Influenza virus inhibitors

Abstract

To provide an agent that substitutes as ethanol to have effect on influenza virus.SOLUTION: An influenza virus inhibitor is prepared with extract from Chinese indigo containing tryptanthrin. The proportion of the extract is 5 ng / ml or more relative to the solvent.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an influenza virus inhibitor using the antiviral action of tryptanthrin contained in blue leaves.

Background Art

[0002] Conventionally, a method for producing a tryptanthrin-containing blue leaf extract disclosed in Patent Document 1 has been known. In this production method, ethanol is added to the sun-dried leaves of Polygonum tinctorium, and this is refluxed for 2 hours. After naturally cooling to room temperature, the leaves are filtered through a mesh filter. The filtrate (blue leaf ethanol extract) thus obtained is subjected to vacuum distillation using a rotary evaporator and concentrated to a predetermined total amount. Then, butylene glycol is added to the concentrated filtrate to dissolve the extract, and a tryptanthrin-containing blue leaf extract is obtained.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] Currently, since the spread of influenza has become a problem, disinfection using ethanol has been utilized. However, since ethanol can also cause rough hands and skin, care is required when using it. For this reason, a drug effective against influenza virus that substitutes ethanol has been demanded.

Means for Solving the Problems

[0005] The influenza virus inhibitor according to the present invention is characterized by using a blue extract extract obtained by extracting limonene from bluegrass. It is preferable to include the blue extract at a ratio of 5 ng / ml or more with respect to the solvent. [Brief explanation of the drawing]

[0006] [Figure 1] This is a photograph used as a substitute for a diagram to show the experimental results. [Figure 2] This is a photograph used as a substitute for a diagram to show the experimental results. [Figure 3] This is a photograph used as a substitute for a diagram to show the experimental results. [Figure 4] This is a photograph used as a substitute for a diagram to show the experimental results. [Figure 5] This is a photograph used as a substitute for a diagram to show the experimental results. [Figure 6] This is a photograph used as a substitute for a diagram to show the experimental results. [Figure 7] This is a photograph used as a substitute for a diagram to show the experimental results. [Examples]

[0007] [Feline Calicivirus (FCV F4) and Culture Conditions] The Crandell Feline Kidney (CRFK) cells used in the study were grown in Eagle's Minimal Essential Medium (EMEM; Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% fetal calf serum (FCS; JRH Biosciences, Lenexa, KS), 1% L-glutamine (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and 1× non-essential amino acids (Thermo Fisher Scientific, Waltham, Massachusetts) at 37°C and 5% CO2.

[0008] FCV was absorbed into the CRFK cells for 1 hour and incubated at 37°C and 5% CO2 for 18-24 hours. After cytopathic effects (CPE) were observed, the supernatant was collected and frozen and thawed only once. Subsequently, cell debris was removed by centrifugation at 8,000 × g at 4°C for 20 minutes. The obtained supernatant was kept at -80°C and used as a virus stock. After infecting CRFK cells grown in a 96-well plate, the stock virus was titrated by calculating the 50% tissue culture infectious dose (TCID50). This stock virus solution contained 1.8 × 10⁶7 Contains TCID50 / mL.

[0009] [Influenza virus and culture conditions] The tests used influenza viruses H1N1 / pdm09 / A / Michigan / 45 / 2015, H3N2 / A / Hong Kong / 4801 / 2014, and B / Phuket / 3073 / 2014 (Yamagata). These influenza viruses were grown in Madin-Darby canine kidney (MDCK) cells obtained from DS Pharma Biomedical (Osaka, Japan). A monolayer of MDCK cells was prepared by culturing the cells in Dulbecco's Modified Eagle Medium (DMEM; Nissui) supplemented with 10% FCS and 1% L-glutamine.

