JP2012097072A - Composition for protecting gastric mucosa, composition for urease activity inhibition, and pharmaceutical preparation comprising the compositions as active ingredients - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical composition composed of the extracts of a plurality of crude drugs, which causes reduced side-effects and has high safety while exhibiting an excellent antibacterial action and has urease inhibitory activity, and a pharmaceutical preparation comprising the pharmaceutical composition.SOLUTION: The composition for urease activity inhibition comprises at least one or more kinds selected from an A group consisting of swertiamarin, its ester, its physiologically admissible salt and their hydrates as an active ingredient. The pharmaceutical preparation comprises the composition as an active ingredient.

Description

  The present invention relates to a composition for inhibiting urease activity, and a pharmaceutical preparation comprising the composition as an active ingredient. More specifically, the present invention relates to a composition for inhibiting urease activity, which contains swell thiamarin as an active ingredient, and a pharmaceutical preparation containing the composition as an active ingredient.

Urease, an enzyme that hydrolyzes urea into carbon dioxide and ammonia, is Klebsiella aerogenes, Sporosarcina pasteurii, Helicobacter pylori, Proteus mirabilis (Proteus mirabilis) ) And other bacteria.
Among these, Helicobacter pylori decomposes urea by urease, neutralizes gastric acid with ammonia generated thereby, itching in the stomach, it is known that it causes diseases such as gastric ulcer and chronic urticaria ing.

  Japanese people have a higher incidence of gastric and duodenal ulcers than Westerners, but it is said that the onset of these diseases and infection with Helicobacter pylori are correlated. Since eradication of Helicobacter pylori entrapped in the stomach is useful for the treatment of gastric ulcers, etc., at present, proton pump inhibitors (PPI) such as omeprazole and lansoprazole and two constituents described later Sterilization by triple combination therapy combining (See Non-Patent Document 1, hereinafter referred to as “Conventional Example 1”).

  On the other hand, instead of using a combination of a proton pump inhibitor (PPI) and two antibiotics used for sterilization in combination with three agents, a Helicobacter pylori activity inhibitor using a crude drug has been proposed ( Patent Document 1: Japanese Patent Laid-Open No. 2004-115536, hereinafter referred to as “Conventional Example 2”).

JP2004-115536 JP2009-263262

http://www.takeda.co.jp/press/article_37653.html

  The triple combination therapy of Conventional Example 1 is an effective method for eradication of Helicobacter pylori. However, there are some problems. First, PPI has side effects that can cause gastric ulcers and cause reflux esophagitis when the stomach wall is weak, and atazanavir sulfate, a reverse transcriptase inhibitor. Concomitant use of theophylline, tacrolimus hydrate, digoxin, methyldigoxin, itraconazole, gefitinib, phenytoin, diazepam, etc. is said to require attention.

In addition, amoxicillin (AMPC) and clasithromycin (CAM) are used for primary sterilization. Instead, as shown by using metronidazole (MNZ), there is a problem of appearance of resistant bacteria, and when resistant bacteria appear, the eradication rate decreases.
For this reason, there is a strong social demand for drugs with few side effects and resistance to resistant bacteria.

The invention of Conventional Example 2 discloses the invention of an anti-Helicobacter pylori activator. This invention inhibits the activity of urease produced by Helicobacter pylori (ATCC 43504 strain) by Ogon, Aloe, Peonies, Kujin, Keihi, Keihi, Otsuka, Nikuzuku, Ryokyo, Sekisha, Enmeiso, Akamegashi, Yeobyi It has been completed by paying attention to this, and is disclosed to have urease inhibitory activity.
However, what is actually disclosed as a formulation is a tablet / capsule containing red-crowned wrinkles and peonies as active ingredients, a granule containing red-necked peas and sweet potatoes as active ingredients, and Ryokyo and Ubai as active ingredients. It is a liquid for internal use.

On the other hand, although Conventional Example 1 describes that the assembly has an effect of inhibiting the growth of Helicobacter pylori, it does not describe that it has an activity to inhibit urease. Moreover, it is known that swell thiamarin contained in the assembly promotes the production of insulin-like growth factor-1 (IGF-1) (Patent Document 2: JP-A-2009-263262, hereinafter “conventional”). Example 3 ”) is not known to have urease inhibitory activity.
Further, from the viewpoint of side effects, it is desirable to search for drugs having urease inhibitory activity using crude drugs as candidates, but in that case, it is necessary to consider that there is no problem in terms of supply amount.

The present invention has been completed under the above environment.
That is, the present invention is for protecting gastric mucosa, comprising as an active ingredient at least one selected from the group A consisting of swell thiamarin, esters thereof, physiologically acceptable salts thereof, and hydrates thereof. It is a composition.
Swell thiamarin is a compound represented by the following formula (I).

Here, the ester of swell thiamarin is preferably tetraacetate, and the physiologically acceptable salt is any one selected from the group consisting of sodium salt, potassium salt, acetate salt and ammonium salt. A salt is preferable, and a hydrate is preferably a monohydrate, dihydrate, or hexahydrate.
Further, the composition includes at least one selected from the group B consisting of berberine represented by the following formula (II), a physiologically acceptable salt thereof, and a hydrate thereof, and the following formula (III) A compound selected from Group A, Group B, and Group C, further comprising at least one selected from Group C consisting of magnolol represented by the following formula: The mixing ratio is preferably 1: (9 to 13) :( 11 to 15) in terms of molar ratio.

