JP2008019193A - Anti-helicobacter pylori agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a safe and effective anti-Helicobacter pylori agent. <P>SOLUTION: The anti-helicobacter pylori agent comprises at least one capsinoid compound. The capsinoid compounds include capsiate, dihydrocapsiate, nordihydrocapsiate, vanillyl octanoate, vanillyl nonanoate, vanillyl decanoate, and a capsinoid compound extracted from a nonpungent capsicum. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a composition having antibacterial or antibacterial activity against Helicobacter pylori bacteria and used for the prevention or improvement of symptoms such as gastritis, gastric ulcer, and duodenal ulcer.

  Helicobacter pylori (Hericobacter pylori or H. pylori) is a gram-negative spiral bacterium first isolated and cultured from patients with chronic gastritis by Warren JR and Marshall BJ in 1983 (non-patent literature) 1). At that time, the stomach was exposed to a low pH by gastric acid, so it was unclear whether the bacteria could survive, but the discovery of this bacterium revealed that the bacteria were also alive in the stomach. Since this bacterium does not grow below pH 4, it is known that it is not an acid-resistant bacterium. By producing strong urease and producing ammonia from urea, it can survive even in the stomach under strong acidity. It is believed that.

  H.pyroli infection is thought to be caused by oral infection. In developing countries, the high infection rate of 70-80% or more already in childhood. On the other hand, infection rates are lower in developed countries than in developing countries. In Japan, the H.pyroli infection rate is about 10-40% for young people in developed countries, but for adults over the age of 40, it is a developing country type and shows a high infection rate (over 80%). These are said to be one of the reasons that they grew up in the poor hygiene situation until 1955.

  Although the relationship between H.pyroli and disease is not completely clear, it is strongly suspected as the causative agent of chronic gastritis isolated for the first time. There are reports that the positive rate of isolated culture is 88% for chronic active inflammation, 64% for gastric ulcer, and 85% for duodenal ulcer (for example, see Non-Patent Document 2). The positive rate of H.pyroli separation culture is as low as 10% or less. In addition, as a result of attempts to sterilize H.pyroli-positive duodenal ulcer patients with antibacterial agents, the recurrence rate after one and a half years for patients who could not be sterilized was as high as 80%. Only 10% of those who did have a recurrence. From these facts, H.pyroli infection is said to be a recurrence factor or healing prolongation factor of gastric / duodenal ulcer. Furthermore, a report has been made on the relationship with gastric cancer (see, for example, Non-Patent Document 3).

For these reasons, attempts have been made to use antibiotics having anti-Helicobacter pylori activity for the prevention and treatment of gastritis or gastric / duodenal ulcers. From the viewpoint of suppressing the growth of Helicobacter pylori, various attempts have been made to use antibiotics as antibacterial agents. Currently, three types of drugs are combined among aminopenicillin, tetracycline, macrolide and bismuth preparations. Combined triple therapy is considered the most effective means. However, administration of these conventional antibiotics has many problems such as safety or recurrence during long-term administration, and the development of effective and safe foods and drugs has been desired.

  In the production of food, it is important to add a favorable seasoning. On the other hand, it is also important in food hygiene to prevent the food from being spoiled or deteriorated. In particular, various means have been taken to control many spoilage bacteria including food poisoning bacteria. Among them, the use of spices is known to have a bacteriostatic action of spoilage bacteria as part of the spices, so it can be said that it has been deeply involved in the preservation of food as well as seasonings. Capsaicin, a spicy component of pepper, which is one of the spices, has been reported to have antibacterial activity. That is, it has been reported that it showed bacteriostatic action against bacteria such as B. cereus and B. subtilis, and also showed antifungal activity against Zygosaccharomyces genus and Mycoplasma agalactiae (for example, Non-Patent Document 4). reference).

