JP5867802B2 - Antibacterial agents and medicines containing them - Google Patents

Description

The present invention relates to an antibacterial agent containing a ume vinegar-derived component such as ume vinegar polyphenol as an active ingredient, and a pharmaceutical product containing the same.

  In recent years, events involving microorganisms have been taken up as major problems in various scenes of daily life. For example, Campylobacter known as food poisoning bacteria and Escherichia coli O-157 sometimes cause serious pathological conditions and can lead to diseases such as Guillain-Barre syndrome and hemorrhagic nephritis. In addition, it has been clarified that microorganisms in the oral cavity cause periodontal disease as well as dental caries, and this is a cause of lifestyle-related diseases, and oral hygiene is an important problem.

  Now, in Japan, plum has long been consumed mainly as plum pickled, but recently it has come to be used in various forms such as plum wine, plum extract, plum juice and confectionery. Plum is positioned as a healthy fruit. Plum pickles are pickled as a pickle. Plum meat extract is made by heating and concentrating plum flesh even when digestive disorders such as indigestion and perishment occur. Etc. are used in folklore medicine. In China, immature ume fruit is crushed into a traditional Chinese medicine called “Toriume” and used to treat digestive diseases. Furthermore, the taste of plums is used as an oral preparation such as toothpaste and tune gum due to its refreshing feeling.

  Thus, it is considered that the antibacterial activity of plums is a major factor in the use of plums as a treatment for digestive diseases and oral preparations, and various research results have been reported. For example, Seiko Nanayama reports that “plum meat extract” has antibacterial activity (see Non-Patent Document 1). Moreover, Otsuka T et al. Reported that glandular and gastric lesions caused by infection of gerbils with Helicobacter pylori can be suppressed by administration of “plum meat extract” (see Non-Patent Document 2). Furthermore, Chen Y et al. Reported the antibacterial activity of “Toriume” (see Non-Patent Document 3).

  Along with this research, the cause of the antibacterial activity of "plum meat extract" and "bird plum" has been studied, and it is thought that organic acids such as citric acid are the main cause. . This is because “plum meat extract” and “bird plum” are processed directly from plum fruit, and these processed products contain a considerable amount of organic acid.

  Actually, the plum acid is about 4 to 6% organic acid content such as citric acid, whereas “plum meat extract” is obtained by concentrating plum flesh by heating, so the organic acid content is usually 50% by weight. %. “Toriume” also contains a considerable amount of organic acids.

  In addition, the mechanism by which antibacterial activity occurs has been studied, and organic acids lower the pH and show bacteriostatic and bactericidal activity. It has been confirmed that the growth of can be suppressed. Therefore, it is generally considered that the reason why “plum meat extract” and “bird plum” have antibacterial activity is that eating them decreases the pH in the intestine and produces antibacterial activity.

  However, considering that even hydrochloric acid in gastric juice with strong acidity is neutralized in the intestines, organic acids are probably also neutralized in the intestines, and organic acids create an acidic environment in the intestines. It is probably impossible to exist and work as such an acid. Therefore, the existence of components exhibiting antibacterial activity other than organic acids is considered.

  However, at present, there are very few studies on components showing antibacterial activity other than organic acids in plum fruit. For example, Miyazawa M et al. Found a substance that inhibits the migration of Helicobacter pylori from immature plum fruit. This substance is a kind of lignan and is (+)-Syringaresinol (see Non-Patent Document 4). However, since the concentration of this substance in ume fruit and its processed products is extremely low, it is estimated that the substance actually contributes little to the antibacterial activity of ume products.

  On the other hand, it is known that about 1000 ppm of polyphenols are present in the fruits of plums, and the inventors have conducted research on this polyphenol. Then, we developed a production method that produces a large amount of fruit-derived polyphenols (plum vinegar polyphenols) from plum vinegar that is generated in large quantities as a by-product during the production of plum dried and processed at a processing cost, and its chemical analysis and health promotion action A patent application has already been filed (see Patent Document 1). In addition, the ume vinegar polyphenol is free of citrus fruit-derived citric acid and salt added in large quantities for the production of umeboshi during the production process. Therefore, plum vinegar polyphenol is an optimal material for examining the activity of polyphenol itself.

  In addition, the inventors conducted safety tests on ume vinegar polyphenols such as rat acute toxicity test, rat 28-day subacute toxicity test, mouse 90-day chronic toxicity test, mutagenicity test such as UMU test and mouse micronucleus test. The ume vinegar polyphenol has been demonstrated to be extremely safe (see Non-Patent Document 5).

