JP2018118959A - Antimicrobial composition having lindera umbellate as source material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antimicrobial composition to norovirus and rotavirus which are possible cause of infectious diarrhea, and mycoplasma which is important as a pathogen of pneumonia.SOLUTION: An antimicrobial composition has a component extracted from Lindera umbellate as an active ingredient, and has proliferation-suppressing activity to at least one microbe selected from virus and mycoplasma. Specifically, in the antimicrobial composition, the virus is at least one selected from rotavirus, norovirus, and influenza virus. More specifically, in the antimicrobial composition containing a component extracted from Lindera umbellate, and food product, medical drug, livestock feed and sanitary article containing the antimicrobial composition, the component extracted from Lindera umbellate contains proanthocyanidin.SELECTED DRAWING: None

Description

  The present invention relates to an antimicrobial composition made from plant black moji. More specifically, the present invention relates to an antimicrobial composition having a growth-inhibiting activity against at least one microorganism selected from viruses and mycoplasma, comprising a black moji extract as an active ingredient, and a food, a medicament, It relates to feed and sanitary products.

  Black mushrooms are deciduous shrubs of the camphor family (Deceae) and are widely distributed from the southern part of Hokkaido to Kyushu. It has been used for toothpicks because of its fragrance. Moreover, the trunk of the black moji is used as a herbal medicine called a shochu, and has also been used as a raw material for folk medicine such as a stomachic medicine. As its component, it is known that proanthocyanidins (also called condensed tannins) are contained. Proanthocyanidins are oligomers or polymers composed of flavan-3-ols such as catechin and epicatechin as units. The bond form between the flavan-3-ol units is the bond at the 4th and 8th positions, which is a B-type proanthocyanidin bond in addition to the 4th and 8th positions, and an ether bond between the 2nd and 7th positions. Those having a form are classified as type A proanthocyanidins. Non-patent document 1 discloses that black moji includes cinnamtannin B1, cinnamtannin B2, cinnamtannin D1 and cinnamtannin D2 which are A-type proanthocyanidins (however, non-patent document 1). In FIG. 1, cinnamtannin B1 is represented as trimer A, cinnamtannin B2 is represented as cinnamtannin B1, cinnamtannin D1 as trigger B, and cinnamtannin D2 as cinnamtannin D1).

  The structural unit of proanthocyanidins, i.e., flavan-3-ol includes at least catechin, epicatechin, gallocatechin, and epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate, etc., and those Furthermore, the structure and properties of individual proanthocyanidins, which are the result of their combination, are very diverse. Pharmacological activity also varies greatly depending on the individual proanthocyanidins.

Effective prophylactic / therapeutic agents for viral diseases and mycoplasmal diseases are generally limited, and development of more diverse and effective prophylactic / therapeutic agents is awaited. The present inventors have already made some proposals in view of these circumstances. That is, in Patent Document 1, antiviral agents containing proanthocyanidins extracted from azuki bean are used as human immunodeficiency virus (HIV), papilloma virus, infectious molluscumoma virus, wart virus, herpes virus, influenza virus, paraviron Influenza viruses, adenoviruses, rhinoviruses, coronaviruses, norwalk viruses, rotaviruses, echoviruses, and enteroviruses have been proposed as target viruses. Although the structure of azuki bean extracted proanthocyanidin used here is not disclosed, based on the m / z value ([M−H] ⁻ ) in Table 2 of Non-Patent Document 3, azuki bean proanthocyanidin is B Presumed to be a type. Moreover, the Example which shows the effectiveness with respect to the norovirus, feline calicivirus, rotavirus, and mycoplasma of the said antiviral agent is not disclosed.

  In patent document 2, the intracellular growth inhibitor of virus which uses epicatechin trimer and / or epicatechin tetramer as an active ingredient, and epicatechin trimer and / or epicatechin tetramer as an active ingredient The Mn-SOD activator is proposed. In paragraph 0023 of the same document, there is a description that “epicatechin x-mer is a compound in which the 4th and 8th positions of the epicatechin molecule are polycondensed”, so that it has a chemical structure other than at least A-type proanthocyanidins. . They are obtained by chemical synthesis. The viruses specifically tested are SARS coronavirus, influenza A virus, and feline calicivirus.

  Non-Patent Document 4 discloses an antiviral growth inhibitory action (for SARS coronavirus and influenza A virus) of proanthocyanidin 4, pentamer fraction derived from azuki bean seeds. However, the structure of the proanthocyanidins has not been identified.

  Non-Patent Document 5 discloses a virus growth-suppressing effect for green tea extract, but the target viruses here are influenza A virus, SARS coronavirus, and feline calicivirus.

  Patent Document 4 discloses an anti-SARS virus agent containing proanthocyanidins or catechins as an active ingredient, a food and drink containing the same, and the like. Specifically, grape seed or apple extracts are used in the SARS coronavirus test, but the extracts are not chemically specified. According to Non-Patent Document 6 and Non-Patent Document 7, since grape seeds and apple extracts contain B-type proanthocyanidins, the proanthocyanidins disclosed in Patent Document 4 are B-type proanthocyanidins. It is estimated to be.

であると推定される。 Patent Document 5 discloses a virus growth inhibitor containing fucodyne or proanthocyanidins as an active ingredient and a functional food containing the same. Specifically, proanthocyanidins extracted from peanut seed coat are said to be effective against AIDS virus and SARS virus. Although the chemical structure of proanthocyanidins is not specified, since it is extracted from peanut seed coat, considering the description of Patent Document 7, the chemical structure of the compound contained therein is, for example, chemical formula (I):

It is estimated that.

  On the other hand, in addition to the above, a plurality of techniques have been disclosed in search of various raw materials for antiviral agents necessary for countermeasures against various viruses.

  That is, Patent Document 3 discloses an anti-influenza virus agent containing an extract such as black moji as an active ingredient and food and drink containing the same, but does not mention chemical identification of the extract ingredient.

  Patent document 6 discloses a production inhibitor of hepatitis C virus, which contains 5 to 10-mer as an active ingredient, particularly of type A and type B proanthocyanidins, and a food and drink composition containing the same.

  In this way, technologies related to antiviral agents using various chemical species and raw materials and foods containing the same have been developed. However, safer and more active anti-viral agents have been developed to combat the diversity of viruses. Viral agents are always needed, and securing antiviral agent resources due to the diversity of raw materials remains a challenge.

  In the case of antibiotics that target bacteria, the target bacteria have a cell structure and a certain amount of common biomolecules, which can be used as targets to inhibit bacterial physiological processes. It often has antibacterial activity (antibacterial spectrum) against species. On the other hand, viruses are very different from bacteria due to their evolutionary origin, and it is rare that the molecules of each type of virus have a common structure, and their life cycle is also highly diverse. Therefore, a substance having antiviral activity for a specific virus rarely has activity for other viruses (Non-patent Document 2), and it is increasingly difficult to secure resources for antiviral agents.

