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Bakuchiol Is a Phenolic Isoprenoid with Novel Enantiomer-selective Anti-influenza A Virus Activity Involving Nrf2 Activation*

  • Masaki Shoji
    Affiliations
    Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan
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  • Yumie Arakaki
    Affiliations
    Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan
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  • Tomoyuki Esumi
    Affiliations
    Institute of Pharmacognosy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan
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  • Shuntaro Kohnomi
    Affiliations
    Department of Neurophysiology, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Kagawa 769-2193, Japan
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  • Chihiro Yamamoto
    Affiliations
    Institute of Pharmacognosy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan
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  • Yutaka Suzuki
    Affiliations
    Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8568, Japan
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  • Etsuhisa Takahashi
    Affiliations
    Division of Enzyme Chemistry, Institute for Enzyme Research, Tokushima University, Tokushima 770-8503, Japan
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  • Shiro Konishi
    Affiliations
    Department of Neurophysiology, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Kagawa 769-2193, Japan
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  • Hiroshi Kido
    Affiliations
    Division of Enzyme Chemistry, Institute for Enzyme Research, Tokushima University, Tokushima 770-8503, Japan
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  • Takashi Kuzuhara
    Correspondence
    To whom correspondence should be addressed: Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan. Tel.: 81-88-602-8477; Fax: 81-88-655-3051
    Affiliations
    Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan
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  • Author Footnotes
    * This work was supported by Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (C) 25460574 (to T. K.), Grant-in-Aid for Young Scientists (B) 26860083 (to M. S.), Tokushima Bunri University for Educational Reform and Collaborative Research Grant TBU2013-2-1 (to M. S.), and the Research Program on Emerging and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development, AMED (to H. K.). The authors declare that they have no conflicts of interest with the contents of this article.
    This article contains supplemental Tables 1 and 2.
Open AccessPublished:October 07, 2015DOI:https://doi.org/10.1074/jbc.M115.669465
      Influenza represents a substantial threat to human health and requires novel therapeutic approaches. Bakuchiol is a phenolic isoprenoid compound present in Babchi (Psoralea corylifolia L.) seeds. We examined the anti-influenza viral activity of synthetic bakuchiol using Madin-Darby canine kidney cells. We found that the naturally occurring form, (+)-(S)-bakuchiol, and its enantiomer, (−)-(R)-bakuchiol, inhibited influenza A viral infection and growth and reduced the expression of viral mRNAs and proteins in these cells. Furthermore, these compounds markedly reduced the mRNA expression of the host cell influenza A virus-induced immune response genes, interferon-β and myxovirus-resistant protein 1. Interestingly, (+)-(S)-bakuchiol had greater efficacy than (−)-(R)-bakuchiol, indicating that chirality influenced anti-influenza virus activity. In vitro studies indicated that bakuchiol did not strongly inhibit the activities of influenza surface proteins or the M2 ion channel, expressed in Chinese hamster ovary cells. Analysis of luciferase reporter assay data unexpectedly indicated that bakuchiol may induce some host cell factor(s) that inhibited firefly and Renilla luciferases. Next generation sequencing and KeyMolnet analysis of influenza A virus-infected and non-infected cells exposed to bakuchiol revealed activation of transcriptional regulation by nuclear factor erythroid 2-related factor (Nrf), and an Nrf2 reporter assay showed that (+)-(S)-bakuchiol activated Nrf2. Additionally, (+)-(S)-bakuchiol up-regulated the mRNA levels of two Nrf2-induced genes, NAD(P)H quinone oxidoreductase 1 and glutathione S-transferase A3. These findings demonstrated that bakuchiol had enantiomer-selective anti-influenza viral activity involving a novel effect on the host cell oxidative stress response.

      Introduction

      An influenza A pandemic caused 50 million deaths worldwide in 1918 (
      • Horimoto T.
      • Kawaoka Y.
      Influenza: lessons from past pandemics, warnings from current incidents.
      ,
      • Itoh Y.
      • Shinya K.
      • Kiso M.
      • Watanabe T.
      • Sakoda Y.
      • Hatta M.
      • Muramoto Y.
      • Tamura D.
      • Sakai-Tagawa Y.
      • Noda T.
      • Sakabe S.
      • Imai M.
      • Hatta Y.
      • Watanabe S.
      • Li C.
      • Yamada S.
      • Fujii K.
      • Murakami S.
      • Imai H.
      • Kakugawa S.
      • Ito M.
      • Takano R.
      • Iwatsuki-Horimoto K.
      • Shimojima M.
      • Horimoto T.
      • Goto H.
      • Takahashi K.
      • Makino A.
      • Ishigaki H.
      • Nakayama M.
      • Okamatsu M.
      • Takahashi K.
      • Warshauer D.
      • Shult P.A.
      • Saito R.
      • Suzuki H.
      • Furuta Y.
      • Yamashita M.
      • Mitamura K.
      • Nakano K.
      • Nakamura M.
      • Brockman-Schneider R.
      • Mitamura H.
      • Yamazaki M.
      • Sugaya N.
      • Suresh M.
      • Ozawa M.
      • Neumann G.
      • Gern J.
      • Kida H.
      • Ogasawara K.
      • Kawaoka Y.
      In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses.
      ), the influenza A virus that originated in swine (H1N1) caused a pandemic in 2009, and avian H5N1 and H7N9 influenza A viruses are highly pathogenic to humans (
      • Horimoto T.
      • Kawaoka Y.
      Influenza: lessons from past pandemics, warnings from current incidents.
      ,
      • Itoh Y.
      • Shinya K.
      • Kiso M.
      • Watanabe T.
      • Sakoda Y.
      • Hatta M.
      • Muramoto Y.
      • Tamura D.
      • Sakai-Tagawa Y.
      • Noda T.
      • Sakabe S.
      • Imai M.
      • Hatta Y.
