Anti-viral agent derived from Alpinia zerumbet against Flavivirus

Abstract

To provide antiviral agents against flaviviruses, such as dengue virus and Japanese encephalitis virus, containing Alpinia zerumbet extract.SOLUTION: The present invention relates to an antiviral agent against flaviviruses, such as dengue virus and Japanese encephalitis virus, containing Alpinia zerumbet extract, the Alpinia zerumbet extract being squeezed juice, soup, and distilled water obtained from Alpinia zerumbet. The Alpinia zerumbet extract can provide antiviral effects against dengue 1, 2, 3, 4 viruses and Japanese encephalitis virus by both mechanisms of directly acting on viruses (virucidal action) and of acting in adsorption and post-adsorption stages.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to an antiviral agent against flavivirus containing Alpinia zerumbet extract.

Background Art

[0002] Viruses of the genus Flavivirus in the family Flaviviridae include Dengue virus, Japanese encephalitis virus, West Nile virus, Yellow fever virus, Zika virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Tick-borne encephalitis virus, etc.

[0003] Among these, Dengue virus is the pathogen of dengue fever, an important mosquito-borne infectious disease in public health. In recent years, the range of Dengue virus infections has been rapidly expanding, and about half of the world's population is at risk of infection. The number of patients has increased 50-fold in 30 years. In addition, there are types 1, 2, 3, and 4 of Dengue virus, and multiple infections can occur. Moreover, antibodies of hetero-types infected in the past act as antibodies that promote amplification (ADE), increasing the risk of severe illness.

[0004] The number of countries where multiple types of Dengue virus have been confirmed was 7 countries 30 years ago, but now it has increased to 127 countries, and the occurrence of severe dengue (dengue hemorrhagic fever, dengue shock syndrome) has also increased. The dengue fever (162 patients, 0 seriously injured, 0 deaths) that prevailed around Yoyogi Park in Tokyo in 2014 is still fresh in memory. However, in the following year, 2015, more than 40,000 patients and about 200 deaths occurred in Taiwan. In addition, as of August 2020, a pandemic has been reported mainly in Southeast Asia, such as in Singapore, where the number of patients has reached a record high of over 25,000. Since infection is caused by the blood-sucking of mosquitoes (Aedes albopictus) living indoors, a relationship with the nesting behavior pattern during the Covid-19 pandemic has been suggested. In Japan too, dengue fever is vulnerable to the effects of globalization and is the most infectious disease that should be most vigilant about after the convergence of the Covid-19 pandemic, which is expected to promote the movement of people and goods.

[0005] In the past, Japanese encephalitis virus was a major public health problem because it caused high mortality and severe sequelae. Currently, in Japan, the number of patients with Japanese encephalitis has been less than 10 per year, but globally, there are reports of 30,000 to 40,000 patients mainly in Asia every year, and it still remains a public health problem.

[0006] Currently, there are no approved specific therapeutic agents or vaccines for dengue virus, and the development thereof is demanded. In addition, although there is a vaccine for Japanese encephalitis, there is no specific therapeutic agent.

[0007] Shell ginger (Alpinia zerumbet, belonging to the genus Alpinia of the Zingiberaceae family), which has been highly valued for its excellent antibacterial and insect repellent effects in Okinawa since ancient times, is known to be effective against HIV (human immunodeficiency virus) and plant viruses, and its antiviral action is expected.

[0008] For example, Patent Document 1 discloses that a specific extract fraction from shell ginger has an excellent control effect against various plant diseases caused by plant virus diseases and the like. However, it has not been clarified that the shell ginger extract has an antiviral effect against flaviviruses such as dengue virus and Japanese encephalitis virus.

