JP3241425B2 - Inhibitor of microbial adhesion to animal cells - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an agent for inhibiting the adhesion of microorganisms to animal cells, comprising a tea extract having an action of inhibiting the adhesion of microorganisms to animal cells.

[0002]

2. Description of the Related Art Many diseases caused by microbial infection of animal cells including human cells are known. For example, a disease of the respiratory system, pharyngitis, tonsillitis, sinusitis, otitis media, bronchitis, pneumonia, etc., Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus pneumoniae (Streptococcus pneumoniae), Haemophilus influenzae (Haemophillus influenza
e), Branhamella-Katarurarisu (Branhamella cata
rrhalis), Chlamydia pneumoniae (Chlamydia pn)
eumoniae), Mycoplasma pneumoniae (Mycopla)
by infection of sma pneumoniae), also, tuberculosis Maikoba
Kuteriumu Chu Velcro cis (Mycobacterium tuber
by infection of culosis), furthermore, whooping cough, Bordetella Bae
It is known that the disease develops due to infection of Ruchtus (Bordetella pertussis).

On the other hand, among skin diseases, impetigo (Tobihi)
Is Staphylococcus aureus
reus), Staphylococcus epidermidis (Staph
The acne of ylococcus epidermidis is Propionibacterium acne
It is known that each disease is caused by the infection of s).

[0004] In visceral diseases, enteritis is caused by Escherichia coli (E. coli), Shigella (Shigella sp.), Salmonella (Salmonella sp.), Vibrio cholera (Vibrio chol).
erae) and other infections.

[0005] Further, as the urogenital disease, urethritis by infection of Escherichia coli (E. coli), also venereal disease is disease by infection of Neisseria gonorrhoeae (Neisseria gonorrhoeae) and Chlamydia trachomatis (Chlamydia trachomatis) It is known.

[0006] Furthermore, in oral diseases, caries (cavities) are affected by Streptococcus mutans.
s), Streptococcus sobrinus
sobrinus) and periodontal disease include Bacteroides gingivalis.

[0007] The cause of these diseases is microbial infection of animal cells including human cells.
The step is believed to be the attachment of the microorganism to the animal cells, and it is clear that if the attachment of this microorganism to the animal cells could be prevented, the infection could also be prevented.

[0008] Periodontal disease is an example of a disease resulting from infection of animal cells with microorganisms as described above.
This periodontal disease, also called alveolar pyorrhea, is a typical dental disease along with dental caries, and is accompanied by periodontal tissue destruction due to chronic inflammation occurring in tissues supporting teeth. In general, inflammation of the gingiva occurs (gingivitis), followed by periodontal ligament, alveolar bone, and other inflammation that spreads to periodontal tissues,
This is considered to be a reaction involving a series of biological reactions that cause the tooth to fall off.

[0009] The main causative factor of periodontal disease is periodontal plaque, and the causative factor is considered to be a causative bacterium resident in the gingival plaque. the first step is believed that the periodontal disease cause bacteria to bind to the pili and gingival epithelial cells of the bacterial cell surface.
After that, mediators such as bacterial endotoxin, tissue destructive enzymes (proteases such as collagenase), peptidoglycan, and teichoic acid release lysosomal enzyme-derived autolytic enzymes, or develop an immune response reaction involving macrophage mobilization. , Causing a pathway of gingival cell destruction and inflammation.

With respect to periodontal disease prevention, research on the development of antimicrobial agents for oral microorganisms and research on inhibitors of collagenase in vivo that are closely related to inflammation have been made. Peripheral drugs have not been found. However, as described above, since the attachment of bacteria to gingival epithelial cells is the first step in the onset of periodontal disease,
It is thought that if this adhesion is suppressed, it is possible to prevent the occurrence of periodontal disease, and effective means for this have been required.

[0011] In addition, enteritis caused by Salmonella, etc. (food poisoning) is expected to be prevented by inhibiting adhesion and colonization of bacteria in the internal organs, as in the concept of periodontal disease prevention. Therefore, effective means for this has been required. In addition, it is considered that the above-mentioned various diseases caused by infection of animal cells with microorganisms can be similarly prevented by suppressing the attachment of microorganisms to animal cells.