      • Watanabe S.
      • Li C.
      • Yamada S.
      • Fujii K.
      • Murakami S.
      • Imai H.
      • Kakugawa S.
      • Ito M.
      • Takano R.
      • Iwatsuki-Horimoto K.
      • Shimojima M.
      • Horimoto T.
      • Goto H.
      • Takahashi K.
      • Makino A.
      • Ishigaki H.
      • Nakayama M.
      • Okamatsu M.
      • Takahashi K.
      • Warshauer D.
      • Shult P.A.
      • Saito R.
      • Suzuki H.
      • Furuta Y.
      • Yamashita M.
      • Mitamura K.
      • Nakano K.
      • Nakamura M.
      • Brockman-Schneider R.
      • Mitamura H.
      • Yamazaki M.
      • Sugaya N.
      • Suresh M.
      • Ozawa M.
      • Neumann G.
      • Gern J.
      • Kida H.
      • Ogasawara K.
      • Kawaoka Y.
      In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses.
      ,
      • Neumann G.
      • Noda T.
      • Kawaoka Y.
      Emergence and pandemic potential of swine-origin H1N1 influenza virus.
      ). Although neuraminidase (NA)
      The abbreviations used are: NA
      neuraminidase
      MDCK
      Madin-Darby canine kidney
      Mx1
      myxovirus-resistant protein 1
      NGS
      next generation sequencing
      NQO1
      NAD(P)H quinone oxidoreductase 1
      Nrf2
      nuclear factor erythroid 2-related factor 2
      NP
      nucleoprotein
      MOI
      multiplicity of infection
      TPCK
      l-(tosylamido-2-phenyl) ethyl chloromethyl ketone
      NS1
      nonstructural protein 1
      rRNA
      ribosomal RNA
      CA
      cytomegalovirus enhancer
      RdRp
      RNA-dependent RNA polymerase
      RLU
      relative light units
      RPKM
      reads per kilobase per million mapped reads
      MTT
      thiazolyl blue tetrazolium bromide
      ME
      marchantin E
      RT-qPCR
      reverse transcription quantitative polymerase chain reaction.
      inhibitors of the influenza virus have been widely used as antiviral drugs (
      • De Clercq E.
      Antiviral agents active against influenza A viruses.
      ,
      • Hayden F.G.
      • Atmar R.L.
      • Schilling M.
      • Johnson C.
      • Poretz D.
      • Paar D.
      • Huson L.
      • Ward P.
      • Mills R.G.
      Use of the selective oral neuraminidase inhibitor oseltamivir to prevent influenza.
      ), adverse effects (
      • Kitching A.
      • Roche A.
      • Balasegaram S.
      • Heathcock R.
      • Maguire H.
      Oseltamivir adherence and side effects among children in three London schools affected by influenza A(H1N1)v, May 2009 - an internet-based cross-sectional survey.
      ,
      • L'Huillier A.G.
      • Ing Lorenzini K.
      • Crisinel P.A.
      • Rebsamen M.C.
      • Fluss J.
      • Korff C.M.
      • Barbe R.P.
      • Siegrist C.A.
      • Dayer P.
      • Posfay-Barbe K.M.
      • Desmeules J.A.
      • H1N1 Pediatric Epidemiology Study Group of Geneva
      ABCB1 polymorphisms and neuropsychiatric adverse events in oseltamivir-treated children during influenza H1N1/09 pandemia.
      ,
      • Chung S.
      • Joung Y.S.
      Oseltamivir (tamiflu) induced depressive episode in a female adolescent.
      ,
      • Hiasa M.
      • Isoda Y.
      • Kishimoto Y.
      • Saitoh K.
      • Kimura Y.
      • Kanai M.
      • Shibasaki M.
      • Hatakeyama D.
      • Kirino Y.
      • Kuzuhara T.
      Inhibition of MAO-A and stimulation of behavioural activities in mice by the inactive prodrug form of the anti-influenza agent oseltamivir.
      ) and the emergence of resistant viral strains (
      • Reece P.A.
      Neuraminidase inhibitor resistance in influenza viruses.
      ,
      • Collins P.J.
      • Haire L.F.
      • Lin Y.P.
      • Liu J.
      • Russell R.J.
      • Walker P.A.
      • Skehel J.J.
      • Martin S.R.
      • Hay A.J.
      • Gamblin S.J.
      Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants.
      ) have been reported. Thus, to prevent and to control future influenza epidemics and pandemics, it is critically important that novel anti-influenza drugs be developed.
      (+)-(S)-Bakuchiol is a naturally occurring phenolic isoprenoid (Fig. 1A) with a chiral tetra-alkylated (all-carbon) quaternary center that has been isolated from the seeds of Psoralea corylifolia L.; this plant (known as Babchi) is used in Chinese and Indian traditional medicine systems to treat a range of diseases (
      • Mehta G.
      • Ramdas Nayak U.
      • Dev S.
      Bakuchiol, a novel monoterpenoid.
      ), such as inflammation due to Acne vulgaris (
      • Poláková K.
      • Fauger A.
      • Sayag M.
      • Jourdan E.
      A dermocosmetic containing bakuchiol, Ginkgo biloba extract, and mannitol improves the efficacy of adapalene in patients with acne vulgaris: result from a controlled randomized trial.
      ), breast and lung cancers (
      • Chen H.L.
      • Feng H.J.
      • Li Y.C.
      [Vitro antitumor activity and synthesis of the key intermediate of bakuchiol].
      ,
      • Chen Z.
      • Jin K.
      • Gao L.
      • Lou G.
      • Jin Y.
      • Yu Y.
      • Lou Y.
      Anti-tumor effects of bakuchiol, an analogue of resveratrol, on human lung adenocarcinoma A549 cell line.
      ), bone loss (
      • Lim S.H.
      • Ha T.Y.
      • Kim S.R.
