JP2005097127A - Antimicrobial agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an antimicrobial agent comprising a component derived from Sparassis crispa. <P>SOLUTION: This antimicrobial agent comprises an extract from a fruit body or a mycelium of the Sparassis crispa as an active ingredient. The antimicrobial agent preferably comprises a fraction extracted from the fruit body or mycelium of the Sparassis crispa with at least one or more kinds of organic solvents as the active ingredient. Furthermore, an alcohol-extracted fraction is obtained from the fruit body or mycelium of the Sparassis crispa with ethanol and a fraction extracted from the resultant fraction by using ethyl acetate is more preferably used as the active ingredient. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an antibacterial agent obtained from Hanabiratake.

  Humans have overcome bacterial infections with antibiotics, but in recent years, bacteria that have acquired resistance to antibiotics have emerged and are again a threat. In particular, methicillin-resistant Staphylococcus aureus (hereinafter referred to as MRSA) occurs in hospitals where antibiotics are frequently used, and it is easy to infect elderly people and patients with reduced immunity after surgery. It may induce infections such as roads and cause death. At present, MRSA has spread throughout the world as a hospital-acquired infectious bacterium and has become a major medical problem. Antibiotics such as vancomycin, which have different mechanisms of action, are effective in treating patients infected with MRSA, but there is a risk that treatment will be lost if the infecting bacteria acquires vancomycin resistance before healing, Treatment is very difficult.

  Various antibacterial agents against MRSA have been reported. For example, vancomycin (for example, refer nonpatent literature 1), teicobranin, arbekacin, etc. are mentioned.

  In addition, natural ingredients derived from hinokitiol, tea catechin, green tea extract (for example, see Non-patent Document 2), persimmon tannin, propolis (for example, see Non-Patent Document 3), chitosan (for example, see Non-Patent Document 4), etc. It has been reported to have anti-MRSA activity.

  Although food poisoning is expected to decrease as hygiene management techniques develop, many food poisoning incidents still occur every year. In recent years, the number of patients per case has increased, and the number of cases tends to increase. As food poisoning bacteria, Staphylococcus aureus, Salmonella, Escherichia coli and the like are known. The outbreaks of pathogenic E. coli O-157 that spread in 1996 indicate the risk of large-scale infections in young children, elderly people, school-related facilities, etc., and are also social problems.

  Wasabi and mustard have long been used as a means for preventing food poisoning, and in recent years, antibacterial components have been identified (see, for example, Non-Patent Document 5). In addition, tea (for example, see Patent Document 1 and Patent Document 2), grass (for example, see Patent Document 3), and lactic acid bacteria-producing substances (for example, see Non-Patent Document 6) have been reported to have antibacterial components. .

  Various synthetic antibacterial agents are also used, but antibacterial activity due to natural components as described above has attracted attention because of the problem of side effects on resistant bacteria and the human body.

  Furthermore, in recent years, it has been clarified that infection caused by Helicobacter pylori (H. pylori) is the etiology of diseases such as chronic gastritis and peptic ulcer, and energetic research is being conducted in the gastrointestinal clinical field. In addition, the number of infected people is enormous, and about half of the world's population is said to be infected with this fungus.

  Helicobacter pylori is a microorganism that inhabits the human gastric mucosa, and epidemiological studies have confirmed that it causes chronic gastritis, gastric ulcers, and duodenal ulcers. It is thought that eradication of H. pylori or suppression of growth is effective for the radical treatment of chronic gastritis and peptic ulcer. Combination therapy with antibiotics and gastric acid secretion inhibitors is generally used as eradication therapy for H. pylori. It came to be used.

  However, the long-term administration of antibiotics has a serious problem of increasing resistant bacteria in addition to its side effects. Synthetic medicines have side effects, and in recent years, attention has been focused on safe substances derived from natural products.

  From such a viewpoint, as a naturally derived substance having safe anti-H. Pylori activity, for example, tea leaf component catechins (see, for example, Patent Document 4), seaweed-derived polysaccharides (for example, Patent Document 5 and Patent Document) 6), fruit polyphenols of Rosaceae plants (see, for example, Patent Document 7), milk or egg white-derived mucin (see, for example, Patent Document 8), extraction of plants belonging to the genus Pepper, Solanum, Ganoderma, Prunus A thing (for example, refer patent document 9) etc. is reported.