[0012]

Therefore, there is a need for the development of a method for suppressing only the attachment of microorganisms to animal cells without causing any problem in terms of safety or the like to the human body or animal living body. I was

[0013]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, a substance that extremely effectively inhibits the attachment of microorganisms to animal cells is contained in a tea extract-based solvent extract. It was found that it existed, and that it was highly secure, and completed the present invention.

That is, a first object of the present invention is to provide an agent for inhibiting the adhesion of microorganisms to animal cells, comprising an aqueous solvent extract from sweet tea as an active ingredient. A second object of the present invention is to provide foods and cosmetics containing the adhesion inhibitor. Further, a third object of the present invention is that
Provided is a method for producing an adhesion inhibitor obtained by extracting bean tea with an aqueous solvent or, after the extraction, subjecting the extract to an adsorption treatment using a synthetic adsorbent to obtain a synthetic adsorbent-adsorbed fraction. It is to be.

The aqueous solvent extract from bean tea (hereinafter, referred to as a "tea extract"), which is an active ingredient of the inhibitor of adhesion of microorganisms to animal cells of the present invention (hereinafter, referred to as an "adhesion inhibitor").
Is produced by extracting sweet tea with an aqueous solvent such as water or water-alcohol according to a conventional method.

The tea (Rubus su) used as an extraction material
avissimus) is a perennial rose of the family Rosaceae, which has been used as sweet tea since ancient times in China, and its leaves or stems, especially leaves dried on weekdays, can be used for extraction.

The aqueous solvent used for the extraction may be water alone or any mixture of water and one or two polar solvents such as lower alcohols such as methanol and ethanol, and acetone. However, since the active ingredient of the present invention cannot be efficiently extracted only with a polar solvent, it is preferable to always use a mixed solution with water and the mixing ratio of the solvent is 90% by volume or less.

In consideration of the fact that the extract is finally incorporated into pharmaceuticals, quasi-drugs, oral preparations and foods, water, ethanol or a mixture thereof is used from the viewpoint of safety. Is preferred.

The ratio of bean tea to the aqueous solvent at the time of extraction is not particularly limited either, but it is generally 2 to 1000 times by weight of the solvent relative to 1 bean tea, and especially 5 to 100% by weight in terms of extraction operation and efficiency. Preferably, it is doubled.

The extraction temperature is conveniently in the range from room temperature to the boiling point of the solvent under constant pressure, and the extraction time is from 10 minutes to 24 minutes.
It is preferable to set the time range.

The thus obtained bean tea extract itself has an activity of inhibiting the adhesion of bacteria to animal cells. Therefore, it can be used as it is as an adhesion inhibitor. It is more preferable to use the obtained adsorption treatment product.

The adsorption treatment with the synthetic adsorbent is carried out by passing a tea extract extract through a column filled with the synthetic adsorbent, removing the non-adsorbed fraction by washing with water, and desorbing with a solvent.

Examples of the synthetic adsorbent used for the adsorption treatment of the tea tea extract include an aromatic synthetic adsorbent produced by polymerizing styrene and divinylbenzene, and a methacrylic adsorbent produced by polymerizing methacrylic acid ester. Synthetic adsorbents are listed, and examples of commercially available products are Diaion HP20,
HP21 (Mitsubishi Kasei), Amberlite XAD
No. 4 (Rohm and Haas, USA) and methacrylic synthetic adsorbents such as Sepabeads HP1MG, HP2MG (Mitsubishi Kasei), Amberlite XAD7, and XAD8 (Rohm and Haas, USA) Can be mentioned.

When the bean tea extract is treated with these synthetic adsorbents, a pretreatment such as removing the organic solvent in the extract by vacuum concentration or the like or diluting it sufficiently with water in order to complete the fractionation. Is preferably performed.

As the desorbing solvent used in the treatment with the synthetic adsorbent, a polar organic solvent such as aqueous ethanol can be used. In order to increase the purity of the active ingredient, the concentration of the organic solvent is adjusted so that the sweet component in the tea is a resin. It is preferable that it is not desorbed from the substrate.