      • Ahn J.
      • Park H.J.
      • Kim S.
      Ethanol extract of Psoralea corylifolia L., and its main constituent, bakuchiol, reduce bone loss in ovariectomised Sprague-Dawley rats.
      ), neurological disorders (
      • Zhao G.
      • Zang S.Y.
      • Zheng X.W.
      • Zhang X.H.
      • Guo L.H.
      Bakuchiol analogs inhibit monoamine transporters and regulate monoaminergic functions.
      ), and oxidative stress (
      • Kim K.A.
      • Shim S.H.
      • Ahn H.R.
      • Jung S.H.
      Protective effects of the compounds isolated from the seed of Psoralea corylifolia on oxidative stress-induced retinal damage.
      ). (+)-(S)-Bakuchiol can be chemically synthesized from (E)-geranic acid in four steps (
      • Esumi T.
      • Yamamoto C.
      • Fukuyama Y.
      A short synthesis of (+)-bakuchiol.
      ). Bakuchiol has been reported to possess a range of biological and pharmacological activities, including anti-microbial (
      • Katsura H.
      • Tsukiyama R.I.
      • Suzuki A.
      • Kobayashi M.
      In vitro antimicrobial activities of bakuchiol against oral microorganisms.
      ), antioxidant (
      • Haraguchi H.
      • Inoue J.
      • Tamura Y.
      • Mizutani K.
      Inhibition of mitochondrial lipid peroxidation by Bakuchiol, a meroterpene from Psoralea corylifolia.
      ,
      • Adhikari S.
      • Joshi R.
      • Patro B.S.
      • Ghanty T.K.
      • Chintalwar G.J.
      • Sharma A.
      • Chattopadhyay S.
      • Mukherjee T.
      Antioxidant activity of bakuchiol: experimental evidences and theoretical treatments on the possible involvement of the terpenoid chain.
      ), anti-inflammatory (
      • Pae H.O.
      • Cho H.
      • Oh G.S.
      • Kim N.Y.
      • Song E.K.
      • Kim Y.C.
      • Yun Y.G.
      • Kang C.L.
      • Kim J.D.
      • Kim J.M.
      • Chung H.T.
      Bakuchiol from Psoralea corylifolia inhibits the expression of inducible nitric oxide synthase gene via the inactivation of nuclear transcription factor-κB in RAW 264.7 macrophages.
      ,
      • Lee S.W.
      • Yun B.R.
      • Kim M.H.
      • Park C.S.
      • Lee W.S.
      • Oh H.M.
      • Rho M.C.
      Phenolic compounds isolated from Psoralea corylifolia inhibit IL-6-induced STAT3 activation.
      ), and anti-tumor (
      • Chen Z.
      • Jin K.
      • Gao L.
      • Lou G.
      • Jin Y.
      • Yu Y.
      • Lou Y.
      Anti-tumor effects of bakuchiol, an analogue of resveratrol, on human lung adenocarcinoma A549 cell line.
      ,
      • Sun N.J.
      • Woo S.H.
      • Cassady J.M.
      • Snapka R.M.
      DNA polymerase and topoisomerase II inhibitors from Psoralea corylifolia.
      ) effects. Bakuchiol was shown to inhibit mitochondrial lipid peroxidation (
      • Haraguchi H.
      • Inoue J.
      • Tamura Y.
      • Mizutani K.
      Inhibition of mitochondrial lipid peroxidation by Bakuchiol, a meroterpene from Psoralea corylifolia.
      ) and to induce apoptosis in human lung adenocarcinoma A549 cells via reactive oxygen species (ROS)-dependent disruption of mitochondrial membrane potential (
      • Chen Z.
      • Jin K.
      • Gao L.
      • Lou G.
      • Jin Y.
      • Yu Y.
      • Lou Y.
      Anti-tumor effects of bakuchiol, an analogue of resveratrol, on human lung adenocarcinoma A549 cell line.
      ). Based on these results, we aimed to determine whether bakuchiol also possessed anti-influenza virus activity.
      Figure thumbnail gr1
      FIGURE 1Effect of bakuchiol on the viability of MDCK cells infected with influenza A virus strains. A, structures of (+)-(S)-bakuchiol (naturally occurring) and (−)-(R)-bakuchiol (not naturally occurring). B, the indicated concentrations of (+)-(S)-bakuchiol or (−)-(R)-bakuchiol in DMSO were mixed with the indicated influenza A virus strains (MOI = 10) or without influenza A virus (virus −) and added to MDCK cells. After incubation for 4 days, the cell viability was determined by naphthol blue black staining. Data are representative of two independent experiments, and the results were reproducible.
      In the present study, we found that (+)-(S)-bakuchiol and (−)-(R)-bakuchiol (a synthetic enantiomer that does not occur naturally; Fig. 1A) inhibited influenza A H1N1 viral infection and growth in Madin-Darby canine kidney (MDCK) cells and also reduced the expression of viral mRNAs and proteins. They reduced the induction of interferon-β (IFN-β) and myxovirus-resistant protein 1 (Mx1) mRNAs by the influenza A virus. (+)-(S)-Bakuchiol showed stronger antiviral activities than (−)-(R)-bakuchiol, indicating that the steric structure was important for these activities. We used an influenza A virus minigenome assay employing a Dual-Luciferase system to analyze mRNA and protein levels, and this unexpectedly revealed that bakuchiol induced host factors that inhibited firefly and Renilla luciferases. Next generation sequencing (NGS) and KeyMolnet analysis revealed an up-regulation of transcriptional regulation by the nuclear factor erythroid 2-related factor (Nrf) pathway, and a Nrf2 reporter assay showed that (+)-(S)-bakuchiol activated Nrf2. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses showed that bakuchiol up-regulated mRNA expression of NAD(P)H quinone oxidoreductase 1 (NQO1) and glutathione S-transferase A3 (GSTA3); these are Nrf2-induced oxidative stress-responsive genes. Taken together, these results indicated that bakuchiol produced novel anti-influenza effects by targeting processes involved in the host oxidative stress response.