Here, the physiologically acceptable salt of berberine is preferably any salt selected from the group consisting of hydrochloride, sulfate, disulfate, and tannate. , Dihydrate, trihydrate, and 5 · 1/2 hydrate are preferable.
The physiologically acceptable salt of magnolol is preferably any salt selected from the group consisting of hydrochloride, sulfate, and ammonium salt.

The present invention is also a composition for inhibiting urease activity, comprising at least two kinds of herbal medicines selected from the group consisting of a powdery extract, an assembly powder extract, and a powdery extract. Here, the composition of the present invention contains a buckwheat extract, an assembly powder extract and a kokuboku powder extract, and more preferably contains a geno shoko powder and a peanut powder.
And in the composition containing these extracts, the mixing ratio of the compounds selected from Group A, Group B, and Group C is 1: (9-13) :( 11-15) More preferably.
In addition, the present invention is a pharmaceutical preparation containing any one of the above-mentioned compositions for protecting gastric mucosa.
Furthermore, this invention is a composition for urease activity inhibition containing the composition for gastric mucosa protection mentioned above. Moreover, this invention is a pharmaceutical formulation containing the said composition for urease activity inhibition.

  According to the present invention, a composition containing swell thiamarin as an active ingredient and having a strong urease activity inhibitory action can be obtained. Moreover, the pharmaceutical formulation which contains this composition as an active ingredient can be obtained.

FIG. 1 is a graph showing the inhibition rate of urease activity by each herbal medicine and Hyakusamaru extract. FIG. 2 is a graph showing the damaged area of the gastric mucosa of gerbils in a normal group (negative control group), a control group (positive control group), a Mitake Hyakusamaru administration group, and a berberine administration group. FIG. 3 is a graph showing bleeding areas of the gastric mucosa of gerbils in a normal group (negative control group), a control group (positive control group), a Mitake Hyakusamaru administration group, and a berberine administration group. FIG. 4 is a graph showing the number of Helicobacter pylori inhabitants in the stomach of gerbils in a normal group (negative control group), a control group (positive control group), a Mitake Hyakusamaru administration group, and a berberine administration group. FIG. 5 is a graph showing the activity of myeloperoxidase (MPO) in the gastric mucosa of gerbils in a normal group (negative control group), a control group (positive control group), a Mitake Hyakusamaru administration group, and a berberine administration group.

The present invention is described in further detail below.
The composition for inhibiting urease activity of the present invention is at least selected from group A consisting of swell thiamarin (C ₁₆ H ₂₂ O ₁₀ , molecular weight 374.34), an ester thereof, a physiologically acceptable salt thereof, and a hydrate. One or more kinds are contained as active ingredients. Swell thiamarin is a glycoside as represented by the above formula (I) and is a compound contained in the assembly described later.
The urease activity inhibiting composition of the present invention is selected from group B consisting of berberine ([C ₂₀ H ₁₈ NO ₄ ] ⁺ , formula amount 336.37), a physiologically acceptable salt thereof, and a hydrate. It further includes at least one or more, and at least one or more selected from group C consisting of magnolol (C ₁₈ H ₁₈ O ₂ , molecular weight 266.34) physiologically acceptable salt and hydrate thereof, This is preferable because of its high urease inhibitory activity.

Berberine is an alkaloid represented by the above formula (II), and is a compound contained in buckwheat described later. Magnolol is a compound represented by the above formula (III), and is contained in the abalone described later.
These esters, physiologically acceptable salts, and hydrates thereof are as described above.
Furthermore, it is because urease inhibitory activity is high that the mixing ratio of the compound selected from said A group, B group, and C group is 1: (9-13) :( 11-15) by molar ratio. Preferably, the activity is highest at 1:11:13.
The composition for inhibiting urease activity of the present invention is not only for commercially available urease derived from peas, ATCC 43504 and other urease obtained from Helicobacter pylori, but also for those obtained from clinical isolates of Helicobacter pylori. It has an inhibitory action.
The present invention is also a composition for inhibiting urease activity, comprising at least two kinds of herbal medicines selected from the group consisting of an extract of a powdered millet powder, an extract of a powdered powder and an extract of a powdered mulberry powder.

(Preparation of herbal extract)
Here, Aubaku is a crude drug obtained from the bark excluding peripheries of yellowfin (Phellodendron Bark) or other related genera (Phellodendron) (Japanese Pharmacopoeia), and the powdered powder is called Aubaku powder (Japanese Pharmacopoeia). An extraction liquid containing the active ingredient berberine can be obtained by adding a predetermined solvent to the powder of oat and extracting it under predetermined conditions. Examples of the predetermined solvent include methanol, ethanol, acetone, ethyl acetate, water, and the like, and it is preferable to use methanol or water because the extraction efficiency of the active ingredient is high. The addition amount of the solvent is preferably 3 to 7 (w / v) from the viewpoint of the extraction efficiency of the active ingredient, and more preferably 4 to 6 (w / v).
Examples of extraction conditions include shaking, Soxhlet extraction, ultrasonic extraction, and the like, and it is preferable to perform ultrasonic extraction in terms of extraction efficiency. The extraction time is preferably about 15 minutes to about 60 minutes from the viewpoint of extraction efficiency, and more preferably about 30 minutes. Moreover, it is preferable from the point of extraction efficiency that extraction temperature shall be 20 to 40 degreeC.

