JP3492061B2 - Helicobacter pylori alcohol dehydrogenase inhibitory composition - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to hobenitine and / or
Also, the present invention relates to a food or drink containing a Helicobacter pylori alcohol dehydrogenase inhibitor composed of a glycoside thereof, and a food or drink containing a Helicobacter pylori antibacterial agent composed of hobenitin and / or a glycoside thereof. Furthermore, the present invention is a Helicobacter pylori alcohol dehydrogenase inhibitor consisting of hobenitin and / or its glycoside, a drug containing this as an active ingredient, and a helicobacter pylori antibacterial agent consisting of hobenitin and / or its glycoside, and The present invention relates to a drug containing this as an active ingredient.

[0002]

[ Prior Art] Helicobacter pylori
bacterium pylori , hereinafter referred to as “H. pylori” or “H. pylori”) was described in Australia in 1983 by B. J. A bacterium (microaerobic gram-negative spiral bacillus) that lives in the stomach, reported by Marshall, and is already one of the important factors for upper gastrointestinal disorders such as gastritis and peptic ulcer Is being recognized by. As a virulence factor of this bacterium, flagella, urease,
Many reports have been made on heat shock protein, superoxide dismutase, catalase, vacuolating toxin, toxin-associated protein, mucinase, adhesion factor, lipopolysaccharide, alcohol dehydrogenase, etc.
Currently, the decisive factor has not been identified.

Clinically, diagnostic methods such as biopsy test and urease breath test, antibacterial treatment methods combining antibiotics, bismuth preparations, H ₂ blockers, proton pump inhibitors and the like have been studied, but they are still being studied. No established methodologies are available. In addition, by devising foods that are taken on a daily basis, Although few attempts have been made to improve infection of pylori, polyphenols contained in tea, flavones and quercetin widely present in the plant kingdom, and flavanones, which are non-naturally occurring flavone reduction products, It is known to inhibit the growth of H. pylori (Beil et al. Arzneim.-Forsch./Drug Res. 45
(I), Nr. 6 697-700 (1995)).

On the other hand, Kemponasi has been used as a Chinese medicine since ancient times in China and is said to be effective for diuresis and thirst, and as a proof of its physiological effect, JP-A-2-786.
In JP 09, a diuretic effect and an anticaries effect using rabbits are reported. Further, Japanese Patent Application Laid-Open No. 4-182318 discloses a food and drink for improving drunkenness containing a solvent extract of kenponashi. In addition, the document “Chem. Pharm. Bull.,
Vol.35, No.11, pp.4597-4604 (1987) '', Hoveniae
The action of a crude drug (water extract) named semen seu fructus on alcohol dehydrogenase in rat liver is disclosed.

However, alcohol dehydrogenase in the stomach and liver of mammals and H. The alcohol dehydrogenases of P. pylori are enzymatically distinct, and Kemponasi has been identified by H. H. pylori that inhibits alcohol dehydrogenase. It is completely unknown that it has the effect of inhibiting the growth of pylori and that its active substance is hobenitin.

[0006] In recent years, the need for prevention rather than treatment of diseases has been emphasized. When suffering from gastric mucosal damage, gastritis, peptic ulcer, and even gastric cancer caused by alcohol, it is natural to want to take some precautionary measures. And it is more desirable that these preventive measures be taken by things that are routinely ingested or used, such as food, rather than by drugs, and it is considered to be one of the causes of gastric mucosal damage, gastritis, peptic ulcer, etc. due to alcohol. There has been a demand for the development of new food materials having an antibacterial effect against existing H. pylori.

[0007]

DISCLOSURE OF THE INVENTION As described above, the present inventors have conducted extensive studies to find a substance that significantly suppresses the growth of Helicobacter pylori and is not harmful to the human body. It was found that the above-mentioned hobenitine has an antibacterial action against Helicobacter pylori by inhibiting alcohol dehydrogenase of Helicobacter pylori, and has completed the present invention.

[0008] In other words, considering the present situation that the virulence factor of H. pylori has not yet been identified, the H. Considering that a means of reducing the growth of P. pylori is also effective, the inventors of the present invention have identified H. We focused on the alcohol dehydrogenase of pylori. H. The alcohol dehydrogenase of pylori is an enzyme that catalyzes the reaction of the bacterium to metabolize a carbon source and produce ethanol from acetaldehyde as its final product. On the other hand, humans ingest alcohol-containing beverages (ie, alcoholic beverages). If excess ethanol is present in the stomach, the Incorporated into pylori, the enzyme catalyzes the reverse reaction of ethanol to acetaldehyde in the cells, and the produced acetaldehyde is released outside the cells.