  Norovirus, the cause of infectious diarrhea, is a single-stranded RNA virus classified in the Caliciviridae family. It causes serious mass infections mainly in winter, and once it develops, there is no effective treatment and simple treatment. There is no disinfection method and it is a problem because of its strong infectivity. Rotavirus, another major cause of infectious diarrhea, is a double-stranded RNA virus that is classified in the reoviridae family. It is extremely infectious and difficult to prevent infection. Is one of the main causes. In Japan, death is rare, but the number of infected people is very large, which is an important issue. Furthermore, mycoplasma is important as a pathogen of pneumonia that is prevalent in winter, and mycoplasma pneumonia often accompanies digestive symptoms such as vomiting and diarrhea. Mycoplasmal diseases are usually treated with antibiotics, but in recent years, the emergence of resistant strains with poor therapeutic effects with antibiotics has become a problem. Non-Patent Document 8 discloses that an extract of Calagatropis processa, a shrub belonging to the genus Calyptaceae, has antimycoplasma activity.

JP 2008-143840 A Japanese Patent Laying-Open No. 2015-214501 JP 2004-059463 A JP 2005-314316 A JP 2007-217410 A JP 2010-059096 A JP 2010-168300 A Japanese Patent No. 5618228

Ezaki N. et al. , “Pharmacological Studies on Linderae umbellae Ramus, IV *. Effects of Condensed Tannin Related Compounds and Pests of Instincted and Stressed-Inst. 1985, 51 (1), p. 34-8. PMID: 17340396. Virology for life sciences-mechanisms of infection and host response, medical applications, David R. Harper, Nanedo, Translated by Kuniada Shimonono and Tsukasa Seya, published February 10, 2015, p.173. Amarowicz R.A. et al. , “Antioxidant activity of extract of adzuki bean and its fractions”. of Food Lipids, 2008, 15 (1), p.119-36. Yoshiyuki Yoshinaka et al., “Analytical Meeting of the Japanese Society for Virology, Abstracts, Vol. 61, p. Yoshiyuki Yoshinaka et al., Japanese Society for Virology Academic Meeting Program, Abstracts, Vol. 62, p.375, published on October 31, 2014, “Inhibition of influenza virus growth by trace components in green tea catechins” Grases F, et al. , “Effect of consumming a supplemented supplement with abundant phenolic compounds on the oxidative status of health human volunters.” J. et al. 2015, 14:94 PMID: 26353756. Mendosa-Wilson AM, et al. , “Chemical composition and antioxidant-proxidant potential of a polyphenolic extract and a proanthocyanidin-rich fraction of skin.” 2016, 2 (2), e00073. PMID: 27441252. Muraina IA, et al. , “Antiplasma activity of some plant specific species from Northern Japan to the current used therapeutic agent.” Pharm Biol. 2010, 48 (10), 1103-7. PMID: 20819025. Guerrero CA, et al. , “Inflammatory and oxidative stress in rotavirus effect.” World J. Virol. 2016, 5 (2), p. 38-62. PMID: Pages 47 to 47 in 27175349. Doubletree JC, et al. "Inactivation of feline calicirus, a Norwalk virus surrogate." Hosp. Infect. 1999, 41 (1), p. 51-7. PMID: 9499965.

  As seen in Patent Document 3 and Non-Patent Document 8, conventional antiviral agents and antimycoplasma agents are mainly those that directly bind to or act on viruses or mycoplasma to inactivate or inhibit their growth. Some of these antiviral agents show their activity only under conditions far from the environment in the host body, and others are unsuitable for use in the human body due to toxicity issues. Moreover, since the disease by the virus cannot be prevented unless it is in principle contacted with a virus or mycoplasma, the use opportunity is limited. Therefore, it is desirable to have an activity of acting on a host cell to suppress the growth of virus or mycoplasma. Regarding viruses, Patent Document 1, Patent Document 2, Non-Patent Document 4, Non-Patent Document 5, Patent Document 5 and An antiviral agent having growth inhibitory activity as described in Patent Document 6 has been proposed.

  Among these, it is desirable that plants can be easily produced as a raw material in view of safety and availability, daily and continuous use during epidemic periods, and use as food. Those containing organically synthesized compounds as active ingredients are not easy to use safely, and at least difficult to use as food.

  Non-Patent Document 5 discloses the feline calicivirus growth inhibitory effect of norovirus substitute virus on green tea extract. However, there is no description regarding the growth inhibitory activity of rotavirus, which is another major cause of viral diarrhea, and mycoplasma, which is the cause of mycoplasmal pneumonia that may be associated with diarrhea.

  Patent Document 3 discloses an anti-influenza virus agent containing an extract of black moji as an active ingredient, and Patent Document 6 discloses a hepatitis C virus production inhibitor containing proanthocyanidins of type A and B as active ingredients. However, it is difficult to easily predict that it will affect other viruses and mycoplasmas.

  Patent Document 1 only exemplifies rotavirus and norwalk virus (classified as norovirus) together with many other viruses, and the effectiveness of the antiviral agent against norovirus, feline calicivirus, rotavirus and mycoplasma. There is no description.

  In addition, Non-Patent Document 4, Patent Document 4 and Patent Document 5 do not describe the effectiveness of the antiviral agent against norovirus, feline calicivirus, rotavirus and mycoplasma.

  In addition, if the active ingredient is completely unknown as in Patent Document 3, the selection and management of raw materials, and the quality control at the time of manufacture, which are usually performed using the existing amount of the active ingredient as an index, can be substantially performed. Not only can it not be done, but of course the mechanism of action is also unclear, which poses problems for proper use. Patent Document 1, Patent Document 2, Non-Patent Document 4, Patent Document 4, and Patent Document 6 describe that the active ingredient is a compound classified as proanthocyanidins, but a single compound The effectiveness of is not specifically exemplified, and compounds classified as proanthocyanidins are rich in diversity, and thus have the same problems as in Patent Document 3. Thus, among various proanthocyanidins derived from natural materials, there is no solution to what is effective against viruses and mycoplasmas, which is an obstacle to industrial use.

  In addition, in order to counter the emergence of various viruses and mycoplasmas and resistant strains, it has always been a challenge to develop safer and more active antiviral agents and to secure antiviral agent resources by diversifying raw materials.

  The present invention has been made to solve the above-described problems, and an object of the present invention is to provide an antiviral / antimycoplasma agent and antiviral / antimycoplasma food using chromomy as a raw material.