The tea tea extract obtained as described above and / or
Alternatively, the synthetic adsorbent-adsorbed fraction can be used in any state, such as an extracted or synthetic adsorbent-treated as-is, a concentrate thereof, or a dried product obtained by removing a solvent from an eluate. It is preferable to make it into a dry product from the viewpoint of storage stability and safety of the organic solvent.

[0027] The adhesion inhibitor of the present invention is prepared by blending the extract of bean tea obtained as described above, its synthetic adsorbent-adsorbed fraction or a mixture thereof with various components conventionally used. .

The adhesion inhibitor of the present invention is a preventive agent for various diseases caused by microorganisms adhering to animal cells, and specifically includes, for example, anti-periodontal agents, food poisoning preventive agents, acne preventive agents, It contains feed additives and the like.

The adhesion inhibitor of the present invention can be prepared in various dosage forms depending on the purpose of use, and may be added to various foods and feeds, including pharmaceuticals and quasi-drugs. Can be. In addition, examples of preferred dosage forms include shampoos, rinses, hair conditioners, hair care products, hair sprays, hair care products such as hair tonics, bath products, soaps, body shampoos, body lotions and other bath products, lotions, emulsions and the like. , Hand creams, skin tonics and other skin care products and various cosmetics.

In particular, preferred dosage forms of the anti-periodontal agent include dental paste used for orthodontic treatment, toothpaste,
Mouthwashes, mouthwashes, mouthwashes, troches, and other oral hygiene agents include, but are not limited to, for example, kneaded bean paste, castella, mizuyokan, dorayaki skin, sponge cake, butter cake, bavaroa, custard Creams, butter creams, custard puttings, cookies, sweet buns, steamed buns, jams, lactic acid bacteria drinks, coffee drinks, coffee jellies, caramels, juices, candies, chocolates, and other dosage forms added to foods.

In the case of preparing these oral hygiene agents and cosmetics, appropriate components usually used depending on the type can be used.

For example, oral hygiene agents include calcium carbonate, dibasic calcium phosphate, silicic anhydride, magnesium carbonate, glycerin, sorbitol, propylene glycol, polyethylene glycol, carboxymethylcellulose, methylcellulose, sodium alginate, carrageenan, carboxyvinyl polymer, dioctyl sulfosuccinic acid Soda, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, butyl parahydroxybenzoate, hinokitiol, allantoin, glycyrrhizin, alcohol, gum arabic, starch, corn starch, saccharin sodium, stevioside, rubusoside, glucose, lactose, magnesium stearate, potassium potassium phosphate , Potassium diphosphate, menthol, eucalyptus oil, peppermint, spare Cement, other such dyes, sodium fluoride, fluorides of sodium monofluorophosphate and the like, lysozyme chloride, anti-inflammatory agents such as azulene, sodium chloride, and the like can be produced by appropriately blending the.

In cosmetics, oils and fats such as vegetable oils, waxes such as lanolin and beeswax, hydrocarbons, fatty acids, higher alcohols and esters, various surfactants, pigments, flavors, vitamins, It can be produced by appropriately blending those commonly used as raw materials for cosmetics, such as animal extract components, ultraviolet absorbers, antioxidants, preservatives and bactericides.

Furthermore, foods and foods using the adhesion inhibitor of the present invention
In the case of producing a feed or the like, an appropriate component usually used depending on the type can be used. For example,
When preparing food, glucose, sorbitol, stevioside, rubusoside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α- tocopherol , sodium erysorbate, glycerin, Propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, gum arabic , carrageenan, casein, gelatin, pectin, agar, vitamin Bs, nicotinamide, calcium pantothenate ,
Amino acids, calcium salts, pigments, flavors, preservatives, etc.
What is used as a usual food raw material can be appropriately compounded.

In preparing a feed, fish protein, soybean meal, rapeseed meal, feather meal, meat bone meal and the like are used as protein sources, and corn, mylo, flour and other carbohydrates or feed fats and oils, soybean oil and soybean meal are used as energy sources.
Fats such as powdered fats and oils, calcium sources such as calcium carbonate,
What is used as a normal feed material such as a phosphorus source, vitamins and minerals can be appropriately blended.