Gennoshouko is a crude drug obtained from the above-ground part (Japanese Pharmacopoeia) of Genranshoun (Geranin thunbergii Siebold et Zuccarini (Geraniaceae)). An extract can also be obtained from the geno shochu powder under the same conditions as those for the above-mentioned oak powder.
The peanut is a herbal medicine obtained from the rhizome of the quercus (Atractylodes japonica Koidzumi ex Kitamura) or the rhizome of the tractor (Atractylodes ovate De Candlle (Compositae)). Japanese Pharmacopoeia). An extract can also be obtained from the peanut powder under the same conditions as the above-mentioned powdered corn.

The assembly is the whole plant in the flowering period of the assembly (Swertia japonica Makino (Gentianaceae)), and the powdered product is called assembly powder (Japanese Pharmacopoeia). Also from the assembly powder, an extract containing an active ingredient can be obtained under the same conditions as the above-mentioned powdered buckwheat powder. This extract contains a glycoside represented by the following formula (I) and swell thiamarin as one of the main components.
Koboku is a crude drug obtained from the bark of Japanese cypress (Magnolia obovata Thunberg, Magnolia officibalis Rehder et al Wilson or Magnolia officinalis Rehder et Wilson var. Biloba Rehder et Wilson (Magnoliaceae)). It is called Koboku end (Japanese Pharmacopoeia). An extract can also be obtained from the powdered powder under the same conditions as the powdered powder. This extract contains magnolol represented by the following formula (III) as one of the main components.

The above-mentioned swell thiamarin, berberine, and magnolol, their esters, their physiologically acceptable salts, and their hydrates can also be obtained by purification according to conventional methods, and commercially available products are purchased. Can also be used.
Further, as a mixture of these extracts, the above-described crude drug extracts may be mixed at a predetermined ratio, and a crude drug preparation containing these extracts is extracted with a predetermined solvent and used. You can also.
Specifically, Mitake Hyakusamaru (registered trademark, Nagano Pharmaceutical Co., Ltd.) is extracted by adding a predetermined solvent, for example, methanol 10 to 20 times (w / v), and using this extract. Can do. Although the above-described method can be used for extraction, it is preferable to perform Soxhlet extraction because of its good berberine extraction efficiency.
The obtained extract (hereinafter referred to as “Homogusamaru extract”) can be used as a sample (Homogusamaru extract) by concentration under reduced pressure using a hot water bath of about 40 to 60 ° C. In use, the Hyakusamaru extract concentrated under reduced pressure is appropriately diluted with a desired solvent.

Hereinafter, the case where an extract containing swell thiamarin is obtained will be described as an example. The above-mentioned assembly is dried to form a powder, or a predetermined amount of commercially available assembly powder is weighed, and 3-7 times (w / v), preferably 4-6 (w / v) times of solvent, For example, methanol is added and, for example, ultrasonic extraction is performed at 20 to 30 ° C. for 15 to 30 minutes.
The obtained extract is filtered, concentrated by, for example, a rotary evaporator, and then freeze-dried according to a conventional method. As described above, an extract containing swell thiamarin can be obtained.
An extract containing berberine or magnolol can be obtained in the same manner. These samples are used as preservation samples for the urease inhibitory activity test, antibacterial test and in vivo gastric mucosa protection test described below.

(Urease inhibition test)
A predetermined medium is prepared, urease producing bacteria are inoculated, and cultured under predetermined conditions. For example, a Columbia HP medium is prepared, and Helicobacter pylori is inoculated therein and cultured at 35 to 39 ° C. under microaerobic conditions. Here, the microaerobic condition means that the carbon dioxide concentration range is in the range of 5 to 8%. The culture temperature is preferably 37 ° C because it is close to the growth conditions in vivo.
After confirming the growth of the bacteria, the scraped bacteria are suspended in a suitable medium, for example, a brain heart infusion medium supplemented with 5 to 15% calf serum, and treated to treat the enzyme produced by the bacteria. Prepare the containing solution.
Specifically, the above suspension is centrifuged at about (15,000 rpm or 126 × g), and the supernatant is removed. An appropriate reducing agent and a chelator, for example, 0.5-2 mM mercaptoethanol and 0.5-2 mM EDTA-2Na are added to the resulting sediment, which is hereinafter referred to as “PB”. .) And, for example, ultrasonic treatment is performed under ice cooling to disrupt the bacteria. The crushed material is precipitated by centrifugation, the supernatant is filtered, and diluted with PB to a predetermined time, for example, 10 to 20 times to prepare an enzyme solution.

An extract from each sample obtained as described above is added to the test enzyme solution thus obtained, and the mixture is allowed to stand under predetermined conditions, and a urease substrate solution is added and mixed. Then, it is made to react for a fixed time under predetermined conditions. Specifically, for example, 10 to 50 μL of the above extract is added to 25 to 200 μL of enzyme solution, placed in a water bath at about 37 ° C. for 10 to 20 minutes, and then PB containing 200 to 600 mM urea is added. To do. Shake immediately after the addition of PB and place again in a water bath at about 37 ° C. for 10-20 minutes, then add 0.5-2N sulfuric acid to stop the reaction.
The solution in which the reaction has been stopped is treated according to the indophenol method, and the inhibition rate of urease is determined from the absorbance at 630 nm. As described above, the urease inhibitory effect can be confirmed.