（式中、ｎ＝１〜６である）で示される、５量体〜１０量体のＡ型プロアントシアニジンである、［４］に記載の抗微生物用組成物である。
［６］更に本発明は、前記Ａ型プロアントシアニジンがシンナムタンニンＤ１、シンナムタンニンＤ２、シンナムタンニンＢ１又はシンナムタンニンＢ２である、［４］に記載の抗微生物用組成物である。
［７］更に本発明は、Ｍｎ−ＳＯＤ発現促進用組成物である、［１］〜［６］のいずれか一つに記載の抗微生物用組成物である。
［８］更に本発明は、［１］〜［７］のいずれか一つに記載の抗微生物用組成物を含む、ロタウイルス性疾患、ノロウイルス性疾患、インフルエンザウイルス性疾患、又はマイコプラズマ性疾患、の予防又は改善用食品である。
［９］更に本発明は、［１］〜［７］のいずれか一つに記載の抗微生物用組成物を含む、ロタウイルス性疾患、ノロウイルス性疾患、インフルエンザウイルス性疾患、又はマイコプラズマ性疾患の、予防若しくは改善用医薬である。
［１０］更に本発明は、［１］〜［７］のいずれか一つに記載の抗微生物用組成物を含む、ロタウイルス性疾患、ノロウイルス性疾患、インフルエンザウイルス性疾患、又はマイコプラズマ性疾患の、予防若しくは改善用飼料である。
［１１］更に本発明は、［１］〜［７］のいずれか一つに記載の抗微生物用組成物を含む、ロタウイルス性疾患、ノロウイルス性疾患、インフルエンザウイルス性疾患、又はマイコプラズマ性疾患の、予防若しくは改善用衛生用品である。 The present invention, which has been made to achieve the above object,
[1] An antimicrobial composition having a growth-inhibiting activity against at least one microorganism selected from viruses and mycoplasma, comprising a black moji extract as an active ingredient.
[2] Further, the present invention provides the antimicrobial composition according to [1], wherein the virus is at least one selected from rotavirus, norovirus, and influenza virus.
[3] Furthermore, the present invention is the antimicrobial composition according to [1] or [2], wherein the chromophore extract component contains proanthocyanidins.
[4] Furthermore, the present invention provides the antimicrobial composition according to [3], wherein the proanthocyanidins are A-type proanthocyanidins.
[5] Further, in the present invention, the A-type proanthocyanidins are represented by the chemical formula (II):

The antimicrobial composition according to [4], which is a pentamer to 10mer A-type proanthocyanidin represented by the formula (where n = 1 to 6).
[6] The present invention further provides the antimicrobial composition according to [4], wherein the A-type proanthocyanidins are cinnamtannin D1, cinnamtannin D2, cinnamtannin B1 or cinnamtannin B2.
[7] Furthermore, the present invention is the antimicrobial composition according to any one of [1] to [6], which is a composition for promoting Mn-SOD expression.
[8] Further, the present invention provides a rotavirus disease, norovirus disease, influenza virus disease, or mycoplasma disease, comprising the antimicrobial composition according to any one of [1] to [7], It is a food for prevention or improvement.
[9] The present invention further relates to a rotavirus disease, norovirus disease, influenza virus disease, or mycoplasma disease, comprising the antimicrobial composition according to any one of [1] to [7]. It is a medicine for prevention or improvement.
[10] Further, the present invention relates to a rotavirus disease, norovirus disease, influenza virus disease, or mycoplasma disease, comprising the antimicrobial composition according to any one of [1] to [7]. It is a feed for prevention or improvement.
[11] The present invention further relates to a rotavirus disease, norovirus disease, influenza virus disease, or mycoplasma disease, comprising the antimicrobial composition according to any one of [1] to [7]. Hygiene products for prevention or improvement.

  The composition for antimicrobials containing the extract of the chromophore of the present invention is a norovirus that is a cause of infectious diarrhea that causes serious outbreaks mainly in the winter, and infectious diarrhea that is extremely infectious and difficult to prevent Can be used in foods, medicines and feeds that have anti-proliferative activity against rotavirus, another major cause, influenza virus that causes outbreaks mainly in winter, and mycoplasma, which is important as a pathogen of pneumonia that prevails in winter It is.

  In addition, since the antimicrobial composition of the present invention has a growth inhibitory activity that acts on the host, it can be used before and after virus infection. It is extremely important as a therapeutic tool for norovirus, rotavirus, influenza virus and mycoplasma infections with limited options for pre-infection prophylaxis and post-infection therapy.

  Furthermore, the antimicrobial composition of the present invention, which has many years of eating experience, is highly safe and easily available, and is made from black moji, is a food, pharmaceutical, Or it can be used as feed. The active ingredients have been clarified to be A-type proanthocyanidins Cinnamtannin D1, Cinnamtannin D2, Cinnamtannin B1 and Cinnamtannin B2, and selection and management of raw materials and quality control during production This is also useful from the point of view. Further, both the active ingredient and the mechanism of action have been clarified, and it is extremely useful from the viewpoint of appropriate use.

FIG. 1 is MALDI-TOFMS data of chromodiproanthocyanidins (PRE) in an antimicrobial composition to which the present invention is applied. FIG. 2 is a chromatogram of a concentrate according to polymerization degree of chromodiproanthocyanidins (PRE) in an antimicrobial composition to which the present invention is applied.

Hereinafter, specific embodiments of the present invention will be described.
The black moji used in the present invention is not particularly limited as long as it belongs to the genus Black moji. (Lindera umbellata (Lindera umbellata). , Especially black moji Thunb.) Is preferred. The use site of the plant is not particularly limited, and examples thereof include leaves, bark, trunks, trunk branches, flowers, fruits, pericarp, pulp, seeds, buds, roots and rhizomes. Among these, a trunk branch is particularly preferable.

The present invention is an antimicrobial composition comprising the above plant extract as an active ingredient. Specifically, the plant is pulverized as it is or to an appropriate size, and extracted using a lower alcohol such as ethanol or isopropanol, acetone, an aqueous solution thereof, water, or the like as a solvent, and the extract is extracted from the chromophore. Get as. In addition, the above plant is pulverized as it is or to an appropriate size and used as a raw material for foods, pharmaceuticals, or feeds as it is, and components extracted by raw materials that are included separately during production, or saliva and mucus when used In addition, a component extracted by digestive juice or other secretion may be used as the extraction component. Preferably, the extract is further fractionated by a column chromatograph, a thin layer chromatograph, a high pressure liquid chromatograph, or the like to obtain a chromodiss extracted component. The carrier used in the chromatograph may be a normal carrier, and is not particularly limited. However, when obtaining an isolated and purified chromodi extract component, a carrier for gel filtration chromatography is preferable, for example, Sephadex LH- 20 (manufactured by GE Healthcare), MCI-GEL CHP-20P (manufactured by Mitsubishi Chemical Corporation), and the like are preferable. On the other hand, in the case of obtaining a crudely purified product as a chromological extract component, the above-mentioned carrier for gel filtration chromatography can be used, but a carrier for acidic substance separation is preferred, for example, Macro-manufactured by Bio-Rad. Prep CM carrier or Diaion HP2MG manufactured by Mitsubishi Chemical Corporation is preferred. More preferably, carboxyl group-modified silica gel is used. For example, Chromatorex COOH series (ACD silica ^{R 2} ) carrier manufactured by Fuji Silysia Co., Ltd. may be mentioned.

  The antimicrobial composition of the present invention includes norovirus, rotavirus, Japanese encephalitis virus, human immunodeficiency virus (HIV), herpes virus, papilloma virus, adenovirus, influenza virus, coronavirus, rhinovirus, enterovirus, echovirus and It can be used in anticipation of growth inhibitory activity against mycoplasma and the like, and in particular, it can be used in anticipation of growth inhibitory activity against norovirus, rotavirus, influenza virus, and mycoplasma. Since no culture methods have been established for human norovirus, methods using feline calicivirus classified in the same Caliciviridae family as alternative viruses are generally employed (Patent Document 8 and Non-Patent Document 10). Therefore, in the present invention, feline calicivirus was used as a substitute virus for Norovirus, and its growth inhibitory activity test was conducted. As a result, it was determined that the black moji extract component that showed feline calicivirus growth inhibitory activity had norovirus growth inhibitory activity.