Since sweet tea has been used as sweet tea in China since ancient times, the extract used in the present invention and the adsorbed fraction obtained by these synthetic adsorption treatments have no problem in terms of safety. However, the compounding amount of the active ingredient in the microbial adhesion inhibitor to animal cells of the present invention is, for example, when used as a periodontal disease agent, 0.000001 in terms of dry weight based on the effect, taste, aroma and color when added. It is preferable to set the concentration range to 0.5%.

[0037]

The adhesion inhibitor of the present invention is a new type of adhesion inhibitor that utilizes the activity of inhibiting the adhesion of pathogenic bacteria to animal cells.
It can be used as a microbial infection inhibitor for preventing diseases caused by infection of animal cells with microorganisms .

[0038]

EXAMPLES Next, an example of a method for producing a bean tea extract as an active ingredient of the present invention and an adsorptive fraction obtained by the treatment with a synthetic adsorbent thereof, an example of a test for inhibiting adhesion to animal cells, and a method for producing microorganisms on animal cells The present invention will be described in more detail with reference to Examples of the production of an adhesion inhibitor and an anti-periodontal agent as examples, but the present invention is not limited to these Examples and the like.

Example 1 Production of hot water extract of tea tea: 100 g of tea tea was placed in a 3000 ml Erlenmeyer flask, 1000 ml of hot water was added, and extraction was carried out in a boiling water bath for 3 hours. This was filtered, and the obtained filtrate was lyophilized to obtain 32.3 g of an extract.

Example 2 Preparation of a mixture of bean tea-ethanol mixture: 100 g of bean tea was placed in a 3000 ml Erlenmeyer flask, 1000 ml of 50% by volume ethanol was added, and the mixture was stirred gently every hour at room temperature and extracted for 24 hours. went. This was filtered, and the obtained filtrate was concentrated under reduced pressure to remove ethanol. Then, water was added and freeze-dried to obtain 31.1 g of an extract.

Example 3 Adsorption treatment of hot water extract of bean tea: 3.2 g of an extract from 10 g of bean tea obtained according to the method of Example 1 was dissolved in 200 ml of water, and this was filled with Diaion HP20. The solution was passed through a column (11 × 210 mm) and adsorbed. After adsorption, the column was washed with 200 ml of water. The adsorbed fraction was desorbed with 10 times the column volume of 50% by volume ethanol, concentrated under reduced pressure, added with water, and freeze-dried to obtain 1.39 g of a sample (50% ethanol eluted fraction).

EXAMPLE 4 Assay for Inhibition Activity of Microorganisms on Cells: Examples 1 and 2
The extract obtained in the above and the fraction eluted with 50% ethanol of Example 3 were assayed for the activity of inhibiting the adhesion of microorganisms to cells by the following method. Table 1 shows the inhibitory activity of microorganisms on normal human fibroblasts, and Table 2 shows the inhibitory activity of microorganisms on normal human gingival cells.
In addition, these inhibitory activities are indicated by a sample concentration (μg / ml) for inhibiting cell adhesion by 50%.

Assay Method: Normal human dermal fibroblasts (NHDF) and normal human gingival fibroblasts (Gin-1) were used as test cells, which were cultured in a 96-well microtiter plate.
The medium was an F-GM medium for NHDF and a Dulbecco's modified Eagle MEM ( D- MEM) medium (1 for Gin-1).
0% FBS). After washing the cultured cells with PBS (-), first, BSA (Bovine seru) was used.
(malbumin: 10 mg / ml PBS) was added and incubated at room temperature for 3 hours to perform blocking to suppress nonspecific adsorption. Thereafter, the cells were washed with PBS, and various types of biotinylated microbial cells (Bacteroides gingivalis (Bacteroides gingivalis; periodontal disease-causing bacterium), Streptococcus mutans (Streptococcucucu
s mutans; cariogenic bacteria) , Streptococcus sobrinus (cariogenic bacteria), salmonella
(Salmonella sp .; enteritis, food poisoning bacteria) and Propionibacterium acnes; added as (Propionibacterium acnes acne causative bacterium)) OD ₅₅₀ as a final concentration of 0.75, at the same time the sample (Tien-cha extract) And 1.5 times at room temperature with BSA (final concentration 5 mg / ml).
Incubated for hours.