(Antimicrobial test)
A predetermined Helicobacter pylori strain that has been cryopreserved is inoculated with a platinum loop into a predetermined medium, and cultured under predetermined conditions to form fungi. The fungus is scraped off to make a suspension, and the turbidity is measured and adjusted to a desired number of bacteria. Applying this suspension onto a predetermined agar medium, culturing under a predetermined condition, placing a disc soaked with a chemical solution of a predetermined concentration, and measuring the antibacterial action due to the formation of a blocking circle it can.
For example, a clinical strain of H. pylori that has been cryopreserved at −80 ° C. is inoculated with a platinum loop on a Columbia HP agar medium and cultured at about 37 ° C. for 2 to 4 days under microaerobic conditions. To form fungus moss and scrape a part of it to make a suspension. The turbidity of this suspension is measured, for example, at OD ₆₀₀ , prepared so that the number of bacteria becomes 0.5-2 × 10 ⁶ cells / 100 μL, applied to Columbia HP agar medium, and 50 μL of chemical solution is soaked. The discs are placed on this agar medium and cultured at about 37 ° C. for 3 days under microaerobic conditions. The strength of the antibacterial action can be evaluated by determining the 50% inhibition concentration from the size of the inhibition circle formed at this time.

Moreover, the colony-forming ability of H. pylori in the inhibition circle can be examined by touching the inhibition circle with a platinum loop, drawing a line on the HI-FBS-TTC medium, and performing streak culture. The streak culture can be performed, for example, at about 37 ° C. for 2 to 4 days under microaerobic conditions.
Furthermore, for example, the colony-forming ability of bacteria can be examined using a 0.1% (w / v) deoxycholic acid-containing medium or the like. For example, the above-mentioned H. pylori is spread on a HP agar medium and cultured under microaerobic conditions at about 37 ° C. for 2 to 4 days. The cultured bacteria are then placed in a platinum loop, and HI-FBS-DCA By drawing a line on the medium, further culturing at about 37 ° C. for 2 to 4 days under microaerobic conditions, and counting the number of colonies formed, the colony forming ability in such a medium can be examined. .

In addition, H. pylori is orally inoculated at a desired number, for example, 2 × 10 ⁸ CFU / 0.5 mL / mouse, and then a desired amount of Mitake Hyakusamaru solution or berberine solution is orally administered for a desired period of time. The degree of damage to the stomach, the number of H. pylori inhabitants in the stomach, and enzyme activities such as myeloperoxidase (MPO) can be measured to examine the effect on the gastric mucosa in vivo. The degree of damage to the gastric mucosa can be evaluated by, for example, visual confirmation under a stereomicroscope. The number of H. pylori in the stomach is, for example, 5-15% equine defibrinated blood, 1-5 μg / mL amphotericin B, 7-11 μg / mL vancomycin, 0. Formed using H. pylori selection plates supplemented with 1-0.64 μg / mL polymyxin B, 1-10 μg / mL trimethoprim, 10-100 μg / mL l, 2, 3, 5-triphenyltetrazolium chloride The number of colonies can be counted and quantified. Commercially available H. pylori selective culture plates may be used.
In addition, the enzyme activity such as myeloperoxidase (MPO) can be determined by administering a drug for a predetermined period, and then laparotomizing a fasted animal under ether anesthesia for a desired period of time, usually about 24 hours. Remove the stomach. As a drug, for example, Mitake Hyakusamaru and Berberine can be prepared and used at 0.5 to 2 grains / kg / day or 0.1 to 0.6 mg / kg / day, respectively.

As the gastric viscosity, a desired region of the removed stomach, for example, a boundary region between the stomach body and the pylorus is punched out using a cork borer or the like to obtain a test piece, and the weight is measured. A predetermined amount of a desired buffer, for example, a 50 mM phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (pH 6.0, hereinafter sometimes referred to as “PBH”) is added to 50 mg of the test piece thus prepared. For example, after adding 1.0 mL and crushing with polytron, etc., this crushing solution is frozen and thawed 2 to 5 times, for example, centrifuged at 2,000 × g for 10 minutes at 4 ° C., and the supernatant is used as a measurement sample. To do.
In this way, 2-10 μL is taken from the measurement sample obtained and the desired concentration, eg 5-20 mM containing 0.167 mg / ml o-dianisidine dihydrochloride and 0.0001-0.001% H ₂ O ₂ . Add 140 to 148 μL of PBH (pH 6.0) and mix. This mixed solution can be measured, for example, at an absorbance of 450 nm to measure MPO activity.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to these examples.

[Preparation of sample extract]
JP JP Owaku powder (berberine concentration 1.21%) is from Horie Seiyaku Co., Ltd., JP JP Koboku powder (Mag roll concentration 2.10%), JP Pharmacy powder powder (Swellthiamarin concentration 3.13%), JP Pharmaco powder powder, JP The burdock root powder was purchased from Nagano Prefectural Pharmaceutical Co., Ltd.
For JP end powder, JP end powder, and JP end powder, 10.0 g was weighed into separate containers, 50 mL of methanol was added, and ultrasonic extraction was performed for 30 minutes.
1.675 g of Japanese geno shoko powder was weighed, 50 mL of methanol was added, and ultrasonic extraction was performed for 30 minutes. 0.50g was weighed out from the Japanese peanut powder, and 50 mL of methanol was added, followed by ultrasonic extraction for 30 minutes.

Hyakusamaru was powdered and passed through a Japanese Pharmacopoeia No. 24 sieve, and 40 g was weighed and taken on a cylindrical filter paper (Whatman, manufactured by GE Healthcare Japan Co., Ltd.). 800 mL of methanol (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and extraction was performed using a Soxhlet extractor in an 80 ° C. water bath for 20 hours.
The obtained extract was filtered through filter paper (No. 4, Whatman), and concentrated under reduced pressure at 50 ° C. with a rotary evaporator to obtain a Hyakusamaru extract. Hyakusamaru extract was subjected to high performance liquid chromatography under the following conditions, and the contents of berberine, magnolol and swellthiamarin were measured.
Standard products of berberine, magnolol and swell thiamarin were purchased from Wako Pure Chemical Industries, Ltd.

