JP5406085B2 - Helicobacter pylori adhesion inhibitor - Google Patents

Description

  The present invention relates to an inhibitor of Helicobacter pylori adhesion containing a tomato seed coat extract as an active ingredient.

  Helicobacter pylori is a helical gram-negative gonococci that floats in gastric mucus or grows attached to gastric epithelial cells. Since 1983 Warren and Marshall et al. Succeeded in the isolation and culture of Helicobacter pylori parasitizing the gastric mucosa of gastritis patients (see Non-Patent Document 1), research has progressed rapidly. It has been revealed that infection is deeply involved in the development of chronic atrophic gastritis, gastric ulcer, gastric cancer, duodenal ulcer and the like. In developed countries, the number of carriers has been declining due to recent improvements in hygiene, but the infection rate in the elderly is still high, and even in Japan, over 70% are said to be infected when over 40 years old.

  Helicobacter pylori eradication improves many of the related pathologies, and long-term infection with Helicobacter pylori is considered a risk factor for the onset, and eradication is the best treatment It is said that. Currently, sterilization therapy is being performed in combination with a proton pump inhibitor and two antibiotics (amoxicillin and clarithromycin), but there are concerns about a decrease in sterilization rate and side effects due to an increase in resistant bacteria. . Therefore, a method for suppressing the adhesion of Helicobacter pylori to the gastric mucosa has been proposed as a preventive and therapeutic method with fewer side effects. Examples of the Helicobacter pylori adhesion inhibitor include Helicobacter pylori fixing inhibitors containing fucoidan as an active ingredient (see Patent Document 1), and Helicobacter pylori adhesion inhibitors containing glucuronic acid-containing fucoidan as an active ingredient (Patent Document 2). Have been proposed).

  Tochinoki (Aesculus turbinata BLUME) seeds, that is, tochinomi, used to be a staple food with acorns as a substitute for rice. In addition, since it can be stored for a long time by drying, it was used as an emergency food in the event of famine. Since the tochino contains a large amount of saponin called escin, research on the physiological function of the extract is being promoted mainly in Europe, and it is used in medicines and cosmetics for the treatment of inflammation and swelling. As for the physiological activity of Tochinomi, the escin derivative isolated from the peeled Tochinomi has an inhibitory action on the increase in blood glucose level (see Non-Patent Document 2) and an inhibitory activity on carbohydrase (See Non-Patent Document 3). It has been known. It is also known that polyphenols obtained from tomato peel, i.e., seeds of tomato seedling, have an inhibitory activity on carbohydrate digestive enzymes (see Non-Patent Document 4). However, it has not been known so far that Tochinoki seed coat extract has an action of suppressing adhesion to gastric mucosa, which is considered to be an early stage of Helicobacter pylori infection.

JP 7-138166 A JP 10-287571 A

Warren J. R., Marshall B., Lancet, 1273-1275 (1983) Hideto Kimura et al., Journal of Japan Society for Food Science and Technology, Vol. 51, 672-679 (2004) Hideto Kimura et al., Japan Society for Food Science and Technology, Vol. 53, 31-38 (2006) Satoshi Ogawa et al., Japan Society for Food Science and Technology, Vol. 56, 95-102 (2009)

  An object of the present invention is to provide a Helicobacter pylori adhesion inhibitor for suppressing gastric inflammation, gastric ulcer, gastric cancer, duodenal ulcer and the like caused by Helicobacter pylori by suppressing adhesion of Helicobacter pylori to the gastric mucosa. is there.

  As a result of diligent research on the above problems, the present inventor has found that an extract of Tochinoki seed coat strongly suppresses the adhesion of Helicobacter pylori to a gastric cancer cell line (MKN45 cells), thereby completing the present invention. did.

  That is, the Helicobacter pylori adhesion inhibitor of the present invention is characterized in that it contains a Tochinoki seed coat extract as an active ingredient.

  The Helicobacter pylori adhesion inhibitor of the present invention is effective for the prevention and treatment of gastritis, gastric ulcer, gastric cancer, duodenal ulcer and the like caused by Helicobacter pylori.

