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Silymarin efficacy against influenza A virus replication.

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Keywords: Silymarin Antiviral Influenza RT-PCR

ABSTRACT

The anti-influenza A/PR/8/34 virus activity of silymarin was evaluated in MDCK cells and investigated the effect of silymarin on synthesis of viral mRNAs. Silymarin was investigated for its antiviral activity against influenza A/PR virus using a cytopathic effect (CPE) reduction method. Silymarin exhibited anti-influenza A/PR/8/34 virus activity of 98% with no cytotoxicity at the concentration of 100 pg/ml reducing the formation of a visible CPE. Oseltamivir also did show moderate antiviral activity of about 52% against influenza A/PR/8/34 virus at the concentration of 100u.g/ml. Furthermore, the mechanism of anti-influenza virus action in the inhibition of viral mRNA synthesis was analyzed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), and the data indicated an inhibitory effect in late viral RNA synthesis compared with oseltamivir in the presence of 100pg/ml of silymarin. Therefore, the potential of silymarin for use in treating influenza virus infection merits greater attention.

[C] 2011 Elsevier GmbH. All rights reserved.

Introduction

Influenza A viruses are respiratory pathogens that can raise to severe illness in humans. In the 20th century, three influenza pandemics occurred: the H1N1 Spanish Flu in 1918, the H2N2 Asian Flu inl957 and the H3N2 Hong Kong Flu in 1968 (Kilbourne 2006). The influenza virus infected patients show symptoms of acute respiratory illness, including fever, cough, and headache (Naffakh and van der Werf 2009). In June 2009, pandemic H1N1 virus spread rapidly throughout the world, which warranted the declaration of "Pandemic (HINl) 2009" by the WHO (WHO 2009). The transmis-sibility of pandemic H1N1 virus is higher than that of the seasonal H1N1 (Dong et al. 2009). There had been over 526,060 infected individuals and at least 6770 confirmed human deaths worldwide in November 15. 2009 (WHO 2009).

Oseltamivir is considered the drug of choice for patients with pandemic influenza for whom drug treatment is recommended because adamantanes seem to be ineffective against pandemic A/H1N1 influenza virus and zanamivir (the other available neuraminidase inhibitor) is contraindicated in people with underlying respiratory conditions and difficult to administer in younger children (Garman and Laver 2004).

Unfortunately, the previously documented resistance to oseltamivir of seasonal influenza viruses has now been reported in individuals infected by pandemic A/H1N1 influenza virus (Stephenson et al. 2009; WHO 2009).

Silymarin, a flavonolignan complex isolated from milk thistle (Silybum marianum L. Gaertn), has been clinically used for its beneficial effects on various liver diseases such as alcohol or drug intoxication, mushroom poisoning and viral hepatitis, whose pathogenesis involves an inflammatory response (Kren and Walterova 2005; Sailer et al. 2001). Also, silymarin is a complex mixture of four flavonolignan isomers, namely silybin, isosilybin, silydianin and silychristin with an empirical formula [C.sub.25][H.sub.22][O.sub.10] (Pradhan and Girish 2006). Among the isomers silybin is the major and most active component and represents about 60-70% (Pradhan and Girish 2006).

Although silymarin has been described to possess antioxidant, immunomodulatory, antiproliferative, antifibrotic, and antiviral activities, its anti-influenza virus activity remains to be studied (Sailer etal. 2001).

In this study we examined the antiviral activity of silymarin against influenza A/PR/8/34 virus. Furthermore, to elucidate the effects of silymarin on influenza A/PR/8/34 virus replication, we investigated RT-PCR analysis.

Materials and methods

Viruses, cells and reagents

Influenza A/PR/8/34 virus was provided by ATCC (American Type Culture Collection, Manassas, VA, USA) and propagated in Madin-Darby canine kidney (MDCK) cells at 37 C. MDCK cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 0.01 % antibiotic-antimycotic solution. Antibiotic-antimycotic solution, trypsin-EDTA, FBS and MEM were supplied by Gibco BRL (Grand Island, NY). Tissue culture plates were purchased from Falcon (BD Biosciences, Franklin Lakes, NJ). Sulforhodamine B (SRB) and silymarin were purchased from Sigma-Aldrich (St. Louis. MO). Silymarin is composed of 30% silybin by high performance liquid chromatography (HPLC). All other chemicals were of reagent grade. Oseltamivir (F. Hofmann-La Roche Ltd, Switzerland) was purchased from a pharmacy in Korea as prescribed by a medical doctor. Stock solutions (10 mg/ml) of the antiviral compounds were made in dimethyl sulfoxide (DMSO) and were subsequently diluted in appropriate culture media. The final DMSO concentration was a maximum of 0.1% maximum, which had no effect on the cell cultures. Therefore, 0.1% DMSO was also added to all no-drug control samples.

Assays of antiviral activity and cytotoxicity

Assays of antiviral activity and cytotoxicity were evaluated by the SRB method using cytopathic effect (CPE) reduction recently reported (Choi et al. 2009a,b). Oseltamivir was used as positive, and DMSO was used as negative control.