      Discussion

      In the present study, we found that (+)-(S)-bakuchiol enhanced the survival of influenza A virus-infected MDCK cells and inhibited influenza A viral infection, growth, and gene expression; in addition, (+)-(S)-bakuchiol reduced the expression of influenza A virus-induced immune response genes in the host cells. We also found that (+)-(S)-bakuchiol induced the activation of Nrf2 and the up-regulation of NQO1 and GSTA3 mRNAs. This is the first report indicating that (+)-(S)-bakuchiol possesses anti-influenza virus activity. We found that the chirality of bakuchiol was important for this activity, and this should be considered when synthesizing bakuchiol derivatives as novel anti-influenza A virus H1N1 drugs.
      We showed that (+)-(S)-bakuchiol had greater anti-influenza activity than (−)-(R)-bakuchiol, suggesting that the chirality of bakuchiol was important for this activity. Although the reason for this is still unclear, (−)-(R)-bakuchiol may have a reduced interaction with the target protein or be more easily degraded in cells, as compared with (+)-(S)-bakuchiol.
      Our preincubation experiment showed that (+)-(S)-bakuchiol inhibited the H1N1 strains of the influenza virus (A/PR/8/34 and A/CA/7/09) but not the H3N2 strain (A/Aichi/2/68) (Fig. 1B, lanes 3 and 4). This may reflect strain differences in viral proteins or in the host cell response.
      The HA and NA viral proteins differ between the H1N1 and H3N2 strains. It has also been reported that an anti-M2 ectodomain monoclonal antibody (clone rM2ss23) inhibited the viral replication of A/Aichi/2/68 and an A/PR/8/34 recombinant variant expressing A/Aichi/2/68-HA and/or M segment strains but did not inhibit the A/PR/8/34 strain (
      • Muto N.A.
      • Yoshida R.
      • Suzuki T.
      • Kobayashi S.
      • Ozaki H.
      • Fujikura D.
      • Manzoor R.
      • Muramatsu M.
      • Takada A.
      • Kimura T.
      • Sawa H.
      Inhibitory effects of an M2-specific monoclonal antibody on different strains of influenza A virus.
      ). HA and M2 were co-localized in infected MDCK cells during virus budding (
      • Rossman J.S.
      • Jing X.
      • Leser G.P.
      • Balannik V.
      • Pinto L.H.
      • Lamb R.A.
      Influenza virus m2 ion channel protein is necessary for filamentous virion formation.
      ), suggesting that strain-dependent differences in HA-M2 interactions might affect the inhibition of viral replication. Therefore, although bakuchiol did not inhibit the functions of A/PR/8/34 HA and M2 proteins (FIGURE 9, FIGURE 10, FIGURE 11), it might affect their interaction while not affecting the A/Aichi/2/68 HA-M2 interactions.
      Bakuchiol induced Nrf2 activation and up-regulated NQO1 and GSTA3 mRNA levels in MDCK cells (Fig. 15), indicating that it influenced the host response to oxidative stress. It has been reported that the host cell responses, including the innate immune response (
      • Wu W.
      • Booth J.L.
      • Duggan E.S.
      • Wu S.
      • Patel K.B.
      • Coggeshall K.M.
      • Metcalf J.P.
      Innate immune response to H3N2 and H1N1 influenza virus infection in a human lung organ culture model.
      ) and the cellular microRNA signature (
      • Terrier O.
      • Textoris J.
      • Carron C.
      • Marcel V.
      • Bourdon J.C.
      • Rosa-Calatrava M.
      Host microRNA molecular signatures associated with human H1N1 and H3N2 influenza A viruses reveal an unanticipated antiviral activity for miR-146a.
      ), differed following infection by H1N1 or H3N2 strains. Therefore, the different effects of bakuchiol on A/PR/8/34 and A/Aichi/2/68 strains may reflect differences in the MDCK host response to oxidative stress following infection with these viruses.
      Nrf2 reporter assay, transcriptome, and RT-qPCR analyses in MDCK cells treated with bakuchiol and A/PR/8/34 showed that bakuchiol induced Nrf2 activation and up-regulated NQO1 and GSTA3 mRNA levels (Fig. 15). NQO1 catalyzes the reduction of various quinones via a two-electron mechanism involving NADH or NADPH, preventing the formation of free radicals and ROS. An increase in the level of ROS activates Nrf2 binding to the NQO1 promoter, increasing NQO1 production (
      • Tsvetkov P.
      • Reuven N.
      • Shaul Y.
      Ubiquitin-independent p53 proteasomal degradation.
      ). Additionally, NQO1 stabilizes p53 in an NADH-dependent manner, promoting accumulation of p53 protein in cells (
      • Tsvetkov P.
      • Reuven N.
      • Shaul Y.
      Ubiquitin-independent p53 proteasomal degradation.
      ). Chen et al. (
      • Chen Z.
      • Jin K.
      • Gao L.
      • Lou G.
      • Jin Y.
      • Yu Y.
      • Lou Y.
      Anti-tumor effects of bakuchiol, an analogue of resveratrol, on human lung adenocarcinoma A549 cell line.
      ) reported that bakuchiol increased p53 expression and induced apoptosis via ROS-dependent reduction of mitochondrial membrane potential in A549 cells. Therefore, we speculate that the up-regulation of NQO1 mRNA by bakuchiol is induced by ROS-dependent Nrf2 activation and increases the level of p53 protein in MDCK cells. Furthermore, Nrf2 up-regulation has been shown to reduce influenza A viral entry and replication (
      • Kesic M.J.
      • Simmons S.O.
      • Bauer R.
      • Jaspers I.