  The present invention relates to an inhibitor of Helicobacter pylori adhesion containing a tomato seed coat extract as an active ingredient. The cinnamon seed coat extract used in the present invention can be extracted from water flea (tochinomi) or its skin with water, a mixture of water and alcohol, or alcohol. In the present invention, the extraction method for obtaining the extract is not particularly limited, and known methods such as extraction by immersion, heat extraction, continuous extraction and supercritical extraction can be used, but hot water extraction is usually preferable. Used.

  After completion of the extraction operation, the extraction residue is removed by a known method such as filtration or centrifugation to obtain an extract. The extract can be concentrated by a known method. For example, a dried extract in powder or solid form can be obtained by drying by a known method such as ventilation drying, vacuum drying, or vacuum freeze drying. Furthermore, a solution in which the extract is dissolved in water and / or alcohol can be purified by a method such as ultrafiltration, adsorption chromatography, reverse phase chromatography, gel filtration chromatography, or the like. As a purification method, adsorption chromatography is particularly preferable. The purified product of the extract thus obtained can also be used in the present invention as a cypress seed coat extract. In addition, the main component of the cypress seed coat extract in this invention is a polyphenol, and the polyphenol content in an extract can be measured by the usual Foline-Ciocalteu method.

  The Helicobacter pylori adhesion inhibitor of the present invention can be prepared by a known method using the above extract or purified product as it is or by appropriately mixing pharmaceutically acceptable additives with the extract or purified product as necessary. It can be manufactured as a preparation. Examples of the preparation include liquids, powders, granules, tablets, microcapsules, soft capsules, and hard capsules. These various preparations are useful as health foods for the purpose of preventing gastritis, gastric ulcer, gastric cancer, duodenal ulcer and the like.

  In addition, the Helicobacter pylori adhesion inhibitor of the present invention can be made into a form of food or drink by blending the above extract or purified product with a food material and food additive, and can be a solid food, cream or jam. It can be used in any form such as semi-fluid food, gel food, and beverage. Examples of such foods and beverages include soft drinks, coffee, tea, milk drinks, lactic acid bacteria drinks, drops, candy, chewing gum, chocolate, gummy, yogurt, ice cream, pudding, chicken pox, jelly, confectionery, cookies, etc. Can be mentioned. These foods and drinks are useful as health foods for the purpose of preventing gastritis, gastric ulcer, gastric cancer, duodenal ulcer and the like.

  Examples of the pharmaceutically acceptable additive, food material and food additive used in the preparation of the above-mentioned preparations and foods and drinks include, for example, excipients, lubricants, disintegrants, binders, solubilizers, and emulsifiers. Antioxidants, preservatives, thickening stabilizers, sweeteners, acidulants, seasonings, colorants, and the like can be used as appropriate.

  Examples of the excipient include lactose, dextrin, corn starch, and crystalline cellulose. Examples of the lubricant include magnesium stearate, sucrose fatty acid ester, polyglycerin fatty acid ester and the like. Examples of the disintegrant include carboxymethyl cellulose calcium, anhydrous calcium hydrogen phosphate, and calcium carbonate. Examples of the binder include starch paste liquid, hydroxypropyl cellulose liquid, gum arabic liquid and the like. Examples of solubilizers include gum arabic and polysorbate 80. Examples of the emulsifier include glycerin fatty acid ester and sucrose fatty acid ester. Examples of the antioxidant include BHT (dibutylhydroxytoluene), BHA (butylhydroxyanisole), ascorbic acid, tocopherol and the like.

  Preservatives include sorbic acid, methyl paraoxybenzoate, sodium sulfite and the like. Examples of the thickening stabilizer include sodium alginate, sodium carboxymethyl cellulose, sodium polyacrylate and the like. Sweeteners include sugar, fructose glucose liquid sugar, honey, aspartame and the like. Examples of the acidulant include citric acid, lactic acid, DL-malic acid and the like. Examples of the seasoning include DL-alanine, sodium 5'-inosinate, sodium L-glutamate and the like. Examples of the colorant include β-carotene, edible tar dye, riboflavin and the like. Examples of the fragrance include mint and strawberry fragrance.