  Furthermore, it has been reported that capsaicin has a bacteriostatic action against the aforementioned H. pylori (see, for example, Non-Patent Document 5), and attention is paid not only to food sanitation but also to action on medically important pathogens. However, large-scale administration of capsaicin (1 mg) to rats increased the occurrence of Shay ulcer due to pyloric ligation and ulcer due to reserpine (for example, see Non-Patent Document 6). Moreover, when pepper was mixed with rabbit diet (5 g / kg BW / day) and given for 12 months, gastric ulcers were observed in all animals (for example, see Non-patent Document 7). Therefore, the problem is that capsaicin is not suitable for safety.

  On the other hand, it is reported that “CH-19 sweet”, a non-pungency fixed variety of pepper selected and fixed by Yazawa et al. As a pepper with less pungency, contains a large amount of novel capsinoids that do not exhibit pungency ( For example, refer nonpatent literature 8). The compounds belonging to these capsinoids (fatty acid esters of vanillyl alcohol, capsiate, dihydrocapsiate, etc., hereinafter simply referred to as “capsinoid compounds”, “capsinoids” or “capsinoids”) are capsaicinoids which are pungent components of red pepper. Unlike (capsaicin, dihydrocapsaicin, etc.), although it does not show a pungent taste, it has been reported to activate immunity, activate energy metabolism, etc. (see Patent Document 1), and is expected to be applied in the future. .

Japanese Patent Laid-Open No. 11-246478 Marshall BJ & Warren JR, THE LANCET, Vol.321, pp.1273-1275, 1984 Blaser M.J., J Infect Dis.Vol.161, pp.626-33, 1990 Nomura A, Stemmermann GN, Chyou PH, Kato I, Perez-Perez GI, Blaser M.J., N Engl J Med.Vol.325, pp.1132-6, 1991 Gal IE. European Food Research and Technology Vol.138, pp.86-92 (1968) Jones NL, Shabib S, Sherman PM. FEMS Microbiol Lett. Vol.146, pp.223-7, 1997 Makara GB, Csalay L, Frenkl R, Somfai Z, Szepeshazi K. Acta Med. Sci. Hung. Vol. 21, pp.213-6, 1965 LEE SO, Taehan Naekwa Hakhoe Chapchi Vol.41, pp.471-81, 1963 Yazawa et al., Journal of Horticultural Society, 58, 601-607, 1989

  It has been reported that capsaicin has a bacteriostatic action against H. pylori, but there is a problem that the amount that can be taken orally is limited due to its strong irritation. Therefore, there is a need for safe and effective anti-Helicobacter pylori agents.

  As a result of intensive studies in view of the above problems, the present inventors have found that an anti-Helicobacter pylori effect is recognized in capsinoids, which are components extracted from hot pepper, and have completed the present invention. That is, the present invention includes the following contents.

(1) An anti-Helicobacter pylori agent comprising one or more capsinoid compounds.
(2) The anti-Helicobacter pylori agent according to (1), wherein the capsinoid compound includes capsiate, dihydrocapsiate, nordihydrocapsiate, vanillyl octoate, vanillyl nonanoate, and vanillyl decanoate.
(3) The anti-Helicobacter pylori agent according to (1) or (2), wherein the capsinoid compound includes a capsinoid compound extracted from a hot pepper.
(4) The anti-Helicobacter pylori agent according to any one of (1) to (3), which is for oral administration.
(5) A pharmaceutical composition for preventing, treating or preventing recurrence of upper gastrointestinal tract diseases, comprising the anti-Helicobacter pylori agent according to any one of (1) to (3) and a physiologically acceptable carrier.
(6) The pharmaceutical composition according to (4), wherein the daily dose of the capsinoid compound is 0.03 to 500 mg / kg body weight.
(7) A health food comprising the anti-Helicobacter pylori agent according to any one of (1) to (3) and used for improving indefinite complaints of the upper gastrointestinal tract.

  According to the present invention, an anti-Helicobacter pylori effect is recognized for capsinoids contained in capsicum, which is a safe food material, and is promising in the fields of pharmaceuticals, health foods, and the like.