      Nrf2 expression modifies influenza A entry and replication in nasal epithelial cells.
      ), and the inhibition of p53 expression increases influenza A viral growth (
      • Turpin E.
      • Luke K.
      • Jones J.
      • Tumpey T.
      • Konan K.
      • Schultz-Cherry S.
      Influenza virus infection increases p53 activity: role of p53 in cell death and viral replication.
      ), suggesting that up-regulation of Nrf2 and p53 would inhibit influenza A viral growth. It has been reported that oltipraz (4-methyl-5(pyrazinyl-2)-1–2-dithiole-3-thione) and D3T (3H-1,2-dithiole-3thione), compounds that possess anti-cancer activities in multiple target organs (
      • Kensler T.W.
      • Groopman J.D.
      • Sutter T.R.
      • Curphey T.J.
      • Roebuck B.D.
      Development of cancer chemopreventive agents: oltipraz as a paradigm.
      ), increase the Nrf2-driven expression of NQO1 (
      • Manandhar S.
      • Cho J.M.
      • Kim J.A.
      • Kensler T.W.
      • Kwak M.K.
      Induction of Nrf2-regulated genes by 3H-1, 2-dithiole-3-thione through the ERK signaling pathway in murine keratinocytes.
      ,
      • Merrell M.D.
      • Jackson J.P.
      • Augustine L.M.
      • Fisher C.D.
      • Slitt A.L.
      • Maher J.M.
      • Huang W.
      • Moore D.D.
      • Zhang Y.
      • Klaassen C.D.
      • Cherrington N.J.
      The Nrf2 activator oltipraz also activates the constitutive androstane receptor.
      ). Therefore, Nrf2 activation could represent one of the anti-influenza A virus H1N1 mechanisms of bakuchiol. However, because the direct target of bakuchiol remains unclear, further studies will be needed to explore this.
      Based on the findings of this study and previous reports, as shown in Fig. 16, we suggest that anti-influenza virus activity by bakuchiol is involved in Nrf2 activation. In conclusion, the findings of the present study demonstrated that bakuchiol produced an enantiomer-selective anti-influenza A virus activity via a novel mechanism involving the host cell response. These data will contribute to the development of novel approaches to the treatment of influenza.
      Figure thumbnail gr16
      FIGURE 16Schematic showing novel mechanisms of anti-influenza virus activity of bakuchiol. This scheme includes results and conclusions of the present study and previous report (
      • Kesic M.J.
      • Simmons S.O.
      • Bauer R.
      • Jaspers I.
      Nrf2 expression modifies influenza A entry and replication in nasal epithelial cells.
      ).

      Author Contributions

      T. K. and M. S. designed the study and wrote the paper. T. E. and C. Y. synthesized and purified chemicals. M. S. and Y. A. performed anti-influenza virus assays. Y. S. performed next generation sequencing. S. Kohnomi and S. Konishi performed channel assays. E. T. and H. K. provided influenza viral strains. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Prof. M. Yamamoto and Dr. T. Suzuki for providing the plasmid pNQO1-ARE-luc.

      References

        • Horimoto T.
        • Kawaoka Y.
        Influenza: lessons from past pandemics, warnings from current incidents.
        Nat. Rev. Microbiol. 2005; 3: 591-600
        • Itoh Y.
        • Shinya K.
        • Kiso M.
        • Watanabe T.
        • Sakoda Y.
        • Hatta M.
        • Muramoto Y.
        • Tamura D.
        • Sakai-Tagawa Y.
        • Noda T.
        • Sakabe S.
        • Imai M.
        • Hatta Y.
        • Watanabe S.
        • Li C.
        • Yamada S.
        • Fujii K.
        • Murakami S.
        • Imai H.
        • Kakugawa S.
        • Ito M.
        • Takano R.
        • Iwatsuki-Horimoto K.
        • Shimojima M.
        • Horimoto T.
        • Goto H.
        • Takahashi K.
        • Makino A.
        • Ishigaki H.
        • Nakayama M.
        • Okamatsu M.
        • Takahashi K.
        • Warshauer D.
        • Shult P.A.
        • Saito R.
        • Suzuki H.
        • Furuta Y.
        • Yamashita M.
        • Mitamura K.
        • Nakano K.
        • Nakamura M.
        • Brockman-Schneider R.
        • Mitamura H.
        • Yamazaki M.
        • Sugaya N.
        • Suresh M.
        • Ozawa M.
        • Neumann G.
        • Gern J.
        • Kida H.
        • Ogasawara K.
        • Kawaoka Y.
        In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses.
        Nature. 2009; 460: 1021-1025
        • Neumann G.
        • Noda T.
        • Kawaoka Y.
        Emergence and pandemic potential of swine-origin H1N1 influenza virus.
        Nature. 2009; 459: 931-939
        • De Clercq E.
        Antiviral agents active against influenza A viruses.
        Nat. Rev. Drug Discov. 2006; 5: 1015-1025
        • Hayden F.G.
        • Atmar R.L.
        • Schilling M.
        • Johnson C.
        • Poretz D.
        • Paar D.
        • Huson L.
        • Ward P.
        • Mills R.G.
        Use of the selective oral neuraminidase inhibitor oseltamivir to prevent influenza.
        N. Engl. J. Med. 1999; 341: 1336-1343
        • Kitching A.
        • Roche A.
        • Balasegaram S.
        • Heathcock R.
        • Maguire H.
        Oseltamivir adherence and side effects among children in three London schools affected by influenza A(H1N1)v, May 2009 - an internet-based cross-sectional survey.
        Euro Surveill. 2009; 14: 19287
        • L'Huillier A.G.
        • Ing Lorenzini K.
        • Crisinel P.A.
        • Rebsamen M.C.
        • Fluss J.
        • Korff C.M.
        • Barbe R.P.
        • Siegrist C.A.
        • Dayer P.
