JP2010043061A - Anti-helicobacter pylori agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a crude medicine and spice exhibiting an antimicrobial action on Helicobacter pylori. <P>SOLUTION: The hydrous ethanol extract of kamala (tentacular of pericarp of Mallotus philippensis), mace (aril of Myristica fragrans), nigella seed (seed of Nigella sativa L.) or Parmelia perlata (lichen) exhibits an antimicrobial action on Helicobacter pylori and is useful as an anti-helicobacter pylori agent. A 1-(5,7-dihydroxy-2H-1-benzopyran-8-yl)-3-phenyl-2-propene-1-one derivative such as rottlerin, etc., contained in a hydrous ethanol extract of kamala and kamala is effective against Helicobacter pylori having resistance to clarithromycin, metronidazole, etc. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to a herbal medicine and a spice that have a bactericidal action against Helicobacter pylori.

Helicobacter pylori (Helicobacter
pylori (hereinafter referred to as H. pylori) is highly related to gastritis, gastric ulcer, duodenal ulcer, etc., and the World Health Organization (HHO) defines H. pylori as a "certain carcinogen". The infected person is thought to reach approximately 40-50% of the world population.
On the other hand, many searches have been made for herbal medicines and spices having anti-Helicobacter pylori action and their extracts, and patents have been filed. Among them, the following are known.

(1) Kusunohagiwa / Kamala / Lottrelin It is known that several types of phloroglucinol derivatives exist in the glandular hair called kamala on the surface of the skin of Mallotus philippensis inhabiting the tropics south of Ryukyu. Yes. Specifically, the red glandular hair of the skin of Kusunohagashi has been used for extermination of tapeworms since ancient times and contains the phloroglucinol derivative lotrelin.
JP 2003-342190 (Patent Document 1) contains at least one of a plant selected from the group consisting of Kusunohagashi, etc., or a juice or water extract or an organic solvent extract obtained therefrom, Anti-Helicobacter pylori compositions are described. In the examples of the publication, there is a description of the action against Helicobacter pylori with respect to the ethanol extract of the leaves of Kusonohagashi, but there is no description of the anti-Helicobacter pylori activity of Kamala and Lotrelin.

(2) Nikuzuku / Nutmeg / Evergreen Tree Nikuzuku (Myristica)
fragrans HOUTTUYN) are nutmeg and mace. Inside the fruit is a dark brown shell wrapped in a bright red mesh-like temporary seed coat. This temporary seed coat is a mace, and the seed in the cracked shell is peeled off the nutmeg.
Japanese Patent Application Laid-Open No. 07-196522 (Patent Document 2) describes an anti-Helicobacter pylori activator characterized by containing one or a plurality of herbal medicines selected from nuts and the like. In the examples of this publication, there is a description of the action against Helicobacter pylori on ethanol extracts of various herbal medicines such as nutmeg, but there is no description of Mace's anti-Helicobacter pylori activity.

(3) Nigella / Black Seed Grass / Ranunculaceae The Nigella sativa L. plant is native to southwestern Asia, the Mediterranean coast and central Europe. It is a dicotyledonous plant, also known as black cumin or black seed grass. Nigella seed, the seed, is used as a general food in Egypt and Turkey.
Cold, asthma, conjunctivitis, infant paralysis, etc.) In addition, chemical and pharmacological studies have been carried out on both black and white oleanders, and essential oils, fatty acids, sterols, terpenoids, saponins, flavonol glycosides, alkaloids, etc. have been isolated as constituents. It has been reported to have antitumor, bronchodilation, antihypertensive, antibacterial and antifungal effects (Non-patent Document 1). However, anti-Helicobacter pylori activity is not known.

(4) Japanese apricot mushroom Japanese Patent Application Laid-Open No. 05-246876 (Patent Document 3) describes an antibacterial agent against gram-positive bacteria as a component extracted from a lichen plant culture containing a Japanese apricot. However, anti-Helicobacter pylori activity is not known.