<HPLC conditions>
Equipment: High-performance liquid chromatograph (Hitachi, Ltd.)
System controller: D-7000
Injector: L-7200
Pump: L-7100
Detector: L-7405
Eluent: Elution was carried out by changing the composition of the mobile phase at 0 to 12 minutes, 12 to 25 minutes, and 25 to 30 minutes. A water / acetonitrile mixture (16: 9) containing 9 mM potassium dihydrogen phosphate and 9 mM sodium 1-octanesulfonate was used except for the mobile phase used in 12-25 minutes. For 12-25 minutes, a water / acetonitrile mixture (56:69) containing 19.7 mM potassium dihydrogen phosphate and 4 mM sodium 1-octanesulfonate was used.
Column: 4.6mm ID x 150mm length Particle size 5μm (ODS)
Flow rate: 1.0mL / min
Column temperature: 40 ° C
Each obtained extract was diluted with methanol (manufactured by Wako Pure Chemical Industries, Ltd.) in the amounts shown in Tables 1 to 6 below, and used for urease inhibition tests.

[Urease activity inhibition test]
(1) Preparation of enzyme solution Using Colombia HP medium (Nihon Becton Dickinson Co., Ltd.), Helicobacter pylori (clinical isolate provided by the Institute of Medical Science, the University of Tokyo) is microaerobic for 5 days. The cells were cultured and grown under conditions (Aneropouch Campiro, manufactured by Mitsubishi Gas Chemical Co., Inc.).
Next, the Helicobacter pylori colonies grown on Colombia HP medium were scraped, and 2-3 mL of brain heart infusion medium containing 10% calf serum (Gemini. Bio. Products) was added and stirred. The bacterial solution was centrifuged at 2,000 rpm.
To the sediment, 2-3 mL of phosphate buffer added with 1 mM mercaptoethanol and 1 mM EDTA-2Na was added and sonicated for 5 minutes under ice cooling. This was then centrifuged at 15,000 rpm and the supernatant was filtered off. The filtered supernatant was diluted 15 times with 20 mM phosphate buffer to obtain an enzyme solution (stock solution).

(2) Measurement of urease activity The activity of urease was measured by the indophenol method. First, 5.0 g of phenol and 25 mg of sodium nitroprusside were weighed and dissolved in 500 mL of water to prepare a phenol / nitropurushid sodium solution (hereinafter referred to as “solution A”). Also, 2.2 g of sodium hydrogen phosphate and 2.5 g of sodium hydroxide are dissolved in about 300 mL of water. To this, 3.0 mL of sodium hypochlorite with an effective chlorine concentration of 10% or more is added, and water is added. A solution of 500 mL (hereinafter referred to as “Solution B”) was prepared.

Take 50 μL of each extract obtained in (1) above in a test tube (n = 3), add 50 μL of the enzyme solution here, mix well, and incubate at 37 ° C. for 15 minutes to add 400 mM urea. 300 μL of the added 100 mM phosphate buffer was added as a substrate solution. The mixture was immediately shaken and incubated at 37 ° C. for 15 minutes, and then 100 μL of 1N sulfuric acid was added to stop the reaction.
In the obtained reaction solution, the above solution A and solution B are dissolved, 3.0 ml of sodium hypochlorite containing 10% or more of effective chlorine is added, 2.5 ml of a solution made up to 500 ml with water is added, and 65 ° C is added. Incubated for 20 minutes in a water bath. Measurement was carried out at a wavelength of 630 nm with an absorptiometer, and the inhibition rate of urease was determined according to the following formula.
Urease activity value (U) = Es-Eb

Es: Absorbance measured using enzyme solution Eb: Absorbance measured using buffer instead of enzyme solution Urease inhibition rate (%) = {(Ub−Us) / Ub} × 100
Ub: Urease activity value measured using water instead of extract solution Us: Urease activity value measured using extract solution Urease inhibitory activity of each sample is shown in Tables 1 to 6.

*反応液が黄色となり、吸光度は−で振り切れたため、100とした。

* Since the reaction solution became yellow and the absorbance was completely shaken by-, it was set to 100.

From Table 6 above, high urease inhibitory activity was seen in Gennosho. On the other hand, moderate urease inhibitory activity and the like were observed in the assembly and peanut (Tables 4 and 5), but the urease inhibitory activity of the buckwheat was weak (Table 2), and the urease inhibitory activity was not found in the amber. (Table 3).
On the other hand, the extract of Hyakusa Maru showed very high urease inhibitory activity (Table 1). For this reason, the contents of swell thiamarin, berberine and magnolol in the extract obtained in Example 1 were measured. The results are shown in Table 7 below.

以上より、上記の各生薬からの抽出物を、それらに含まれるスウェルチアマリン、ベルベリン及びマグノロールの量比が、上記表７に示す割合となるように混合することによって、これらの抽出物単独では得られない、相乗的な高いウレアーゼ阻害活性を示すことが明らかになった。

From the above, by mixing the extracts from each of the herbal medicines so that the quantitative ratio of swell thiamarin, berberine and magnolol contained in them is the ratio shown in Table 7 above, these extracts alone It became clear that it shows the synergistic high urease inhibitory activity which cannot be obtained by.

