JP7321410B2 - Norovirus inactivation method - Google Patents

Description

The present invention relates to a method for inactivating norovirus using the antiviral action of tryptanthrin contained in indigo leaves.

Conventionally, a method for producing a tryptanthrin-containing indigo leaf extract disclosed in Patent Document 1 is known. In this production method, ethanol is added to the sun-dried leaves of knotweed indigo, and the mixture is refluxed for 2 hours, naturally cooled to room temperature, and then filtered through a mesh filter. The filtrate (indigo leaf ethanol extract) thus obtained is distilled under reduced pressure using a rotary evaporator and concentrated to a predetermined total volume. Then, butylene glycol is added to the concentrated filtrate to dissolve the extract to obtain a tryptanthrin-containing indigo leaf extract.

Re-table 2012/124743

Currently, food poisoning and infectious diseases caused by norovirus are a problem, and since there is no antiviral agent effective against this norovirus, an early solution is desired. On the other hand, it is necessary to prevent side effects from appearing on the human body.

In the norovirus inactivation method according to the present invention, a tryptanthrin-containing indigo extract is used. For example, Aomori indigo extract is diluted with 100% ethanol, and Aomori indigo extract solution containing 0.002 μM tryptanthrin is used.

It is a drawing substitute photograph which showed the experimental result. It is a drawing substitute photograph which showed the experimental result.

In the tests according to the present invention, feline calicivirus (FCV), which has similar properties to norovirus, was used.
(Experimental result)
1. Preparation of feline calicivirus (FCV) Feline calici FCV strain F4 and feline kidney-derived cell line (CRFK) cells were prepared with 10% fetal calf serum (FCS; JRH Biosciences, Lenexa, Kans.), 1% L-glutamine, and 1% Eagle's minimum essential medium (EMEM; Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with fetal bovine serum (Wako Pure Chemical Industries, Osaka, Japan) and 1× non-essential amino acids (Thermo Fisher Scientific, Waltham, MA) were used. and cultured.

FCV was inoculated into CRFK monolayer cells with a virus solution (20 μl of stock FCV solution diluted in 2 ml of EMEM without FCS) and incubated at 37° C. in a 5% CO ₂ environment for 1 hour with tapping every 15 minutes. bottom.

After that, the virus solution was replaced with EMEM supplemented with 10% FCS, and the cells were continuously incubated at 37° C., 5% CO ₂ for 18 to 24 hours. After confirming the cytopathic effect, the supernatant was collected, freeze-thawed once, and centrifuged at 8,000 xg at 4°C for 20 minutes to remove cell debris. The resulting supernatant was used as virus stock. Stock viruses were used to infect CRFK cells grown in 96-well plates before calculating the 50% tissue culture infectious dose (TCID50).

2. Effect of Aomori Indigo Extract on Virus Viability CRFK cells were seeded in 96-well plates (1×105 cells/well) and incubated at 37° C., 5% CO ₂ for 12 to 18 hours. . Aomori indigo extract was diluted with 100% ethanol, and 25 μl of Aomori indigo extract solution containing 0.002 μM tryptanthrin was incubated with 75 μl of FCV for 10 minutes at room temperature. Ethanol was used as a control. Ten-fold serial dilutions of the reaction mixture were made in EMEM without FCS and inoculated onto CRFK cells.

After incubating for 1 hour at 37°C in 5% _CO2 environment with tapping every 15 minutes, cells were washed 3 times with EMEM without FCS and incubated at 37°C for 5 minutes in EMEM supplemented with 5% FCS. Incubated for 72 hours in % _CO2 environment. Cells were stained with staining solution (0.5% gentian violet, 50% ethanol, 0.9% NaCl and 1.85% paraformaldehyde) for 15 minutes and observed for cell viability.

FIG. 1 shows the results of the above experiment. The indigo extract shows its effect at a dilution concentration of 10 ⁻⁶ , but the ethanol finally shows its effect at a dilution concentration of 10 ⁻⁸ . Thus, it was found that the indigo extract was effective in reducing the virus titer even at a concentration nearly 100 times higher than that of ethanol. That is, this indigo extract is considered to be effective in reducing the virus titer of norovirus.

As shown in FIG. 2, indigo extract and ethanol have no effect on cells.