On the other hand, in the gastric ulcer medical care guideline described later, a triple combination therapy of two antibacterial agents (amoxicillin and clarithromycin) and one proton pump inhibitor (lansoprazole and omeprazole) for suppressing gastric acid is recommended for sterilization. However, an increase in clarithromycin-resistant Helicobacter pylori used in triple-drug therapy has been a problem, and studies on herbal medicines and their components effective against clarithromycin-resistant Helicobacter pylori have been made (Patent Documents 4 and 5). ).

JP2003-342190 JP 07-196522 JP 05-246876 JP2006-124296 JP2007-217384

Phytother.Res., 17, 299-305 (2003)

Helicobacter pylori causes chronic inflammation in the stomach, resulting in chronic atrophic gastritis and the origin of gastric cancer. According to the guidelines for gastric ulcer treatment by the research team of the Ministry of Health, Labor and Welfare, It is decided to give priority to the sterilization treatment. About 70% of Japanese people are over 40 years old and infected with Helicobacter pylori, and there are many cases of gastric cancer worldwide, and it is important for Japanese to eliminate Helicobacter pylori from the stomach. It is a problem.
About 50% of the sterilization therapy according to the gastric ulcer medical care guidelines have observed some side effects such as loose stool, diarrhea and other gastrointestinal symptoms and taste abnormalities. More seriously, it has recently been found that only about half of patients can be sterilized because of the increase in resistant bacteria that do not die with antibiotics. If sterilization treatment is not appropriate, resistant bacteria may increase.

In South Asia such as Pakistan, the infection rate of H. pylori is close to 100% reflecting hygiene, but the incidence of gastric cancer is much lower than in Japan. This may be due to differences in bacterial side virulence factors such as the structure and product of H. pylori, and host side virulence factors produced by host gastric epithelial cells and immunocompetent cells due to infection. The inventors focused on differences in lifestyle habits, especially on dietary habits, and traditional medicines used in daily life. Therefore, spices and traditional Pakistani medicines taken daily in the South Asian region have antibacterial activity against Helicobacter pylori, thus inhibiting the activity of Helicobacter pylori and preventing chronic inflammation caused by the fungus even if it is not completely sterilized. The antibacterial action against various Helicobacter pylori and other traditional spices and traditional medicines was examined. As a result, Kusunohagashi (Mallotus
Anti-Helicobacter pylori activity in kamala on the skin of philippensis, mace made from fruit of Myristica fragrans HOUTTUYN, Nigella sativa L. seeds and water-containing ethanol extracts Furthermore, the present inventors have found that there are compounds having an antibacterial action stronger than amoxicillin among the components of the extract, and that the compounds therein are also effective against resistant H. pylori. Hereinafter, the present invention will be described in detail.

The camara, mace, nigera seed, and ume tree used in the present invention are as follows.
<Kamala> Kusunohagashi (Mallotus)
As long as it is a glandular hair called kamala on the surface of the skin of philippensis, there are no particular restrictions on the origin of the raw material, Kusonohagashi.
<Mace> Nikuzuku (Myristica
fragrans HOUTTUYN) is a temporary seed coat called a mace, and there are no particular restrictions on the origin of the nutmeg, the raw material.
<Nigella Seed> As long as it is Nigella sativa L., also known as black cumin, a seed of black seeds, it is not particularly limited as long as it is called Nigella Seed.
<Umeno Moke> Although it will not be limited if it is a Umeno Moke of the Umeno Moke department of lichen, 1 type of Umeno Moke called Parmelia perlata Eschha is preferable.
In the present invention, the above-mentioned camara, mace, nigera seed and umenochoke may be used as an extraction material per se or pulverized product.

Examples of the alcohol used for obtaining the extract of the present invention include lower alcohols having 1 to 4 carbon atoms, specifically, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, These water-containing alcohols containing t-butanol or a mixture thereof or water are mentioned. Preferable examples include hydrous alcohols containing up to about 60% by volume of water. Further preferred are hydrous ethanol having an alcohol concentration of 40 to 95% by volume, particularly hydrous ethanol having an alcohol concentration of 60 to 80% (W / W).
These extraction solvents may be used 1 to 50 times (volume), preferably 2 to 10 times (volume) with respect to the extraction material.