  The antimicrobial composition referred to in the present invention has a growth inhibitory activity against the above-mentioned microorganisms. The antiproliferative activity referred to in the present invention is different from inactivation in which the activity is lost by binding directly to the virus or mycoplasma or the like. Or the activity which suppresses the proliferation of a virus or mycoplasma in the host cell which parasitized mycoplasma. When using virus or mycoplasma inactivation or using direct action on the virus or mycoplasma to protect the host, the active substance directly before the virus or mycoplasma infects or parasitizes the target cell It is not effective without acting, and when administered to a living body, its effective timing is limited. In contrast, when using the antimicrobial composition of the present invention and utilizing the growth inhibitory activity, it is sufficient to act on the host cell, and it is effective even when administered at the time of infection and after infection. Can also be administered prophylactically.

  Furthermore, the present invention is the antimicrobial composition, wherein the chromophore extract component comprises proanthocyanidins. That is, by fractionating the chromophore extract using the column chromatograph, chromodiproanthocyanidin (may be abbreviated as “PRE”) is obtained, and this is used as a chromodisy extract component containing proanthocyanidin. As a result of various microbial growth inhibitory activity tests using cultured cell lines, PRE showed growth inhibitory activity for rotavirus, feline calicivirus, Japanese encephalitis virus and mycoplasma. Furthermore, we have found that oral administration of PRE improves diarrhea in rats due to rotavirus.

（式中、ｎ＝０〜１０以上）で表される二量体、三量体からオリゴマー、ポリマーまでの分子量分布を持った形で存在し、その構成単位は例えば、化学式（ＩＶ）−１〜４：

で表されるフラバン−３−オールであるカテキン、エピカテキン、ガロカテキン及びエピガロカテキンに加えて、カテキンガレート、エピカテキンガレート、ガロカテキンガレート、及びエピガロカテキンガレート並びにそれらの光学異性体から成る。 Proanthocyanidins referred to in the present invention are classified into condensed tannins among polyphenols. For example, chemical formula (III):

(Wherein n = 0 to 10 or more), and present in a form having a molecular weight distribution from a trimer to an oligomer or polymer, and the structural unit thereof is, for example, chemical formula (IV) -1 ~ 4:

And catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate and optical isomers thereof, in addition to catechin, epicatechin, gallocatechin and epigallocatechin represented by

で表されるＡ型プロアントシアニジンに分類される。 Proanthocyanidins are mainly bonded to B-type proanthocyanidins represented by the chemical formula (III) and 4- and 8-positions, which are bonded between the 4- and 8-positions due to the difference in the bonding mode between the structural units. In addition, it has a bonding mode in which an ether bond is formed between the 2-position and the 7-position, for example, the chemical formula (V):

It is classified into A type proanthocyanidins represented by

（式中、ｎ＝０〜７）で表されるＡ型プロアントシアニジンが、ＰＲＥには含まれていることを見出した。 Furthermore, the present invention is preferably an antimicrobial composition wherein the proanthocyanidins are A-type proanthocyanidins. That is, the present inventors individually quantified proanthocyanidins in PRE using LCMS. As a result, it was confirmed that A-type proanthocyanidins were contained in the PRE. Moreover, when PRE was confirmed by MALDI-TOFMS, a peak presumed to be an 11-mer from a trimer of type A proanthocyanidins was obtained. On the other hand, in the LCMS analysis of PRE, cinnamtannin B1 and cinnamtannin D1 of type A proanthocyanidin trimer and cinnamtannin B2 and cinnamtannin D2 of type A proanthocyanidin tetramer were detected. Considered together, these four molecules are polymers in which the flavon-3-ol unit is sequentially bonded in the same manner in a carbon-carbon bond at one place, chemical formula (VI):

It was found that A-type proanthocyanidins represented by (wherein n = 0 to 7) are contained in PRE.

（式中、ｎ＝１〜６）で表される、５量体〜１０量体のＡ型プロアントシアニジンを含む抗微生物用組成物である。即ち発明者らは、ＰＲＥを、カラムクロマトグラフを用いて分画して、重合度別に濃縮した画分を複数得て、増殖抑制活性試験を行い、前記Ａ型プロアントシアニジンの５量体〜１０量体を濃縮した画分がウイルス増殖抑制作用を顕著に示すことを、本発明者等は見出したのである。 In the present invention, it is more preferable that the A-type proanthocyanidins have the chemical formula (II):

It is an antimicrobial composition containing pentamer to 10mer A-type proanthocyanidins represented by (wherein n = 1 to 6). That is, the inventors fractionated PRE using a column chromatograph to obtain a plurality of fractions concentrated according to the degree of polymerization, conducted a growth inhibitory activity test, and obtained the pentamer to 10 type A proanthocyanidins. The present inventors have found that a fraction enriched in a mer exhibits a virus growth inhibitory action.

  Furthermore, the present invention provides the antimicrobial composition wherein the A-type proanthocyanidins are cinnamtannin D1, cinnamtannin D2, cinnamtannin B1 or cinnamtannin B2. That is, the present inventors have analyzed from the PRE, the trimer cinnamtannin B1 and cinnamtannin D1, which are a kind of type A proanthocyanidins, and the tetramer cinnamtannin B2 and cinnamtannin D2 in the PRE. These were extracted from Chromody and isolated and purified using a chromatograph. As a result of conducting a growth inhibitory activity test on these, it was found that the virus growth inhibitory activity was remarkably exhibited.

  Furthermore, the present invention is an antimicrobial composition which is a composition for promoting Mn-SOD (manganese superoxide dismutase) expression. That is, in order to elucidate the mechanism of action, the present inventors added PRE to the culture medium of African green monkey-derived Vero cells, cultured for 24 hours, extracted proteins from the cells, Western blotted, Mn -The amount of SOD present was quantified. As a result, it was confirmed that the amount of Mn-SOD present by the PRE treatment was increased. It is known that reactive oxygen species induced by an infected virus may play an important role in the expression of virus action on host cells (Non-patent Document 9). Therefore, PRE is thought to suppress viral growth as a result by inducing the expression of Mn-SOD that detoxifies its reactive oxygen species.

  Furthermore, the present invention is a food for preventing or improving rotavirus disease, norovirus disease, influenza virus disease, or mycoplasma disease, comprising the antimicrobial composition. That is, the antimicrobial composition of the present invention can be taken as a food, and as a result, as a food for preventing or improving rotavirus disease, norovirus disease, influenza virus disease, or mycoplasma disease. Function. Food includes non-alcoholic beverages such as juice, fermented beverages such as alcoholic beverages and yogurt, solid confectionery such as tablet confectionery and candy, chewable confectionery such as gum and gummy confectionery, and vitamins, minerals, amino acids, proteins, etc. This includes forms such as dietary supplements. Functionally, foods for specified health use (so-called “Tokuho”), functional nutritional foods, functional labeling foods and the like are included.