  In addition, the inventors have obtained a polyphenol extract obtained from ume vinegar, an enzyme inhibitor having an α-amylase inhibitory action and an α-glucosidase inhibitory action effective for the treatment and prevention of obesity and diabetes using the same, food Patents relating to the composition, the food composition for specified health use, the quasi-drug composition, and the pharmaceutical composition have already been filed (see Patent Document 1). In addition, the inventors have already filed a patent application by finding that the polyphenol extract obtained from ume vinegar has an effect of suppressing an increase in blood pressure (see Patent Document 2).

JP 2009-137929 A Japanese Patent Application No. 2011-36710

Seiko Nanayama, “Antimicrobial Substances in Foods, Umeboshi, Antibacterial Activity of Plum Meat Extract”, Journal of Japanese Society of Food Microbiology 12: 211-217 (1996) Otsuka T, Tsukamoto T, Tanaka H, Inada K, Utsunomiya H, Mizoshita T, Kumagai T, Katsuyama T, Miki K, Tatematsu M., “Suppressive effects of fruit-juice concentrate of Prunus mume Sieb. Et Zucc. , Ume) on Helicobacter pylori-induced glandular stomach lesions in Mongolian gerbils. ", Asian Pac J Cancer Prev. 6: 337-41 (2005) Chen Y, Wong RW, Seneviratne CJ, Hags U, McGrath C, Samaranayake LP, Kao R., "The antimicrobial efficacy of Fructus mume extract on orthodontic bracket: a monospecies-biofilm model study in vitro.", Arch Oral Biol. 56 : 16-21 (2011) MIYAZAWA M, Utsunomiya H, Inada K, Yamada T, OKUNO Y, Tanaka H, Tatematsu M. "Inhibition of Helicobacter pylori motility by (+)-Syringaresinol from unripe Japanese apricot." Biol Pharm Bull. 29 (1): 172- 3. (2006) Yusuke Shiga, Takenori Tsuchida, Takehiro Hara, Kunihiro Kishida, Masanobu Maeda, Kazuhisa Miyashita, Maki Fujiwara, Hayako Yamanishi, Fumiko Yano, Takahiko Mitani, "A Study of the Safety of Plum Vinegar Polyphenol Extract", Bulletin of Department of Biophysics, Kinki University .28 p31-40 2011

This invention makes it a subject to provide the antibacterial agent which is safe and strong in antibacterial activity while promoting the utilization of the ume vinegar produced as a by-product when manufacturing a ume processed food. Another object is to provide pharmaceuticals and quasi drugs with strong antibacterial activity.

As a result of intensive studies, the inventors have found that ume vinegar polyphenol has a high antibacterial activity and have completed this invention.

That is, the antibacterial agent of the present invention contains ume vinegar polyphenol. Further, medicines of the present invention, quasi drugs, is intended to include antibacterial agents of the present invention.

Since the antibacterial agent of this invention has been confirmed to have high antibacterial activity, it can be added to general foods to prevent food corruption or administered as a medicine in the digestive tract, urinary tract, etc. It can be expected to sterilize abnormally propagated bacteria. Moreover, since the antibacterial agent of the present invention is derived from food, it is guaranteed to have high safety, and it can be expected that it can be used for a long period of time. Moreover, since the antibacterial agent of this invention uses the ume vinegar generated as a by-product at the time of umeboshi manufacture as a raw material, the resources conventionally discarded as industrial waste can be used effectively.

FIG. 1 is a graph showing the antibacterial activity of ume vinegar polyphenols against 8 strains of enteric bacteria. FIG. 2 is a graph showing antibacterial activity of ume vinegar polyphenol aglycone against 8 intestinal bacteria. FIG. 3 is a graph showing the antibacterial activity of citric acid against E. coli. FIG. 4 is a graph showing the antibacterial activity of neutralized citric acid against E. coli.

1. Antimicrobial agents antimicrobial agent of the present invention is intended to include vinegar polyphenols obtained from vinegar as an active ingredient. The details will be described below.

(1) Plum vinegar polyphenols Plum vinegar polyphenols contained in ume vinegar have many types, and their structures include glycosides whose aglycone part is hydroxycinnamic acid, and derivatives of hydroxycinnamic acid, such as esters of hydroxycinnamic acid and sugar / organic acid. Accounts for the majority. Such a plum vinegar polyphenol can be manufactured by the method of patent document 1, for example. In addition, the density | concentration of the polyphenol contained in the ume vinegar polyphenol extract manufactured by patent document 1 cannot be measured correctly. However, when measured by the foreign-thiocult method using gallic acid as a standard substance, it is known that polyphenol is contained in the ume vinegar polyphenol extract by about 10 to 15%.