The extraction temperature can be arbitrarily set between room temperature and the boiling point of the solvent, and may be performed by immersing the extraction material in the extraction solvent with shaking or non-shaking or refluxing. When the extraction material is immersed under shaking or non-shaking, it may be performed for 1 hour to 72 hours, preferably about 24 to 48 hours. Moreover, when immersing in non-shaking, it is preferable to carry out under ultrasonic irradiation. Moreover, when extracting under reflux of an extraction solvent, it is preferable to heat to reflux for 30 minutes to several hours.
The extraction operation is preferably repeated a plurality of times, for example, about 2 to 5 times for the same material.

The extract obtained by removing solids from the extraction mixture may be concentrated by a conventional method to obtain an extract. Concentration is preferably performed at a low temperature and under reduced pressure, and may be performed until the extract is dried.
The extract may be used as it is to prepare the composition of the present invention, but may also be used as a powder or lyophilized product. A method known in the art can be adopted as the method for forming these solids.

The extract may be subjected to a purification treatment before and after concentration. The purification treatment may be performed alone or in combination with chromatographic methods, ion-exchange chromatographic methods, partition extraction with a solvent, and the like. For example, as a chromatographic method, a column chromatography, a thin layer chromatography, a high performance liquid chromatography, a centrifugal liquid chromatography or the like using a normal phase or reverse phase carrier or an ion exchange resin, or a combination thereof is performed. Is mentioned. In this case, purification conditions such as a carrier and an elution solvent can be appropriately selected according to various chromatographies.

Among the compounds obtained by purifying Kamala ethanol extract using column chromatography, the compounds exhibiting anti-Helicobacter activity are: Lotrelin, 1- (5,7-dihydroxy-2,2,6-trimethyl-2H-1 -benzopyran-8-yl) -3-phenyl-2-Propen-1-one
(red compound).

The present invention provides the following general formula [1] containing lotrerin:
“In the formula, R ₁ and R ₂ are the same or different alkyl groups which may be substituted, R ₃ represents a hydrogen atom or a hydroxyl group, and n represents an integer of 1 to 5,”
An anti-Helicobacter pylori agent containing 1- (5,7-dihydroxy-2H-1-benzopyran-8-yl) -3-phenyl-2-propen-1-one derivative represented by the formula: .

Examples of the alkyl group for R ₁ and R ₂ include linear or branched lower alkyl groups having 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl and hexyl groups. Can be mentioned.
Examples of the alkyl substituent of R ₁ include a phenyl group substituted with a group such as a methyl group, a hydroxyl group, and an acetyl group, an alkadiene group having 2 to 10 carbon atoms and an alkenyl group substituted with a methyl group and the like.
Examples of the substituent for the alkyl of R ₂ include an alkenyl group having 2 to 10 carbon atoms substituted with a methyl group or the like.