  Furthermore, the present invention is a medicament for preventing or improving rotavirus disease, norovirus disease, influenza virus disease, or mycoplasma disease, or feed for prevention or improvement, comprising the antimicrobial composition. That is, the antimicrobial composition of the present invention can be administered orally or parenterally (that is, injection, transdermal, transmucosal) as a pharmaceutical, and tablets, powders, granules, fine particles, Dosage forms such as granules, solutions, suspensions, emulsions, capsules and the like can be employed. These preparations can be formulated according to a conventional method together with other pharmaceutically acceptable additives, for example, carriers such as excipients, binders and lubricants. In addition, sweeteners, colorants and the like can be added and taken. These preparations can be carried out by the method described in the Japanese Pharmacopoeia General Rules for Preparations.

  In the antimicrobial composition of the present invention, the chromophore extract component used as an active ingredient ingests about 0.1 to 2 g, preferably about 0.2 to 1 g per day, based on its microbial growth inhibitory activity. In general, the content of the active ingredient to be taken immediately before each meal is set 1 to 3 times a day, preferably 3 times a day, and it may be designed as a preparation for ingesting the dose.

  Moreover, when it contains PRE itself, PRE can also be diluted with a suitable solvent and can also be prepared as a liquid composition. Alternatively, PRE itself may be mixed with an appropriate carrier, preferably fatty acid triglyceride, and filled in soft capsules or the like in the liquid state.

  When the antimicrobial composition of the present invention is used as a feed, it can be produced, for example, by mixing it with livestock feed or pet food. The produced feed can be administered to livestock, mammals, chickens or pets.

  In the formulation as the above food, medicine or feed, commonly used excipients, binders, disintegrants, lubricants, stabilizers, surfactants, solubilizers, reducing agents, buffering agents, Adsorbents, fluidizers, antistatic agents, antioxidants, sweeteners, flavoring agents, cooling agents, light-blocking agents, flavoring agents, fragrances, fragrances, coating agents, plasticizers, etc. Two or more kinds can be appropriately selected and added.

  Specific examples of such formulation additives include crystalline cellulose, low-substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, croscarmellose sodium, maltodextrin, ethylcellulose, lactose, sorbitol, silicic anhydride, and silicic acid. Magnesium, hydroxypropylcellulose, stearic acid, oleic acid, liquid paraffin, dicalcium phosphate, dibutyl sebacate, macrogol, propylene glycol, corn starch, starch, pregelatinized starch, gelatin, popidone, crospovidone, glycerin, polysorbate 80, citrus Acid, acesulfame potassium, aspartame, sodium carbonate, talc, magnesium stearate, calcium stearate, etc. That.

  In addition, in the formulation as an antimicrobial composition of the present invention, other active ingredients such as vitamins and minerals within the range of quality and quantity that do not impair the antimicrobial activity of the active ingredient chromomy extract. Can also be added.

  Moreover, it can be set as the antimicrobial sanitary goods with respect to the said microorganisms by adding the antimicrobial composition of this invention to sanitary goods, such as a spray, a mask, soap, wet tissue, lotion, and cream.

  Examples of the present invention will be described in detail below, but the scope of the present invention is not limited to these examples.

Example 1. Production of Chromodiproanthocyanidin (PRE) The trunk branch of Kuromoji (Lindera umbellata Thumb.) Is extracted with 30% ethanol of 3 times weight. Ethanol was removed using a rotary evaporator, the ethanol concentration was adjusted to 5% or less, and filtration was performed using filter paper. The filtrate was passed through a column of 1/10 carboxyl group-modified silica gel (“ACD silica” manufactured by Fuji Silysia) packed with distilled water, and the column was washed with 10 times the column volume of distilled water. The components retained in 30% ethanol were eluted, ethanol was removed using an evaporator, and the mixture was dried under reduced pressure using a freeze dryer (RLEII-103, manufactured by Kyowa Vacuum Technology Co., Ltd.) to prepare PRE.

Example 2 Production of proanthocyanidin concentrate from Kuromoji hot water extract Add 10 times the amount of water to the trunk branch of Kuromoji, heat extract at 98 ° C for 3 hours, and dry under reduced pressure in a freeze dryer to extract hot water of Kuromoji I got a thing. A 2-fold amount of 100 mL of 30% methanol is added to the suspension, centrifuged at 1700 × g for 10 minutes, the supernatant is taken, and a Sephadex LH-20 (manufactured by GE Healthcare) column (φ50 ×) filled with 30% methanol. 270 mm). Subsequently, the column was washed with 4 volumes of 30% methanol, 4 volumes of 70% methanol, and 4 volumes of 100% methanol. Eluted with 4 volumes of 70% acetone and dried in vacuo to a proanthocyanidin concentrate.

Example 3 Concentration by degree of polymerization PRE 0.75 g was dissolved in 6 volumes of ethanol, and passed through a column (φ30 mm × 300 mm) filled with Sephadex LH-20 with ethanol. Elution of ethanol and methanol in order using 2.1 L each and then 0.9 L of 70% acetone in turn, fractions every 300 mL, F1 as the first eluted fraction, and a total of 17 fractions F1 to F17 in order. Obtained. F1 to F3, F4 to F6, F7 and F8, F12 to F14, and F15 to F17 fractions are mixed together, respectively, F1-3 fraction, F4-6 fraction, F7-8 fraction, F12 -14 fraction and F15-17 fraction. F9, F10, and F11 fractions were used as F9 fraction, F10 fraction, and F11 fraction as they were. Each fraction was dried under reduced pressure with a rotary evaporator to obtain each fraction.

Example 4 Proliferation inhibitory activity test of feline calicivirus (norovirus alternative virus) Human norovirus has not been established as a culture method, so the method using feline calicivirus classified in the same caliciviridae as an alternative virus is generally adopted. (Patent Document 8). Therefore, a growth inhibitory activity test was performed using feline calicivirus.
As the feline calicivirus, F9 strain (purchased from ATCC (American Type Culture Collection)) was used. CRFK cells (derived from fetal fetal kidney, purchased from ATCC) were used as the cultured cells, the medium was Dulbecco's minimal essential medium (DMEM) containing high glucose, 10% fetal bovine serum (Sigma Aldrich) and PSm (100 U / mL penicillin). , 100 μg / mL streptomycin, manufactured by Nacalai Tesque) was used and cultured at 37 ° C. in the presence of 5% CO ₂ . The virus stock solution was infected at room temperature for 60 minutes under the condition that the amount of virus per cell was 0.1 (MOI (multiplicity of infection) = 0.1), and the medium was collected 48 hours after infection. After filtration through a 0.45 μm filter (Millipore), the solution was dispensed and stored at −70 ° C.
The virus growth inhibitory activity test was performed by adding PBS (−) (Mg ²⁺ , Ca ²⁺ -free 0.05 mol / L phosphate buffer, 0.15 mol / L with 1% bovine serum albumin (Boehringer Ingelheim). A virus solution of 100 PFU / 0.2 mL was prepared with NaCl, pH 7.0), and inoculated into each well (6 well plate, manufactured by Corning) in which 90% monolayer was formed, and then at room temperature for 60 minutes. Infected. After infection, the cells were cultured for 3 days in DMEM (containing 2% fetal bovine serum and 1.1% methylcellulose) to which various concentrations of specimens were added. After cultivation, methylcellulose is removed, cells are fixed / stained with 2.5% crystal violet (30% ethyl alcohol, 1% ammonium oxalate), washed / decolored three times with PBS (-), and the number of plaques is counted. Then, the IC ₅₀ value (concentration (μg / mL) that inhibits the growth of feline calicivirus by 50%) of the test substance that reduces plaque by 50% was determined.