Prior Art Documents

Patent Documents

[0009] [Patent Document 1] Japanese Unexamined Patent Application Publication No. 2020-002016 [Summary of the Invention] [Problems to be Solved by the Invention]

[0010] Therefore, an object of the present invention is to provide a safe therapeutic agent, prophylactic agent, and disinfectant for flaviviruses including dengue virus and Japanese encephalitis virus, for which almost no antiviral action has been reported yet, and to provide an antiviral agent for flaviviruses containing Alpinia zerumbet extract. [Means for Solving the Problems]

[0011] The present invention is an antiviral agent for flaviviruses containing Alpinia zerumbet extract. In particular, it is a medicine having an antiviral effect against dengue virus and Japanese encephalitis virus. The Alpinia zerumbet extract is squeezed juice, boiled juice, or distilled water obtained from Alpinia zerumbet, and the antiviral agent according to the present invention is characterized by containing these Alpinia zerumbet extracts. [Effects of the Invention]

[0012] The antiviral agent for flaviviruses such as dengue virus and Japanese encephalitis virus containing Alpinia zerumbet extract has an antiviral effect against dengue virus types 1 to 4 and Japanese encephalitis virus by both a mechanism that directly acts on the virus (virucidal action) and a mechanism that acts at a stage after adsorption. [Brief Description of the Drawings]

[0013]

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Mode for Carrying Out the Invention

[0014] To confirm the effects of the present invention, experiments were conducted under the following conditions.

[0015] 1. Extract of Alpinia zerumbet The extract of Alpinia zerumbet was obtained by the following three methods. (1) Squeezed juice The stems and leaves of Alpinia zerumbet (also known as "Gettou" in the Zingiberaceae family, Alpinia genus, hereinafter simply referred to as "Alpinia zerumbet") were put into a juicer (model YBK-2, manufactured by Yabiku Agricultural Machinery Works), and without adding water, the liquid extracted from Alpinia zerumbet was collected, and impurities were removed by a centrifuge (6000 rpm, for 5 minutes). The supernatant was filtered through filter paper and a filter with a pore size of 0.22 μm (manufactured by TPP). The obtained liquid was designated as "squeezed juice". (2) Boiled juice 10 L of water was added to 7.6 kg of the stems and leaves of Alpinia zerumbet and boiled for 2 hours. Then, the liquid from which plant residues were removed was sterilized by filtration and designated as "boiled juice". (3) Distilled water The above-mentioned boiled juice was distilled and separated into an oil layer and a water layer. The water layer obtained by the first hour of distillation among the distillates was sterilized by filtration and designated as "distilled water".

[0016] 2. Alpinia zerumbet components The following components contained in Alpinia zerumbet were used. (1) 5,6-Dehydrokawain (manufactured by Fujifilm Wako Pure Chemical Corporation) (2) 7,8-dihydro-5,6-dehydrokawain (manufactured by Fujifilm Wako Pure Chemical Corporation) (3) Proanthocyanidin

[0017] The squeezed juice was put into a membrane with a fractional molecular weight of 3,500 as a dialysis membrane (manufactured by Thermo Fisher Scientific, SnakeSkin Pleated Dialysis Tubing) and dialyzed against distilled water (4 liters × 3 times). The inner dialysis solution obtained was freeze-dried, and the resulting dried product was designated as "Proanthocyanidin". The purity assay of Proanthocyanidin generally followed the method using 4-dimethylaminocinnamaldehyde (DMAC) described in the report by Oki (Oki, Tomoyuki et al., Nippon Shokuhin Kagaku Kogaku Kaishi Vol.60, No6, 301-309 (2013)). That is, an aqueous solution of proanthocyanidin for calibration curve (10 mg / ml) obtained by separate purification was diluted 20-fold with assay solution A (ethanol: methanol: 2-propanol = 90:5:5), and the resulting solution was further diluted 2-fold with assay solution B (assay solution A: water = 95:5) to create a 2-fold dilution series, which was used as the calibration curve sample. A freeze-dried product obtained from the squeezed juice and dissolved in distilled water at 5 mg / ml was diluted 20-fold with assay solution A (ethanol: methanol: 2-propanol = 90:5:5) to obtain sample X for measurement. Additionally, sample X for measurement was diluted 2-fold and 4-fold with assay solution B to obtain samples Y and Z for measurement. Concentrated hydrochloric acid (3 ml) was added to the above assay solution A (27 ml), and DMAC (30 mg) was added to the solution cooled on ice for 15 minutes, followed by dissolution by stirring. The solution was stored on ice until immediately before use (DMAC solution). 40 μl of the calibration curve and measurement samples were dispensed into the wells of a 96-well microplate, and 200 μl of the DMAC solution was added to the wells containing the samples using an 8-channel micropipettor. Subsequently, a plate seal was affixed to the top of the plate, and the plate was placed in a plate reader with the internal temperature set to 30°C and stirred. After leaving it for 20 minutes, the absorbance at 640 nm of each well was measured. A calibration curve was created from the absorbance values and concentrations obtained from the calibration curve samples. From the average concentration of the proanthocyanidin concentration contained in measurement samples X, Y, and Z and the concentration at which the freeze-dried product was dissolved (5 mg / ml), the purity of proanthocyanidin in the freeze-dried product obtained from the squeezed juice was converted to 78.8%.