Thereafter, the cells were washed with PBS (-),
After adding SA (10 mg / ml) and incubating at room temperature for further 0.5 hours, the cells were washed again with PBS, and then streptavidin-alkaline phosphatase (100%).
(0-fold dilution) and BSA (5 mg / ml) were added and incubated at room temperature for 1 hour. After the incubation was completed, the cells were washed with PBS (-), and an alkaline phosphatase substrate was added to start a color reaction.
The absorption at 405 nm was measured with a microplate reader. The inhibition rate (%) was calculated using the following formula, and the 50% inhibitory concentration was determined from the result.

[0045]

A1: When the sample and the biotinylated bacteria were administered A0: When the sample and the PBS were administered B1: When the PBS and the biotinylated bacteria were administered B0: When only the PBS was administered

[0047]

[0048]

Example 5 Livestock Feed: (Composition) (Parts by Weight) Corn 65.3 Soybean Meal 15.95 Fish Meal 6.0 Alfalfa Meal 2.0 Tallow 3.0 NaCl 0.3 CaCO ₃ 6.0 CaHPO ₄ 1.0 Mineral mixture ⁽¹⁾ 0.1 Vitamin AD ⁽²⁾ 0.1 Vitamin B ⁽³⁾ 0.2 Tengcha extract ^*** 0.05 ────────── ───────────────── Total amount 100.0 *** Adsorbed fraction of bean tea extract obtained in Example 3 (fraction eluted with 50% ethanol) (1) Manganese A mixture of 8.0 g, 0.6 g of iron, 0.06 g of copper, 5.0 g of zinc, and 0.1 g of iodine. (2) This drug contains vitamin I at 1000 IU / g and vitamin D
3 contains 2000 IU / g. (3) This drug contains 1 g of vitamin B1 2.0 g, vitamin B2 10.0 g, vitamin B6 2.0 g, nicotinic acid
Contains 2.0 g, pantothenic acid 4.0 g, choline 120 g, and folic acid 1.0 g.

Example 6 Feed for laying hens: (Composition) (parts by weight) Corn 69.1 Soybean oil cake 18.1 Fish meal 4.0 Animal fats and oils 0.5 Food salt 0.25 Vitamin and minerals 8. 0 Teng tea extract ^*** 0.05 ─────────────────────────── Total amount 100.0 *** Obtained in Example 3 Fraction extracted from extracted bean tea extract (fraction eluted with 50% ethanol)

Example 7 Toothpaste: (composition) (parts by weight) dibasic calcium phosphate 42.0 glycerin 18.0 carrageenan 0.9 sodium lauryl sulfate 1.2 sodium saccharin 0.09 butyl paraoxybenzoate 0.005 bean tea Extract ^*** 0.05 Fragrance 1.0 Water remaining ─────────────────────────── Total 100.0 *** Adsorbed fraction of bean tea extract obtained in Example 3 (fraction eluted with 50% ethanol)

Example 8 Mouthwash: (composition) (parts by weight) Sodium lauryl sulfate 0.8 glycerin 7.0 sorbitol 5.0 ethyl alcohol 15.0 sweet tea extract ^** 0.05 1-menthol 0 .05 Perfume 0.04 Saccharin sodium 0.1 Water balance Total amount 100.0 ** Example 2 Bean tea extract obtained in

Example 9 Dental paste: (composition) (parts by weight) water 32.3 sodium benzoate 0.5 sodium saccharin 0.2 polyethylene glycol 600 25.0 carboxymethylcellulose 1.5 thyroid-244 3.0 Zeo 49 35.0 Sodium lauryl sulfate 1.45 Flavoring agent 1.0 Teng tea extract ^* 0.05 ─────────────────────────── Total amount 100.0 * Teng tea extract obtained in Example 1