        • Posfay-Barbe K.M.
        • Desmeules J.A.
        • H1N1 Pediatric Epidemiology Study Group of Geneva
        ABCB1 polymorphisms and neuropsychiatric adverse events in oseltamivir-treated children during influenza H1N1/09 pandemia.
        Pharmacogenomics. 2011; 12: 1493-1501
        • Chung S.
        • Joung Y.S.
        Oseltamivir (tamiflu) induced depressive episode in a female adolescent.
        Psychiatry Investig. 2010; 7: 302-304
        • Hiasa M.
        • Isoda Y.
        • Kishimoto Y.
        • Saitoh K.
        • Kimura Y.
        • Kanai M.
        • Shibasaki M.
        • Hatakeyama D.
        • Kirino Y.
        • Kuzuhara T.
        Inhibition of MAO-A and stimulation of behavioural activities in mice by the inactive prodrug form of the anti-influenza agent oseltamivir.
        Br. J. Pharmacol. 2013; 169: 115-129
        • Reece P.A.
        Neuraminidase inhibitor resistance in influenza viruses.
        J. Med. Virol. 2007; 79: 1577-1586
        • Collins P.J.
        • Haire L.F.
        • Lin Y.P.
        • Liu J.
        • Russell R.J.
        • Walker P.A.
        • Skehel J.J.
        • Martin S.R.
        • Hay A.J.
        • Gamblin S.J.
        Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants.
        Nature. 2008; 453: 1258-1261
        • Mehta G.
        • Ramdas Nayak U.
        • Dev S.
        Bakuchiol, a novel monoterpenoid.
        Tetrahedron Lett. 1966; 7: 4561-4567
        • Poláková K.
        • Fauger A.
        • Sayag M.
        • Jourdan E.
        A dermocosmetic containing bakuchiol, Ginkgo biloba extract, and mannitol improves the efficacy of adapalene in patients with acne vulgaris: result from a controlled randomized trial.
        Clin. Cosmet. Investig. Dermatol. 2015; 8: 187-191
        • Chen H.L.
        • Feng H.J.
        • Li Y.C.
        [Vitro antitumor activity and synthesis of the key intermediate of bakuchiol].
        Yao Xue Xue Bao. 2010; 45: 467-470
        • Chen Z.
        • Jin K.
        • Gao L.
        • Lou G.
        • Jin Y.
        • Yu Y.
        • Lou Y.
        Anti-tumor effects of bakuchiol, an analogue of resveratrol, on human lung adenocarcinoma A549 cell line.
        Eur. J. Pharmacol. 2010; 643: 170-179
        • Lim S.H.
        • Ha T.Y.
        • Kim S.R.
        • Ahn J.
        • Park H.J.
        • Kim S.
        Ethanol extract of Psoralea corylifolia L., and its main constituent, bakuchiol, reduce bone loss in ovariectomised Sprague-Dawley rats.
        Br. J. Nutr. 2009; 101: 1031-1039
        • Zhao G.
        • Zang S.Y.
        • Zheng X.W.
        • Zhang X.H.
        • Guo L.H.
        Bakuchiol analogs inhibit monoamine transporters and regulate monoaminergic functions.
        Biochem. Pharmacol. 2008; 75: 1835-1847
        • Kim K.A.
        • Shim S.H.
        • Ahn H.R.
        • Jung S.H.
        Protective effects of the compounds isolated from the seed of Psoralea corylifolia on oxidative stress-induced retinal damage.
        Toxicol. Appl. Pharmacol. 2013; 269: 109-120
        • Esumi T.
        • Yamamoto C.
        • Fukuyama Y.
        A short synthesis of (+)-bakuchiol.
        Synlett. 2013; 24: 1845-1847
        • Katsura H.
        • Tsukiyama R.I.
        • Suzuki A.
        • Kobayashi M.
        In vitro antimicrobial activities of bakuchiol against oral microorganisms.
        Antimicrob Agents Chemother. 2001; 45: 3009-3013
        • Haraguchi H.
        • Inoue J.
        • Tamura Y.
        • Mizutani K.
        Inhibition of mitochondrial lipid peroxidation by Bakuchiol, a meroterpene from Psoralea corylifolia.
        Planta Medica. 2000; 66: 569-571
        • Adhikari S.
        • Joshi R.
        • Patro B.S.
        • Ghanty T.K.
        • Chintalwar G.J.
        • Sharma A.
        • Chattopadhyay S.
        • Mukherjee T.
        Antioxidant activity of bakuchiol: experimental evidences and theoretical treatments on the possible involvement of the terpenoid chain.
        Chem. Res. Toxicol. 2003; 16: 1062-1069
        • Pae H.O.
        • Cho H.
        • Oh G.S.
        • Kim N.Y.
        • Song E.K.
        • Kim Y.C.
        • Yun Y.G.
        • Kang C.L.
        • Kim J.D.
        • Kim J.M.
        • Chung H.T.
        Bakuchiol from Psoralea corylifolia inhibits the expression of inducible nitric oxide synthase gene via the inactivation of nuclear transcription factor-κB in RAW 264.7 macrophages.
        Int. Immunopharmacol. 2001; 1: 1849-1855
        • Lee S.W.
        • Yun B.R.
        • Kim M.H.
        • Park C.S.
        • Lee W.S.
        • Oh H.M.
        • Rho M.C.
        Phenolic compounds isolated from Psoralea corylifolia inhibit IL-6-induced STAT3 activation.
        Planta Med. 2012; 78: 903-906
        • Sun N.J.
        • Woo S.H.
        • Cassady J.M.
        • Snapka R.M.
        DNA polymerase and topoisomerase II inhibitors from Psoralea corylifolia.
        J. Nat. Prod. 1998; 61: 362-366
        • Takahashi E.
        • Kataoka K.
        • Indalao I.L.
        • Konoha K.