[0010] This influenza virus was absorbed into MDCK monolayer cells for 1 hour and incubated in DMEM at 34°C under a 5% CO2 environment with 0.1% bovine serum albumin (BSA; Wako), 1% antibiotic-antifungal agent (Abx; ×100 stock, Thermo Fisher Scientific), and 0.6 μg / mL TPCK-treated trypsin (Thermo Fisher Scientific). Next, CPE was monitored for 4-6 days, after which the culture supernatant was collected and centrifuged at 8,000 × g for 20 minutes. The supernatant was stored at -80°C as a virus stock until use. The titer of the stock virus was evaluated by TCID50. For H1N1 / pdm09 / A / Michigan / 45 / 2015, the titer was 1.0 × 10⁶. 5 For TCID50 / mL, H3N2 / A / Hong Kong / 4801 / 2014, use 1.0 × 10 4 For TCID50 / mL, B / Phuket / 3073 / 2014 (Yamagata), use 1.0 × 10 5 It contains TCID50 / mL.

[0011] [Cytopathic effects (CPE) and plaque assay of FCV] 25 microliters of Aomori indigo extract diluted with ethanol and 25 μL of ethanol alone were incubated with 75 μL of FCV at room temperature for 10 minutes, followed by 10-fold serial dilutions with serum-free EMEM. These dilutions were then used to infect a CRFK monolayer in a 96-well microplate at 37°C and 5% CO2 for 1 hour. The dilutions in the wells were then replaced with EMEM medium supplemented with 5% FCS, 1% L-glutamine, and 1% non-essential amino acids. The plates were further incubated at 37°C and 5% CO2 for 48 hours, and CPE was evaluated by staining with Gentian violet.

[0012] FCV titers were calculated by plaque assay. Specifically, serially diluted mixtures of Aomori indigo extract and FCV were absorbed into CRFK cells in a 12-well microplate at 37°C and 5% CO2 for 1 hour. Next, EMEM medium containing 10% FCS and 1% carboxymethylcellulose (Sigma-Aldrich, St. Louis, Missouri) was overlaid and incubated at 37°C and 5% CO2 for 30 hours. These wells were fixed with 3.7% formaldehyde and stained with gentian violet. Plaques were then counted and expressed as plaque-forming units (PFUs).

[0013] [Cytopathic effects (CPE) and plaque assay of influenza virus] 90 μL of Aomori indigo extract diluted with ethanol, or 90 μL of ethanol alone, were incubated with 10 μL of influenza virus at 37°C and 5% CO2 for 1 hour. The resulting solutions were then serially diluted 10-fold in DMEM containing 0.1% BSA and 0.6 μg / mL TPCK-treated trypsin. These dilutions were then infecting a MDCK monolayer in a 96-well microplate at 34°C and 5% CO2 for 1 hour. The dilutions in the wells were then replaced with DMEM containing 0.1% BSA, 1% Abx, and 0.6 μg / mL trypsin-treated TPCK. The plates were incubated at 34°C and 5% CO2 for 48–72 hours, and CPE was evaluated by staining with Gentian violet.

[0014] The influenza virus titer was calculated by plaque formation assay. Specifically, a serial dilution mixture of the ao-mori blue extract and influenza virus was absorbed onto MDCK cells in a 12-well microplate at 34 °C and 5% CO2 for 1 hour. Next, DMEM medium supplemented with 0.1% BSA, 1% Abx, 0.6 μg / mL of TPCK-treated trypsin, and 1% carboxymethyl cellulose was overlaid and incubated at 34 °C and 5% CO2 for 30 hours. The wells were fixed with 3.7% formaldehyde and stained with Gentian violet. Then, the plaques were counted and expressed as PFU.

[0015] [Experimental Results] (1) Effect of ao-mori blue extract on FCV The experimental results are shown in Figure 1. FCV (1.8×10 7 TCID50 / mL) was incubated with ethanol-diluted ao-mori blue extract (5 ng / mL) and ethanol without ao-mori blue extract at a ratio of 3:1 for 10 minutes at 37 °C, and then the titer of infectious virus was measured.

[0016] The virus titer of the treatment sample using ao-mori blue extract was one-tenth of the virus titer of the ethanol-treated sample without ao-mori blue extract, indicating that ao-mori blue extract may inactivate FCV. The effect of ao-mori blue extract on FCV was examined several times by CPE assay and plaque assay, and a stable effect of ao-mori blue extract was obtained.