The Aomori indigo extract solution containing 0.002 μM tryptanthrin of the present invention is supplied to an object or space that may be an infection route by spraying or the like. This allows inactivation of norovirus.

The Aomori indigo extract to be used is extracted, for example, as follows.

Indigo plants that can be used in the present invention include, for example, Tate indigo (Polygonaceae), Ryukyu indigo (Polygonaceae), Ezo indigo (Ward; Brassicaceae), Mountain indigo (Euphorbiaceae), Indian indigo (Fabaceae), and the like. It is an indigo plant. In particular, Tateran, an annual plant belonging to the knotweed family, is readily available and rich in tryptanthrin, which is a unique component, and therefore is preferable for use.

In addition, the indigo plant to be used is not particularly limited in its origin or cultivation method, and may be naturally occurring indigo plants, cultivated ones, or mutants obtained by breeding these indigo plants by conventional methods. . In addition, the cyanobacterium used in the present invention may be a culture obtained by tissue culture, callus culture, cell culture, or the like. When using a plant as a raw material for extraction, a part or the whole of the plant can be used. Also, the plant body may be in a state containing water, in a frozen state, in a dry state, or in a mixture thereof. From the viewpoint of ease of handling, it is desirable to use the dry state.

In the heat-drying step, only indigo leaves are collected from the indigo plant harvested as described above, and heat-dried. In heat drying, indigo leaves are placed in the drying chamber of the drying device. Then, the indigo leaves are agitated while blowing high-temperature air heated by a burner. The amount of air is set to the extent that the indigo leaves fly in the drying room, so that the entire indigo leaves are uniformly exposed to the air.

Next, a predetermined amount of the heat-dried indigo leaves is put into a container, and a solution of d-limonene is put into the container. The solution of d-limonene is preferably placed in the container to such an extent that the indigo leaves are completely immersed. Then, after slowly stirring the inside of the container, the mixture is held at normal temperature for a certain period of time. Since d-limonene volatilizes when the temperature is high, it is preferable to keep the temperature below room temperature.

The immersion time is preferably 20 hours or more and 30 hours or less. It was found that triptanthrin can be sufficiently extracted at 20 hours, 24 hours and 30 hours, and there is no significant difference in the amount of extraction at these times compared to 48 hours and 72 hours. In other words, d-limonene can extract a sufficient amount of tryptanthrin in a short period of time. When the extraction was carried out for a short time of 20 hours or more and 30 hours or less, almost no chlorophyll was eluted, and the coloration of the extract was not observed with the naked eye, and could be evaluated as colorless and transparent.

In contrast, in the case of extraction with ethanol, the amount of tryptanthrin extracted was less than that of d-limonene after 24 hours of extraction. In addition, in the case of extraction with ethanol, the degree of coloring due to chlorophyll was strong, and the extract turned blackish green.

[Extraction efficiency and coloring]
An extraction experiment using d-limonene and ethanol was performed on indigo leaves of Shin-indigo. As the experimental conditions, 1.2 L of d-limonene solution (Wako Pure Chemical Industries, special reagent grade) was added to 100 g of dried new indigo leaves, and all the indigo leaves were immersed in the d-limonene solution, and this was stirred for 24 hours. , 48 hours, 72 hours, and 96 hours.

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

As a result, in the extraction with d-limonene, when the extraction time was 24 hours, the tryptanthrin content of the extract was 81 μg/g. When the extraction time is 48 hours and 72 hours, the tryptanthrin content of the extract is around 98 μg/g. From this experimental result, it was found that even if 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 of 24 hours extraction time. From this experimental result, it was found that triptanthrin can be rapidly extracted up to a predetermined amount in about 24 hours. Moreover, the extract obtained after the extraction time was 24 hours was colorless and transparent, and the appearance of chlorophyll could not be visually confirmed.

On the other hand, in the extraction with ethanol, the extraction time was 24 hours, and the tryptanthrin content was 73 μg/g, which was slightly smaller than that in the case of d-limonene. The amount of tryptanthrin extracted was 137 μg/g at 48 hours and 232 μg/g at 72 hours, and the amount of triptanthrin extracted increased in proportion to time. Strongly, the extract turned blackish green.

