JP2001328941A - Antiviral agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antiviral agent containing the active ingredient which is revealed to be contained in the concentrate of the extract from Ribes rubrum L. SOLUTION: This antiviral agent contains a cyanidin glycoside and exhibits antiviral activity against influenza A or B. The above cyanidin glycoside consists of cyanidin-3-rhamnoglucoside or cyanidin-3-glucoside. Further, this antiviral agent contains delphinidin-3-rhamnoglucoside or delphinidin-3-glucoside and exhibits antiviral activity against influenza A or B.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to influenza A
The present invention relates to an antiviral agent having antiviral activity against influenza B or influenza B.

[0002]

2. Description of the Related Art In recent years, there has been increasing interest in the physiologically active functions of substances extracted from plants, and utilization of plant extracts as various physiologically active agents has been studied.

For example, edited by Susumu Kimura et al., "Chemistry of Food Change" (Korin, 1995), edited by Masayasu Inoue, "Reactive Oxygen and Medical Food: The Search for Contact between Molecular Background and Medical Food" (Kyoritsu Shuppan) , 1996) report that anthocyanins, one of the polyphenols in food, or anthocyanidins, their aglycones, have various physiological activities such as an antioxidant effect, a carcinogenesis-suppressing effect, an immunostimulatory effect, and an antibacterial effect. Have been. The anthocyanins, Rubus,
Fruits that exhibit a deep purple to black color such as currants and grapes,
It is contained in large amounts in seeds such as black rice and black beans, and the fruits or seeds have attracted attention as physiologically active foods.

[0004] Also, recently, Eldabury (Sambuc)
us nigra L. (Japanese name: Elderberry) The extract has been reported to suppress influenza virus growth in vitro and to alleviate the symptoms of patients during an influenza B epidemic (Zakkei-Lones Z, "Eldaberry (Sambu during influenza B Panama outbreak)
cus nigra L .; ) In vitro suppression of various strains of influenza virus and reduction of symptoms by extracts ", J. Biol. Alert. Complete. Med.
1, p. 361-369, 1995).

[0005] The present inventors have previously reported that the wild species of Saxifragaceae, Kuroburi, native to Heilongjiang Province of the People's Republic of China (Rive)
s nigrum L) fruit extract (supplier: Kuro Gohonpo Co., Ltd., trade name: "Kurokarin", hereinafter referred to as "Kurofusa extract") has antiviral activity and antibacterial activity And proposed to use a concentrated extract of Kuroburo extract as an antiviral and antibacterial agent.
1-118279).

However, as an antiviral / antibacterial agent, it is more effective to extract the active ingredient in the concentrate and clarify the structure thereof, and to use the active ingredient, than to use the above-mentioned concentrated extract of the black bolling extract as it is. Is desired.

[0007]

SUMMARY OF THE INVENTION In view of the foregoing, an object of the present invention is to provide an antiviral agent containing an active ingredient which has been clarified to be contained in a concentrated extract of black tasting extract.

[0008]

Means for Solving the Problems The present inventors have repeatedly studied the components having an antiviral activity contained in the concentrated solution of the black tasting extract and found that the components are contained in the concentrated solution of the black tasting extract. The present inventors have found that a glycoside of anthocyanidin (anthocyanin) exhibits antiviral activity and is effective as an antiviral agent, and thus completed the present invention.

Therefore, the antiviral agent of the present invention is characterized by containing a cyanidin glycoside, particularly cyanidin-3-rhamnoglucoside or cyanidin-3-glucoside. Further, the antiviral agent of the present invention is characterized by containing a delphinidin glycoside, delphinidin-3-rhamnoglucoside or delphinidin-3-glucoside.

The above-mentioned cyanidin glycosides such as cyanidin-3-rhamnoglucoside or cyanidin-3-glucoside, and delphinidin glycosides such as delphinidin-3-rhamnoglucoside or delphinidin-3-glucoside are influenza A or influenza. Since it has antiviral activity against type B, it can be used as an antiviral agent against influenza A or influenza B.