[0017] (2) Effect of ao-mori blue extract on influenza virus The experimental results are shown in Figure 2. Influenza virus H1N1 / pdm09 / A / Michigan / 45 / 2015 was incubated at 37 °C for 1 hour at a ratio of 1:9 under three conditions: ao-mori blue extract (5 ng / mL) dissolved in 10% ethanol, 10% ethanol without ao-mori blue extract, or only medium. Then, the titer of infectious virus was measured.

[0018] As a result, the viral titer decreased to one-tenth with 10% ethanol, but with Aomori indigo extract, the viral titer decreased to one-tenth even further compared to the ethanol-treated sample without Aomori indigo extract.

[0019] Specifically, when the virus solution was diluted at a ratio of 1:100, a 10% ethanol solution failed to inhibit the replication of influenza A virus. On the other hand, an Aomori indigo extract solution containing 5 ng / ml of Aomori indigo extract was able to inhibit the replication of influenza A virus.

[0020] Furthermore, when the virus solution was diluted at a ratio of 1:1000, a 10% ethanol solution could inhibit the replication of influenza A virus, but the effect was slight. On the other hand, a 5 ng / ml Aomori indigo extract could inhibit the replication of influenza A virus even at this dilution ratio.

[0021] Furthermore, when the virus solution was diluted at ratios of 1:10000, 1:100000, and 1:1000000, both the 10% ethanol solution and the 5 ng / ml Aomori indigo extract were able to inhibit the replication of influenza A virus.

[0022] Next, the effect of Aomori indigo extract on the infectivity of influenza A virus was evaluated by plaque assay. The Aomori indigo extract (5 ng / mL) completely suppressed viral plaque formation (see Figure 3).

[0023] The experimental conditions shown in Figure 3 were as follows: 90 μL of Aomori indigo extract diluted with ethanol, or 90 μL of ethanol alone, were incubated with 10 μL of influenza virus at 37°C and 5% CO2 for 1 hour. The resulting solutions were then serially diluted threefold in DMEM containing 0.1% BSA and 0.6 μg / mL TPCK-treated trypsin. These dilutions were then infected with MDCK in a 96-well microplate at 34°C and 5% CO2 for 1 hour. The dilutions in the wells were then replaced with semi-solid DMEM containing 0.1% BSA, 1% Abx, and 0.6 μg / mL trypsin-treated TPCK. The plates were incubated at 34°C and 5% CO2 for 48-72 hours, and CPE was evaluated by staining with Gentian violet.

[0024] From these experimental results, it was confirmed that the 5 ng / ml Aomori indigo extract inhibited replication in all virus solution dilution ratios ranging from 1:30 to 2430 (30, 90, 270, 810, 2430). In contrast, plaques were observed in the 10% ethanol solution at virus solution dilution ratios ranging from 1:30 to 270. Inhibition of influenza A virus replication was confirmed at virus solution dilution ratios of 1:810 and 2430. As a comparative example, when influenza A virus alone was measured, plaque formation was observed in all virus solution dilution ratios ranging from 1:30 to 2430 (30, 90, 270, 810, 2430).

[0025] These experimental results confirmed that a 5 ng / ml Aomori indigo extract inhibited the replication of influenza A virus even at a high concentration of 1:30 when the virus solution was diluted.

[0026] Next, as shown in Figure 4, the dose-dependence of Aomori indigo extract (0.05-5 ng / mL) was confirmed by plaque assay. 0.05 ng / mL of Aomori indigo extract showed similar antiviral effects to 0.1% ethanol. The PFU of 0.5 ng / mL of Aomori indigo extract was slightly reduced compared to the 1% ethanol-treated sample, but no significant difference was observed. 5 ng / mL of Aomori indigo extract significantly reduced the viral PFU compared to 10% ethanol.

[0027] At doses of 0.5 ng / ml and 0.05 ng / ml of Aomori indigo extract, the PFU / ml levels were approximately the same as those of the 1% ethanol solution and the 0.1% ethanol solution. However, at a dose of 5 ng / ml, the amount of infectious influenza A virus was reduced to an almost undetectable level. In contrast, the 10% ethanol solution did not show a significant reduction in viral load compared to the 1% and 0.1% ethanol solutions. From these experimental results, it can be understood that even at a dose of 50 ng / ml, Aomori indigo extract can reduce the amount of infectious influenza A virus to an almost undetectable level.