(2) Plum vinegar polyphenol hydrolyzate Plum vinegar polyphenol hydrolyzate is converted to sugar or organic acid and aglycone part (plum vinegar polyphenol aglycone) by a known method using an enzyme such as alkali, acid, amylase or esterase. It has been hydrolyzed. Hydrolysis conditions need not be particularly limited, but conditions under which the aglycone part is totally destroyed are not preferred.

(3) Plum vinegar polyphenol aglycone Plum vinegar polyphenol aglycone is a product obtained by removing sugar and organic acid from the ume vinegar polyphenol hydrolyzate and purifying the aglycone portion. The purification method includes, for example, subjecting the hydrolyzate to column chromatography composed of a synthetic adsorbent (eg, Diaion HP20 manufactured by Mitsubishi Chemical Corporation), washing the column with deionized water, and then adding the plum vinegar polyphenol aglycone adsorbed on the column to methanol or Examples include elution with ethanol and the like, and eluate is concentrated to dryness.

In addition, the ume vinegar polyphenol may be liquid or solid, and may be used as a mixture alone or in combination with another substance. When used as a mixture, it may be subjected to spray drying, freeze drying, addition of a shaping agent such as dextrin, and the like. In the case of liquid, unnecessary components such as sugar, salt, organic acid, etc. can be removed by concentrating by a known method such as ultrafiltration.

  Moreover, ume vinegar polyphenol etc. contain many types of polyphenols. Therefore, a specific polyphenol may be used after being selected by a known means such as filtration, column treatment, or solvent washing.

2. Pharmaceuticals, etc. The pharmaceuticals and quasi drugs of this invention contain the antibacterial agent of this invention. Incidentally, pharmaceuticals, the content of the antibacterial agent definitive in quasi drugs, can be freely set in consideration of the antibacterial antimicrobial agents and use thereof to be used.

(1) Drugs Drugs are stipulated in the Pharmaceutical Affairs Law and include both ethical drugs and over-the-counter drugs (OTC). Moreover, if the disease used as the object is a pharmaceutical including a conventional antibacterial agent , for example, a cold medicine, an intestinal regulating agent, etc., it can be used without particular limitation. In addition, for example, pills, liquids, powders, granules, tablets, capsule tablets, troches, syrups, dry syrups, suspensions, emulsions, elixirs and other oral preparations, injections And non-oral agents such as suppositories, liquids for external use, and coating agents such as ointments, but are not limited thereto.

  In the case of producing the pharmaceutical product of the present invention as an oral preparation, known excipients, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, flavoring agents, coloring agents, etc. At the same time, it may be manufactured by a known manufacturing method.

  In addition, when the pharmaceutical product of the present invention is produced as a parenteral agent, it is a diluent such as distilled water for injection, physiological saline diluent, glucose aqueous solution, known disinfectant, preservative, stabilizer, isotonic agent. In addition to the stabilizer, preservative, and soothing agent, it may be produced by a known method.

(2) Quasi-drugs Quasi-drugs are stipulated in the Pharmaceutical Affairs Law, and include, for example, deodorants (deodorants), hair restorers, medicated cosmetics, nutritional supplements (supplements), etc. Although it is mentioned, it is not limited to these.

(3) Cosmetics Cosmetics are stipulated in the Pharmaceutical Affairs Law. For example, makeup cosmetics such as lipsticks and foundations, basic cosmetics such as lotions, hair tonics, perfumes, toothpastes, shampoos, rinses, bodies So-called toiletry products such as soaps and bathing agents for washing water are included, but the present invention is not limited to these.

(4) Foods Food means human general foods, health functional foods (specific health foods, nutritional functional foods), health foods, nutritional supplements, and the like. As food, specifically, processed fishery products such as kamaboko, chikuwa, hanpen, processed meat products such as sausage, ham, and wine, agricultural processed products such as tofu, fried chicken, konjac, western confectionery, Japanese confectionery, bread, cake Sweets such as ki, jelly, pudding, snacks, cookie, gum, candy, ramune, noodles such as raw noodles, Chinese noodles, buckwheat, udon, sauce, soy sauce, dressing, mayonnaise, sauce, honey, powder Seasoning such as candy and syrup, curry powder, mustard powder, pepper powder and other spices, jam, marmalade, chocolate spread, pickles, soy sauce, sprinkles, various vegetables and fruit canning Dairy products such as vegetables and fruits, seeds, butter, yogurt, fruit juice, vegetable juice, whey drink, soft drink, health tea, beverages such as medicinal liquor, etc. Nourishing reinforcing tablets for health for the purpose of (supplement), etc., beverages, but such food products such health conscious granules and the like can be exemplified, but the invention is not limited thereto.