Example 5 FIG. Rotavirus (G1 strain) growth inhibitory activity test Rotavirus (G1 strain) and sensitive cell line MA104 (African green monkey kidney cells) were purchased from ATCC. The cells were cultured in DMEM supplemented with 10% fetal calf serum and PSm at 37 ° C. in the presence of 5% CO _{2. The} virus stock solution was cultured in DMEM supplemented with 0.1% bovine serum albumin. After washing twice, infection was carried out at MOI = 0.1 in the same manner as in Example 4 above, and the medium was collected 48 hours after infection, filtered through a 0.45 μm filter, dispensed, and stored at −70 ° C.
The virus growth inhibitory activity of the specimen contains 1% bovine serum albumin after washing a cultured cell in which 90% monolayer is formed on a 12-well plate twice with DMEM supplemented with 0.1% bovine serum albumin. Virus diluted with PBS (−) was infected with MOI = 0.3 to 0.5 at room temperature for 60 minutes, and after infection, 4 times in the same medium containing various concentrations of samples and 2.5 μg / mL trypsin. Incubation was performed for 5 days. After culturing, the cells are fixed / stained with 2.5% crystal violet solution, washed / decolored three times with PBS (−), sterilized and dried under ultraviolet light to stain viable cells and inhibit cell death by 50%. The concentration of the test substance was determined as an IC ₅₀ value.

Example 6 Mycoplasma Growth Inhibitory Activity Test The mycoplasma inhibition test was carried out by M. cerevisiae isolated from Vero-E64 cells (African green monkey kidney cells) in the experiment. Salivarium was used. M. on a 6-well plate (Corning). Samples of various concentrations were added to Vero cells that were continuously infected with Salivalium, and the medium was collected 48 hours later, and 10 μL each was applied to (P9) Mycoplasma PPLO agar medium (manufactured by Nikken Biomedical Research Institute). And cultured for 2 days at 37 ° C. in a 5% CO ₂ incubator. The amount of mycoplasma in 1 mL is calculated as CFU (Colony Forming Unit)) / mL, and the IC ₅₀ value of the test substance that reduces CFU by 50% (concentration that inhibits the growth of mycoplasma by 50% (Inhibition concentration) , (Μg / mL)).

Example 7 Japanese encephalitis virus growth inhibitory activity test Japanese encephalitis virus (JEV, Nakayama strain, distributed from the Department of Microbiology, Kobe University School of Medicine) uses Vero (African green monkey kidney cells) and is 10 in Dulbecco's minimum essential medium (DMEM). Using a medium supplemented with fetal bovine serum and PSm, the cells were cultured at 37 ° C. in the presence of 5% CO ₂ . In the virus stock solution, cultured cells in which 90% monolayer was formed were infected at MOI = 0.1, and the culture supernatant was collected 48 hours later. The supernatant of the collected culture broth was serially diluted 10-fold with PBS (−) supplemented with 1% bovine serum albumin, and 0.2 mL of each dilution of each stage was inoculated into each well (6-well plate). After 60 minutes of infection at 25 ° C., the cells were cultured for 7 days in DMEM (containing 5% fetal calf serum) supplemented with 1.0% methylcellulose. After culturing, methylcellulose was removed, the cells were stained with 2.5% crystal violet, washed / decolored 3 times with PBS (−), and the number of plaques was measured. The analyte concentration at which the number of plaques was reduced by 50% was determined as the IC ₅₀ value.

Example 8 FIG. Influenza virus growth inhibitory activity test Influenza A virus, PR8 strain was purchased from ATCC. For virus plaque assay, MDCK cells were prepared in monolayer culture at 37 ° C in the presence of 5% CO _{2 in} Eagle's minimum nutrient medium (MEM) supplemented with 10% fetal bovine serum and antibiotics on 6-well plates. . After washing the cells once with MEM supplemented with 0.1% BSA, the virus stock solution was diluted ^10-6 times with MEM supplemented with 0.1% BSA, 200 μL (about 100 PFU) of which was inoculated into each well, and at room temperature. Infected for 60 minutes. After infection, in a medium mixed with an equal amount of 2% agar aqueous solution kept at 45 ° C. and doubling concentration of MEM (containing 0.2% BSA, 50 μg / mL trypsin, 200 μg / mL streptomycin, 200 U / mL penicillin) Samples added to each concentration were overlaid on the cells and cultured at 37 ° C. for 3 days.
After incubation, the cells were fixed with 2 mL of a methanol: acetic acid = 5: 1 solution, the agar layer was removed, the cells were stained with the 2.5% crystal violet solution described above, washed with PBS (−) three times, and plaques were removed. The concentration of the test substance that inhibits virus growth by 50% was determined as the IC ₅₀ value.

Example 9 Rotavirus Pharmacological Test LEW / CrlCrlj rats (4 days old) purchased from Charles River, Japan were orally inoculated with rotavirus purchased from ATCC, and 1 hour later, the PRE prepared in Example 1 was 70 μg per day. Orally administered daily at regular times. The stool properties 7 days after the inoculation were confirmed, and the presence or absence of diarrhea was confirmed.

Example 10 Measurement of the effect of PRE on Mn-SOD inducing activity PRE was added to Vero cells cultured in 6-well plates (2.5 mL medium per well, 2 × 10 ⁶ cells, purchased from ATCC) at 0 or 100 μg / mL The cells were cultured at 37 ° C., and after 24 hours, all cells were collected in 0.3 mL of polyacrylamide electrophoresis (abbreviated as SDS-PAGE) sample buffer, and all proteins were separated by SDS-PAGE. Next, the gel after electrophoresis was stained with Coomassie Brilliant Blue R250 (CBB, Sigma-Aldrich) and then decolorized with 7% acetic acid. Images were taken with a CCD camera (Bio-rad) and analyzed using image analysis software (Image Lab software, Bio-rad). For the measurement of Mn-SOD induction, after separating all the above-mentioned proteins by SDS-PAGE, the proteins are transferred to a PVDF membrane (Polyvinylidene fluoride (Polyvinylidene fluoride) membrane, manufactured by Millipore) and tubulin as a primary antibody. (Tubulin: indicator of increase / decrease in total cellular protein, manufactured by Santa Cruz) and Mn-SOD antibody (intracellular antioxidant factor, manufactured by Stressgen), secondary antibody was anti-rabbit labeled with alkaline phosphatase Using a goat antibody (manufactured by Santa Cruz), detection was performed by color development with NBT (nitroblue tetrazolium salt, manufactured by Boehringer), and immunoblotting was performed. The relative expression level was measured from image analysis as described above.