Example 10 Emulsion: (Composition) (parts by weight) Stearic acid 2.5 Cetyl alcohol 1.5 Vaseline 5.0 Liquid paraffin 10.0 Polyoxyethylene (10 mol) monooleate 2. 0 Polyethylene glycol 1500 3.0 Triethanolamine 1.0 Tengcha extract ^** 0.05 Appropriate amount of fragrance Preservatives Appropriate amount Purified water balance 水─────────────── Total amount 100.0 ** Teng tea extract obtained in Example 2

Example 11 Lotion: (composition) (parts by weight) 95% ethanol 50.0 sodium lauryl sulfate 0.06 hydrogenated castor oil ethylene oxide (40 mol) adduct 0.45 bean tea extract ^* 0.05 purification Water balance Perfume and pigment Suitable amount 色素 Total amount 100.0 * In Example 1 The obtained bean tea extract

Example 12 Lozenges: (Composition) (parts by weight) Gum arabic 6 Glucose 73 Tengcha extract ^* 0.02 Potassium phosphate 0.2 Potassium phosphate 0.1 Lactose 17 Fragrance 0.1 Magnesium stearate Residual amount ────────────────────────── Total amount 100 * Extract of bean tea obtained in Example 1 object

EXAMPLE 13 13 GAM: (composition) (parts by weight) Gum base 20 Calcium carbonate 2 Stevioside 0.1 Teng tea extract ^*** 0.01 Lactose 76.89 Fragrance 1.0% ─────────────────────── Total amount 100.0 ^*** Adsorbed fraction of bean extract extracted in Example 3 (50% ethanol eluted fraction) Minutes)

Example 14 Diet: (Composition) (parts by weight) Frozen Condensed Unshu Mandarin Juice 5.0 Fructose-Glucose Liquid Sugar 11.0 Citric Acid 0.2 L-Ascorbic Acid 0.02 Tengcha Extract ^*** 0.001 Fragrance 0.2 Color element 0.1 Water remaining amount ─────────────────────────── Total amount 100. 0 *** Adsorbed fraction of bean tea extract obtained in Example 3 (fraction eluted with 50% ethanol)

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61K 7/00 A61K 7/26 7/26 9/44 9/44 9/68 9/68 A61P 1/02 A61P 1/02 31 / 04 31/04 A23L 2/26 (56) References JP-A-58-36368 (JP, A) JP-A-58-28245 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB A61K 35/78 A23G 3/30 A23K 1/16 304 A23L 2/52 A61K 7/00 A61K 7/26 A61K 9/44 A61K 9/68 A61P 1/02 A61P 31/04 BIOSIS (DIALOG) CA (STN ) MEDLINE (STN)

Claims (9)

(57) [Claims] 1. An agent for inhibiting the adhesion of microorganisms to animal cells, comprising an aqueous solvent extract from sweet tea as an active ingredient. 2. An aqueous solvent extract from sweet tea is extracted from the tea extract with an aqueous solvent extract, and then subjected to an adsorption treatment using a synthetic adsorbent to be adsorbed on the synthetic adsorbent, and then the solvent is extracted with a solvent. 2. The method according to claim 1, which is obtained as an adsorbed fraction of a desorbed synthetic adsorbent. 3. The method according to claim 1, which comprises 0.00001 to 0.5% of an active ingredient. 4. The agent for inhibiting adhesion of a microorganism to animal cells according to claim 1, which is an anti-periodontal agent. 5. The anti-periodontal agent according to claim 4, wherein the dosage form is an oral hygiene agent. 6. The food additive according to claim 1, which is a food additive.
Item 3. Inhibition of adhesion of microorganisms to animal cells according to any one of Items 3
Harmful agents. 7. A cosmetic comprising the agent for inhibiting the adhesion of microorganisms to animal cells according to any one of claims 1 to 3. 8. A feed additive according to claim 1, wherein the feed additive is a feed additive.
Item 3. Inhibition of adhesion of microorganisms to animal cells according to any one of Items 3
Harmful agents. 9. A substance that inhibits microorganisms from adhering to animal cells, wherein after extracting bean tea with an aqueous solvent, the extract is subjected to an adsorption treatment using a synthetic adsorbent to obtain an adsorbed fraction of the synthetic adsorbent. Manufacturing method.