Since this contributes to gastric mucosal disorders,
It is considered as one of the pathogenic factors of Helicobacter pylori. Moreover, if the activity of this enzyme is inhibited even when ethanol is not ingested, the metabolic pathway is suppressed, resulting in H. It is known that the growth of pylori is suppressed. That is, the inhibition of the activity of this enzyme means that H. pylo
It is expected to suppress the growth of ri and to prevent or improve upper gastrointestinal diseases such as gastritis and peptic ulcer, and further prevent gastric cancer. A preventive or ameliorating effect on gastric mucosal disorders caused by acetaldehyde produced by Pylori is also expected.

Therefore, the present invention relates to H. elegans consisting of hobenitin and / or its glycosides. It is an object of the present invention to provide a food or drink containing a pylori alcohol dehydrogenase inhibitor, or a food or drink having a preventive or ameliorating effect on gastric mucosal disorders caused by alcohol containing the inhibitor. The present invention also relates to H. elegans consisting of hobenitin and / or its glycosides. py
It is an object of the present invention to provide a food or drink containing a loli antibacterial agent, or a food or drink containing the antibacterial agent and having an action of preventing or improving gastritis or peptic ulcer.

The present invention further relates to H. elegans consisting of hobenitin and / or its glycosides. pylori alcohol dehydrogenase inhibitor, or a preventive or therapeutic agent for gastric mucosal disorders caused by alcohol containing the same as an active ingredient, or H. pylori consisting of fobenitin and / or its glycoside. It is an object of the present invention to provide a pylori antibacterial agent or a preventive or therapeutic agent for gastritis and peptic ulcer containing the same as an active ingredient.

[0012]

Hovenitine (3'-methoxy 3,4 ', 5,5', 7-pentahydroxyflavanone) of the present invention, which is a flavonoid contained in kenponashi, was extracted and purified from kenponashi. It may be one that is chemically synthesized. The hobenitin of the present invention may also be in the form of glycoside.
Further, as hovenitin and / or its glycoside, hovenitin and / or an extract containing the glycoside thereof, which is appropriately extracted from a natural product containing the hovenitine and / or its glycoside, or the natural product in the form of powder It is also possible to use the prepared one.

Examples of natural products containing hobenitin and / or glycosides thereof include, for example, Hovenia dulcis Thunber of the buckthorn family.
The fruits, seeds, peduncles or fruit branches of g) can be used. These parts of Kemponasi are used for food and are also marketed as dried fruits in China. Considering that the intended use of the present invention is pharmaceuticals and foods, it is desirable to have food experience.

The extract is obtained by extraction with water or a mixed solvent of water and a polar solvent. The solvent used for the extraction may be water or a lower alcohol such as methanol, ethanol or isopropanol, a hydrophilic organic solvent such as ketones such as acetone, or any mixture of two or more thereof, but the final use is Considering that it is a medicine or food, it is preferable to use water, ethanol, or a mixture thereof in terms of safety.

The ratio of the raw material and the solvent at the time of extraction is not particularly limited, either.
000 times by weight, especially 20 to 100 in terms of extraction operation and efficiency
Weight times are preferred. The extraction temperature is conveniently in the range of room temperature to the boiling point of the solvent under normal pressure, and the extraction time is preferably in the range of 10 minutes to 48 hours, although it varies depending on the extraction temperature.

In the present invention, selective enrichment for containing more hovenitin or isolation of hobenitin can be carried out. As a treatment method therefor, column treatment using a synthetic adsorbent, liquid-liquid extraction based on solubility, selection using an ultrafiltration membrane, precipitation of hovenitin using solubility, and hovenitine other than the same principle The precipitation removal of the impurities can be mentioned. The hovenitin and / or its glycoside-containing kenponasi extract obtained as described above is an extracted state, or a concentrated product thereof, a solvent-free dried product, or an extract containing hovenitine and It is possible to use the glycosides obtained by concentrating or isolating the glycosides as they are, or in any state such as dried products from which the solvent has been removed.