(5) Feed The feed is the food of animals other than humans. Examples of animals other than humans include pets such as dogs and cats, ornamental birds such as canaries and parakeets, ornamental fish such as goldfish and tropical fish, domestic animals such as cows, pigs, sheep and horses, poultry such as chickens, Examples include, but are not limited to, cultured fish such as yellowtail, red sea bream, and flounder.

  Hereinafter, the present invention will be described more specifically based on examples. However, these examples do not limit the invention described in the claims in any way.

1. Measurement of antibacterial activity of ume vinegar polyphenol The antibacterial activity of ume vinegar polyphenol was examined based on the amount of harmful intestinal bacteria grown. Specifically, it investigated as follows.

(1) Plum vinegar polyphenol, sample strain and liquid medium Plum vinegar polyphenol was prepared by an industrial process according to the production method described in Patent Document 1 (Lot. 100525). Intestinal bacteria include Citrobacter freundii (NBRC NO. 12681), Enterobacter aerogenes (NBRC NO. 13534), Enterobacter cloacae (NBRC NO. 13535), Escherichia coli (NBRC NO. 15034), Klebsiella oxytoca (NBRC NO. 102593). ), Proteus hauseri (NBRC NO. 3851), Proteus mirabilis (NBRC NO. 10567), and Salmonella enterica (NBRC NO. 105726). Further, the liquid medium used was a standard medium autoclaved by dissolving 5 g of casein pancreatic digested peptone (manufactured by Oriental Yeast), 2.5 g of yeast extract (manufactured by Oriental Yeast), and 1 g of glucose in 1 L of deionized water.

(2) Antibacterial activity test One platinum loop was planted in a liquid medium from a storage slant culture of each bacterium and allowed to stand for 12 hours, then transplanted to a liquid medium again and pre-cultured for 4 hours. The cell suspension obtained by the preculture was diluted with a liquid medium so that the OD ₆₃₀ value was 0.1, and 100 μl was dispensed into each well of a 96-well plate. In each well of the 96 well-plate containing the diluted bacterial cell suspension, use a liquid medium so that the final concentration of each well is 1000 μg / ml, 2000 μg / ml, 3000 μg / ml, 4000 μg / ml, and 5000 μg / ml. 100 μl of the dissolved ume vinegar polyphenol solution was added. In addition, 5 holes were prepared for each concentration of the same plum vinegar polyphenol. Further, a control containing no ume vinegar polyphenol was prepared in the same manner.

The 96-well plate was statically cultured in an incubator adjusted to 37 ° C., and the OD ₆₃₀ value was measured with a microplate reader every 10 hours until 10 hours. Then, the average value of the OD ₆₃₀ values after 10 hours was converted to a growth rate by converting the average value of the control to 100%. The relationship between ume vinegar polyphenol concentration and growth rate is shown in FIG.

  From FIG. 1, it was confirmed that ume vinegar polyphenols suppressed growth in 7 strains excluding Enterobacter aerogenes among 8 intestinal bacteria used in this experiment in a wide concentration range of 1250 to 5000 μg / ml. Moreover, it has confirmed separately that the ume vinegar polyphenol used in this experiment does not contain a free type organic acid and salt. Therefore, it has been found that the ume vinegar polyphenol used in this experiment does not use the antibacterial activity of organic acids or salt, but has its own antibacterial activity.

2. Measurement of antibacterial activity of ume vinegar polyphenol aglycone It has been found that ume vinegar polyphenols are mainly composed of hydroxycinnamic acid derivatives. Then, the ume vinegar polyphenol was alkali-hydrolyzed, sugar and organic acid were removed from the ume vinegar polyphenol hydrolyzate, and the antibacterial activity of the obtained ume vinegar polyphenol aglycone was examined. Specifically, it investigated as follows.

(1) Plum vinegar polyphenol and liquid medium for measuring antibacterial activity Plum vinegar polyphenol was the same as in Example 1. As the liquid medium, a modified standard medium in which MOPS buffer (pH 7.0) was added to the standard medium of Example 1 so that the final concentration was 100 mM was used. This is because the prepared plum vinegar polyphenol aglycone is made into an acidic solution in order to improve its stability, which greatly affects the pH of the standard medium. It has been separately confirmed that the added MOPS has no effect on the growth of bacteria.