[Examination of antibacterial action]
(1) Samples, etc. Products that are usually marketed as gastrointestinal drugs and contain multiple preparations with antibacterial Chinese medicine and Japanese herbal medicine (Homogusamaru, Nagano Prefectural Pharmaceutical Co., Ltd.). The antibacterial action of herbal medicine against Helicobacter pylori was investigated.
Columbia HP agar was purchased from Nippon Becton Dickinson Co., Ltd. and used. Heart Infusion (HI), Trypticase Soy Broth (TBS) medium, and Mueller Hinton medium purchased from Eiken Chemical Co., Ltd. were used. The HI medium containing serum was prepared by adding 10% (v / v) fetal bovine serum (FBS, manufactured by Gemini. Bio. Products).
If necessary, triphenyltetrazolium chloride (TTC, manufactured by Tokyo Chemical Industry Co., Ltd.) 5 × 10 ⁻⁴ % (v / v), or deoxycholic acid (DCA, manufactured by Wako Pure Chemical Industries, Ltd.) 0.1% ( w / v) Added. The LB medium was prepared by adding 100 mL of distilled water to 0.5 g of NaCl, 0.5 g of yeast extract, and 1 g of tryptone.

Extraction was performed by adding 100 mL of methanol to 5 g of Hyakusamaru, and the methanol extract (containing 4.13% berberine, 3.77% magnolol, 0.33% swell thiamarin) was subjected to the test.
As a traditional Japanese herbal medicine, a stock solution of agate extract (containing 8.8% berberine, manufactured by Nippon Powder Co., Ltd.) and a dried extract of Kokuboku (containing 0.695% magnolol, manufactured by Nippon Powder Co., Ltd.) were used. Each of these was dissolved in 50 mL of water and adjusted to 100 mg / mL to obtain a stock solution.

In addition, an assembly powder extract (containing 9.86% swell thiamarin, manufactured by Nippon Powder Co., Ltd.) was prepared by preparing an aqueous solution of 66% assembly extract (170 mg / mL). For the geno shochu powder extract, a 33.5 mg / mL aqueous solution was prepared using 66% geno shochu extract to obtain a geno shochu stock solution. The peanut powder extract (manufactured by Nippon Powder Co., Ltd.) was adjusted to a 82 mg / mL aqueous solution using 73.6% peanut extract and used as a stock solution.
Commercially available berberine, magnolol, and swell thiamarin (all manufactured by Wako Pure Chemical Industries, Ltd.) were used as standard products of active ingredients of crude drugs. Berberine chloride was adjusted to 10 mg / mL, and magnolol and swellthiamarin were adjusted to 1 mg / mL methanol solutions, respectively. In addition, clarithromycin (manufactured by Wako Pure Chemical Industries, Ltd.) was adjusted to a 2 mg / mL methanol solution to prepare a stock solution.
Helicobacter pylori (Helicobacter pylori) was used after obtaining 1 strain from the University of Tokyo Medical Research Institute and 2 strains from RIKEN (no stock name, JCM12095).

(2) Measurement of antibacterial effect The antibacterial effect was measured by the disk method, and the diameter of the inhibition circle was measured and evaluated.
The bacterial solution was adjusted as follows. Helicobacter pylori HI-FBS frozen coagulum frozen at -80 ° C was inoculated on the entire surface of Columbia HP agar with platinum ears, and a microaerobic gas pack (Mitsubishi Gas Chemical Co., Ltd.) was used. Then, the cells were cultured at 37 ° C for 3 days. Next, 2 mL of HI-FBS was added onto this medium to scrape the fungus and make a suspension.
The turbidity of this suspension was measured at OD _600, were prepared as the number of bacteria is ^106/100 [mu] L. Apply 100 μL of this bacterial solution to Columbia HP agar medium and place a disk (paper disk antibiotic test, 8 mm in diameter (manufactured by ADVANTEC Toyo Corp.)) impregnated with 50 μL of the chemical solution on this agar medium. The cells were cultured at 0 ° C for 3 days under microaerobic conditions.
As a negative control, another plate in which a 10-fold diluted bacterial solution was applied to Columbia HP agar medium was prepared, and cultured at the same time, and the number of colonies was counted.
For one specimen, the experiment was performed three times or more under the same conditions, and the inhibition circle was measured each time to obtain the average value and the deviation.

Using Escherichia coli and Staphylococcus aureus H209P as positive subjects, the same experiment was conducted to measure the antibacterial effect. E. coli is cultured in LB medium and S. aureus is cultured in TSB medium, and the turbidity of each bacterial solution is measured at OD ₆₀₀ , and prepared at 10 ⁶ cells / 100 μL as in Helicobacter pylori, The disc was placed on the Mueller Hinton medium, and cultured at 37 ° C overnight, and the diameter of the inhibition circle was measured. The results are shown in Table 8.

All of the powder extract, powder extract and powdered genoder extract formed a blocking circle against S. aureus, two strains of Helicobacter pylori, and exhibited antibacterial action against these bacteria. In Awaku and Kokuboku, there was a difference in the size of the inhibition circle between the standard product and the extract, and the inhibition circle of the extract was larger.
In the case of the assembly powder extract, inhibition circles were formed against E. coli, S. aureus and 2 strains of H. pylori, but no formation of inhibition circles was observed at any concentration in the standard swell thiamarin. It was. In addition, the peanut powder extract did not form a blocking circle for any of the bacteria used. On the other hand, in the preparation, formation of inhibition circles was observed for 2 strains of H. pylori. In the Aoba powder extract containing almost the same amount of berberine as that of the preparation, formation of a larger inhibition circle than that of the preparation was observed. In S. aureus, no inhibition circle was formed when the formulation was used.
On the other hand, Helicobacter pylori was smaller than the inhibition circle formed when the extract was used, but the inhibition circle was also formed in the preparation, and it was confirmed that it had an antibacterial action.