The specific compound of general formula [1] is illustrated below.
・ 1- [6-[(3-Acetyl-2,4,6-trihydroxy-5-methylphenyl) methyl] -5,7-dihydroxy-
2,2-dimethyl-2H-1-benzopyran-8-yl] -3-phenyl-2-propen-1-one (Rottlerin)
・ 1- [6-[(3-Acetyl-2,4,6-trihydroxy-5-methylphenyl) methyl] -5,7-dihydroxy-
2,2-dimethyl-2H-1-benzopyran-8-yl] -3- (4-hydroxyphenyl) -2-propen-1-one
・ 1- [6-[(3-Acetyl-2,4,6-trihydroxy-5-methylphenyl) methyl] -5,7-dihydroxy-
2,2-dimethyl-2H-1-benzopyran-8-yl] -3- (3,4-dihydroxyphenyl) -2-propen-1-one
・ 1- (5,7-Dihydroxy-2,2,6-trimethyl-2H-1-benzopyran-8-yl) -3-phenyl-2-propen- 1-one
・ 1- [5,7-Dihydroxy-2,2-dimethyl-6- (3-methyl-2-buten-1-yl) -2H-1-benzopyran-8-yl] -3- (4-hydroxyphenyl) -2-propen-1-one
・ 1- [6- (3,7-Dimethyl-2,6-octadien-1-yl) -5,7-dihydroxy-2,2-dimethyl-2H-
1-benzopyran-8-yl] -3- (4-hydroxyphenyl) -2-Propen-1-one (Mallotophilippen C)
・ 1- [6- (3,7-Dimethyl-2,6-octadien-1-yl) -5,7-dihydroxy-2,2-dimethyl-2H-1-
benzopyran-8-yl] -3- (3,4-dihydroxyphenyl) -2-propen-1-one (Mallotophilippen D)
・ 3- (3,4-Dihydroxyphenyl) -1- [6- (3,7-dimethyl-2,6-octadien-1-yl) -5,7-
dihydroxy-2,2-dimethyl-2H-1-benzopyran-8-yl] -2-propen-1-one
・ 1- [2-Methyl-2- (4-methyl-3-pentenyl) -5,7-dihydroxy-6- (3-methyl-2-butenyl)-
2H-1-benzopyran-8-yl] -3- (3,4-dihydroxyphenyl) -2-propen-1-one (Mallotophilippen
E)

In order to produce the compound of the general formula [1], for example, it can be extracted and purified from Kusonohagashi plants containing Kamala by the method described in Table 2005/087871, or can be produced by a known synthesis method. Examples of known production methods include the method described in J. Org. Chem., 2008, 73 (11), 4313-4316.

The anti-Helicobacter pylori agent of the present invention can be used as pharmaceuticals and foods.
In the case of a pharmaceutical product, it is combined with excipients, adjuvants, additives, etc., and various pharmaceutical preparations such as solutions, suspensions, syrups, elixirs, extracts, powders, granules, fine granules, It can be set as a tablet and a capsule.
When it is used as a food, it may be processed as it is or after adding various nutritional components into a granular form, granular form, paste form or the like, or may be added to various foods.

1- (5,7-dihydroxy-2H-1-benzopyran, comprising hydrous ethanol extract and lottorin of kumnohagashi pericarp (camara), nutmeg temporary seed coat (mae), black seed seed (nigella seed) and genus mushroom -8-yl) -3-phenyl-2-propen-1-one derivatives have antibacterial activity against H. pylori and are useful as anti-Helicobacter pylori agents.
Furthermore, rotrelin is also effective against H. pylori resistant to antibacterial drugs used for sterilization therapy, specifically, Clarislot machine-resistant H. pylori, Metronidazole-resistant H. pylori, and the like.

Hereinafter, although the manufacture example of this invention is demonstrated with Kamara and a test example is demonstrated about an anti- Helicobacter pylori action, this invention is not limited to this.
Production Example 1
3 kg of 70% ethanol was added to 1.5 kg of Kamala, and ultrasonic extraction was performed 3 times at room temperature for 24 hours. After filtering through a filter, it was dried under reduced pressure and further freeze-dried to obtain 120 g of an extract.