[0018] Method for preparing calibration curve proanthocyanidin aqueous solution: The squeezed juice of Alpinia zerumbet stems and leaves (120 ml) was applied to a reverse phase carrier (Sep-Pak C18 (35 cc) manufactured by Waters Japan, Ltd.), washed with ultrapure water (100 ml), and then eluted with 20% acetonitrile (100 ml) to obtain a fraction. The acetonitrile contained in the obtained fraction was distilled off using an evaporator (50 °C, 15 minutes), and the remaining solution was placed in a dialysis membrane with a molecular weight cut-off of 10,000 (manufactured by Spectrum) and dialyzed against distilled water (4 liters × 4 times). The obtained inner dialysis solution was freeze-dried (for 4 days) to obtain an Alpinia zerumbet-derived proanthocyanidin purified product (492.7 mg). This purified product was used as the calibration curve proanthocyanidin aqueous solution.

[0019] 3. Viruses used To confirm the antiviral effect, the following flaviviruses were used. (1) Dengue virus type 1 (DENV1) Hawaiian strain (prototype) (2) Dengue virus type 2 (DENV2) New Guinea B strain (prototype) (3) Dengue virus type 3 (DENV3) H-87 strain (prototype) (4) Dengue virus type 4 (DENV4) H-241 strain (prototype) (5) Japanese encephalitis virus Beijing strain (vaccine strain)

[0020] 4. Cells and others used (1) Cells BHK-21 cells (hamster kidney cells) Vero cells (African green monkey kidney cells) Hep2 cells (HeLa deviation) (human laryngeal cancer cells) The cells were cultured in an incubator (37 °C, 5% CO2). (2) Culture medium Eagle’s MEM (3) Culture medium used for subculture MEM supplemented with 8% FBS (fetal bovine serum) (4) Culture medium used for culture medium MEM supplemented with 2% FBS (fetal bovine serum) (5) Antibodies for immunostaining A. Primary antibody: anti DENV1 polyclonal hyperimmune mouse antibody (prepared) anti DENV2 polyclonal hyperimmune mouse antibody (prepared) anti DENV3 polyclonal hyperimmune mouse antibody (prepared) anti DENV4 polyclonal hyperimmune mouse antibody (prepared) anti Beijing polyclonal hyperimmune rabbit antibody (prepared) B. Secondary antibody: Peroxidase conjugated anti mouse antibody (American qualex, CA) Peroxidase conjugated anti rabbit antibody (American qualex, CA) C. Chromogenic reagent: diaminobenzidine (sigma, MO)