(3) Examination of Helicobacter pylori colony-forming ability in the inhibition circle Also, Helicobacter pylori is cultured according to the procedure of the method for measuring the antibacterial effect, and the inhibition circle is formed by placing discs of clarithromycin, berberine, and alum extract I let you. Thereafter, the inside of the inhibition circle was touched with a platinum loop, a line was drawn on the HI-FBS-TTC medium, and streaked. As a control, the same operation was performed for the moss outside the inhibition circle. The plate prepared as described above was cultured at 37 ° C for 3 days under microaerobic conditions.
Moreover, the disk immersed in said chemical | medical solution was set | placed on each plate, it culture | cultivated on the same conditions, and the presence or absence of the colony formation ability of the microbe inside and outside the inhibition circle was investigated over time until the 1st-4th day. The results are shown in Table 9.

As a result of confirming the colony-forming ability of H. pylori in the inhibition circle, the streaked bacteria grew from the fungus in the inhibition circle. However, none of the fungi collected from streak circles and streak-cultured by the disc impregnated with clarithromycin, berberine, and agate powder extract did not grow.
H. pylori is known to change to a spherical cocoid form when the surrounding environment is no longer suitable for its survival. It could not be determined whether or not the growth was due to the antibacterial action of the antibacterial drug.

(4) Fungal colony-forming ability in medium containing 0.1% (w / v) deoxycholic acid
Looking at the antibacterial spectrum of each drug used in experiments with E. coli, S. aureus and H. pylori, Helicobacter pylori is not the same Gram-negative E. coli but the Gram-positive S. aureus. Similar antibacterial spectrum was shown. From this, the presence or absence of Helicobacter pylori growth in a deoxycholic acid-containing medium that suppresses the growth of Gram-positive bacteria was examined.
Helicobacter pylori cryopreserved at −80 ° C. was spread on an HP agar medium and cultured at 37 ° C. for 3 days under microaerobic conditions. Subsequently, the cultured bacteria were put on a platinum loop, a line was drawn on the HI-FBS-DCA medium, and cultured at 37 ° C. for 3 days under microaerobic conditions. For E. coli and S. aureus, LB agar medium Ueno colony was caught with platinum ears, streaked on HI-FBS-DCA medium, and cultured overnight at 37 ° C.
In addition, the same culture was carried out using HI-FBS medium as a target. The results are shown in Table 10.

  As shown in Table 10, in the deoxycholic acid-containing medium, E. coli could grow, but S. aureus and Helicobacter pylori did not grow. In contrast, all the fungi grew on the control medium containing no deoxycholic acid. This suggests that even with the same Gram-negative bacteria, Helicobacter pylori and E. coli may have different permeability in the outer membrane.

  As mentioned above, the antimicrobial action with respect to Helicobacter pylori was seen in the formulation A currently used as a gastrointestinal medicine, and this suggested the possibility that it could be used as an aid for sterilization treatment. In addition, in the preparations mixed with each extract, the antibacterial action against S. aureus H209P was attenuated, but the specific growth inhibitory effect against H. pylori was maintained. Furthermore, the antibacterial action of the Aoba powder extract and Koboku powder extract was found to have a synergistic effect that exceeded the antibacterial effects of the main components (berberine and magnolol).

[Investigation of effects on Helicobacter pylori in vivo]
(1) Animals and Reagents, etc. 6-week-old male gerbils (Japan SLC Co., Ltd., 6-week-old) and body weight 40-50 g were purchased from Shimizu Experimental Materials Co., Ltd. As the Helicobacter pylori strain, TN2GF4 strain (clinical isolate, Vac A and Cag A positive) was used. Mitake Hyakusamaru (manufactured by Nagano Pharmaceutical Co., Ltd.) was used as a sample, and berberine was used as a control.
Mitake Hyakusamaru was dissolved in a solvent with the same composition as the 1st solution of the 15th revised Japanese Pharmacopoeia Dissolution Test pH 1 (about pH 1.2) to give a solution of 1 / 10mL. Berberine was dissolved in a solvent having the same composition as the first solution of the 15th revised Japanese Pharmacopoeia dissolution solution (pH 1.2) to make a 0.03 mg / 10 mL solution.

(2) Test method 20 male gerbils were acclimated and cultivated for one week under conditions of a temperature of 22 ± 1 ° C and a light / dark cycle of 12/12 hours. Divided into 5 animals.
Except for the negative control group (indicated as “normal” in FIGS. 2 to 5), Helicobacter pylori was orally inoculated with 2 × 10 ⁸ CFU / 0.5 mL / animal, and then Mitake Hyakusamaru solution (1 tablet) / kg / day) or berberine (0.3 mg / kg / day) was administered continuously for 4 weeks.