  The amount of the tomato seed coat extract of the present invention blended in the above-mentioned various preparations and 100% by mass of food and drink is usually about 0.001 to 50% by mass, preferably about 0.01 to 20% by mass in terms of solid content. More preferably, it is about 0.1 to 10% by mass.

  When these various preparations and foods and drinks are taken orally, the daily dose of the tomato seed coat extract of the present invention is preferably in the range of about 1 to 3000 mg / kg in terms of solid content. May be taken once to several times. However, the actual dose is determined in consideration of the purpose and the situation of the intaker (gender, age, symptoms, etc.).

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited at all by these.

[Experiment 1: Preparation of tomato seed coat extract and measurement of polyphenol content]
<Experiment 1-1: Preparation of Tochinoki seed coat extract>
10 g of tomato seed coat (water content 11.5%) was pulverized in a mortar, 200 ml of distilled water was added, and the mixture was heated to reflux for 2 hours. After filtering the extract, 200 ml of distilled water was added to the residue, and the same operation was performed again. The extract was concentrated under reduced pressure using a rotary evaporator to obtain 1.56 g of a dried product. This extract was subjected to column chromatography using a Diaion HP-20 (inner diameter: 30 mm, length: 300 mm, manufactured by Nippon Nensui) column, and eluted sequentially with 500 ml of distilled water and 500 ml of methanol. The resulting methanol-eluted fraction was concentrated under reduced pressure to obtain 0.67 g of a dried product. This was subjected to ODS column chromatography using a Chromatolex ODS1024T column (inner diameter 30 mm, length 300 mm, manufactured by Fuji Silysia Chemical Ltd.), the column was washed with 500 ml of 5% methanol, and then eluted with 500 ml of 50% methanol. The obtained 50% methanol-eluted fraction was concentrated under reduced pressure to obtain 0.54 g of Tochinoki seed coat extract.

<Experiment 1-2: Polyphenol content in Tochinoki seed coat extract>
A portion of the tomato seed coat extract obtained in Experiment 1-1 was dissolved in purified water, diluted as appropriate, and polyphenols were prepared by a conventional Foline-Ciocalteu method using (−)-epicatechin (reagent) as a standard substance. The content was measured. As a result, the total amount of polyphenols in 0.54 g of the tomato seed coat extract obtained in Experiment 1-1 was 0.44 g (in terms of (−)-epicatechin). This indicates that the cypress seed coat extract contains polyphenol as a main component.

  In addition, the Tochinoki seed coat extract dissolved in a 10% ethanol solution based on solid mass so as to be 4 mg / ml was further diluted appropriately with a solvent used and used in the following experiments.

[Experiment 2: Anti-Helicobacter pylori action of Tochinoki seed coat extract]
First, the antibacterial action against Helicobacter pylori was examined for the Tochinoki seed coat extract prepared in Experiment 1-1.
<Experiment 2-1: Preparation of Helicobacter pylori solution>
Helicobacter pylori ATCC 43504 strain was inoculated into BBL Brucella medium (Becton Dickinson) containing 10% fetal bovine serum (manufactured by Hyclone), and an oxygen scavenger (“Aneropac microaerobic”, manufactured by Mitsubishi Gas Chemical) ) At 37 ° C. for 5 to 7 days. The culture solution whose absorbance at a wavelength of 550 nm reaches about 0.7 is centrifuged (7,000 rpm, 5 minutes) to recover the cells and suspended in phosphate buffered saline (hereinafter abbreviated as “PBS”). The operation of turbidity and centrifugation again was repeated twice to wash the cells. The washed cells were suspended in PBS, the amount of cells was evaluated by measuring the absorbance at a wavelength of 550 nm, and the concentration was adjusted to obtain a Helicobacter pylori solution. Incidentally, the bacterial cell concentration in the bacterial solution having an absorbance at a wavelength of 550 nm of 0.1 corresponds to approximately 3 × 10 ⁶ CFU / ml.