Production Example 2
55 g of the dried extract was subjected to silica gel column chromatography, and 10% ethyl acetate / hexane eluate (fraction 1; 2 g), 20% ethyl acetate / hexane eluate (fraction 2; 5 g), 30% ethyl acetate / hexane Eluate (fraction 3; 8 g), 50% ethyl acetate / hexane eluate (fraction 4; 15 g), 80% ethyl acetate / hexane eluate (fraction 5; 12 g), 30% methanol / ethyl acetate eluate (Fraction 6; 5 g) was obtained.
A 500 mg hexane-insoluble fraction of fraction 2 was purified by normal phase thin layer chromatography using dichloromethane to obtain compound 1 (30 mg).
Fraction 2 of 500 mg of hexane dissolved fraction was subjected to reverse phase chromatography and separated into 20 fractions in acetonitrile / acetone / water (1: 1: 0.5) system. The 13th fraction (40 mg) was purified by normal phase thin layer chromatography using 5% acetone-hexane to obtain Compound 2 (10.2 mg).
Fraction 3 was purified by crystallization from hexane to obtain compound 3 (3.0 g).
2 g of fraction 4 was subjected to silica gel column chromatography with 100% dichloromethane, separated into 5 fractions with methanol / dichloromethane, fraction 4-1 (200 mg),
Fraction 4-2 (100 mg), Fraction 4-3 (1 g), Fraction 4-4 (150 mg), Fraction 4-5 (300 mg) were obtained.
Fraction 4-3 (1 g) was subjected to reverse phase chromatography and separated into 10 fractions in an acetonitrile / acetone / water (1: 1: 0.5) system. The fifth fraction (100 mg) was subjected to reverse phase silica gel thin layer chromatography and purified with an acetonitrile / acetone / water (1: 1: 1.5) system to give compound 4 (75 mg). The seventh fraction was purified by normal phase thin layer chromatography using 8% acetone / chloroform to obtain Compound 5 (200 mg).
Fraction 5 (5 g) was washed with methanol, and the insoluble fraction was subjected to reverse phase thin layer chromatography and purified with an acetonitrile / acetone / water (1: 1: 0.5) system to obtain compound 5 (1 g).

Each compound was identified from the physical property values (NMR, mass spectrometry, etc.) of each compound.
The name and chemical structure of each compound are shown below.

・ Compound 1
5,7-Dihydroxy-8-methyl-6-prenylflavanone
(5,7-dihydroxy-8-methyl-6-prenylflavanone)

・ Compound 2
3'-prenylrubranine

・ Compound 3
1- (5,7-Dihydroxy-2,2,6-trimethyl-2H-1-benzopyran-8-yl) -3-phenyl-2-propen-1-one
(red compound)

Compound 4
Isorottlerin

Compound 5
Rottlerin

Physical properties Dark red powder
¹ H NMR (CDCl ₃ ,
400 MHz) δ1.53 (6H, s, -OC- (CH ₃ ) ₂ ), 2.08 (3H, s, CH ₃ -Ar),
2.70 (3H, s, -CO- (CH ₃ ), 3.80 (2H, s, Ar-CH ₂ -Ar),
5.47 (1H, d, J = 10 Hz, Ar-CH = CH-), 6.65 (1H, d, J = 10 Hz, Ar-CH = CH-), 7.41-7.61
(5H, m, -C ₆ H ₆ ), 7.82 (1H, d, J = 16 Hz, -CO-CH = CH-C ₆ H ₆ ),
8.18 (1H, d, J = 16 Hz, -CO-CH = CH-C ₆ H ₆ ). EI-MS m / z: 515.9
[M +] (Cald for C ₃₀ H ₂₈ O ₈ : 516.1). Other
important peaks at m / z 500.8 (25%), 427.9 (20%), 334.9 (15%), 321.9 (35%),
306.9 (70%), 166.9 (100%).

Test example 1
In order to examine the antibacterial activity of the extract of the present invention, MBC measurement (minimum bactericidal concentration) against standard Helicobacter pylori strain ATCC43504 was measured. MBC was measured by the agar plate method. Helicobacter pylori was inoculated into 10 mL of Brucella broth (manufactured by Becton Dickinson), cultured at 37 ° C. for 1-2 days on a constant-temperature shaker, and maintained. The absorbance of the shaking cultured Helicobacter pylori bacterial solution, was measured by a spectrophotometer, it was diluted to approximately 0.1 (Helicobacter pylori 10 ⁸ / mL). 0.1 mL of this was added to 0.9 mL of extract solution and mixed for 60 minutes. The extract solution was diluted 50% sequentially from 50 μg / mL. And 0.1mL of the mixture is commercial selective Pylori agar plates (Far East,
(Tokyo). The concentration of the lowest extract that does not form colonies on the agar was defined as MBC. Amoxicillin was used as a control drug. The results are shown in Table 1.

Test example 2
Compounds 1-5 were tested for anti-Helicobacter pylori activity in the same manner as in Test Example 1. The results are shown in Table 2.