[0021] 5. Test procedures and results (1) Serial dilutions (diluent: 2% FBS added to MEM, 50 μL each) of Alpinia speciosa extracts (squeezed juice (10-fold dilution (x10)), decoction, distilled water) sterilized by filtration (0.8 μm filter, Millipore) were added to 50 μL of virus solutions (DENV1, 2, 3, 4 viruses, Japanese encephalitis virus) adjusted to 100 ffu per well (「ffu」 indicates focus forming unit, and the number indicates the number of virus particles capable of forming a focus). After mixing and standing at 37°C for 1 hour, 25 μL / well of the mixture was inoculated onto BHK-21 cells. After standing for one and a half hours, 100 μL of diluent was added to each well. Furthermore, after standing at 37°C for 48 hours, immunostaining (Figure 1) was performed, and the virus infectivity titer was measured by the focus method (Method A in Figure 2). Figure 3 is a microscopic photograph (magnification approximately 7 times) taken after immunostaining of a plate inoculated with 12.5 μL / well of virus solution (Dengue 2 virus) without adding Alpinia zerumbet extract and a plate inoculated with 12.5 μL / well of virus solution (Dengue 2 virus) mixed with 12.5 μL of squeezed juice (10-fold dilution (x10)). The dark-looking areas in the photograph are virus-infected cells that have formed foci. Then, with the number of foci formed when no Alpinia zerumbet extract was added taken as 100%, the antiviral effect was determined by the focus formation rate. (2) Figures 4 to 8 are graphs showing the results of experiments on Dengue 1, 2, 3, 4 viruses and Japanese encephalitis virus in (1) (the serial dilutions of the boiled juice and distilled water were only experimented up to the measured values of the 3 7 dilution). As shown in the graphs of Figures 4 to 8, all the Alpinia zerumbet extracts of squeezed juice, boiled juice, and distilled water had an antiviral effect against Dengue 1, 2, 3, 4 viruses and Japanese encephalitis virus, and a dose-dependent relationship was observed with the antiviral effect. Note that only the squeezed juice had a high antiviral effect, so it was serially diluted based on the one that had been diluted 10-fold in advance (because no foci were formed at dilutions less than 10-fold). (3) Next, in order to reduce the influence of the Alpinia zerumbet extract, the Alpinia zerumbet extract used in the experiment in (1) above was diluted 100-fold and then inoculated onto the virus, and the virus infectivity titer was measured by the focus method (Method B in Figure 2). By diluting the Alpinia zerumbet extract 100-fold to reduce the influence on the cells, it was confirmed whether the antiviral action of the Alpinia zerumbet extract acted after the virus adsorbed to the cells or directly on the virus. (4) Filter-sterilized (0.8um filter, Millipore) extracts of Alpinia zerumbet (squeezed juice (10-fold dilution (x10)), decoction, distilled water) were serially diluted (diluent: MEM supplemented with 2% FBS, 50 μL each), added to 50 μL of virus solution (Dengue 2 virus) adjusted to 10,000 ffu / well (100-fold infectious titer), and mixed. After standing at 37°C for 1 hour, to reduce the effect on cells, the mixture was diluted 100-fold (squeezed juice (1000-fold dilution (x1000)), decoction (100-fold dilution (x100)), distilled water (100-fold dilution (x100))), and then the 100-fold diluted mixture was inoculated onto BHK-21 cells. After inoculation, the cells were allowed to stand for one and a half hours, and then 100 μL of the diluent was added to each well. Then, after standing at 37°C for 48 hours, immunostaining was performed, and the virus infectious titer was measured by the focus method. Taking the number of focus formations without the addition of Alpinia zerumbet extract as 100%, the antiviral effect was determined by the focus formation rate. (5) Figures 9 to 11 are graphs showing the results of the experiment on Dengue 2 virus in (4). The significant differences in the figures were determined by t-test and represented in the graph as p < 0.05*, p < 0.01**. From the graphs in Figures 9 to 11, significant differences were observed in squeezed juice 10x3 3 ~3 6 , decoction 3 3 ~3 5 , distilled water 3 0 . In particular, squeezed juice and decoction are considered to exhibit antiviral effects in two stages: a mechanism that directly acts on the virus to inactivate it and a mechanism that acts at the stage after the virus adsorbs to cells to suppress virus infection and inactivate it. Since the antiviral effect of squeezed juice was high, it was serially diluted based on the pre-diluted 10-fold sample (dilutions less than 10-fold did not form foci).