[0028] Furthermore, the effects of Aomori indigo extract on influenza A subtype H3N2 (Hong Kong) (Figure 5) and influenza B lineage Yamagata (Figure 6) were evaluated using a CPE assay. The infectivity of both viruses was reduced by the Aomori indigo extract. These results indicate that Aomori indigo extract inhibits the infectivity of influenza A and B viruses.

[0029] The conditions for the experimental results shown in Figure 5 are as follows: Influenza virus H3N2 / A / Hong Kong / 4801 / 2014 was incubated at 37°C for 1 hour under three conditions: with Aomori indigo extract (2.5 ng / mL) dissolved in 5% ethanol, with 5% ethanol without Aomori indigo extract, or with culture medium alone. The resulting mixtures were then serially diluted four-fold in DMEM containing 0.1% BSA and 0.6 μg / mL TPCK-treated trypsin. These dilutions were then used to infect MDCK cells in well microplates at 34°C and 5% CO2 for 1 hour. The dilutions in the wells were then replaced with DMEM containing 0.1% BSA, 1% Abx, and 0.6 μg / mL trypsin-treated TPCK. The plates were incubated at 34°C and 5% CO2 for 48–72 hours, and CPE was evaluated by staining with Gentian violet.

[0030] Furthermore, the conditions for the experimental results shown in Figure 6 are as follows: Influenza B / Phuket / 3073 / 2014 (Yamagata) was incubated at 37°C for 1 hour under three conditions: with Aomori indigo extract (5 ng / mL) dissolved in 10% ethanol, with 10% ethanol without Aomori indigo extract, or with culture medium alone. The resulting dilutions were then serially diluted five-fold in DMEM containing 0.1% BSA and 0.6 μg / mL TPCK-treated trypsin. These dilutions were then used to infect MDCK cells in well microplates at 34°C and 5% CO2 for 1 hour. The dilutions in the wells were then replaced with DMEM containing 0.1% BSA, 1% Abx, and 0.6 μg / mL trypsin-treated TPCK. The plates were incubated at 34°C and 5% CO2 for 48-72 hours, and CPE was evaluated by staining with Gentian violet.

[0031] [Aomori Indigo Extract Extraction Method] The Aomori indigo extract used in this invention is extracted, for example, as follows.

[0032] Indigo plants include, for example, Polygonum tinctorium (Polygonaceae), Ryukyu indigo (Acanthaceae), Ezo indigo (Ward; Brassicaceae), mountain indigo (Euphorbiaceae), and Indian indigo (Fabaceae). Polygonum tinctorium, in particular, is a preferred choice for use because it is readily available and rich in tryptanthrin, a unique component.

[0033] Furthermore, there are no particular restrictions on the origin or cultivation method of the indigo plant used; it can be naturally growing indigo, cultivated indigo, or mutant strains obtained by breeding using conventional methods. In addition, the indigo plant used in this invention may be a culture obtained by tissue culture, callus culture, cell culture, etc. When using the plant body as the raw material for extraction, part or all of the plant body may be used. The plant body may be in a moist state, a frozen state, a dry state, or a mixture of these. For ease of handling, it is preferable to use the dry state.

[0034] In the heat drying process, only the indigo leaves are collected from the harvested indigo plants as described above and then heat-dried. For heat drying, the indigo leaves are placed in the drying chamber of the drying apparatus. Then, high-temperature air heated by a burner is blown in while stirring the indigo leaves. The airflow is set to a level that causes the indigo leaves to fly around in the drying chamber, and the air is blown evenly over the entire indigo leaf.

[0035] Next, a predetermined amount of the heat-dried indigo leaves is placed into a container, and a d-limonene solution is added to the container. It is preferable to add enough d-limonene solution to completely immerse the indigo leaves in the container. After slowly stirring the contents of the container, it is kept at room temperature for a certain period of time. Since d-limonene will volatilize at high temperatures, it is preferable to keep it at or below room temperature.