  On the other hand, mushrooms have been used for food since ancient times, but recently the physiological activity of the ingredients has been clarified, and the usefulness of krestin, lentinan, schizophyllan (all trade names), etc. has been recognized as pharmaceuticals. Agaricus, Meshimakobu, Ganoderma lucidum, etc. have been tried to use dried or extracted fruit bodies and mycelium as health food materials in anticipation of antitumor action. As an antibacterial agent obtained from mushrooms, a novel sesquiterpene compound extracted and isolated from the fruit body of Hoontake (for example, see Patent Document 10), an antibiotic called ganomycin obtained from the genus Canoderma (for example, Patent Document 11) Is known.

  Hanabiratake belonging to these mushrooms is a very few mushroom that grows on larch. It is an edible mushroom that is chewy and features a pure white color and a shape like leaf peony. Up until now, it has been said that this bamboo shoot has slow growth and is extremely difficult to artificially cultivate, but recently, a new cultivation method that can be cultivated in a relatively short period of time has been established and can be supplied on a commercial scale. It has become.

  A method for extracting β-glucan with a solvent is proposed for the bamboo shoot, and the use of the extract in the pharmaceutical field is proposed (for example, see Patent Documents 12 and 13).

So far, it has been suggested that the mycelium of Hanabiratake has antibacterial activity against Escherichia coli, but the antibacterial activity of the fruit body of Hanabiratake has not been known so far.
Hiroki Kora et al., “Basic knowledge of bactericidal and antibacterial properties”, Ohm, 2000, p. 129-130 "Medical Tribune", Medical Tribune, August 2, 1996 "About anti-MRSA active substances in propolis", Bee Science, Bee Science Research Facility, Tamagawa University, 16 (4), 175-177 (1995) Edited by Atsushi Nishino, "Science of Antibacterial Agents" Part 2, Industrial Research Committee (1997), p. 98-101 JP-A-2-276562 JP-A-8-38133 JP 2002-249436 A Edited by Atsushi Nishino, "Science of Antibacterial Agents" Part 2, Industrial Research Committee (1997), p. 108-110 “Food Technology”, Jan, 1989, p. 164-167 Japanese Patent Laid-Open No. 5-139972 JP-A-6-247861 JP 7-138166 A Japanese Patent Laid-Open No. 11-180888 JP 2000-229865 A JP 2000-154146 A JP-A-9-216880 Special table 2002-538215 gazette JP 2000-217543 A JP 2002-125460 A

  The object of the present invention is to obtain an effective antibacterial agent against bacteria from the moss.

  For these reasons, the inventors of the present invention conducted screening for natural substances having antibacterial activity by paying attention to Hanabiratake and found that they have antibacterial activity against various fungi and reached the present invention.

  That is, the gist of the present invention is an antibacterial agent characterized by comprising an extract of the fruit body or mycelium of the leaflet, preferably at least one kind from the fruit body or the mycelium of the leaflet. A fraction extracted with the above organic solvent is an active ingredient, and more preferably, an extract is obtained from the fruit body or mycelium of Hanabiratake using alcohol, and then ethyl acetate is used from this fraction. The extracted fraction is the active ingredient. Furthermore, the antibacterial agent of the present invention preferably exhibits antibacterial activity against opportunistic infectious bacteria, food poisoning bacteria or Helicobacter pylori.

  The antibacterial agent of the present invention has antibacterial activity especially against causative bacteria of opportunistic infections, causative bacteria of food poisoning or H. pylori. Furthermore, Hanabiratake, which is derived from the antibacterial agent of the present invention, is an edible mushroom. The antibacterial agent of the present invention can be used as an oral administration agent or can be included in a meal. Can be selected.

  Hereinafter, the present invention will be described in detail.

  Hanabiratake is a mushroom that occurs specifically in conifers such as larch at an altitude of over 1,000 meters, and has been said to be a "phantom mushroom" because it is difficult to find. Until now, its cultivation has been difficult and generally not well known, but in recent years, an artificial cultivation method has been established and mass production has been achieved.