<Experiment 2-2: Evaluation of anti-Helicobacter pylori action of Tochinoki seed coat extract>
The anti-Helicobacter pylori action was evaluated by measuring the minimum growth inhibitory concentration (hereinafter referred to as “MIC”) exhibited by Tochinoki seed coat extract against Helicobacter pylori.

  Test samples prepared by adjusting the tomato seed coat extract prepared by the method of Experiment 1-1 to a concentration of 1.0, 1.2, 1.4, 1.6, 1.8, or 2.0 mg / ml, respectively To 0.5 ml of the solution, 4.5 ml of Brucella medium containing 10% horse serum (manufactured by Invitrogen) and 1% agar (hereinafter abbreviated as “brucella agar medium”) was added to prepare an agar plate. Next, 50 μl of the bacterial solution obtained by diluting the Helicobacter pylori bacterial solution prepared in Experiment 2-1 with PBS so that the absorbance at a wavelength of 550 nm is approximately 0.3 is dropped on the agar plate. Spread on the surface. After culturing at 37 ° C. for 7 days under microaerobic conditions, the presence or absence of bacterial growth was confirmed under a stereomicroscope, and the minimum concentration at which bacterial growth was not observed was defined as MIC. The results are shown in Table 1.

  The growth of Helicobacter pylori was clearly observed on Brucella agar plates containing 100 and 120 μg / ml of Tochinoki seed coat extract. However, these observed colonies were smaller than the control colonies using media without the tomato seed coat extract. When the concentration of Tochinoki seed coat extract was 140 and 160 μg / ml, colonies could not be detected on the agar plate, and very small colonies were detected by observation with a stereomicroscope. On the other hand, the growth of Helicobacter pylori could not be confirmed even by observation with a stereomicroscope when the concentration of Tochinoki seed coat extract was 180 and 200 μg / ml. Therefore, the MIC for Helicobacter pylori was found to be 180 μg / ml. This result indicates that the cypress seed coat extract has an antibacterial action against Helicobacter pylori.

[Experiment 3: Helicobacter pylori adhesion inhibitory effect of Tochinoki seed coat extract]
Next, the effect of inhibiting the adhesion of Helicobacter pylori to the extract of Tochigi seed coat was examined for the Tochigi seed coat extract prepared in Experiment 1-1. The Helicobacter pylori adhesion-suppressing action was determined using the amount of Helicobacter pylori adhered to MKN45 cells using MKN45 cells, a human gastric cancer cell line, in the presence of various concentrations of Tokinoki seed coat extract. ).
<Experiment 3-1: Helicobacter pylori adhesion inhibition test to MKN45 cells>
MKN45 cells prepared using RPMI 1640 medium containing 10% fetal bovine serum were seeded at 1 × 10 ⁵ cells / well in a 96-well microplate (manufactured by Becton Dickinson) at 37 ° C., 5% CO _2. It was cultured for 3 days under the environment. Next, an equal amount of Helicobacter pylori solution prepared by the method of Experiment 3-1 and adjusted to have an absorbance at a wavelength of 550 nm of about 0.2 with PBS and a test sample solution adjusted to each concentration with PBS are mixed. And preincubated at 37 ° C. for 30 minutes. Next, after removing the medium from each well of the 96-well microplate, 100 μl of each sample solution containing Helicobacter pylori was added to each well and incubated at 37 ° C. for 2 hours.

After removing the sample solution containing Helicobacter pylori from each well, 4% paraformaldehyde solution was added and fixed by treatment at room temperature for 1 hour. After washing each well with PBS containing 0.05% Tween 20, a 3% hydrogen peroxide solution was added and treated at room temperature for 30 minutes. After washing, biotin-labeled-rabbit anti-Helicobacter pylori polyclonal antibody (manufactured by Virostat) was added and reacted at room temperature for 60 minutes. After washing, horseradish peroxidase (HRP) -labeled streptavidin (manufactured by Dako) was added and reacted at room temperature for 60 minutes. After washing, color was developed using a kit (trade name “Quanta Blue ^™ Fluorogenic Peroxidase Substrate Kit” (Pierce), and the fluorescence intensity was measured to evaluate the amount of Helicobacter pylori adhering to MKN45 cells by an average of 5 points.