[0036] The immersion time is preferably between 20 and 30 hours. Sufficient tryptanthrin can be extracted at 20, 24, and 30 hours, and it was found that the amount extracted at these times did not differ significantly from that at 48 and 72 hours. In other words, d-limonene allows for the extraction of a sufficient amount of tryptanthrin in a short time. Furthermore, with extraction at a short time of 20 to 30 hours, chlorophyll hardly leaches out, and the extract is not colored to the naked eye and can be evaluated as colorless and transparent.

[0037] In contrast, with ethanol extraction, the amount of tryptanthrin extracted was less than that of d-limonene after 24 hours of extraction. Furthermore, with ethanol extraction, the degree of chlorophyll coloration was stronger, resulting in a blackish-green extract.

[0038] [Extraction efficiency and coloring] Extraction experiments were conducted on new indigo leaves using d-limonene and ethanol. The experimental conditions involved adding 1.2 L of d-limonene solution (Wako Pure Chemical Industries, reagent grade) to 100 g of dried new indigo leaves, immersing all the leaves in the solution, and maintaining agitation while keeping at room temperature for 24, 48, 72, and 96 hours. The amount of tryptanthrin extracted at each extraction time was measured.

[0039] As a comparative example, 1.2 L of ethanol solution (Wako Pure Chemical Industries, reagent grade) was added to 100 g of dried new indigo leaves, and all the leaves were immersed. The solution was kept at room temperature while stirring, and the amount of tryptanthrin extracted was measured at 24 hours, 48 ​​hours, 72 hours, and 96 hours.

[0040] The results showed that in d-limonene extraction, the tryptanthrin content in the extract was 81 μg / g when the extraction time was 24 hours. When the extraction time was 48 hours and 72 hours, the tryptanthrin content in the extract was approximately 98 μg / g. From these experimental results, it was found that even when the extraction time was doubled to 48 hours and 72 hours, the amount of tryptanthrin extracted did not increase proportionally, and there was not much difference from the case with a 24-hour extraction time. From these experimental results, it was found that tryptanthrin can be rapidly extracted to a predetermined amount in about 24 hours. In addition, the extract obtained after 24 hours was colorless and transparent, and the appearance of chlorophyll could not be confirmed visually.

[0041] On the other hand, extraction with ethanol yielded a tryptanthrin content of 73 μg / g after 24 hours of extraction, which was slightly less than the amount extracted with d-limonene. Furthermore, the amount of tryptanthrin extracted increased proportionally with time, reaching 137 μg / g after 48 hours and 232 μg / g after 72 hours. However, in all extraction time cases from 24 to 72 hours, chlorophyll was extremely prominent, resulting in a dark green extract.

[0042] From the experimental results above, it was found that by extracting indigo leaves with d-limonene for 24 hours, the appearance of chlorophyll can be suppressed and tryptanthrin can be extracted efficiently.

[0043] In the following experiment, the change in the amount of tryptanthrin extracted was measured over short periods of 1, 5, 10, 15, 20, 25, and 30 hours. In this experiment, the amount of tryptanthrin extracted was compared using three solvents: n-hexane (Wako Pure Chemical Industries, reagent grade), the above-mentioned ethanol, and the above-mentioned d-limonene. Specifically, 5 mL of the extraction solvent was added to 0.5 g of indigo leaves, and cold maceration was carried out at room temperature in the dark according to the cold maceration method of General Rules 15 of the Japanese Pharmacopoeia. Subsequently, extracts were collected at 1, 5, 10, 15, 20, 25, and 30 hours, filtered through a membrane filter, and prepared as samples for HPLC.

[0044] HPLC instrument and analytical conditions Detector: SPD-20A (SHIMADZU) Liquid transfer pump: LC-20AD (SHIMADZU) Degassing unit: DGU-20A3R (SHIMADZU) Column: COSMOSIL 5PE-MS 4.6×250mm (nacalai tesque) Mobile phase: 40% acetonitrile (Sigma-Aldrich) Flow rate: 0.7 ml / min Detection wavelength: 250nm

[0045] Five hours after the start of cold maceration, the tryptanthrin content in the n-hexane extract, d-limonene extract, and ethanol extract was 18.0188 μg / ml, 36.2266 μg / ml, and 38.6007 μg / ml, respectively. The extraction amounts from d-limonene and ethanol were approximately twice the extraction amounts from n-hexane.

