHCl 3 extracts inhibited the growth of influenza A H1N1 virus, and that the CHCl 3 extract possessed the strongest activity.
Together, these results demonstrated the inhibition of influenza A viral infection and growth by extracts from the stems of J. multifida. The variation in the inhibitory effects of the extracts may be due to the polarities of the phytochemical constituents present in the stems.

Discussion
In the present study, the H 2 O extract from the stems of J. multifida strongly increased survival of influenza A virus-infected MDCK cells (Fig. 1) and inhibited influenza A viral infection (Fig. 3), whereas the CHCl 3 extract demonstrated the strongest inhibition of influenza A viral growth (Fig. 4) compared with the other crude extracts. These results indicated that the different polarities of the H 2 O and CHCl 3 crude extracts produced inhibition of viral infection or growth by different mechanisms. Infection with the influenza virus begins with binding to the surface of a host cell by viral hemagglutinin (HA), viral surface protein [19]. The influenza virus invades host cells by endocytosis, and the viral genome is then released into the host's cytoplasm through fusion of the viral membrane with the host endosomal membrane via HA cleavage. The influenza viral genome replicates in the host nucleus using viral RNA polymerase. The virions bud and are released from the membrane of the host cell using viral NA. In the cell viability and viral infection assays, the H 2 O extract from the stems of J. multifida inhibited influenza A viral binding to host cells when the influenza virus and extracts were co-incubated in advance. In contrast, in the viral growth assay, the CHCl 3 extract inhibited influenza A viral replication in host cells. We therefore propose that the H 2 O extract may include compounds that inhibit influenza A viral binding to host cells surface, endocytosis, membrane fusion, or uncoating by inhibiting viral HA, while the CHCl 3 extract may include compounds that inhibit influenza viral replication in host cells by inhibiting viral RNA polymerase or NA activities.
Previously, chemical studies of the stems of J. multifida led to the isolation of lathyrane-type diterpenoids [20][21][22][23][24], jatrophane-type diterpenoids [11,22], and coumarino-type lignoids [22]. The lathyrane-type diterpenoids, multifidone, multifidanol, and multifidenol, showed cytotoxicity and antibacterial activity [24]. The jatrophane-type diterpenoid, jatrophone, was reported to possess a wide range of biological effects such as 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. H 2 O, EtOAc, Hex, or CHCl 3 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 CHCl 3extract 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 cytotoxicity and antitumor activity [25,26]. However, the anti-influenza activity of the phytochemical constituents of J. multifida has not been yet investigated. The lathyrane-type diterpenoids from Euphorbia micractina showed the anti-HIV activity [27]. Dang et al. recently reported that the abietane-type tricyclic phenolic diterpenoids, (+)-podcarpic acid and (+)-totarol, inhibited influenza A H1N1 viral infection (A/PR/8/34) [28]. Therefore, the diterpenoids contained in J. multifida may confer the anti-viral effects such as anti-influenza and anti-HIV activities. It is expected that anti-influenza compounds will be isolated from the active crude extracts in our ongoing work.

Conclusions
The findings of the present study demonstrated that the most polar extract, the H 2 O extract, from the stems of J. multifida increased survival of MDCK cells infected with the influenza A H1N1 virus and showed the strongest inhibition of influenza A H1N1 viral infection in MDCK cells. Of the EtOAc, Hex, and CHCl 3 extracts, the CHCl 3 extract showed the strongest inhibition of influenza A H1N1 viral growth in MDCK cells. These results indicated that the stems of J. multifida could be used as a herbal medicine for the treatment of influenza and may be a source of candidate compounds for novel antiinfluenza drug development.