Example 11 Analysis of PRE for anthocyanidins PRE analysis of proanthocyanidins for PRE was performed by Agilent 1260 Infinity, Agilent 6150 LCMS (liquid chromatograph mass spectrometer, manufactured by Agilent). The ion source was observed by ESI and negative mode. Column is poroshell 120 EC-C18 φ: 3.0 × 150 mm, particle size: 2.7 μm (manufactured by Agilent), column temperature is 40 ° C., flow rate is 0.5 mL / min, and solution A is 0.05% formic acid solution The liquid B was changed as follows by using acetonitrile with 0.05% formic acid as a mobile phase and changing the ratio of the liquid B as follows. 0 minutes: 5%, 12 minutes: 20%, 15 minutes: 70%, 16 minutes: 5%, 17 minutes: 5%. Catechin and epicatechin were obtained by obtaining mass chromatograms of m / z = 289, cinnamtannin B1 and cinnamtannin D1 were m / z = 863, cinnamtannin D2 and cinnamtannin B2 were m / z = 1115 and obtained peaks. The area value was quantified, and the concentration of each substance was calculated by comparison with a calibration curve obtained from the standard.

Example 12 Analysis by MALDI-TOFMS (Matrix Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometer) Using a matrix solution containing CHC (α-cyano-4-hydroxycinnamic acid), PRE was prepared by MALDI-TOFMS ultraflexstream (manufactured by Bruker Daltonics) ) Using a conventional method.

Example 13 Preparation of preparations Catechin and epicatechin were obtained from Nacalai Tesque, and procyanidin C1 and cinnamtannin A2 were obtained from PhytoLab GmbH & CO. Obtained from KG.
20 kg of the trunk branch of black moji was extracted with 60% acetone, the extract was concentrated, and the precipitate was filtered off to obtain a concentrated extract (726.2 g). This was applied to a column packed with Sephadex LH-20, and eluted by passing through the methanol aqueous solution while changing the methanol concentration from 0% to 100%. Fraction 1 (51 g), fraction 2 ( 67 g), fraction 3 (111 g) and fraction 4 (215 g). Fraction 3 was applied to a column packed with MCI-GEL CHP-20P (Mitsubishi Chemical Co., Ltd.), eluted with 30% methanol and purified repeatedly, and cinnamtannin B2 (1.2 g) and cinnamtannin D2 ( 4.5 g) was isolated and purified to prepare a sample. Fraction 3 was subjected to Sephadex LH-20 and eluted with 60% methanol and purified repeatedly, and cinnamtannin B1 (4.5 g) and cinnamtannin D1 (13.5 g) were isolated and purified. It was a standard product.

Example 14 Analysis by HPLC (high performance liquid chromatograph) using a Diol column (Analysis of PRE fractions separated by Sephadex LH20)
According to the method described in the literature of Kelm et al. (Kelm MA, et al., J. Agric. Food Chem. 2006, 54 (5), p. 1571-6. PMID: 16506802). That is, it was carried out by HPLC (Lachrom Elite, manufactured by Hitachi) equipped with a fluorescence detector. Column is Inertsil WP300 Diol 5 μm (4.6 × 250 mm) (GL Science), column temperature is 30 ° C., flow rate is 0.8 mL / min, solution A is acetic acid: acetonitrile (2:98), solution B is acetic acid : Water: methanol (2: 3: 95) was used as the mobile phase, and the ratio of the B liquid was changed as follows. 0 minutes: 0%, 35 minutes: 40%, 40 minutes: 40%, 45 minutes: 0%, 55 minutes: 0%. The excitation wavelength of the fluorescence detector was 276 nm, the measurement wavelength was 316 nm, and the chromatogram was obtained. As shown in the literature of Kelm et al., Under these conditions, proanthocyanidins of each degree of polymerization are eluted in order from the smallest degree of polymerization, and the amount of proanthocyanidins of each degree of polymerization can be known. For measurement of multivalent ions, liquid A was acetic acid: acetonitrile (1: 999), liquid B was methanol: water: acetic acid (949: 50: 1), and the ratio of liquid B was the same as above. A mixer was connected after the above-mentioned column, water was mixed at 0.5 mL / min, and it was measured by connecting it to a mass spectrometer (Agilent 6150, manufactured by Agilent).

  Example 15. A 10% weight aqueous solution of 33% or 66% ethanol was added to the trunk branch of black moji, followed by extraction, followed by drying under reduced pressure to obtain a black momodiethanol extract.

<Results and discussion>
[1] Viral and Mycoplasma Growth Inhibitory Activity of Chromodiproanthocyanidins (PRE) Chromodiproanthocyanidins (PRE) were obtained from chromomoly extracts using an ACD silica packed column (Example 1). About this, the growth inhibitory activity test using the cultured cell system was done (Examples 4-8). PRE showed an IC ₅₀ of 30 μg / mL or less for rotavirus, feline calicivirus, Japanese encephalitis virus, influenza virus, and mycoplasma, and showed growth inhibitory activity (Table 1).

In addition, a proanthocyanidin concentrate was obtained from a hot water extract of black moji using a Sephadex LH-20 column (Example 2). As a result of carrying out the growth inhibitory activity test similarly about this concentrate (Examples 4-7), although activities are lower than PRE obtained in Example 1 except Japanese encephalitis virus, rotavirus and feline calici Regarding the virus, Japanese encephalitis virus and mycoplasma, IC ₅₀ of 30 μg / mL or less was shown, and the activity was recognized (Table 2).

[2] Viral growth inhibitory activity of chromody extract Cromodi hot water extract and chromodiethanol extract were obtained (Example 2), and the results of a similar growth inhibition activity test were conducted on this extract (Example 4 ( Feline calicivirus), Example 8 (influenza virus)), showing an IC ₅₀ of 100 μg / mL or less, indicating growth inhibitory activity (Tables 3 and 4).

[3] Quantification of each component of proanthocyanidins in PRE Various types of proanthocyanidins in PRE were individually quantified (Example 11). As a result, it was revealed that these proanthocyanidins are contained in the PRE (Table 5).

  Table 6 summarizes the binding mode of proanthocyanidins and the distinction between catechin or epicatechin units.

に示す。 The structure of proanthocyanidins is represented by chemical formulas (VII) to (VIII):

Shown in

[4] MALDI-TOFMS analysis of PRE The PRE extracted from Kuromoji was confirmed by MALDI-TOFMS (Example 11). As shown in FIG. 1, peaks from trimers to 11-mers were obtained. For example, sodium adduct ion m / z = 887.2 of trimeric cinnamtannin B1 and cinnamtannin D1 (both monoisotopic masses are 864.2) is detected as a peak. Further, for example, sodium adduct ion m / z = 1175.3 of tetramer cinnamtannin B2 and cinnamtannin D2 (both monoisotopic masses are 1152.3) is detected as a peak. Similarly, for the 5-11 mer, the actual values obtained by MALDI-TOFMS agree well with the theoretical values calculated from the expected molecular formula (Table 7).

（式中、ｎ＝０〜７を表す。）で表されるＡ型のプロアントシアニジン）が、ＰＲＥには含まれていることが確認された。 The difference in the mass of the main peak in the MALDI-TOFMS spectrum (FIG. 1) is 288, which corresponds to the difference in structure in which the flavan-3-ol unit is polymerized at one carbon-carbon bond. Considering that LCMS analysis detected trimeric cinnamtannin B1 and cinnamtannin D1, and tetramer cinnamtannin B2 and cinnamtannin D2, 4th and 8th positions were considered. These four molecules (Cinnamtannin B1, Cinnamtannin D1, Cinnamtannin B2 and Sinnamtannin B1 and Cinnamtannin B1) are combined in one place with an ether bond between the 2-position and the 7-position in addition to the carbon-carbon bond of Namtannin D2) is a polymer in which flavan-3-ol units are bonded in the same manner in one carbon-carbon bond (ie, chemical formula (VI):

(In the formula, n represents 0 to 7) A type proanthocyanidins) was confirmed to be contained in the PRE.