  The fruit body of Hanabiratake used in the present invention may be natural or artificially cultivated. As the method of artificial cultivation, it can be carried out by preparing a fungus bed for artificial cultivation (for example, see JP-A-11-56098, JP-A-2002-369621, JP-A-2002-125460). .

  In the present invention, a mycelium of agaricus can also be used. The mycelium can be obtained by a liquid culture method. As a carbon source used in the medium, a commonly used carbon source such as dextrin and glycerol can be used in addition to a monosaccharide such as glucose. Moreover, although an inorganic or organic nitrogen source can be used as the nitrogen source, it is preferable to use an organic nitrogen source from the viewpoint of the growth rate. Moreover, adding growth factors such as trace elements and vitamins as needed is no different from normal culture. The culture temperature is 15 ° C to 30 ° C, preferably 18 ° C to 28 ° C, and most preferably 20 ° C to 25 ° C. The pH is most preferably 2.5 to 8.0, preferably 3.0 to 7.0, 3.5 to 5.0. It is preferable to add an insoluble component to the medium component because it can be grown uniformly. The culture period can be set to several days to several weeks depending on the strain.

  The bamboo shoot obtained in this way may be transferred to the next extraction step as it is, or may be dried and then transferred to the extraction step. Moreover, it can grind | pulverize and use.

The active ingredient in the present invention is contained in the bamboo shoot, but it is desirable to extract and concentrate the active ingredient because it is low in the concentration of the raw bamboo shoot or dried product, and thus has low activity. The active substance can be obtained by extraction with an organic solvent or an aqueous solution. Examples of the organic solvent include alcohol, acetonitrile, acetate ester, DMSO, dioxane, glycol, ether, THF, acetone, methylene chloride, chloroform, hexane, cyclohexane and the like. As the aqueous solution, pure water, an acid aqueous solution, an alkaline aqueous solution, a salt solution, or the like can be used. These solvents can be used alone or in admixture of two or more for extraction, but chloroform, alcohol or ethyl acetate is preferred.
Although there is no restriction | limiting in particular in the quantity of the solvent used for extraction, It is preferable to use 2-20 times amount with respect to the weight of Hanabira bamboo. If the amount is less than twice, the operability is poor, and if the amount is more than 20 times, the work efficiency is poor.
The extraction can also be performed multiple times using one or more solvents. In the case of performing multiple times, the extraction may be performed from Hanabira bamboo, or the extracted fraction obtained from Hanabira bamboo may be further extracted. Moreover, it can carry out combining them.

  The temperature during the extraction operation is not particularly limited but is preferably 10 to 60 ° C. The extraction efficiency is poor at 10 ° C. or lower, and the algal bloom changes at 60 ° C. or higher, and the extract is colored. Although there is no restriction | limiting in particular also in extraction time, About 1 hour-3 days are preferable. The amount of extraction is less than 1 hour, and work efficiency is low after 3 days. In addition, extraction can be performed while standing, but extraction efficiency can be increased by stirring or shaking.

  The extracted fraction thus obtained contains an active ingredient having antibacterial activity against bacteria. If the active ingredient is to be used at a high concentration, the active ingredient may be extracted and concentrated. Concentration of the active ingredient can be achieved by simply drying, but a method such as extraction using an organic solvent may also be used.

  This active ingredient can be used as it is or processed into an easy-to-use form as an antibacterial agent. Hanabiratake is an edible mushroom, and the antibacterial agent of the present invention can be used as an oral administration agent or included in a meal. Therefore, it is possible to select various dosing methods suitable for the patient. It can also be mixed with food and cosmetics to prevent bacterial growth.

  Bacteria for which the antibacterial agent of the present invention is particularly effective include causative bacteria for opportunistic infections, causative bacteria for food poisoning, and Helicobacter pylori.