[0022] 6. Experiment to confirm the difference in antiviral effect according to the inoculation time (1) To examine at what timing the Alpinia speciosa extract acts on the virus, the following experiment was conducted by dividing the timing of adding the Alpinia speciosa extract into three: a) before the virus infects the cells, b) when the virus adsorbs to the cells, and c) after the virus infects the cells. a. 12.5 μL each of serially diluted Alpinia speciosa extracts (filtered and sterilized (0.8 μm filter, Millipore) squeezed juice (10-fold dilution (x10)) and decoction) were added to BHK-21 cells. After standing at 37°C for 1 hour, the added Alpinia speciosa extract was removed from the BHK-21 cells, and 12.5 μL of a virus solution (Dengue 2 virus) adjusted to 100 ffu / well was inoculated into the BHK-21 cells. After inoculation, after standing for one and a half hours, 100 μL of a diluent was added to each well. Furthermore, after standing at 37°C for 48 hours, immunostaining was performed, and the virus infectivity titer was measured by the focus method. Taking the number of focus formations when no Alpinia speciosa extract was added as 100%, the antiviral effect was determined by the focus formation rate. b. 12.5 μL each of serially diluted Alpinia speciosa extracts (filtered and sterilized (0.8 μm filter, Millipore) squeezed juice (10-fold dilution (x10)) and decoction) were added to BHK-21 cells. After standing at 37°C for 1 hour, without removing the added Alpinia speciosa extract, 12.5 μL of a virus solution (Dengue 2 virus) adjusted to 100 ffu / well was inoculated into the BHK-21 cells. After inoculation, after standing for one and a half hours, 100 μL of a diluent was added to each well. Furthermore, after standing at 37°C for 48 hours, immunostaining was performed, and the virus infectivity titer was measured by the focus method. Taking the number of focus formations when no Alpinia speciosa extract was added as 100%, the antiviral effect was determined by the focus formation rate. 12.5 μL of the virus solution (Dengue 2 virus) adjusted to 100 ffu / well was inoculated onto BHK-21 cells. After standing at 37°C for 1 hour, the inoculated virus solution (Dengue 2 virus) was removed from the BHK-21 cells, and the BHK-21 cells were washed with phosphate-buffered saline (PBS). Then, 12.5 μL of each serial dilution of the Alpinia zerumbet extract (filtered and sterilized (0.8 μm filter, Millipore) squeezed juice (10-fold dilution (x10)) and decoction) was added to the BHK-21 cells. After inoculation, the cells were left standing for one and a half hours, and then 100 μL of the dilution solution was added to each well. Furthermore, after standing at 37°C for 48 hours, immunostaining was performed, and the virus infection titer was measured by the focus method. The number of focus formations without the addition of the Alpinia zerumbet extract was set as 100%, and the antiviral effect was determined by the focus formation rate.

[0023] (2) Figures 12 to 14 are graphs showing the experimental results from A to U for the squeezed juice (10-fold dilution (x10)) in (1), and Figures 15 to 17 are graphs showing the experimental results from A to U for the decoction in (1). It was confirmed that both the squeezed juice (10-fold dilution (x10)) and the decoction exerted an effect on the virus before infection and during the adsorption stage, suppressing virus growth, but had little effect on suppressing virus growth after the virus adsorption stage.