(Capsinoid compound)
In the present invention, the capsinoid compound refers to a fatty acid ester of vanillyl alcohol, and typical components thereof include capsiate, dihydrocapsiate, nordihydrocapsiate, which are identified as components contained in red peppers, Furthermore, various linear or branched fatty acids and fatty acid esters of vanillyl alcohol having the same fatty acid chain length as those of capsiate such as vanillyl decanoate, vanillyl nonanoate, vanillyl octanoate and nordihydrocapsiate. contains. Capsiate (hereinafter may be abbreviated as “CST”), dihydrocapsiate (hereinafter abbreviated as “DCT”) and nordihydrocapsiate (hereinafter abbreviated as “NDCT”), respectively. It can be represented by the following chemical formula.

  Capsinoids are mostly contained in plants belonging to the genus Capsicum (hereinafter referred to as “capsicum”), and therefore can be prepared by purifying and separating from capsicum plants and / or fruits. The capsicum used for purification is not particularly limited as long as it contains capsinoid and may be derived from capsicum varieties having conventional pungent taste typified by `` Nikko '' and `` Five Colors ''. Is preferred. Among them, spicy varieties such as “CH-19 Ama”, “Manganji”, “Fushimi Akanaga”, shiso, green pepper, and the like contain a lot of capsinoid compounds and can be suitably used. In particular, “CH-19 sweet” which is a spicy taste varieties is more preferable because the content of the component is high. In this specification, the term “CH-19 sweet” means a group of varieties including “CH-19 sweet” varieties and progeny related varieties derived from “CH-19 sweet”. Purification and separation of capsinoid compounds can be carried out by solvent extraction, various types of chromatography such as silica gel chromatography, high-performance liquid chromatography for preparation, etc., which are well known to those skilled in the art, alone or in combination. For example, the method described in Patent Document 1 described above can be used.

  Moreover, said capsinoid compound can also be synthesize | combined by the transesterification which used the corresponding fatty acid ester and vanillyl alcohol as a starting material like the above-mentioned patent document 1, for example. Alternatively, it can be synthesized by other reaction methods well known to those skilled in the art based on the structural formula. Furthermore, capsinoids can be easily prepared by a synthetic method using an enzyme. For example, according to the method described in JP 2000-31598 A, Kobata et al. (Biosci. Biotechnol. Biochem., 66 (2), 319-327, 2002), a fatty acid ester corresponding to a desired compound and / or A desired capsinoid compound can be easily obtained by utilizing the reverse reaction of lipase using a fatty acid-containing compound such as triglyceride and vanillyl alcohol as a substrate.

  When used in the present invention, the capsinoid compound may be any of the above-described extracted products and synthetic products, and may be a single capsinoid compound or a mixture of two or more. Furthermore, the capsinoid compound to be used may contain a free fatty acid, vanillyl alcohol, or the like, which is a decomposition product thereof.

(Anti-Helicobacter pylori)
The following examples of the present invention revealed for the first time that capsinoid compounds have a protective effect against oral infection of gerbils with Helicobacter pylori. The capsinoid compound according to the present invention shows a significant decrease in the number of bacteria in the stomach at a dose of 100 mg / kg body weight, shows a tendency to decrease that was not significant at 500 mg / kg body weight, and at a dose of 100 mg / kg body weight or more. It is shown to have a protective effect. Since Helicobacter pylori was first isolated and cultured from patients with chronic gastritis, its relationship with various upper gastrointestinal diseases has been clarified. Therefore, the anti-Helicobacter pylori agent of the present invention is useful as a pharmaceutical or health food. It is thought that. According to the results of experiments by the present inventors, the protective effect of capsaicin, which is a pungent component of red pepper used as a positive control, was also verified. As a result, an effect comparable to that of capsinoid 100 mg / kg body weight at 10 mg / kg body weight. It could be confirmed. These results suggest that there is a concentration difference of about 10 times between capsaicin and capsinoid in terms of the growth inhibitory effect of Helicobacter pylori. Helicobacter pylori is considered to have a sufficient advantage by adjusting the administration concentration. Based on the reports that gastric ulcers increase due to experimental large-scale exposure to capsaicin (Non-Patent Documents 6 and 7 listed above), it is highly possible that long-term intake of capsaicin may cause an increase in gastric ulcers. In terms of safety, capsinoids may be superior to capsaicin.