In the present invention, the foods and drinks include foods for specified health use, functional foods, food materials, etc. in addition to general foods, and food materials are used as raw materials for producing foods. However, the method of use thereof is not particularly limited. The H. elegans comprising the hovenitin of the present invention and / or its glycosides. pylori alcohol dehydrogenase inhibitor or H. pylori. When the pylori antibacterial agent is used as a general food or drink, hobenitin and / or its glycoside or an extract containing them, such as an extract of kenponashi, is used alone or together with various components usually used in foods. It is prepared by blending.

In the production of these foods, various ingredients can be used depending on the type thereof, such as glucose,
Fructose, sucrose, maltose, sorbitol, stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α
-Tocopherol, sodium erythorbate, 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 B , Nicotinamide, calcium pantothenate, amino acids,
Calcium salts, pigments, flavors, preservatives and the like which are commonly used as raw materials for foods can be appropriately mixed and produced.

The form of the produced food can be any food form such as a solid food, a creamy or jam-like semi-liquid food, a gel-like food, a beverage, for example, a capsule, a granule, a tablet, Soft drinks, juices, coffee, tea, beverages such as liqueurs, candy, candy, chewing gum, candy, chocolate, and confectionery such as gummy can be prepared by using forms such as drinks and any commonly used base material. it can.

In the food and drink of the present invention, H. elegans comprising the hovenitin of the present invention and / or its glycosides. pylori alcohol dehydrogenase inhibitor or H. pylori. The amount of the pylori antibacterial agent to be added can be selected from a wide range without particular limitation, because the kenponashi extract is not toxic to the human body, but from the viewpoint of effective concentration and economical efficiency, it is 0. 00001% to 5%, especially 0.00
It is preferably 1% to 1%.

The H. elegans comprising the hovenitin of the present invention and / or its glycosides. pylori alcohol dehydrogenase inhibitor or H. pylori. pylori antibacterial agent, food for specified health use,
When used as a functional food, hobenitin and / or its glycosides or extracts containing these, such as kenposhi extract alone, or usually, in combination with a carrier or various components used in the production of food, It can be used in the form of powder, granule, paste, capsule, syrup, solid, gel, liquid, suspension, emulsion and the like.

The specified health food and functional food of the present invention are:
For the purpose of improving infection of H. pylori by inhibiting alcohol dehydrogenase, that is, for suppressing the growth of H. pylori existing in the stomach, it is unlikely to cause gastrointestinal mucosal disorders such as gastritis, gastric ulcer, duodenal ulcer, etc. The present invention can be used not only for healthy people but also for patients with gastrointestinal mucosal disorders or the like for the purpose of controlling or reducing the effect. Further, in order to exert the effect in the human body, as fobenitin, 1 mg to 100 mg, preferably 10 mg to 50 mg per day, and 1 mg to 50 mg as a preventive effect.
Those designed to be taken in the mg range are desirable.

The H. elegans comprising the hovenitin of the present invention and / or its glycosides. pylori alcohol dehydrogenase inhibitor or H. pylori. When the pylori antibacterial agent is used as a food material, hobenitin and / or its glycoside or an extract containing them, for example, a kaempferi extract is used alone or with a carrier or various components usually used for food production. The combination can be used as a food material.

The H. elegans comprising the hovenitin of the present invention and / or its glycosides. pylori alcohol dehydrogenase inhibitor or H. pylori. When the pylori antibacterial agent is used as a pharmaceutical, the dosage form may be any dosage form as long as it is conveniently administered orally, for example, powders, granules, tablets, capsules, pills. , Troches, liquid solutions for internal use, suspensions, emulsions, syrups, etc., and these can be used alone or in combination depending on the symptoms.

These various preparations are usually used in the technical field of pharmaceutical preparation such as an excipient, a binder, a preservative, an oxidative stabilizer, a disintegrating agent, a lubricant, a flavoring agent, etc. as a main ingredient according to the purpose according to a conventional method. It is possible to formulate with known auxiliary agents that can be used. Although the dose varies depending on the purpose of administration and the situation of the recipient (sex, age, weight, etc.), it is usually 1 mg to 100 mg per day as fovenitin for an adult, orally when administered orally. The dose can be administered within the range of 10 mg to 50 mg and the preventive effect within the range of 1 mg to 50 mg.