[0046] The tryptanthrin content in the n-hexane extract increased to 31.1338 μg / ml after 30 hours from the start (16.2836 μg / ml), approximately doubling compared to the content after 1 hour. However, at that point, a significant increase in content was observed in the ethanol and limonene extracts. Compared to the hexane extract (31.1338 μg / ml), the ethanol extract reached 73.0444 μg / ml and the limonene extract reached 96.9976 μg / ml, indicating extraction efficiencies 2.35 times and 3.12 times higher, respectively.

[0047] On the other hand, when comparing the ethanol extract and the d-limonene extract, a change in extraction efficiency was observed starting 10 hours after the start of cold maceration. After 20 hours, the ethanol extract yielded 58.3826 μg / ml, while the d-limonene extract yielded 72.2422 μg / ml, showing a difference of approximately 1.3 times in extraction efficiency.

[0048] Furthermore, significant differences in color were observed among the extracts. After 30 hours, the n-hexane extract showed a strong yellowish-greenish-brown color, while the ethanol extract showed a pale greenish-black color. The d-limonene extract was a pale yellow, almost colorless. In addition, it was found that the d-limonene extract had a higher tryptanthrin content compared to the n-hexane and ethanol extracts during short-time extraction. In other words, it was found that d-limonene allows for efficient extraction of tryptanthrin in a short time, and that a colorless extract can be obtained.

[0049] [Bacteriostatic activity] A d-limonene extract of tryptanthrin from indigo leaves exhibits high bacteriostatic activity. Both tryptanthrin and d-limonene are known to have the same effect individually. It is also conceivable to use a mixture of tryptanthrin and d-limonene. However, experimental results have shown that an extract obtained by extracting tryptanthrin from indigo leaves with d-limonene exhibits significantly greater bacteriostatic activity compared to the use of these substances individually or in mixtures.

[0050] The bacteriostatic activity was measured as follows. 38g of Pearlcore® Mueller-Hinton S agar medium (Eiken Chemical) was dissolved in 1000ml of purified water and autoclaved at 115°C for 30 minutes (ES-245, TOMY). 20mL to 25mL of this mixture was dispensed into one petri dish (Cell Culture Dish 100mm x 20mm Style, NEST) to serve as the culture medium. Yeast (Saccharomyces cerevisiae) was used as the test organism. The number of bacteria was counted using a hemocytometer (C-Chip DHC-N01 NanoEnTek inc.) and was determined to be 107 CFU.

[0051] Four test solutions were prepared: (1) tryptanthrin in DMSO solution, (2) d-limonene alone, (3) tryptanthrin in DMSO solution + d-limonene alone, and (4) d-limonene extract of indigo leaf tryptanthrin (8.33 w / v%) from dried indigo leaves of Aomori Indigo Industry Cooperative. Solutions (1) to (3) were prepared at concentrations ranging from 10 μM to 0.001 μM, and solution (4) was prepared by diluting the extract with DMSO from the stock solution to a 10⁻⁶ dilution. 180 μL of liquid medium (185 μL blank), 5 μL of bacterial suspension, and 5 μL of sample were dispensed into each well of a 96-well microplate and incubated at 38°C for 3 hours. The bacteriostatic activity of each test solution was measured using a microbial colorimetric detection kit (Microbial Viability Assay Kit-WST, Dojin Chemical). Specifically, 10 μL / well of the chromogenic reagent WST-8 was added to the culture medium, and the absorbance at 450 nm was measured after incubation for 1 hour to obtain the bacteriostatic activity value. The MIC50 value was then calculated from its concentration dependence. Since Saccharomyces cerevisiae was used as the test bacterium, the chromogenic reagent was prepared on a case-by-case basis by diluting it 8-fold with DMSO.