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

In the next experiment, changes in the amount of tryptanthrin extracted were measured for short periods of 1, 5, 10, 15, 20, 25 and 30 hours. In this experiment, three kinds of solvents, ie, n-hexane (Wako Pure Chemical Industries, special reagent grade), the above ethanol, and the above d-limonene were used as extraction solvents, and the amounts of tryptanthrin extracted were compared. Specifically, 5 mL of an extraction solvent was added to 0.5 g of indigo leaves, and the mixture was soaked at room temperature in a dark place according to the cold soaking method of General Rule 15 of the Japanese Pharmacopoeia. After that, extracts were collected successively at 1, 5, 10, 15, 20, 25 and 30 hours, filtered with a membrane filter, and used as samples for HPLC.

HPLC device and analysis condition detector: SPD-20A (SHIMADZU)
Liquid sending pump: LC-20AD (SHIMADZU)
Degassing unit: DGU-20A3R (SHIMADZU)
Column: COSMOSIL 5PE-MS 4.6 x 250 mm (nacalai tesque)
Mobile phase: 40% acetonitrile (Sigma-Aldrich)
Flow rate: 0.7 ml/min
Detection wavelength: 250 nm

Five hours after the start of cold immersion, the tryptanthrin contents in the n-hexane extract, d-limonene extract and ethanol extract were 18.0188 μg/ml, 36.2266 μg/ml and 38.6007 μg/ml, respectively. . The extraction amounts with d-limonene and ethanol were about twice that with n-hexane.

The tryptanthrin content in the n-hexane extract was approximately doubled to 31.1338 μg/ml 30 hours after the start compared to the content (16.2836 μg/ml) one hour after the start (16.2836 μg/ml). In the ethanol extract and the limonene extract, a marked increase in the content was observed, compared to the hexane extract (31.1338 μg/ml), the ethanol extract was 73.0444 μg/ml, and the limonene extract was 96.9976 μg/ml. It was found that 2.35-fold and 3.12-fold extraction efficiencies were obtained with ml, respectively.

On the other hand, in a comparison between the ethanol extract and the d-limonene extract, a change in the extraction efficiency was observed from 10 hours after the start of cold immersion, and 20 hours after the start of cold immersion, the ethanol extract was 58.3826 μg / ml, d - The limonene extract was 72.2422 μg/ml, and a difference in extraction efficiency of about 1.3 times was observed.

Furthermore, a large difference in color tone was observed between the above extracts. After 30 hours, the color tone of the extract was yellowish greenish brown in the n-hexane extract, and pale greenish black in the ethanol extract. The d-limonene extract was almost colorless and pale yellow. In addition, in the short-time extraction, it was found that the tryptanthrin content of the d-limonene extract was higher than that of the n-hexane extract and the ethanol extract. That is, it was found that d-limonene can efficiently extract tryptanthrin in a short time and yield a colorless extract.

[Bacteriostatic activity]
The d-limonene extract of indigo leaf tryptanthrin provides high bacteriostatic activity. Originally, both tryptanthrin and d-limonene alone are known to have the same effect. A mixture of tryptanthrin and d-limonene may also be used. However, as a result of experiments, it was found that the extract obtained by extracting tryptanthrin from indigo leaves with d-limonene exerts a remarkable bacteriostatic action compared to the single use or mixed use thereof.

Bacteriostatic activity was measured as follows.
Pearlcore (registered trademark) Mueller Hinton S agar medium (Eiken Chemical) 38 g was dissolved in 1000 ml of purified water, autoclaved (ES-245, TOMY) at 115 ° C. for 30 minutes, and placed in one petri dish (Cell Culture Dish 100 mm × 20 mm Style , NEST) and 20 mL to 25 mL of this was used as a medium. Yeast (Saccharomyces cerevisiae) was used as a test fungus. The number of bacteria was counted using a hemocytometer (C-Chip DHC-N01 NanoEnTek Inc.) and set to 107 CFU.

(1) DMSO solution of tryptanthrin, (2) d-limonene solution alone, (3) DMSO solution of tryptanthrin + d-limonene solution alone, (4) d-limonene extract of indigo leaf tryptanthrin (Aomori Indigo industry cooperative dried indigo leaves 8.33 w / v %) (1) to (3) at concentrations from 10 μM to 0.001 μM, (4) from extract undiluted solution to 10-6 diluted solution Prepared by dilution in DMSO. 180 μL of liquid medium (185 μL of blank), 5 μL of bacterial solution, and 5 μL of sample were dispensed per 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 Doujin Kagaku). That is, 10 μL/well of the chromogenic reagent WST-8 was added to the above culture medium, and the absorbance at 450 nm after further incubation for 1 hour was measured as the bacteriostatic activity value, and the MIC50 value was calculated from its concentration dependence. Since Saccharomyces cerevisiae was used as the test bacterium, the coloring reagent was diluted 8-fold with DMSO and used as a fresh preparation.