(Pharmaceutical composition)
The anti-Helicobacter pylori agent of the present invention can be used as a pharmaceutical composition alone or in combination with other compounds in the presence of a physiologically acceptable carrier. Use of a dosage form suitable for administration to humans or animals, preferably for prevention, treatment or prevention of recurrence of upper gastrointestinal diseases, more preferably for prevention, treatment or prevention of recurrence of gastritis, gastric ulcer or duodenal ulcer Used as a composition. Dosage forms or compositions suitable for administration to such subjects are well known to those skilled in the art. The pharmaceutical composition of the present invention is preferably administered orally. Physiologically acceptable carriers include various organic or inorganic carrier materials commonly used as pharmaceutical materials, such as excipients, lubricants, binders and disintegrants in solid formulations, or solvents and solutions in liquid formulations. Adjuvants, suspending agents, isotonic agents, buffers, soothing agents and the like can be mentioned. If necessary, additives such as conventional preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents and the like can be used in appropriate amounts.

  Examples of the excipient include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid and the like. Examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like. Examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, sodium carboxymethylcellulose and the like. Examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl starch sodium, L-hydroxypropyl cellulose, and the like. Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like. Examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like. Examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; for example, polyvinyl alcohol, polyvinylpyrrolidone, carboxy Examples include hydrophilic polymers such as sodium methylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose. Examples of isotonic agents include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol and the like. Examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate and the like. Examples of soothing agents include benzyl alcohol. Examples of the preservative include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Examples of the antioxidant include sulfite, ascorbic acid, α-tocopherol and the like.

  The pharmaceutical composition of the present invention can be produced by a conventional method such as mixing, kneading, granulation, tableting, coating, sterilization, emulsification, etc., depending on the form of the preparation. In addition, regarding manufacture of a formulation, the Japanese Pharmacopoeia formulation general rules can be referred.

  According to the guidelines for sterilization indications by the Japan Helicobacter Society, A) Diseases recommended for sterilization treatment include gastric ulcer / duodenal ulcer, gastric MALT lymphoma, and B) Diseases for which eradication treatment is desirable include endoscopy for early gastric cancer. Stomach after mirror mucosal resection, atrophic gastritis, gastric hyperplastic polyp, C) Non-ulcer dyspepsia (NUD), Gastro-Esophageal Reflux Disease (GERD) The names of most upper gastrointestinal diseases are listed (Study Group on the development of gastric ulcer treatment guidelines based on evidence). From these situations, the pharmaceutical composition of the present invention is useful for preventing or ameliorating upper gastrointestinal diseases.

  The dosage of the pharmaceutical composition of the present invention varies depending on the type and condition of the above gastrointestinal tract disease, the age, weight, constitution, physical condition, dosage form, administration method, administration period, etc. of the subject. (Body weight about 60 kg) 1 mg to 100 g, preferably 1 mg to 50 g, most preferably 2 mg to 30 g (capacity per day of 0.03 to 500 mg / kg body weight) of capsinoid compound of the present invention per person, generally per day It is possible to select appropriately within the range of the above), which is administered once or divided into several times. Of course, as described above, the dosage varies depending on various conditions. Therefore, a dosage smaller than the dosage may be sufficient, or the dosage may need to be administered beyond the range.