Test example 3
(1) In the antimicrobial susceptibility test of clarithromycin and metronidazole against Helicobacter pylori, the E test method was used to evaluate at the minimum inhibitory concentration (MIC). The E test method was performed using a Mueller-Hinton agar medium containing 5% sheep blood and mixing at 37 ° C. for 72 hours under microaerobic conditions. The MIC was judged from the numerical values described at the position where the formed inhibition zone and the strip crossed by placing a strip-shaped strip coated with a drug in a concentration gradient on an agar medium coated with bacteria. When the MICs of clarithromycin and metronidazole were 1 mg / L or more or 8 mg / L or more, respectively, they were regarded as clarithromycin-resistant bacteria or metronidazole-resistant bacteria.

(2) Obtain the minimum bactericidal concentration (MBC) of clarithromycin and metronidazole using the strain determined to be clarithromycin-resistant bacteria (CR) or metronidazole-resistant bacteria (MR) by the MIC obtained by the E test method. (Table 3).

In ATCC 43504, a standard strain sensitive to clarithromycin, the MBC of clarithromycin was 6.25 mg / L, and in MB, the MBC of clarithromycin was 100 mg / L or higher. Therefore, the MBC of Kamala extract, compound 3 and compound 5 in CR is 15.6-31.2 mg / L, 25-100 mg / L, 3.12-6.25 mg / L, respectively. It clearly shows that the anti-H. Pylori activity in CR of 3 and compound 5 is strong (Table 4).

As for metronidazole, MIC of metronidazole was 0.5 mg / L and MBC was 500 mg / L by E test method using a strain (392A) isolated from a Japanese susceptible to metronidazole. The MBC of metronidazole in the standard strain ATCC 43504 resistant to metronidazole was 2000 mg / L or more. Therefore, the MBC of Kamala extract, compound 3 and compound 5 in MR is 15.6-31.2 mg / L, 25-100 mg / L, 3.12-6.25 mg / L, respectively, indicating that Kamala extract, compound 3 and It clearly shows that the anti-H. Pylori activity at MR of compound 5 is strong (Table 5).

Water-containing ethanol extracts and lottorin of Kusonohagashi pericarp (camara), nutmeg temporary seed coat (mae), black seed seed (nigella seed) and agaric are useful as anti-helicobacter pylori agents.
In addition, 1- (5,7-dihydroxy-2H-1-benzopyran-8-yl) -3-phenyl-2-propen-1-one derivatives such as lotus relin contained in an aqueous ethanol extract of Kamala and Kamala and the like are It is also effective against Helicobacter pylori resistant to Clarislo machine, metronidazole, etc., and by adding or adding these components, it is possible to develop pharmaceuticals, foods and beverages with enhanced antibacterial activity.

Claims (5)

Anti-Helicobacter pylori agent selected from pericarp gland hair (Kamala) of Kusonohagashi, temporary seed coat of macaque (Mace), black seed seed (Nigera Seed) or alcohol extract of Prunus vulgaris. The anti-Helicobacter pylori agent according to claim 1, which is an alcoholic extract of pericarp gland hair (camara) of Kusunohagashi. The anti-Helicobacter pylori agent according to claim 2, wherein the compound exhibiting anti-Helicobacter pylori activity in the alcoholic extract is rottlelin. General formula
“In the formula, R ₁ and R ₂ are the same or different alkyl groups which may be substituted, R ₃ represents a hydrogen atom or a hydroxyl group, and n represents an integer of 1 to 5,”
The anti- Helicobacter pylori agent containing 1- (5,7-dihydroxy-2H-1-benzopyran-8-yl) -3-phenyl-2-propen-1-one derivative represented by these. R _{1 of} 1- (5,7-dihydroxy-2H-1-benzopyran-8-yl) -3-phenyl-2-propen-1-one derivative is an alkyl substituted with an alkenyl group or a substituted phenyl group The anti-Helicobacter pylori agent according to claim 4, wherein the group R ₂ is an alkyl group which may be substituted with an alkenyl group.