[0024] Experiment to confirm the antiviral effects of 7.5,6-Dehydrokawain (DK) and 7,8-dihydro-5,6-dehydrokawain (DDK) (1) Serial dilutions of 5,6-Dehydrokawain (DK) and 7,8-dihydro-5,6-dehydrokawain (DDK), which are known to be contained in Alpinia zerumbet (50 μL of each dilution solution: 2% FBS added to MEM), were added to 50 μL of the virus solution (Dengue 2 virus) adjusted to 100 ffu per well to form a mixture. After standing at 37°C for 1 hour, 25 μL / well of the mixture was inoculated onto BHK-21 cells. After inoculation, the cells were left standing for one and a half hours, and then 100 μL of the dilution solution was added to each well. Furthermore, after standing at 37°C for 48 hours, immunostaining was performed, and the virus infection titer was measured by the focus method. The focus formation number without the addition of 5,6-Dehydrokawain (DK) and 7,8-dihydro-5,6-dehydrokawain (DDK) was set as 100%, and the antiviral effect was determined by the focus formation rate. (2) Figure 18 is a graph showing the results of the experiment in (1). A dose-dependent relationship was observed between 5,6-Dehydrokawain (DK) and 7,8-dihydro-5,6-dehydrokawain (DDK) and the antiviral effect.

[0025] 8. Experiment to confirm the antiviral effect of proanthocyanidins (1) A serial dilution solution (dilution solution: 2% FBS added to MEM, 50 μL each) of proanthocyanidins (purified from the squeezed juice of Alpinia zerumbet to a purity of 78.8%) known to be contained in Alpinia zerumbet was added to 50 μL of a virus solution (Dengue virus type 2) adjusted to 100 ffu per well to form a mixed solution. After standing at 37 °C for 1 hour, 25 μL / well of the mixed solution was inoculated onto BHK-21 cells. After inoculation and standing for one and a half hours, 100 μL of the dilution solution was added to each well. Furthermore, after standing at 37 °C for 48 hours, immunostaining was performed, and the virus infection titer was measured by the focus method. The focus formation number without the addition of proanthocyanidins was set as 100%, and the antiviral effect was determined by the focus formation rate. (2) Figure 19 is a graph showing the results of the experiment in (1). A dose-dependent relationship was observed between proanthocyanidins and the antiviral effect.

[0026] 9. Experiment to confirm the antiviral effect of the ultrafiltration fraction of Alpinia zerumbet extract (1) The decoction, which is the extract of Alpinia speciosa, was ultrafiltered using an Amicon (registered trademark) Ultra-4 10K centrifugal filter device (manufactured by Merck Millipore Ltd., Ireland). The fraction filtered by the filter device (molecular weight ≤ 10 kDa) was obtained as the low-molecular-weight fraction, and the fraction remaining on the filter device (molecular weight ≥ 10 kDa) was obtained as the high-molecular-weight fraction. To 50 μL of the virus solution (Dengue 2 virus) adjusted to 100 ffu per well, 50 μL of the serial dilutions (diluent: MEM supplemented with 2% FBS, 50 μL each) of this fraction (high-molecular-weight fraction (molecular weight ≥ 10 kDa), low-molecular-weight fraction (molecular weight ≤ 10 kDa)) were added and mixed. After standing at 37°C for 1 hour, 25 μL / well of the mixture was inoculated onto BHK-21 cells. After standing for one and a half hours after inoculation, 100 μL of the diluent was added to each well. Furthermore, after standing at 37°C for 48 hours, immunostaining was performed, and the virus infectivity titer was measured by the focus method. Then, taking the number of focus formations when the fraction was not added as 100%, the antiviral effect was determined by the focus formation rate. (2) Figures 20 to 22 are graphs showing the results of the experiment in (1). Figure 20 shows the results of measuring the antiviral effects of the high-molecular-weight fraction (1-fold dilution) and the low-molecular-weight fraction (1-fold dilution), which are the ultrafiltered fractions of the decoction. Figure 21 shows the results of measuring the antiviral effects of the high-molecular-weight fraction (100-fold dilution) and the low-molecular-weight fraction (100-fold dilution). Figure 22 shows the results of measuring the antiviral effects of the high-molecular-weight fraction (1-fold dilution) and the high-molecular-weight fraction (100-fold dilution). When comparing the high-molecular-weight fraction and the low-molecular-weight fraction, which are the ultrafiltered fractions of the decoction, it was confirmed that the high-molecular-weight fraction has a higher antiviral effect. Also, the high-molecular-weight fraction of the decoction is considered to exhibit an antiviral effect in two stages: a mechanism of directly acting on the virus and inactivating it, and a mechanism of suppressing virus infection that acts at the stage after the virus has adsorbed to the cells and inactivates it.