[0052] Experimental Result 1 The MIC50 values ​​(ng / μL) obtained from the inhibition rates of each test solution (1) to (4) are shown below. Bacteriostatic effect against Saccharomyces cerevisiae (1) Tryptanthrin DMSO solution 460 (2) d-limonene 300 (3) Tryptanthrin DMSO solution + d-limonene 560 (4) d-limonene extract of indigo leaf tryptanthrin 3

[0053] In the individual solutions of test solutions (1) and (2), the MIC50 value of the tryptanthrin-only solution (1) was 460 ng / μL, and the MIC50 value of the d-limonene-only solution was 300 ng / μL. In (3), which used an equal volume mixture of (1) and (2), the MIC50 value was higher than that of each solution used individually, at 560 ng / μL. From these experimental results, no enhancement of the bacteriostatic effect was observed when the tryptanthrin solution and the d-limonene solution were mixed.

[0054] On the other hand, when (4) was used to extract tryptanthrin from indigo leaves with d-limonene, its MIC50 value was 3 ng / μL, which was nearly 100 times stronger than (1) or (2) used alone. This significantly enhanced effect indicates that the bacteriostatic action of the d-limonene extract of indigo leaf tryptanthrin is greatly increased compared to when each is used alone.

[0055] According to the chart measured by the HPLC instrument described above, several compounds were measured near tryptanthrin in the d-limonene extract, suggesting that these compounds have a positive effect on the bacteriostatic action. In contrast, in the ethanol extraction, a large number of compounds were measured in different quantities than in the d-limonene extraction. These compounds are not thought to contribute to the bacteriostatic action and are presumed to be the cause of discoloration. From these results, it can be seen that the tryptanthrin extract obtained by limonene extraction is superior to that obtained by ethanol extraction in terms of bacteriostatic action.

[0056] Furthermore, ascorbic acid may be added during the extraction of limonene as described above. The amount of ascorbic acid added should be 0.01% or more, as its solubility in d-limonene is 0.01% or less, so that ascorbic acid crystals precipitate and a saturated state is maintained. It is also preferable to add the ascorbic acid at the same time as adding the indigo leaves. Experimental results showed that the above 24-hour extraction does not affect the amount of tryptanthrin extracted.

[0057] In this invention, ascorbic acid, which is almost insoluble in d-limonene, was added for the following reasons: Limonene undergoes photoreaction, oxidative modification, and auto-oxidation. Ascorbic acid exists in large quantities in the air as the oxidized form dehydroascorbic acid and has a higher reactivity with hydroxyl radicals than coexisting limonene or components of indigo leaves, thus functioning as an antioxidant that suppresses the oxidative modification of coexisting compounds.

[0058] Furthermore, when tryptanthrin extract is used to dye fabric, if it is air-dried in sunlight after dyeing, the photoreaction of ascorbic acid produces radicals that decompose the pigments causing "uneven dyeing," inducing fading. In other words, ascorbic acid functions as a bleaching agent.

[0059] Furthermore, during the aforementioned air drying under sunlight, the limonene also undergoes oxidation and photoreactions to produce volatile products and unstable, highly reactive products such as dicarbonyls. These limonene oxides can be decomposed by ascorbic acid.

[0060] Furthermore, since the products derived from limonene are far more reactive with radicals than the alkaloids, such as tryptanthrin, the photoreaction of ascorbic acid to decompose tryptanthrin hardly needs to be considered. In other words, the effect of adding ascorbic acid on the antibacterial active ingredients of indigo leaves is low.

[0061] Thus, it was found that adding ascorbic acid during tryptanthrin extraction produces extremely useful effects on the tryptanthrin extract.

[0062] In the above embodiment, tryptanthrin was extracted by immersing indigo leaves in a limonene solution. However, the limonene solution may also be brought into contact with the indigo leaves by dripping or spraying the limonene container onto the leaves, and the tryptanthrin may be extracted while circulating these drops. In other words, since the extraction is performed by the limonene solution coming into contact with the indigo leaves, the method of contact that creates such a state is not limited to these examples.

Claims

1. An influenza virus inhibitor for influenza A or B, containing an indigo extract solution obtained from indigo plants using limonene.

2. The influenza virus inhibitor according to claim 1, wherein the indigo extract is contained in a solvent at a concentration of 5 ng / ml or more and 50 ng / ml or less.

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