        • Fujii K.
        • Chida J.
        • Mizuno D.
        • Fujihashi K.
        • Kido H.
        Oral clarithromycin enhances airway immunoglobulin A (IgA) immunity through induction of IgA class switching recombination and B-cell-activating factor of the tumor necrosis factor family molecule on mucosal dendritic cells in mice infected with influenza A virus.
        J. Virol. 2012; 86: 10924-10934
        • Shoji M.
        • Takahashi E.
        • Hatakeyama D.
        • Iwai Y.
        • Morita Y.
        • Shirayama R.
        • Echigo N.
        • Kido H.
        • Nakamura S.
        • Mashino T.
        • Okutani T.
        • Kuzuhara T.
        Anti-influenza activity of c60 fullerene derivatives.
        PLoS One. 2013; 8: e66337
        • Iwai Y.
        • Murakami K.
        • Gomi Y.
        • Hashimoto T.
        • Asakawa Y.
        • Okuno Y.
        • Ishikawa T.
        • Hatakeyama D.
        • Echigo N.
        • Kuzuhara T.
        Anti-influenza activity of marchantins, macrocyclic bisbibenzyls contained in liverworts.
        PLoS One. 2011; 6: e19825
        • Kuchipudi S.V.
        • Tellabati M.
        • Nelli R.K.
        • White G.A.
        • Perez B.B.
        • Sebastian S.
        • Slomka M.J.
        • Brookes S.M.
        • Brown I.H.
        • Dunham S.P.
        • Chang K.C.
        18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells.
        Virol. J. 2012; 9: 230
        • Wu Z.L.
        • Ethen C.
        • Hickey G.E.
        • Jiang W.
        Active 1918 pandemic flu viral neuraminidase has distinct N-glycan profile and is resistant to trypsin digestion.
        Biochem. Biophys. Res. Commun. 2009; 379: 749-753
        • Zhu L.
        • Li Y.
        • Li S.
        • Li H.
        • Qiu Z.
        • Lee C.
        • Lu H.
        • Lin X.
        • Zhao R.
        • Chen L.
        • Wu J.Z.
        • Tang G.
        • Yang W.
        Inhibition of influenza A virus (H1N1) fusion by benzenesulfonamide derivatives targeting viral hemagglutinin.
        PLoS One. 2011; 6: e29120
        • Ozawa M.
        • Fujii K.
        • Muramoto Y.
        • Yamada S.
        • Yamayoshi S.
        • Takada A.
        • Goto H.
        • Horimoto T.
        • Kawaoka Y.
        Contributions of two nuclear localization signals of influenza A virus nucleoprotein to viral replication.
        J. Virol. 2007; 81: 30-41
        • Murakami S.
        • Horimoto T.
        • Yamada S.
        • Kakugawa S.
        • Goto H.
        • Kawaoka Y.
        Establishment of canine RNA polymerase I-driven reverse genetics for influenza A virus: its application for H5N1 vaccine production.
        J. Virol. 2008; 82: 1605-1609
        • Kanematsu S.
        • Tanimoto K.
        • Suzuki Y.
        • Sugano S.
        Screening for possible miRNA-mRNA associations in a colon cancer cell line.
        Gene. 2014; 533: 520-531
        • Sato H.
        • Ishida S.
        • Toda K.
        • Matsuda R.
        • Hayashi Y.
        • Shigetaka M.
        • Fukuda M.
        • Wakamatsu Y.
        • Itai A.
        New approaches to mechanism analysis for drug discovery using DNA microarray data combined with KeyMolnet.
        Curr. Drug Discov. Technol. 2005; 2: 89-98
        • Kang M.I.
        • Kobayashi A.
        • Wakabayashi N.
        • Kim S.G.
        • Yamamoto M.
        Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes.
        Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2046-2051
        • Zhao J.
        • Moore A.N.
        • Redell J.B.
        • Dash P.K.
        Enhancing expression of Nrf2-driven genes protects the blood brain barrier after brain injury.
        J. Neurosci. 2007; 27: 10240-10248
        • Kato H.
        • Takeuchi O.
        • Sato S.
        • Yoneyama M.
        • Yamamoto M.
        • Matsui K.
        • Uematsu S.
        • Jung A.
        • Kawai T.
        • Ishii K.J.
        • Yamaguchi O.
        • Otsu K.
        • Tsujimura T.
        • Koh C.S.
        • Reis e Sousa C.
        • Matsuura Y.
        • Fujita T.
        • Akira S.
        Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses.
        Nature. 2006; 441: 101-105
        • Kato H.
        • Takeuchi O.
        • Mikamo-Satoh E.
        • Hirai R.
        • Kawai T.
        • Matsushita K.
        • Hiiragi A.
        • Dermody T.S.
        • Fujita T.
        • Akira S.
        Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5.
        J. Exp. Med. 2008; 205: 1601-1610
        • Holzinger D.
        • Jorns C.
        • Stertz S.
        • Boisson-Dupuis S.
        • Thimme R.
        • Weidmann M.
        • Casanova J.L.
        • Haller O.
        • Kochs G.
        Induction of MxA gene expression by influenza A virus requires type I or type III interferon signaling.
        J. Virol. 2007; 81: 7776-7785
        • Seitz C.
        • Frensing T.
        • Höper D.
        • Kochs G.
        • Reichl U.
        High yields of influenza A virus in Madin-Darby canine kidney cells are promoted by an insufficient interferon-induced antiviral state.
        J. Gen. Virol. 2010; 91: 1754-1763
        • von Itzstein M.
        The war against influenza: discovery and development of sialidase inhibitors.
        Nat. Rev. Drug Discov. 2007; 6: 967-974
        • Chen J.
        • Lee K.H.
        • Steinhauer D.A.
        • Stevens D.J.
        • Skehel J.J.
        • Wiley D.C.
        Structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation.