Further, the drug of the present invention improves the infection of Helicobacter pylori by suppressing the growth of Helicobacter pylori present in the stomach, or hardly causes gastrointestinal mucosal disorders such as gastritis, gastric ulcer and duodenal ulcer. It can be expected to have the effect of doing or reducing. Furthermore, H. pylori is so-called acute gastric mucosal lesion such as vomiting, abdominal pain, redness of gastric mucosa, edema, erosion, gastric atony and atrophic gastritis, chronic gastritis such as superficial gastritis, MALT (mucosa-associated).
Lymphoid tissue) lymphoma, risk of gastric cancer development, involvement in gastric polyps, pharyngeal disease, periodontal disease, etc. are also suggested, and it is expected that the significance of the present invention will expand as the research progresses in the future.

[0027]

EFFECT OF THE INVENTION Pylori has an enormous number of people who are infected with 70% of the population aged 40 and over in Japan, and is an important bacterial infection that is highly likely to be involved in an extremely large number of upper gastrointestinal tract diseases. Is becoming apparent. For example, medical
Journal of Australia (Med. J. Aust.) Vol. 142, pp. 436-439 (1985) and American Journal of Gastroenterology (A.
m. J. Gastroentel.) Vol. 82, 192-199 (1.
987), H. piler
It has been reported that oral administration of i caused acute gastritis.

Also, gastroenterology (Gastr
oentel. 94, 33-40 (1988)
, Peptic ulcer patients were treated with antibiotics and H. p
It was also reported that the removal of Y. ylori improved the gastric tissue image. The relationship between pylori and gastritis and peptic ulcer is drawing attention. Further, Lancet, pp. 1437-1442 (1988).
H.). patients with pylori-positive peptic ulcer
It has been reported that the recurrence rate of peptic ulcer is higher than that of negative patients, and to prevent and treat recurrent peptic ulcer,
H. It has been revealed that the eradication of pylori is essential.

Therefore, H. H. pylori by inhibiting the alcohol dehydrogenase. pylori
Hovenitin of the present invention having an antibacterial action against H.
It is effective for preventing or ameliorating upper gastrointestinal diseases such as gastritis / peptic ulcer and gastric cancer due to infection with P. pylori. To inhibit alcohol dehydrogenase of P. pylori, H. pyl
It is effective in preventing or improving gastric mucosal disorders caused by acetaldehyde produced by ori.

Furthermore, since Kenponasi has been used as a Chinese medicine since ancient times in China, the Kenponashi extract of the present invention containing hobenitin is used as a food for daily intake to improve infection of H. pylori, or It can also be used for the prevention and amelioration of gastric mucosal damage due to Helicobacter pylori, gastritis, peptic ulcer, and gastric cancer. Moreover, since the hovenitin of the present invention has a small amount and an antibacterial action against H. pylori, it can be easily used as a medicine or a food for specified health use.

Furthermore, the Kemponasi extract of the present invention is safe, has a good flavor, and has a H. H. pylori by inhibition of alcohol dehydrogenase. Since it has an antibacterial effect against pylori, it is an excellent food material for developing a new type of food, and it is possible to provide foods and drinks that are aware of the antibacterial effect against Helicobacter pylori and that match the taste of consumers. it can. From this point of view, it can be said that it is valuable to find a new action in the conventionally known Kemponi extract.

[0032]

EXAMPLES Next, a method for producing the Japanese quince extract and its active ingredient according to the present invention, H. pylori alcohol dehydrogenase inhibitory activity test, H. pylori. Pylori growth inhibitory activity test, and alcohol dehydrogenase inhibitory action or H. The present invention will be described in more detail with reference to Examples relating to the production of foods having an inhibitory effect on the growth of pylori bacteria, but the present invention is not limited to these Examples.

Example 1. Example of production of extract To 500 g of dried dried Kemponasi ( Hovenia dulcis ) drug substance (fruit with fruit pattern), 10 L of 50% by volume ethanol was added, and extraction was performed at room temperature for 48 hours. This was filtered, the obtained filtrate was concentrated under reduced pressure to remove ethanol, and syrup 2 prepared so that the solid content became 50% by weight.
10 g were obtained.

Example 2. Example of production of extract 10 L of water was added to 500 g of dried dried venom (Hovenia dulcis) medicinal material (fruit with a fruit pattern) to give 95
Extraction was carried out at 0 ° C for 1 hour. This was filtered, and the obtained filtrate was concentrated under reduced pressure to obtain 160 g of syrup prepared so that the solid content was 50% by weight.