[5] Virus growth inhibitory activity test of preparations In order to confirm the virus growth inhibitory activity of the substances contained in PRE, with respect to the preparations of Table 8 prepared in Example 13, rotavirus, feline calicivirus and influenza The virus was subjected to a growth inhibitory activity test. As a result, strong virus growth inhibitory activity was observed for cinnamtannin B1 and cinnamtannin B2 for rotavirus and cinnamtannin B1 for feline calicivirus. Moreover, strong virus growth inhibitory activity was recognized by cinnamtannin D1 and cinnamtannin D2 with respect to influenza virus.

[6] Concentration of PRE according to polymerization degree using gel filtration column In addition, in order to examine the virus growth inhibitory activity of proanthocyanidins having a polymerization degree of 5 or more, PRE is separated by a Sephadex LH-20 packed column. Fractionation (Example 3). As a result, particularly high virus growth inhibitory activity was confirmed in the F10 and F11 fractions for rotavirus and feline calicivirus and in the F11 fraction for influenza virus (Table 9).

Of the fractions in Table 9, F7-8 fraction, F9 fraction, F10 fraction, F11 fraction and F12-14 fraction were analyzed by HPLC using a Diol column (Example 14). As shown in FIG. 2, the F10 and F11 fractions of the highly active fraction are pentamer, hexamer, heptamer, octamer, 9mer, and 10mer polymers. It was confirmed to contain. Among them, the pentamer, hexamer, and heptamer were also subjected to LCMS peak of multivalent ions ([M-2H] ²⁻ : m / z = 719, [M-2H], respectively). ^2- : m / z = 863 and [M-2H] ^2- : m / z = 1007) were confirmed.

[7] Rotavirus pharmacology test LEW / CrlCrlj rats were orally inoculated with rotavirus, and PRE was administered daily after infection (Example 9). As a result, 7 days after inoculation, 9 out of 9 animals developed diarrhea in the group not administered PRE. In contrast, only 1 out of 9 animals in the group that received PRE developed diarrhea. The administration of PRE suppressed diarrhea symptoms due to rotavirus. The virus growth inhibitory activity at the individual level of PRE and the effect of improving diarrhea which is a symptom of rotavirus disease were confirmed.

[8] Analysis of mechanism of action: Mn-SOD expression analysis To elucidate the mechanism of action, PRE was added to the culture medium of African green monkey-derived Vero cells and cultured for 24 hours, and proteins were extracted from the cells. The abundance of Mn-SOD (manganese superoxide dismutase) was quantified by Western blot. The Western blot image was analyzed, the band density was digitized, and normalized with the tubulin band density, and the change in the expression level of Mn-SOD was calculated (Example 10). As a result, it was confirmed that the amount of Mn-SOD increased by 1.25 times by the PRE treatment. In general, it is known that reactive oxygen species induced by an infected virus may play an important role in the process of propagation in viral host cells, and PRE is responsible for the expression of Mn-SOD that detoxifies it. It was suggested that the induction suppresses the growth of the virus.

Example 16 <Composition example of composition>
Hereinafter, a composition containing PRE obtained from Example 1 of the present invention, chromodium hot water extract obtained from Example 2, F11 fraction obtained from Example 3, or chromodium powder was produced. The mixing ratio of each component is mass%.
1. Alcoholic beverage 50% ethanol 32.0%
Sugar 6.0%
Fragrance 0.2%
PRE 0.1%
Water 61.7%
2. Juice Fruit juice 5.0%
Fructose glucose liquid sugar 1.0%
Fragrance 0.2%
PRE 0.1%
Citric acid 0.2%
Water 93.5%
3.飴 Sugar 55.0%
Minamata 29.65%
Fragrance 0.25%
Water 15.0%
Kuromoji hot water extract 0.1%
4). Tablets Sugar 74.7%
Glucose 20.0%
Fragrance 0.1%
F11 fraction 0.1%
L-ascorbic acid 0.1%
Water 5.0%
5. Yogurt drink Fermented milk 15.0%
Fructose glucose liquid sugar 15.0%
Lactic acid 1.0%
Fragrance 0.1%
PRE 0.1%
Water 68.8%
6). Gummy Candy Juice 30.0%
Gelatin 9.0%
Sugar 25.0%
Chromoji powder 1.0%
Water 35.0%
7). Spray ethanol 25.0%
Fragrance 1.0%
Kuromoji hot water extract 0.1%
Water 73.9%
8). Mouthwash ethanol 1.0%
Fragrance 0.1%
Cetylpyridinium chloride 0.25%
Kuromoji hot water extract 0.1%
Water 98.55%
9. Chewing gum Gum base 25.0%
Xylitol 35.0%
Minamata 38.0%
Fragrance 1.0%
Kuromoji hot water extract 1.0%

  The anti-microbial composition containing the chromophore extract component of the present invention is a norovirus that is a cause of infectious diarrhea that causes serious outbreaks mainly in winter, and infectious diarrhea that is extremely infectious and difficult to prevent infection. To foods, medicines, feeds, and hygiene products that have anti-proliferative activity against rotavirus, another major cause, influenza virus that causes outbreaks mainly in winter, and mycoplasma, which is important as a pathogen of pneumonia that prevails in winter Is expected to be used.

Claims (11)

  An anti-microbial composition having a growth-inhibiting activity against at least one microorganism selected from viruses and mycoplasma, comprising a black moji extract as an active ingredient.   The antimicrobial composition according to claim 1, wherein the virus is at least one selected from rotavirus, norovirus, and influenza virus.   The antimicrobial composition according to claim 1 or 2, wherein the chromomy extract component comprises proanthocyanidins.   The antimicrobial composition according to claim 3, wherein the proanthocyanidins are A-type proanthocyanidins. The A-type proanthocyanidins have the chemical formula (II):

The antimicrobial composition according to claim 4, which is a pentamer to 10mer A-type proanthocyanidin represented by (wherein n = 1 to 6).   The antimicrobial composition according to claim 4, wherein the A-type proanthocyanidins are cinnamtannin D1, cinnamtannin D2, cinnamtannin B1 or cinnamtannin B2.   The antimicrobial composition according to any one of claims 1 to 6, which is a composition for promoting Mn-SOD expression.   A food for the prevention or improvement of rotavirus disease, norovirus disease, influenza virus disease, or mycoplasma disease, comprising the antimicrobial composition according to any one of claims 1 to 7.   A medicament for preventing or ameliorating rotavirus disease, norovirus disease, influenza virus disease, or mycoplasma disease, comprising the antimicrobial composition according to any one of claims 1 to 7.   A feed for preventing or improving rotavirus disease, norovirus disease, influenza virus disease, or mycoplasma disease, comprising the antimicrobial composition according to any one of claims 1 to 7.   A sanitary article for prevention or amelioration of rotavirus disease, norovirus disease, influenza virus disease, or mycoplasma disease, comprising the antimicrobial composition according to any one of claims 1 to 7.

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