        Cell. 1998; 95: 409-417
        • Chen J.
        • Skehel J.J.
        • Wiley D.C.
        N- and C-terminal residues combine in the fusion-pH influenza hemagglutinin HA(2) subunit to form an N cap that terminates the triple-stranded coiled coil.
        Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 8967-8972
        • Skehel J.J.
        • Wiley D.C.
        Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin.
        Annu. Rev. Biochem. 2000; 69: 531-569
        • Deyde V.M.
        • Xu X.
        • Bright R.A.
        • Shaw M.
        • Smith C.B.
        • Zhang Y.
        • Shu Y.
        • Gubareva L.V.
        • Cox N.J.
        • Klimov A.I.
        Surveillance of resistance to adamantanes among influenza A(H3N2) and A(H1N1) viruses isolated worldwide.
        J. Infect. Dis. 2007; 196: 249-257
        • Furuse Y.
        • Suzuki A.
        • Oshitani H.
        Large-scale sequence analysis of M gene of influenza A viruses from different species: mechanisms for emergence and spread of amantadine resistance.
        Antimicrob. Agents Chemother. 2009; 53: 4457-4463
        • Balannik V.
        • Wang J.
        • Ohigashi Y.
        • Jing X.
        • Magavern E.
        • Lamb R.A.
        • Degrado W.F.
        • Pinto L.H.
        Design and pharmacological characterization of inhibitors of amantadine-resistant mutants of the M2 ion channel of influenza A virus.
        Biochemistry. 2009; 48: 11872-11882
        • Honda A.
        • Mizumoto K.
        • Ishihama A.
        Minimum molecular architectures for transcription and replication of the influenza virus.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 13166-13171
        • Cheung P.P.
        • Watson S.J.
        • Choy K.T.
        • Fun Sia S.
        • Wong D.D.
        • Poon L.L.
        • Kellam P.
        • Guan Y.
        • Malik Peiris J.S.
        • Yen H.L.
        Generation and characterization of influenza A viruses with altered polymerase fidelity.
        Nat. Commun. 2014; 5: 4794
        • Hayes J.D.
        • McMahon M.
        NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer.
        Trends Biochem. Sci. 2009; 34: 176-188
        • Manandhar S.
        • Cho J.M.
        • Kim J.A.
        • Kensler T.W.
        • Kwak M.K.
        Induction of Nrf2-regulated genes by 3H-1, 2-dithiole-3-thione through the ERK signaling pathway in murine keratinocytes.
        Eur. J. Pharmacol. 2007; 577: 17-27
        • Klaassen C.D.
        • Reisman S.A.
        Nrf2 the rescue: effects of the antioxidative/electrophilic response on the liver.
        Toxicol. Appl. Pharmacol. 2010; 244: 57-65
        • Johnson J.
        • Maher P.
        • Hanneken A.
        The flavonoid, eriodictyol, induces long-term protection in ARPE-19 cells through its effects on Nrf2 activation and phase 2 gene expression.
        Invest. Ophthalmol. Vis. Sci. 2009; 50: 2398-2406
        • Muto N.A.
        • Yoshida R.
        • Suzuki T.
        • Kobayashi S.
        • Ozaki H.
        • Fujikura D.
        • Manzoor R.
        • Muramatsu M.
        • Takada A.
        • Kimura T.
        • Sawa H.
        Inhibitory effects of an M2-specific monoclonal antibody on different strains of influenza A virus.
        Jpn. J. Vet. Res. 2012; 60: 71-83
        • Rossman J.S.
        • Jing X.
        • Leser G.P.
        • Balannik V.
        • Pinto L.H.
        • Lamb R.A.
        Influenza virus m2 ion channel protein is necessary for filamentous virion formation.
        J. Virol. 2010; 84: 5078-5088
        • Wu W.
        • Booth J.L.
        • Duggan E.S.
        • Wu S.
        • Patel K.B.
        • Coggeshall K.M.
        • Metcalf J.P.
        Innate immune response to H3N2 and H1N1 influenza virus infection in a human lung organ culture model.
        Virology. 2010; 396: 178-188
        • Terrier O.
        • Textoris J.
        • Carron C.
        • Marcel V.
        • Bourdon J.C.
        • Rosa-Calatrava M.
        Host microRNA molecular signatures associated with human H1N1 and H3N2 influenza A viruses reveal an unanticipated antiviral activity for miR-146a.
        J. Gen. Virol. 2013; 94: 985-995
        • Tsvetkov P.
        • Reuven N.
        • Shaul Y.
        Ubiquitin-independent p53 proteasomal degradation.
        Cell Death Differ. 2010; 17: 103-108
        • Kesic M.J.
        • Simmons S.O.
        • Bauer R.
        • Jaspers I.
        Nrf2 expression modifies influenza A entry and replication in nasal epithelial cells.
        Free Radic. Biol. Med. 2011; 51: 444-453
        • Turpin E.
        • Luke K.
        • Jones J.
        • Tumpey T.
        • Konan K.
        • Schultz-Cherry S.
        Influenza virus infection increases p53 activity: role of p53 in cell death and viral replication.
        J. Virol. 2005; 79: 8802-8811
        • Kensler T.W.
        • Groopman J.D.
        • Sutter T.R.
        • Curphey T.J.
        • Roebuck B.D.
        Development of cancer chemopreventive agents: oltipraz as a paradigm.
        Chem. Res. Toxicol. 1999; 12: 113-126
        • Merrell M.D.
        • Jackson J.P.
        • Augustine L.M.
        • Fisher C.D.
        • Slitt A.L.
        • Maher J.M.
        • Huang W.
        • Moore D.D.
        • Zhang Y.
        • Klaassen C.D.
        • Cherrington N.J.
        The Nrf2 activator oltipraz also activates the constitutive androstane receptor.
        Drug Metab. Dispos. 2008; 36: 1716-1721