(2) Preparation of ume vinegar polyphenol aglycone To 50 mg of ume vinegar polyphenol was added 4 ml of a 1N aqueous sodium hydroxide (Sigma) solution previously substituted with nitrogen and reacted at 37 ° C. for 24 hours. After completion of the reaction, 0.27 ml of 85% orthophosphoric acid (Nacalai Tesque) and 5 ml of ethyl acetate were added to the reaction solution and centrifuged at 4 ° C. and 3000 rpm for 1 minute to collect the supernatant. To the residue from which the supernatant was collected, 5 ml of ethyl acetate was added again, followed by centrifugation in the same manner, and the supernatant was collected. The first and second supernatants were mixed and dried at 60 ° C. under nitrogen atmosphere to obtain ume vinegar polyphenol aglycone.

(3) Antibacterial activity test The preculture and the proliferation test were conducted in the same manner as in Example 1 except that the medium was different and that ume vinegar polyphenol aglycone was used. Since ume vinegar polyphenol aglycone is not dissolved in the medium, it was dissolved in DMSO and then added to the medium (final concentration of DMSO is 1%). The result is shown in FIG.

  From FIG. 2, it was confirmed that the ume vinegar polyphenol aglycone had a concentration-inhibiting effect on all the fungi in a concentration-dependent manner, and that there was not much difference in antibacterial activity depending on the bacterial species. Moreover, it was confirmed that ume vinegar polyphenol aglycone has antibacterial activity even in a concentration range 15 times lower than that of ume vinegar polyphenol. From these facts, we inferred that aglycone mainly works on the antibacterial activity of ume vinegar polyphenols, not sugars or organic acids.

  From Example 1 and Example 2, it was proved that both ume vinegar polyphenol and ume vinegar polyphenol aglycone have antibacterial activity. This indicates that ume vinegar polyphenols can exhibit stronger antibacterial activity in vivo even when subjected to hydrolysis by enzymes produced by intestinal bacteria and the like.

3. Antibacterial activity when citric acid is neutralized Plum contains about 5% citric acid, and citric acid was thought to be the main antibacterial effect of plum. This is because it was considered that when citric acid was added, the pH of the medium was lowered to suppress the growth of bacteria. For example, as shown in FIG. 3, citric acid was added to a standard medium so as to have a concentration of 0.3125 to 5%, and the growth of Escherichia coli (NBRC NO. 15034) was examined. I couldn't see it.

  Therefore, the antibacterial activity of citric acid was examined by adjusting the pH of the medium supplemented with citric acid to near neutral with NaOH and examining the growth activity of the bacteria in the medium. As a result, for example, as shown in FIG. 4, growth inhibition of Escherichia coli (NBRC NO. 15034) was not observed at all, and conversely, by adding citric acid, the growth rate of the bacteria increased. The same results were obtained for the 8 strains of intestinal bacteria. From this result, it was inferred that the added citric acid was nutritional when neutralized. From this, it is presumed that under the neutral pH condition, the antibacterial action of plums is mainly due to substances other than citric acid, such as the above polyphenols.

4). Confirmation of antibacterial activity against bacteria related to dental caries On the other hand, when a separate experiment was conducted, eight bacterial strains involved in dental caries, namely Streptococcus mutans (ATCC 25175), Streptococcus anginosus (ATCC 33397), Streptococcus intermedius ( Antibacterial against ume vinegar polyphenols against the growth of ATCC 27335), Streptococcus constellatus (ATCC 27823), S. Streptococcus oralis (ATCC 35037), Streptococcus salivarius (ATCC 7073), Streptococcus sanguinis (ATCC 10556), Streptococcus pyogenes (ATCC 12344) It was found to be active (data not shown). In addition, as described above, it was also found that antibacterial activity is increased by alkaline hydrolysis of ume vinegar polyphenol (data not shown).

The antibacterial agent of the present invention can be easily prepared from ume vinegar generated as a by-product during the production of umeboshi, is inexpensive and has been confirmed to be highly safe. Therefore, it can be applied in a wider field than conventional antibacterial agents .

Claims (3)

  An antibacterial agent containing ume vinegar polyphenol as an active ingredient.   An antibacterial drug comprising the antibacterial agent according to claim 1.   An antibacterial quasi-drug comprising the antibacterial agent according to claim 1.

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