  Opportunistic infections are conditions in which microorganisms with relatively low pathogenicity cause injuries, illnesses, and infect people who have weakened immunity for other reasons. In many cases, patients are infected in hospitals, and it is said to be a nosocomial infection. The causative organisms of opportunistic infections include Haemophilus influenzae, β-streptococci, Pseudomonas aeruginosa, Streptococcus pneumoniae, Klebsiella pneumoniae, Serratia, Katarum, Staphylococcus aureus (including MRSA), Candida, Entero Examples include Bacter, Enterococcus faecalis, Proteus vulgaris, and Morganella morganii.

  Food poisoning is caused by ingesting foods that have propagated bacteria. In many cases, the cause is a toxin produced by a pathogen. Salmonella, Vibrio parahaemolyticus, Vibrio barnificus, Vibrio cholerae, Staphylococcus aureus, Shigella flexneri, Salmonella typhi, Clostridium perfringens, Clostridium botulinum, Examples include Campylobacter, pathogenic Escherichia coli, Yersinia, Listeria monocytogenes, and Providencia alcalifaciens.

  As described above, Helicobacter pylori is a causative bacterium of chronic gastritis, gastric ulcer, and duodenal ulcer.

  Next, the present invention will be described specifically by way of examples.

  In addition, the antibacterial activity with respect to various bacteria about the fraction obtained in the Example was investigated as follows.

  That is, the extracted fraction dissolved in the solution was soaked in an antibiotic test paper disk (manufactured by Advantech) having a diameter of 6 mm and dried. Various bacterial cultures were diluted and 100 μl was smeared on an agar medium. Dry paper disc on agar medium ((1) is TSA medium, Nippon Becton Dickinson, (2)-(15) is TSB medium, (12) is 3% NaCl LB medium, (16) is 5% Muller Hinton medium supplemented with sheep blood (manufactured by Nippon Becton Dickinson) was cultured for 1 to 5 days in an incubator at 37 ° C., and the growth inhibition circle formed around the paper disk was observed (H. pylori was Culture under microaerobic conditions).

A list of various bacteria used in this experiment is shown below.
(1) MRSA2932 strain (Nagoya University Hospital clinical isolate)
(2) Escherichia coli (RIMD0509939) Enterohemorrhagic Escherichia coli (EHEC) O157
(3) Escherichia coli Enteropathogenic E. coli (EPEC)
(4) Escherichia coli (RIMD0509266) Toxigenic E. coli (ETEC)
(5) Escherichia coli K12 strain DH5α
(6) Salmonella enteritidis (RIMD1933006)
(7) Shigella flexneri (RIMD3102002)
(8) Providencia alcalifaciens (RIMD1656001) Fukui food poisoning strain (9) Proteus vulgaris (RIMD1643003)
(10) Morganella morganii (RIMD1642003)
(11) Klebsiella pneumoniae (RIMD1102001)
(12) Vibrio parahaemolyticus (RIMD2210633)
(13) Bacillus subtilis (RIMD0225012)
(14) Staphylococcus aureus (RIMD3109007)
(15) Enterococcus faecalis (RIMD3116001)
(16) Helicobacter pylori (ATCC43504)
* RIMD is the strain storage number at the Research Institute for Microbial Diseases, Osaka University.
Example 1
Component extraction was performed from 5 kg of Hanabira bamboo harvested by artificial cultivation with 20 L of ethanol at room temperature for 24 hours. This operation was repeated 5 times, and then component extraction was performed 3 times in the same manner using 20 L of acetone from Hanabiratake after ethanol extraction. The obtained ethanol solution and acetone solution were mixed and concentrated under reduced pressure. From the resulting concentrate, the dissolved portion was extracted in turn using chloroform and then ethyl acetate, and the solvent was distilled off to obtain 51 g of a chloroform soluble portion and 1 g of an ethyl acetate soluble portion. Further, the remaining extraction solvent was distilled off, and water was added to separate only the dissolved portion to obtain 120 g of a water-soluble portion.

  An appropriate amount of each of the obtained soluble components was impregnated into a paper disk, dried, and then used for an antibacterial activity test.

  The test results are shown in Table 1.