[0011]

Next, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. FIGS. 1 to 3 are diagrams showing the results of high-performance liquid chromatography of components contained in the black tasting extract.

In the present embodiment, first, an Amberlite XAD resin column (manufactured by Organo Co., Ltd., trade name; ) To adsorb the anthocyanin dye onto the resin column and remove the non-adsorbed component (fraction A).
And fractionated. Next, the component adsorbed on the resin column was
Eluted with 0% ethanol. 3 (manufactured by Toyo Roshi Kaisha Co., Ltd.), and the filtrate was dried at 40 ° C. under reduced pressure to obtain a dried crude pigment. Next, 99.9% of the dried crude pigment was added.
It was dissolved in ethanol and fractionated into an ethanol non-dissolved component (fraction B) and a dissolved component. On the other hand, strongly adsorbed components that were not eluted with 70% ethanol from the resin column were eluted with 99.9% ethanol, and dried under reduced pressure to obtain a dried powder under reduced pressure (fraction C).

Next, the dissolved component obtained by fractionation into the fraction B and the dissolved component was dried again under reduced pressure, dissolved in 99.9% ethanol, and precipitated by adding 10 times volume of diethyl ether. The precipitate was freeze-dried to obtain a freeze-dried powder (fraction D).

The fraction A is a mixture of a saccharide, an organic acid and a small amount of a pigment component, the fraction B is a mixture of tannin and chlorogenic acid, and the fraction C is a decomposed product (aglycone)
And a mixture of highly hydrophobic dyes. The fraction D was an anthocyanin pigment.

Next, the fraction D was subjected to Kieselgel (Kie
sel Gel) 60 thin-layer chromatography (trade name, manufactured by Merck) using n-butanol: acetic acid: distilled water = 4: 1: 5 as a developing solvent, A '~
It was fractionated into 6 fractions of F '. The Rf value of each fraction is A ′:
0.78, B ': 0.65, C': 0.47, D ':
0.41, E ': 0.28, F': 0.16.

Next, the purified powder of the fractions A ′ to F ′ was dissolved in distilled water to obtain an aqueous solution of 10,000 μg / ml, and the aqueous solution was diluted stepwise to prepare a sample solution.

Next, the MD is placed on a 24-hole plastic plate.
CK cells (epithelial-like cells established from the kidney of an adult Cocker Spaniel dog (female)) were cultured in a monolayer, and influenza virus type A (A / PR / 8/34 strain) and influenza virus type B (B / Gifu / 2/73 strain) were inoculated at 50 PFU / well in 12 wells. After adsorbing the virus for 1.5 hours at 37 ° C., the cells were
After washing three times with EM medium, the sample solution prepared as above was mixed with 1 unit / ml of trypsin (Sigma,
Type XIII) and 0.8% agar (Difco,
(Agar Noble) in two layers. For comparison, a control was prepared by layering on an overlay agar medium containing only the trypsin and agar without the sample solution. After 48 hours, the number of plaques in each well was measured, and the ratio of the number of plaques in the sample solution at each dilution ratio to the number of plaques in the control (average value in two wells) was determined.

The IC ₅₀ was determined from the concentration (μg / ml) of the sample solution in which the number of plaques showed a 50% decrease with respect to the control. The sample solution having a lower IC ₅₀ value has a higher antiviral activity. Table 1 shows the results.

[0019]

[Table 1]

From Table 1, it is clear that the fractions D ', E' and F 'in particular have excellent anti-influenza virus activity.

Then, next, the above fraction D was separated into six fractions A ′ to F ′ by high performance liquid chromatography,
Fractions D ', E', F 'were purified. Next, fraction D ',
E 'and F' were further subjected to high performance liquid chromatography and compared with a chromatogram of a standard anthocyanin reagent (Funakoshi). Fraction D ′ separated from fraction D,
The chromatograms of E ′ and F ′ are shown in FIGS.

