Introduction

The pandemic influenza H1N1 virus has recently spread worldwide. The appearance of an influenza virus more virulent than the pandemic H1N1 is now predicted. The amantadine and neuraminidase inhibitors zanamivir and oseltamivir have been used for the treatment and prevention of influenza virus infection [1–3], but the appearance of viruses resistant to them has been reported [1, 4–6]. It is therefore important to develop new types of anti-influenza virus agents with anti-influenza virus actions different from those of the known agents.

In a series of studies on the development of bioactive components from natural sources, we found that a methanol extract from the rhizome of Alpinia officinarum is effective in inhibiting tumor promotion in 12-O-tetradecanoylphorbol-13-acetate-induced inflammatory ear edema in mice [7]. Diarylheptanoids isolated from Alpinia officinarum have been shown to exhibit biological activities such as antioxidant activity [8, 9], cytotoxic activity [10], suppressive activity of inducible nitric oxide synthase expression [11], and inhibitory activity of biosynthesis of prostaglandin and leukotriene [12, 13]. Although a variety of biological activities associated with diarylheptanoids have been demonstrated, antiviral activity by diarylheptanoids has not been reported. In the present study, we examined the potential anti-influenza virus activity of diarylheptanoids in vitro.

Materials and methods

Chemicals

Bovine serum albumin was purchased from Sigma–Aldrich Japan, Tokyo, Japan. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was purchased from Sigma, Japan. Sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO), N,N-dimethylformamide, and ribavirin were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Diarylheptanoids (1–10) were isolated from the rhizome of Alpinia officinarum as described previously [10, 14].

Cell and viruses

Madin–Darby canine kidney (MDCK) cells, which were provided by Dr. H. Ochiai (Toyama University, Japan), were grown and maintained in Eagle’s minimum essential medium (EMEM; Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 8 and 2% heat-inactivated fetal calf serum, respectively [15]. The influenza virus A/PR/8/34 (H1N1) was provided by Dr. H. Ochiai (Toyama University, Japan) and propagated in MDCK cells [15].

Antiviral and cytotoxic assay

The anti-influenza virus activity of 10 diarylheptanoids was examined using the MTT assay. Briefly, MDCK cells grown in 96-well plates were infected with 60 plaque-forming units/50 µl of influenza A/PR/8/34 (H1N1) virus at 37°C for 1 h. The cells were overlaid with 50 µl of maintenance EMEM containing 0.1% bovine serum albumin and various concentrations (0, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 50 µg/ml) of one of the following compounds, and maintained in a humidified atmosphere containing 5% CO2 for 3–4 days. All diarylheptanoids were dissolved in DMSO and diluted with culture medium to make the various final concentrations. The concentration of DMSO in each medium was less than 1%. Ribavirin was dissolved in distilled water and used as a control. For the MTT assay, 10 µl of MTT (7.5 mg/ml) solution in phosphate-buffered saline was added to octuplicate wells with each concentration of diarylheptanoids and incubated for 4 h at 37°C in a CO2 incubator. The crystallized formazan in the plates was dissolved by the addition of 100 µl of 20% (w/v) SDS/50% (v/v) N,N-dimethylformamide. Absorbance was measured at two wavelengths (540 and 690 nm) in a computer-controlled microplate reader (Bio-Rad, Tokyo, Japan). The 50% effective antiviral concentration (EC50) was the concentration that reduced virus-induced cell destruction by 50%, as described previously [16]. The cytotoxicity of diarylheptanoids (1 and 3–8) and ribavirin was assessed by MTT assay using mock-infected MDCK cells exposed to each diarylheptanoid (0, 15, 30, 50 or 45, 60, and 80 µg/ml) in duplicate wells for 3–4 days as described above. The 50% cytotoxic concentration (CC50) was determined as the concentration that reduced cell destruction by 50% in the three independent experiments [16]. The cytotoxicity of all diarylheptanoids used was also assessed by the detection of morphological changes in the infected cells under the microscope. The maximum non-cytotoxic concentration (MNCC) of diarylheptanoids was defined as the highest concentration at which obvious morphological change was not observed in more than 50% of octuplicate wells.

Results and discussion

Diarylheptanoids (1–10, Fig. 1) were examined for their anti-influenza virus activity and cytotoxicity in vitro. As shown in Table 1, the EC50 value of each diarylheptanoid examined for influenza virus was clearly lower than the CC50 and/or MNCC of each diarylheptanoid. DMSO at 1%, which was used as a maximum concentration to dissolve diarylheptanoids in the culture medium, was not cytotoxic. The therapeutic indexes (CC50/EC50) of 7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-4E-hepten-3-one (3) and (5S)-5-hydroxy-7-(4″-hydroxyphenyl)-1-phenyl-3-heptanone (8) were more than 11.7 and 114.3, respectively, and influenza virus was more susceptible to 3 and 8 than to the other diarylheptanoids. In this assay, the EC50 value of ribavirin, used as a control, was similar to the results reported previously [15, 17]. The EC50 values of the diarylheptanoids examined were similar to or lower than that of ribavirin, and they were demonstrated to show potential antiviral activity against influenza virus in vitro. This is the first evidence demonstrating the anti-influenza virus activity of diarylheptanoids in vitro. Various kinds of diarylheptanoids have been isolated from Alpinia officinarum [10, 12–14, 18, 19]. Studies of the relationship between their structures and their anti-influenza activities may be worthwhile to obtain much more effective anti-influenza virus diarylheptanoids.

Fig. 1
figure 1

Structures of diarylheptanoids (1–10) from the rhizomes of Alpinia officinarum

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Table 1 Anti-influenza virus activity and cytotoxicity of diarylheptanoids
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