  As shown in Table 2, tomato seed coat extract did not substantially affect the adhesion of Helicobacter pylori to MKN45 cells at a concentration of less than 3.1 μg / ml. However, at a concentration of 3.1 μg / ml or more, the adhesion to MKN45 cells is suppressed in a concentration-dependent manner, and the relative adhesion amount in the case of not containing the tomato seed coat extract is 54.6% at a concentration of 12.5 μg / ml, It decreased to 39.6% at a concentration of 50 μg / ml.

<Experiment 3-2: Effect of concentration of Tochinoki seed coat extract used for adhesion inhibition test on Helicobacter pylori>
For the purpose of confirming that the decrease in the adhesion amount of Helicobacter pylori depending on the concentration of Tochinoki seed coat extract in the results of Experiment 3-1 is not due to the antibacterial action of Tochinoki seed coat extract, Helicobacter pylori and Tochinoki seed coat The contact time of the extract was set to be the same as in Experiment 3-1, and the survival rate (colony forming ability) of Helicobacter pylori was examined.

  The Helicobacter pylori solution obtained by the method of Experiment 2-1 was adjusted with PBS so that the absorbance at 550 nm was about 0.2, and mixed in equal amounts with the test sample solution adjusted to each concentration. This mixture was incubated at 37 ° C. for 2.5 hours (a total of 30 minutes of preincubation in Experiment 3-1 and 2 hours of infection time for MKN45 cells). Next, a test sample solution containing Helicobacter pylori was appropriately diluted with PBS, and 50 μl was dropped on a Brucella agar plate and spread on the plate surface. After culturing at 37 ° C. for 5 days, the number of colonies on the plate surface was counted. Table 3 shows the relative number of colonies in the liquid treated in the presence of the extract of tomato seed coat with each concentration when the number of colonies without the addition of the tomato seed coat extract was 100%.

  As shown in Table 3, when Helicobacter pylori and the Tochigi seed coat extract were contacted for 2.5 hours, the relative colony count of Helicobacter pylori did not include the Tochinoki seed coat extract at a concentration of 50 μg / ml. It decreased to about 70% when treated with the solution and to about 10% at 100 μg / ml. However, at a concentration of 25 μg / ml or less, the relative colony number of Helicobacter pylori did not substantially decrease, and no antibacterial action against Helicobacter pylori was observed. This result means that the decrease in the fluorescence intensity due to the low concentration of cypress seed coat extract in Experiment 3-1, that is, the effect of inhibiting the adhesion of Helicobacter pylori to MKN45 cells is not due to the antibacterial effect. .

  From the results of Experiments 1 to 3, Tochinoki seed coat extract containing polyphenol as a main component not only has antibacterial activity against Helicobacter pylori, but also to gastric mucosa even at a low concentration that does not show antibacterial activity. It has been found that it has the effect of suppressing the adhesion of Helicobacter pylori.

[Experiment 4: Preparation of Tochinoki seed coat extract and food and drink]
(Preparation Example 1)
<Preparation of tablets>
10 parts by weight of L-ascorbic acid, 80 parts by weight of maltose (registered trademark “Sanmalto”, sold by Hayashibara Shoji Co., Ltd.), 9 parts by weight of Tochinoki seed coat extract obtained by the method of Experiment 1-1, and sucrose stearate 1 After mixing the respective parts by mass uniformly, tableting was carried out by a conventional method to obtain tablets containing the tomato seed coat extract. This product is an easily ingestible tablet that has excellent solubility and can be supplemented with vitamin C. In addition, since the Tochinoki seed coat extract suppresses the adhesion of Helicobacter pylori to the gastric mucosa, it can be advantageously used as a health food for preventing gastritis, gastric ulcer, gastric cancer, duodenal ulcer and the like.