As a result, fraction D 'was a mixture of cyanidin glycoside, fraction E' was a mixture of cyanidin-3-rhamnoglucoside and cyanidin-3-glucoside, which are cyanidin glycosides, and fraction F ' Was identified as a mixture of delphinidin-3-rhamnoglucoside and delphinidin-3-glucoside, which are glycosides of delphinidin. In addition, the reason why the peak of each fraction is divided into two is due to the difference in glycosides.

Thus, a mixture of cyanidin glycosides in fraction D ', a mixture of cyanidin-3-rhamnoglucoside and cyanidin-3-glucoside in fraction E', and delphinidin-3-rhamnoside in fraction F ' It is clear that mixtures of glucoside and delphinidin-3-glucoside are all effective as anti-influenza virus agents.

Next, the purified powders of the fractions E ′ and F ′ were each dissolved in a 0.1% formic acid solution to obtain 10,000 μg /
A sample solution was prepared by serially diluting the resulting solution. Further, the purified powders of the fraction E ′ and the fraction F ′ were each dissolved in a 0.1% formic acid solution to obtain 5000 μg / 0.5 m
1 solution was mixed in equal amounts to prepare a sample solution that was serially diluted. Above except for using each sample solution, in the exactly same manner as in the above-described method to obtain the IC ₅₀ of the fraction E 'alone, a fraction F' alone and fraction E ', F' mixture. The formic acid solution was used to prevent the virus activity of each fraction from decreasing over time. Table 2 shows the results.

[0025]

[Table 2]

From Table 2, it is clear that the mixture of fractions E 'and F' shows the same anti-influenza virus activity as the case of the single use, and is effective as an anti-influenza virus agent.

The anti-influenza virus agent of the present embodiment is preferably a cyanidin glycoside, particularly cyanidin-3-rhamnoglucoside or cyanidin-3-glucoside, or delphinidin-3-rhamnoglucoside which is a delphinidin glycoside. Delphinidin-3-glucoside may be contained alone or as a mixture of two or more thereof, and the glycoside is not limited to the component separated from the extract of black tasting.

The anti-influenza virus agent of the present embodiment may contain the glycoside alone or as a mixture of two or more, such as a throat lozenge (troche), a gargle (gargle), processed fish meat, It can be administered in the usual form such as jam, confectionery, various drinks and the like.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of high-performance liquid chromatography of components contained in an extract of Kurobou currant extract.

FIG. 2 is a view showing the results of high performance liquid chromatography of components contained in a Kurobou stalk extract.

FIG. 3 is a view showing the results of high performance liquid chromatography of components contained in a Kurobou stalk extract.

[Explanation of symbols]

 None.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Tatsuo Suzuya 2-1-1-1, Midorigaoka-Higashi, Asahikawa-shi, Hokkaido Asahikawa Medical University (72) Itsuro Yoshida 2-1-1-1, Midorigaoka-Higashi, Asahikawa-shi, Hokkaido Asahikawa Medical University (72) Inventor Masahiro Ogasawara Hokkaido Midorigaoka Higashi 2-1-1-1 Asahikawa Medical University Asahikawa Medical University F-term (reference) 4B018 LB01 LB03 LB05 LB06 LB08 MD52 ME09 ME14 MF01 4C057 BB02 DD01 KK09 4C086 AA01 AA01 EA02 NA14 ZB33

Claims (3)

[Claims] 1. An antiviral agent comprising a cyanidin glycoside and having antiviral activity against influenza A or influenza B. 2. The method according to claim 1, wherein the cyanidin glycoside is cyanidin-3.
The antiviral agent according to claim 1, which is -rhamnoglucoside or cyanidin-3-glucoside. 3. An antiviral agent comprising delphinidin-3-rhamnoglucoside or delphinidin-3-glucoside and having antiviral activity against influenza A or influenza B.