(Application to health food)
The anti-Helicobacter pylori agent of the present invention can be administered or ingested in the form of a functional food or a health food (supplement), for example, tablets (including sugar-coated tablets and film-coated tablets), powders, granules, It can be safely taken orally as capsules (including soft capsules), syrups, liquids and the like. In order to obtain a supplement, according to a known method, the active ingredient capsinoid compound is, for example, an excipient (eg, lactose, sucrose, starch, etc.), a disintegrant (eg, starch, calcium carbonate, etc.), a binder (eg, , Starch, gum arabic, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose, etc.) or a lubricant (eg, talc, magnesium stearate, polyethylene glycol 6000, etc.) etc. Coating is carried out by a known method for the purpose of masking, enteric or persistent. Alternatively, it can be taken in the form of a general food and drink, for example, frozen drinks such as soft drinks, jelly, confectionery and ice cream, dairy products such as yogurt and milk, various emulsified foods and drinks such as pudding, mousse, bavaria and dressing Although it is conceivable to add the capsinoid compound according to the present invention to a product, it is not limited to these. Thereby, it can be used to improve indefinite complaints (NUD) of the upper gastrointestinal tract in which no ulcer is observed. Specific indefinite complaints include upper abdominal pain, heartburn, burping, nausea, upper abdominal fullness, loss of appetite, and the like. Although it is a common disease that is said to be recognized in about 30% of the general population, Helicobacter pylori infection is assumed as one of the causes of this NUD, and the intake of the health food of the present invention has a preventive / improving effect. It is thought that it is demonstrated.

1. A male gerbil (SPF) purchased at 5 weeks of age from the experimental animal Seak Yoshitomi was preliminarily raised for 8 days and used for the experiment. Gerbils were bred throughout the pre-breeding period and experimental period in an infected animal breeding room (lighting time 7:00 to 19:00, ventilation rate 18 times / hour) at a room temperature of 24 ± 3 ° C. and a relative humidity of 55 ± 15%. Two to three gerbils / cage were used. Drinking water was supplied with sterilized distilled water in a water supply bottle, and feed was freely given radiation sterilized solid feed (CMF, oriental yeast) with a feeder. In addition, individual identification of gerbils was performed by a pigment (picric acid solution) coating method.

2. Preparation of Infectious Bacteria and Bacterial Solution A standard strain of H. pylori (43504 strain was purchased from ATCC) was used as an infectious strain. Brain-heart infusion (BHI) medium (Lot. No. 48001, Eiken Equipment Co.) containing 10% fetal bovine serum (Lot. No. 4391F, Dainippon Pharmaceutical) was added to the preserved strain and reconstituted. And cultured at 37 ° C. under microaerobic conditions (using Campipack, Becton Dickinson, Lot. No. 4204653) for a concentration of about 2.6 × 10 ⁴ CFU / ml.

3. How to infect H. pylori
We fasted from 24 hours before H. pylori infection until 4 hours after infection. To this fasted gerbil, 2.0 ml of H. pylori culture was orally administered in two portions (5.2 × 10 ⁴ CFU / animal). The infection date was day0.

4). Preparation and administration of test substance Capsinoids were synthesized according to the method described in Kobata et al. (Biosci. Biotechnol. Biochem., 66 (2), 319-327, 2002) (total capsinoid amount (purity) 86.7). %, Capsiate 54%, dihydrocapsiate 26.3%, nordihydrocapsiate 6.4%). The capsinoid was left overnight at room temperature and allowed to warm to room temperature. Then, it weighed in a tube, corn oil was added, and it mixed well using the vortex, and prepared to the designated density | concentration. The dosing solution was prepared at the beginning, and divided into portions each day and stored at -20 ° C. The dosing solution was returned to room temperature on the day before administration, and was well homogenized using a vortex. The administration was carried out orally once a day using a sonde at a predetermined time (between 9 am and 10 am). A prescribed amount of corn oil was administered to the control group. Capsaicin was stored refrigerated and prepared and administered in the same manner as capsinoids.