The effect of silymarin on influenza virus-induced CPE was observed. Briefly, MDCK cells were seeded onto a 96-well culture plate at a concentration of 2 x [10.sup.4] cells per well. Next day, medium was removed and washed with PBS. Then, 0.09 ml of diluted virus suspension and 0.01 ml of medium supplemented with trypsin-EDTA containing silymarin of 100 [micro]-g/ml were added. After incubation at 37 [degrees]C in 5% [CO.sub.2] for 2 days, the morphology of cells was observed under microscope of 32 x 10 magnifications (AXIOVERT10, ZEISS, Germany), and images were recorded.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis

MDCK cells were seeded onto a 96-well culture plate at a concentration of 2 x [10.sup.4] cells per well. After 24 h, medium was removed and the cells were washed with PBS. Subsequently, 0.09 ml of diluted virus suspension and 0.01 ml of medium supplemented with trypsin-EDTA containing silymarin or oseltamivir of 100 [micro] -g/ml were added. After incubation at 37 [degrees]C in 5% [CO.sub.2] for 48 h, the next step was performed.

Total RNA was extracted from cells as described elsewhere (Chomczynski and Sacchi 1987). The parallel expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was tested under the same PCR conditions as an internal standard. Randomly primed cDNA obtained by reverse transcription (RT)-PCR was amplified (Gen-Amp system 2700; Perkin-Elmer, CT, USA) in a PCR mixture (50u-l) that contained membrane (M) or GAPDH gene primers: M gene, 5-AGTGAGCGAGGACTGCAGCGT-3' and 5'-TAGCYTTAGCYGTRGTGCTGGC-3'; GAPDH, 5'-CCCA-TCACCATCTTCCAGGAGC-3' and 5'-CCAGTGAGCTTCCCTTCAGC-3'. The products were electrophoresed and visualized by ethidium bromide staining.

Results and discussion

Antiviral activity of silymarin against influenza A/PR/8/34 virus

Silymarin was investigated for its antiviral activity against influenza A/PR/8/34 virus and MCDK cell viability. The antiviral assays demonstrated that silymarin possessed strong antiviral activity of about 98% against influenza A/PR/8/34 virus at the concentration of lOO [micro] -g/ml and antiviral activity of about 45% at the same virus at the concentration 100 [micro] -g/ml (Fig. 1). Oseltamivir also did show moderate antiviral activity of about 52% against influenza A/PR/8/34 virus at the concentration of 100 [micro] -g/ml and weak antiviral activity of less than 40% at under of 100 [micro] -g/ml and oseltamivir were not toxic to MDCK cells with cell viability of about 100% at the concentration of 100 [micro] -g/ml (Fig. 1).

[FIGURE 1 OMITTED]

The effect of silymarin on influenza A/PR/8/34 virus-induced CPE

After 2 day infections of MDCK cells with influenza A/PR/8/34 virus, there was no difference between the mock cells (Fig. 2A) or cells treated with 100 [micro].g/ml silymarin (Fig. 2C) or oseltamivir (Fig. 2E) in terms of typical spread-out shapes and normal morphology. The cell proliferation was not significantly affected under the 100 [micro] -g/ml concentrations of silymarin. Infection with influenza A/PR/8/34 virus in the absence of silymarin resulted in a severe CPE (Fig. 2B). The addition of silymarin on infected MDCK cells inhibited the formation of a visible CPE (Fig. 2D). However, the addition of oseltamivir in influenza A/PR/8/34 virus-infected MDCK cell was weakly prevented CPE (Fig. 2F). Thus, this result showed that the CPE of the virus infection was prevented by above-mentioned concentrations of silymarin.

[FIGURE 2 OMITTED]

Effect of silymarin on synthesis of viral mRNAs

The viral mRNA syntheses assessed by PCR analysis of the M gene were inhibited completely at silymarin 100 [micro].g/ml after 48 h infection, while oseltamivir (100 [micro] -g/ml) exhibited a small amount of the viral mRNA synthesis product band at 48 h after infection (Fig. 3). The amplification of house keeping gene GAPDH from the same sample was positive in the PCR analyses (Fig. 3). These findings indicate that silymarin does decrease viral mRNA synthesis.

[FIGURE 3 OMITTED]

The development of new antiviral agents for influenza is receiving much greater attention because of the frequent emergence of antiviral resistance during oseltamivir, its association with clinical failure in immunocompromised hosts, and the emergence of new pandemic subtypes of influenza A virus (Beigel and Bray 2008; Hayden 2001, 2004; Ison et al. 2006).

The present study describes the cytotoxicity and antiviral activity of silymarin. Silymarin was shown to exhibit anti-influenza A/PR/8/34 virus activity against influenza A/PR/8/34 virus reducing the formation of a visible CPE. These results are similar to the effects of quercetin 3-rhamnoside on influenza virus (Choi et al. 2009a,b).

To provide information whether the silymarin prevent viral mRNA synthesis. RT-PCR analysis was performed with silymarin. The data indicated an inhibitory effect in late viral RNA synthesis compared with oseltamivir in the presence of 100[micro]g/ml of silymarin (Fig. 3). Similar result has been reported previously (Choi et al. 2009a,b).

In conclusion, silymarin is a mixture of small molecules that can efficiently inhibit influenza A/PR/8/34 virus replication. Therefore, silymarin is an alternative to agents for treating influenza virus infections. Their potent anti-influenza virus activity in vitro warrants further studies to evaluate whether silymarin treatment can also result in antiviral activity in vivo.

Acknowledgments

No conflict to disclose.

References

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J.H. Song (a), H J. Choib (b)

(a) Department of Herbal Resources, Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan 570-749, Republic of Korea.

(b) Daejeon Health Sciences College, 77-3 Gayang2-Dong, Dong-Gu, Daejeon 300-711, Republic of Korea

* Corresponding author. Tel.: +82 42 670 9369: fax: +82 426709166.

E-mail address: rerived@empal.com (H.J. Choi).

doi:10.1016/j.phymed.2011.01.026
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Author:Song, J.H.; Choib, H J.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9SOUT
Date:Jul 15, 2011
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