Example 3. Production Example of Active Ingredient 200 g of syrup (solid content 100 g) obtained in Example 1 was diluted to 5 L with water, and passed through a column (5 × 30 cm) packed with Diaion registered trademark HP20 (Mitsubishi Kasei),
The active ingredient was adsorbed. After adsorption operation, wash with 2L of water,
It was further washed with 1 L of 30% by volume ethanol. The active ingredient was desorbed with 2 L of ethanol and concentrated and dried under reduced pressure to obtain 1.8 g of a solid powder. Next, Develosil-
ODSHG-5 (Nomura Chemical) HPLC column (5 × 30
(cm) were separated and fractionated, and 200 mg of the active ingredient was isolated and purified using the alcohol dehydrogenase inhibitory activity described below as an index. When the structure was determined by various instrumental analysis, the active ingredient was hovenitin (3'-methoxy 3,4 ', 5,5', 7-pentahydroxyflavanone).

Example 4. Production Example of Active Ingredient 200 g of syrup obtained in Example 2 (100 g of solid content) was diluted to 5 L with water and passed through a column (5 × 30 cm) packed with Diaion registered trademark HP20 (Mitsubishi Kasei),
The active ingredient was adsorbed. After adsorption operation, wash with 2L of water,
It was further washed with 1 L of 30% by volume ethanol. The active ingredient was desorbed with 2 L of ethanol, and this was concentrated and dried under reduced pressure to obtain 1.2 g of a solid powder.

Example 5. Alcohol dehydrogenase inhibition
Harmful activity test H. Purification and activity measurement of pylori alcohol dehydrogenase was performed by Kaihovaara, P et al. (Alco
hol. Clin. Exp. Res. , Vol. 18,
1220, (1994)) using ethanol as a substrate and measuring the increase in absorbance at 340 nm. From the increase in absorbance (A) when the sample solution was added and the increase in absorbance (B) when water was used instead of the sample solution (control), the inhibition rate (%) was determined by the following formula. Inhibition rate (%) = (B−A) / B × 100

Using the above reaction system, the alcohol dehydrogenase inhibitory activities of the extracts obtained in Examples 1, 2, 3 and 4 and the active ingredient of the present invention were measured. This result is the first
Shown in the table.

Table 1 Test sample Final concentration (μg / ml) Inhibition rate (%) Kemphonashi extract of Example 1 1000 72 Kemponasi extract of Example 2 1000 51 HP20 column adsorbed component of Example 3 20 90 Example Hovenitin 20 95 of Example 3 HP 20 column adsorption component 20 84 of Example 4

Example 6. Helicobacter pylori growth inhibition test 1 ml of Brucell containing serially diluted 2-fold test sample
1 × 10 ^{4 in} a medium. f. u. H. pylori
NCTC11637 strain was inoculated and cultured at 37 ° C. under microaerobic condition for 7 days. After the completion of the culture, the mixture was centrifuged, and 0.2 ml of a 10% urea solution (pH 6) containing 0.01% phenol red was added to the obtained precipitate. If the cells are growing,
It immediately turns red due to the action of urease, but when growth is suppressed, it remains yellow with no change in color tone.
In this way, the H. The minimum inhibitory concentration (MIC) for P. pylori was determined. The results are shown in Table 2.

[0041] The following are examples of actual food production.

Example 7.

Example 8.

Example 9.

Example 10.

Example 11.

Example 12

Example 13.

Example 14

Example 15.

Example 16.

Example 17

Example 18.

Example 19

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ryuji Tanaka 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka Suntory Ltd. Biomedical Research Center (56) Reference JP-A-7-309733 (JP, A) ) JP-A-4-134026 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) A23L 1/30 A61K 31/70 A61K 35/78 CA (STN) BIOSIS (DIALOG)

Claims (4)

(57) [Claims] 1. A Helicobacter pylori alcohol dehydrogenase inhibitor comprising hobenitin and / or its glycoside. 2. A preventive or therapeutic agent for gastric mucosal disorders caused by alcohol, which comprises the Helicobacter pylori alcohol dehydrogenase inhibitor according to claim 1 as an active ingredient. 3. A Helicobacter pylori antibacterial agent comprising hobenitin and / or its glycoside. 4. A preventive or therapeutic agent for gastritis or peptic ulcer, which comprises the Helicobacter pylori antibacterial agent according to claim 3 as an active ingredient.