5. Group configuration number of animals: 7 mice / group

6). Test item 1) Body weight The day of infection was defined as day 0, and measurements were taken on day-7, -3, 0, 4, 7, 11, 14, 18, 21, 25 and 28 with a scale.
2) General symptoms Changes in general symptoms were observed daily and entered on a record sheet.
3) Feed intake Feed was replenished twice every Monday and Thursday. The measurement days of feed intake were day-3, 0, 4, 7, 11, 14, 18, 21, 25, and 28.
4) Evaluation of gastric damage A gerbil was sterilized on day 29 under ether anesthesia with 80% alcohol and then laparotomized with sterilized scissors, and the stomach was aseptically removed. The stomach was opened along the great bay and stretched on the cork board. This specimen was placed under a stereomicroscope, and the area of damage was reported by Naka et al (Naka et al J. Physiology, Vol. 95, pp. 443-451, 2001).
5) Bacterial count measurement The stomach was put into a centrifuge tube containing 5 ml of phosphate buffer (PBS (−)) and homogenized with a polytron homogenizer. 0.1 ml of the homogenate was smeared on a pore Vi helico-agar medium (Eiken, Lot. No. 53002) and cultured for 7 to 10 days under the microaerobic condition at 37 ° C. similar to the pre-culture. Colonies were counted.

7). The statistically processed body weight and the number of colonies were calculated as an average value ± standard error for each group. In addition, in order to test the statistical significance of each group with respect to the control group, analysis of variance (ANOVA) was performed using analysis software (Stat View, Abacus Inc., USA), and after confirming equal variance, Fisher's PLSD A multiple comparison test, which is a method, was performed to compare between groups. The case of p <0.05 was considered statistically significant.

8). Results The results above are shown in FIG. 1 and FIG. FIG. 1 shows the change in the body weight of gerbils during the experiment with H. pylori infection as Day 0 as an average value and standard deviation. In the figure, CSNs represents capsinoid compounds, CAP represents capsaicin, and the next number represents the dose (mg / kg). As a result, the weight change of the gerbil was not significantly different between the capsinoid and capsaicin administration groups as compared to the control group (Vehicle). There was also no difference in general symptoms and food intake recorded during the experiment.

  On the other hand, FIG. 2 shows the average number and standard deviation of the number of Helicobacter pylori colonies in the stomach homogenate after administration of the capsinoid compound for 35 days. The administration groups shown on the horizontal axis are the same as in FIG. Note that * indicates p <0.05. From the results shown in FIG. 2, the capsinoid compound showed a significantly lower colony value compared to the colony number after culture of the gastric homogenate solution in the control group at a dose of 100 mg / kg. The capsinoid compound-administered group showed a low value for the number of colonies even at a dose of 500 mg / kg, although no statistically significant difference was shown. Furthermore, as a result of dissecting gerbils 4 weeks after infection and observing the stomach with a stereomicroscope, no disorder was observed in all groups including the control group. From these results, it was found that the capsinoid compound has an infection-protecting effect against oral infection with H. pylori at the above dose.

It is the graph which showed the weight change of the gerbil during an experiment period. It is the graph which showed the colony number of Helicobacter pylori in the stomach homogenate after administration of a capsinoid compound for 35 days.

Claims (7)

  1. An anti-Helicobacter pylori agent comprising one or more capsinoid compounds.   The anti-Helicobacter pylori agent according to claim 1, wherein the capsinoid compound comprises capsiate, dihydrocapsiate, nordihydrocapsiate, vanillyl octoate, vanillyl nonanoate, vanillyl decanoate.   The anti-Helicobacter pylori agent according to claim 1 or 2, wherein the capsinoid compound contains a capsinoid compound extracted from a hot pepper.   The anti-Helicobacter pylori agent according to any one of claims 1 to 3, which is for oral administration.   A pharmaceutical composition for preventing, treating or preventing recurrence of upper gastrointestinal tract diseases, comprising the anti-Helicobacter pylori agent according to any one of claims 1 to 3 and a physiologically acceptable carrier.   The pharmaceutical composition according to claim 4, wherein the daily dose of the capsinoid compound is 0.03 to 500 mg / kg body weight. A health food comprising the anti-Helicobacter pylori agent according to any one of claims 1 to 3 and used for improving indefinite complaints of the upper gastrointestinal tract.

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