(2-1) Damage to gastric mucosa After completion of drug administration or 28 days after H. pylori inoculation, the animals were killed with ether and the stomach was removed. The excised stomach was incised along a large fistula and fixed on a cork board with a pin, and then the degree of damage was observed under a dissecting microscope (× 10), and the damage area and bleeding area (mm ² ) were measured.
(2-2) Number of Helicobacter pylori inhabitants in the stomach After the completion of drug administration, the stomach after the observation in Example 4 was placed in 20 mL of PBS (pH 7.0) and crushed with polytron. This crushed solution was used as a stock solution, which was diluted 10-fold with PBS (pH 7.0).
100 μL each of the stock solution and the 10-fold diluted solution was applied to H. pylori selective culture plates (Nissui Pharmaceutical Co., Ltd.) and cultured at 37 ° C. for 7 to 8 days under microaerobic conditions. After completion of the culture, the number of colonies was counted using colonies on the plate as H. pylori. The number of viable bacteria in H. pylori was expressed in CFU / stomach.

(2-3) Change in Myeloperoxidase (MPO) Activity After completion of drug administration, the animals fasted for 24 hours were opened under ether anesthesia. The whole body was perfused with physiological saline, and then the stomach was removed, and the MPO activity was measured by the method of Krawisz et al.
That is, the stomach is incised along the large vagina, fixed with a pin on the cork plate, and then the gastric body and pyloric boundary region is punched out with a cork borer (inner diameter 9 mm) to obtain a test piece. Wet weight (mg) was measured.
To 50 mg of each test piece, 0.5 mL hexadecyltrimethylammonium bromide (manufactured by Sigma) containing 50 mM phosphate buffer (pH 6.0, hereinafter sometimes referred to as “PB”) was added 1.0 mL, and after crushing with polytron, This crushed solution was freeze-thawed three times and centrifuged at 2,000 × g for 10 minutes at 4 ° C., and the supernatant was used as a sample for measurement.

Take 5 μL of each sample, add 0.167 mg / ml o-dianisidine dihydrochloride (manufactured by Sigma) and 145 μL of 10 mM PB (pH 6.0) containing 0.0005% H ₂ O ₂ , and mix the absorbance at 450 nm. Changes were measured using a microplate reader (Molecular Devices). MPO activity was expressed as μmol H ₂ O ₂ / min / mg protein.
The amount of protein in the sample was measured using a BCA protein assay kit (Pierce).

(3) Results (3-1) Damage to gastric mucosa FIG. 2 shows the damage area and FIG. 3 shows the bleeding area. In the gastric mucosa, hyperplastic changes, ulcers and erosions were observed. Although the damage area tended to be slightly lower in the berberine administration group, no significant difference was found between the control group and the Mitake Hyakusamaru administration group.
In addition, the area of bleeding was significantly reduced in the Mitake Hyakusa-maru administration group, indicating that Mitake Hyakusa-maru suppressed bleeding of gastric ulcers caused by Helicobacter pylori infection.
(3-2) Number of Helicobacter pylori in the stomach After drug administration, H. pylori was detected in all of the control group, Mitake Hyakusamaru administration group, and berberine administration group. Number of bacteria, about 5.0 × 10 ⁴ CFU / stomach in the control group, the other two groups is about 0.9x10 ² CFU / stomach had become significantly less compared to the control group.
From the above, it was shown that both Mitake Hyakusamaru and Berberine reduce the number of Helicobacter pylori.

(3-3) Change in MPO activity The MPO activity of samples prepared from the stomachs of gerbils in each group tended to be slightly higher in the Mitake Hyakusamaru administration group than in the control and slightly lower in the berberine administration group. But no significant difference was observed.
From the above, in vivo, Mitake Hyakusamaru greatly reduced the bleeding area, although it did not reduce the mucosal damage area. It was also shown that the number of Helicobacter pylori inhabitants in the stomach decreased to the same extent as berberine.
It has been suggested that Mitake Hyakusamaru has a protective effect against gastric mucosal damage caused by Helicobacter pylori infection.

  The present invention is useful in the field of medicine.

Claims (8)

  A composition for protecting gastric mucosa, comprising as an active ingredient at least one selected from the group A consisting of swell thiamarin, esters thereof, physiologically acceptable salts thereof, and hydrates thereof.   From at least one selected from the group B consisting of berberine, physiologically acceptable salts thereof, and hydrates thereof, and magnolol, physiologically acceptable salts thereof, and hydrates thereof The mixture ratio of the compound selected from Group A, Group B and Group C is 1: (9-13) :( 11-15). The composition for protecting gastric mucosa according to claim 1, wherein   3. The gastric mucosa protective composition according to claim 1, comprising an extract from at least two kinds of herbal medicines selected from the group consisting of powdered algae powder, powdered ginger powder, powdered powdered powder, powdered powder and powdered powder. Composition.   The composition for protecting gastric mucosa according to claim 3, comprising extracts from powdered mackerel powder, powdered ginger powder, powdered juniper powder, powdered powder and powdered powder.   And at least one selected from the group A consisting of swell thiamarin, its physiologically acceptable salt, and hydrate in the composition, berberine, their physiologically acceptable salt, and their A mixing ratio of at least one selected from Group B consisting of hydrates and at least one selected from Group C consisting of Magnolol physiologically acceptable salts thereof and hydrates thereof, The composition for protecting gastric mucosa according to claim 4, wherein the composition is in a molar ratio of 1: (9 to 13) :( 11 to 15).   The pharmaceutical formulation containing the composition for gastric mucosa protection in any one of Claims 1-5.   A composition for inhibiting urease activity, comprising the composition for protecting gastric mucosa according to any one of claims 1 to 5.   A pharmaceutical preparation comprising the composition for inhibiting urease activity according to claim 7.

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