(Preparation Example 2)
<Preparation of chewing gum>
3 parts by weight of gum base was heated and melted until soft, and 7 parts by weight of hydrous crystal trehalose (registered trademark "Treh", sold by Hayashibara Shoji Co., Ltd.) and 0.5 parts by weight of Tochinoki seed coat extract obtained by the method of Experiment 1-1 Added. Further, 0.1 parts by mass of vitamin E and appropriate amounts of coloring agent and flavoring agent were mixed, and then kneaded, molded, and packaged by a conventional method to obtain a chewing gum containing a Tochinoki seed coat extract. This product has good texture and flavor. In addition, since the Tochinoki seed coat extract suppresses the adhesion of Helicobacter pylori to the gastric mucosa, it can be advantageously used as a health food for preventing gastritis, gastric ulcer, gastric cancer, duodenal ulcer and the like.

(Preparation Example 3)
<Preparation of health supplement beverage>
Isomerized sugar 40 parts by weight, maltitol (trade name “powder mabit”, Hayashibara Corporation sales) 2 parts by weight, rice vinegar 10 parts by weight, apple vinegar 6 parts by weight, citric acid 2 parts by weight, malic acid 2 parts by weight Then, 2 parts by mass of concentrated apple fruit juice and 2 parts by mass of Tochinoki seed coat extract obtained by the method of Experiment 1-1 were mixed and dissolved to prepare a beverage. This product has good flavor and taste. In addition, since the tomato seed coat extract suppresses the adhesion of Helicobacter pylori to the gastric mucosa, it can be advantageously used as a health supplement drink for preventing gastritis, gastric ulcer, gastric cancer, duodenal ulcer and the like.

(Preparation Example 4)
<Preparation of tube oral nutrition>
Hydrous crystal trehalose (registered trademark "Treh", Hayashibara Shoji Sales) 30 parts by weight, sodium glutamate 3 parts by weight, glycine 2 parts by weight, salt 2 parts by weight, citric acid 2 parts by weight, magnesium carbonate 0.3 parts by weight, A blend consisting of 1 part by weight of calcium lactate, 4 parts by weight of Tochinoki seed coat extract prepared by the method of Experiment 1-1, 0.02 parts by weight of thiamine and 0.02 parts by weight of riboflavin was prepared. This blend was divided into 30 g portions in laminated aluminum bags and heat sealed to prepare tube oral nutrition. This product can be dissolved in about 250 to 500 ml of sterile water in a bag and used as a nutrient by tube or orally. In addition, since the Tochinoki seed coat extract suppresses the adhesion of Helicobacter pylori to the gastric mucosa, it can be advantageously used as a liquid for preventing gastritis, gastric ulcer, gastric cancer, duodenal ulcer and the like.

  The Helicobacter pylori adhesion inhibitor of the present invention is safe for the human body, and when taken orally in the form of food, can effectively inhibit the adhesion of Helicobacter pylori to the gastric mucosa, It is useful for the prevention of gastritis, gastric ulcer, gastric cancer, duodenal ulcer caused by Helicobacter pylori infection.

Claims (3)

It is extracted from Tochigi seed coat, contains Tochinoki seed coat extract containing polyphenol as the main ingredient, and is administered to the stomach by drinking from the oral route, and suppresses the adhesion of Helicobacter pylori to the stomach mucosa ,
A Helicobacter pylori adhesion inhibitor , wherein the tomato seed coat extract is administered at a concentration of 12.5 μg / ml or more . The tomato seed coat extract is
(A) pulverizing the seed coat of torch and forming an extract by adding water and alcohol or adding alcohol and heating;
(B) obtaining a concentrate by concentrating the extract;
The Helicobacter pylori adhesion inhibitor of Claim 1 formed by the extraction process which has this. In the extraction step, after the step (b),
(C) The Helicobacter pylori adhesion inhibitor of Claim 2 which has the refinement | purification process by column chromatography.