[0027] 10. Cytotoxicity test (1) To test the cytotoxicity of Alpinia speciosa extracts (pressed juice (10-fold diluted (x10)), boiled juice, distilled water), each of BHK-21 cells, Vero cells, and Hep2 cells was adjusted to 5000 cells / well and cultured in a 96-well plate starting 24 hours before adding the Alpinia speciosa extracts. Then, serial dilutions of the Alpinia speciosa extracts (pressed juice (10-fold diluted (x10)), boiled juice, distilled water) (diluent: MEM supplemented with 2% FBS) were added at 10 μL per well, and after culturing for 48 hours, the live cell count reagent SF (WST-8, nacalai tesque, Kyoto) was added at 10 μL per well. After standing for 2 hours, the absorbance was measured at a wavelength of 450 nm. The absorbance without the addition of Alpinia speciosa extracts was set as 100%, and the cell viability rate of each was determined. (2) Figures 23 to 25 are graphs showing the results of cytotoxicity tests on BHK-21 cells, Vero cells, and Hep-2 cells for each of the Alpinia speciosa extracts (pressed juice (10-fold diluted (x10)), boiled juice, distilled water). No toxicity was observed for any of the pressed juice (10-fold diluted (x10)), boiled juice, or distilled water against BHK-21 cells and Hep-2 cells. Toxicity dependent on concentration against Vero cells was observed for the pressed juice at high concentrations, but not at 10x3 2 times or higher. No toxicity against Vero cells was observed for the boiled juice or distilled water. From the above results, it was confirmed that the antiviral effect of the Alpinia speciosa extracts (pressed juice (10-fold diluted (x10)), boiled juice, distilled water) using BHK-21 cells was not due to toxicity to BHK-21 cells.

[0028] 11. Discussion All of the Alpinia speciosa extracts, namely the pressed juice, boiled juice, and distilled water, showed an antiviral effect against dengue virus type 2. In particular, the pressed juice showed a very high antiviral effect at dilution concentrations (10x3 1 ~3 5 ) where no cytotoxicity was observed. Its mechanism of action was an antiviral effect through direct action on the virus (virucidal action) and mechanisms acting at the adsorption stage and after adsorption. In addition, the extract of Alpinia zerumbet showed antiviral activity against dengue virus type 1, dengue virus type 3, and dengue virus type 4 in any of the squeezed juice, boiled extract, and distilled water. Regarding Japanese encephalitis virus, antiviral effects were observed in the squeezed juice and boiled extract of the Alpinia zerumbet extract, but a virus enhancement reaction was observed in the distilled water. Therefore, since the Alpinia zerumbet extract exhibits antiviral effects against dengue virus types 1, 2, 3, and 4, it can be safely used as an antiviral agent against dengue virus containing the Alpinia zerumbet extract.

Claims

**Claim 1** An antiviral agent against Japanese encephalitis virus, comprising Alpinia zerumbet extract. **Claim 2** An antiviral agent against Japanese encephalitis virus, comprising squeezed juice of Alpinia zerumbet. **Claim 3** An antiviral agent against Japanese encephalitis virus, comprising decocted juice of Alpinia zerumbet. **Claim 4** An antiviral agent against Japanese encephalitis virus, comprising decocted juice of Alpinia zerumbet obtained by concentration with an ultrafiltration membrane having a cut-off molecular weight of 10 kDa or more. **Claim 5** An antiviral agent against dengue virus, having 5,6-Dehydrokawain (DK) or 7,8-dihydro-5,6-dehydrokawain (DDK) as an active ingredient.

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