Experimental result 1
The MIC50 values (ng/μL) obtained from the respective inhibition rates of test solutions (1) to (4) are shown below.
Bacteriostatic action 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

In the test solutions (1) and (2) alone, the MIC50 value of the tryptanthrin alone solution (1) was 460 ng/μL, and the MIC50 value of the d-limonene alone solution was 300 ng/μL. . The MIC50 value of (3), in which an equivalent mixed solution of (1) and (2) was used, was 560 ng/μL, which was higher than the MIC50 value when each was used alone. From the results of this experiment, even if the tryptanthrin solution and the d-limonene solution were used in combination, no enhancement of the bacteriostatic effect was observed.

On the other hand, when tryptanthrin is extracted from indigo leaves with d-limonene (4), its MIC50 value is 3 ng/μL, which is about 100 times more effective than single use (1) or (2). It was found that the bacteriostatic action of the d-limonene extract of indigo leaf tryptanthrin was significantly increased compared to when each of them was used alone.

According to the chart measured by the above HPLC device, in the d-limonene extract, multiple compounds were measured near tryptanthrin, and it is speculated that these compounds have a favorable effect on the bacteriostatic action. be done. In contrast, extraction with ethanol measured a number of compounds at different fractions than in the case of d-limonene. These compounds do not contribute to the bacteriostatic action and are presumed to be the causative agents of coloration. From these results, it can be seen that the tryptanthrin extract obtained by limonene extraction is superior to ethanol extraction in terms of bacteriostatic action.

Further, ascorbic acid may be added during the extraction of limonene. Since the solubility of ascorbic acid in d-limonene is 0.01% or less, the amount of ascorbic acid to be added should be 0.01% or more at which crystals of ascorbic acid are precipitated, and a saturated state should be maintained. In addition, ascorbic acid is preferably added at the same time as indigo leaves are added. As a result of the experiment, it was found that the 24-hour extraction did not affect the amount of tryptanthrin extracted.

The reason for adding ascorbic acid, which is almost insoluble in d-limonene, in the present invention is as follows. Whereas limonene undergoes photoreaction, oxidative denaturation, and autoxidation, ascorbic acid exists in large amounts in the air as oxidized dehydroascorbic acid, and reacts with hydroxyl radicals more than coexisting limonene and components of indigo leaves. It functions as an antioxidant that suppresses oxidative degeneration of coexisting compounds due to its high potency.

In addition, when the tryptanthrin extract is used for dyeing cloth, when it is air-dried in sunlight after the dyeing, the production of radicals due to the photoreaction of ascorbic acid decomposes the pigment that causes "uneven dyeing" in the dyeing. It acts and induces fading. That is, ascorbic acid functions as a bleaching agent.

Furthermore, in the air drying under sunlight, the limonene also generates volatile products and unstable highly reactive products such as dicarbonyl due to oxidation and photoreaction, but these limonene oxides are ascorbic Can be decomposed by acid.

Since the product from limonene is much more reactive with radicals than alkaloids, which are the active ingredients of tryptanthrin, the decomposition of tryptanthrin due to the photoreaction of ascorbic acid should not be considered. Also good. That is, the addition of ascorbic acid has little effect on the antibacterial active ingredients of indigo leaves.

Thus, it was found that the addition of ascorbic acid during the tryptanthrin extraction brings about extremely useful effects on the tryptanthrin extract.

In the above embodiment, the tryptanthrin was extracted by immersing the indigo leaves in the limonene solution. The tryptanthrin may be extracted while the droplets are circulated. That is, since extraction is performed by bringing the limonene solution into contact with indigo leaves, the contact method is not limited to these as long as it forms such a state.

Claims (1)

A method for inactivating norovirus using a tryptanthrin-containing indigo extract consisting of a d-limonene extract of indigo leaf tryptanthrin for cells infected with norovirus (however, methods for treating humans are excluded).