実施例２
６０℃で６時間熱風乾燥して得たハナビラタケ乾燥粉末５００ｇからクロロホルム５Ｌを用いて、室温、静置状態で２４時間、成分抽出を行った。抽出溶液を分離して、同様の操作を５回繰り返した。さらに、クロロホルム抽出後のハナビラタケ粉末から酢酸エチル５Ｌで３回、次にメタノール５Ｌで３回、同様に抽出操作を行った。それぞれの抽出溶媒を減圧下に留去して、クロロホルム可溶部２０ｇ、酢酸エチル可溶部１ｇ、メタノール可溶部５５ｇを得た。

Example 2
Component extraction was carried out for 24 hours at room temperature in a stationary state from 500 g of dried powder of Hanabiratake obtained by drying with hot air at 60 ° C. for 6 hours. The extraction solution was separated and the same operation was repeated 5 times. Furthermore, extraction operation was similarly performed from 5 L of ethyl acetate and then 3 times with 5 L of methanol from Hanabira bamboo powder after chloroform extraction. Each extraction solvent was distilled off under reduced pressure to obtain 20 g of a chloroform soluble part, 1 g of ethyl acetate soluble part, and 55 g of methanol soluble part.

  An appropriate amount of a solution of these soluble parts was impregnated into a paper disk, dried, and then used for an antibacterial activity test.

  The test results are shown in Table 2.

以上の結果からハナビラタケの酢酸エチル抽出成分中に抗菌活性のあることが明らかとなった。
実施例３
実施例1において得られたクロロホルム抽出画分を分取用薄層クロマトグラフィープレート（シリカゲル６０、Merck社製）にアプライし、クロロホルム／メタノール＝８／２溶媒を用いて展開した。展開したプレートは展開距離に応じて２０等分し、シリカゲルを回収した。回収したシリカゲルからクロロホルムを用いて画分を回収し、濃縮した。得られた濃縮液を適当量ペーパーディスクに含浸し、乾燥した後、抗MRSA活性試験に用いた。
展開距離の順から３、４および１２、１３番目の画分を含浸したペーパーディスクの周囲に発育阻止円が形成された。結果を表3にまとめた。

From the above results, it was clarified that the ethyl acetate extract component of Agaricus has antibacterial activity.
Example 3
The chloroform extract fraction obtained in Example 1 was applied to a preparative thin layer chromatography plate (silica gel 60, manufactured by Merck) and developed using chloroform / methanol = 8/2 solvent. The developed plate was divided into 20 equal parts according to the development distance, and the silica gel was collected. Fractions were collected from the collected silica gel using chloroform and concentrated. An appropriate amount of the obtained concentrated liquid was impregnated into a paper disk, dried, and then used for an anti-MRSA activity test.
A growth-inhibiting circle was formed around the paper disk impregnated with the third, fourth, twelfth and thirteenth fractions in the order of the development distance. The results are summarized in Table 3.

以上の結果から、ハナビラタケのクロロホルム抽出成分中に抗MRSA活性のあることが明らかとなった。

From the above results, it was clarified that the chloroform extract of Hanabiratake has anti-MRSA activity.

Claims (10)

An antibacterial agent characterized by comprising an active ingredient of a fruit body or mycelium of a garlic mushroom. The antibacterial agent according to claim 1, wherein the active ingredient is a fraction extracted from at least one kind of organic solvent from the fruit body or mycelium of the flower. The antibacterial agent of Claim 1 which uses the fraction extracted from the fruit body or mycelium of a garlic bamboo using ethyl acetate as an active ingredient. The antibacterial agent of Claim 1 which uses the fraction extracted from the fruit body or mycelium of a garlic bamboo using chloroform as an active ingredient. The antibacterial agent according to any one of claims 1 to 4, wherein the active ingredient has antibacterial activity against bacteria. 6. The antibacterial agent according to claim 5, wherein the bacterium is a causative bacterium of human infection. 6. The antibacterial agent according to claim 5, wherein the bacterium is a causative bacterium of an opportunistic infection. The antibacterial agent according to claim 5, wherein the bacterium is methicillin-resistant Staphylococcus aureus (MRSA). 6. The antibacterial agent according to claim 5, wherein the bacterium is a causative bacterium of food poisoning. The antibacterial agent according to claim 5, wherein the bacterium is H. pylori.