JPH10175877A - Urease activity inhibitor of helicobacter pyrroli - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicine having inhibiting action to activity of urease produced by Helicobacter pyrroli and effective as a preventive or curing agent for gastritis, gastric ulcer, duodenal ulcer, etc., by containing an extract of Saigon cinnamon as an active component. SOLUTION: This urease activity inhibitor is obtained by subjecting dried substances of leaves, barks, branches, roots, etc., of Saigon cinnamon as an evergreen tree belonging to family Lauraceae to a pretreatment such as shredding or crushing to obtain a powder-like state and extracting the resultant powder of 1 kg with 5-50 L of water or a mixture of water and a lower alcohol such as methanol or ethanol at 30-100 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel Helicobacter pylori.
i) urease activity inhibitors, more particularly gastritis,
The present invention relates to a drug having an activity of inhibiting the activity of urease produced by Helicobacter pylori, which is effective as an agent for preventing and treating gastric ulcer and duodenal ulcer.

[0002]

2. Description of the Related Art Campylobacter pylori (Campylobacter pylori) was obtained from a biopsy tissue of a gastric mucosa of a patient with gastritis or gastric ulcer in 1983.
ylobacter pylori is detected at a high rate [“Lanset” No. 12
73-1275 (1983)], it has been gradually revealed that this bacterium is involved in the development of gastritis or gastric / duodenal ulcer. This bacterium was later proved to belong to a different genus from other bacteria of the genus Campylobacter, and was renamed Helicobacter pylori (hereinafter sometimes simply referred to as "H. pylori"), resulting in gastritis or gastric / duodenal ulcer disease It has become clear clinically that this is closely related to

[0003] The H. pylori is a gram-negative spiral bacillus that preferentially infects the gastric mucosa, particularly the pyloric region, which is the exit of the stomach, and is a flagellated microaerobic bacterium. H. pylori itself is weak against acid, but unlike other bacteria, it has urease on the body surface, so it decomposes urea in the stomach from the host into ammonia to neutralize stomach acid, Growth is possible.

[0004] For such H. pylori, NIH (N
national Institutes of Hea
lth) issued a recommendation statement in February 1994, stating that "peptic ulcer cases positive for Helicobacter pylori should be eliminated regardless of whether they are first-time or recurrent", and the WHO (World Health Organi).
zation) found in 1994 that Helicobacter pylori induced a high rate of carcinogenesis.

As a mechanism by which H. pylori colonizes and proliferates in the stomach and exerts pathogenicity, H. pylori reaches the stomach orally like other Escherichia coli and swims through the mucus layer using flagella. In the stomach, it reaches the mucous membrane layer and adheres (adheres), where urease produced by itself decomposes urea derived from the host, produces ammonia, neutralizes stomach acid, prepares a favorable living environment and starts proliferation However, in this case, the ammonia synthesized by H. pylori peels mucus covering the surface of the mucous membrane of the stomach and exposes the mucous membrane, so that the mucous membrane is exposed to gastric acid and inflammation generally occurs. It is considered.

[0006] When the H. pylori inhabits the mucous membrane in this way, it produces interleukins (bioactive substances), and as a result, lymphocytes increase and act on neutrophils (all of them are leukocytes). Brings activation. Activation of neutrophils produces hypochlorous acid, which reacts with ammonia to produce monochloramine, which has a strong cell-damaging effect. Inflammation is caused in this manner. At this time, it is thought that when the secretion of stomach acid becomes excessive due to stress or the like, the mucous membrane is further damaged and the inflammation progresses to ulcer.

[0007] By the way, various attempts have been made to utilize an antibiotic having an anti-Helicobacter pylori activity for the prevention and treatment of gastritis or gastric / duodenal ulcer thus caused. For example, penicillin, β-lactam agents such as ampicillin, erythromycin, macrolide agents such as clarithromycin, aminoglycoside agents such as streptomycin, or antibiotics such as tetracycline agents, and strong antibacterial activity against H. pylori such as bismuth preparations and administering those that exhibit, also, recently, H ₂ receptor antagonists, in anti-peptic ulcer agent, such as proton pump inhibitors, are also being developed drugs having both antibacterial activity against H. pylori .

However, conventional administration of antibiotics and the like has been problematic in terms of safety during long-term administration.
There is a risk of recurrence, and there is also a risk that resistant bacteria may develop. Although some of the conventional drugs have been applied clinically, their evaluation has not been constant, and no effective drug that is safe and can be administered for a long time has not been found so far. Under such circumstances, there has been a demand for an effective and safe drug effective for preventing and treating gastritis or gastric / duodenal ulcer.

On the other hand, as the urease activity inhibitor, for example, hydroxamic acid (Japanese Patent No. 1432756) and its mutants and disulfides are known, but the experience of using it in the diet has been long and its safety has been confirmed. It has not been known that spices, especially extracts obtained from calyx, have a strong urease inhibitory effect.

[0010]

Under such circumstances, the present invention has an activity of inhibiting the activity of urease produced by H. pylori, and is safe and capable of long-term administration.
The purpose of the present invention is to provide a drug effective as an agent for preventing and treating gastritis, gastric ulcer, duodenal ulcer and the like.

[0011]

Means for Solving the Problems The present inventors have conducted intensive studies to develop a urease activity inhibitor of H. pylori that is safe and can be administered for a long period of time, and as a result, it has been widely used as a cinnamon in food spices and the like. The extract of cabbage, which is used as a component of various Chinese herbs, including Annaka-san, which is famous as a therapeutic agent for neurogenic gastritis, has an activity of inhibiting the activity of urease produced by H. pylori. Have been found to be suitable for the above purpose, and the present invention has been completed based on this finding.

[0012] That is, the present invention provides a urease activity inhibitor of Helicobacter pylori, which comprises a cinnamon extract as an active ingredient.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the urease activity inhibitor of the present invention, cinnamon (Cinnamomi Cortex) which is a raw material of a cinnamon extract used as an active ingredient
Is an evergreen tree of the camphoraceae family, and has a unique aroma in its leaves and branch roots. The essential oil obtained therefrom has long been used as cinnamon (Cinnamon) for flavoring foods. Keihi is also used as a component of Chinese herbal medicine in many Chinese herbal medicines such as Annaka-san, Onkyo-to, Oren-to, Kakkon-to, Keishi-to, and Keishi-ka-shakuyaku-to. As described above, Keihi has long been used in food spices and Chinese herbs, and there is no problem with its safety.

The method for extracting cinnamon is not particularly limited, but a method in which highly polar substances are extracted at a high concentration is preferable. As the extraction solvent, an aqueous solvent such as water or a mixture of water and alcohol is suitable. As the alcohol, a lower alcohol such as methanol or ethanol is preferable.

Next, a preferred example of the processing for extracting caffeine will be described. First, the leaves, bark, twigs, roots, etc. of dried cabbage are sufficiently dried, and then pre-processed such as shredding and crushing.
Make powder. Next, about 5 to 50 liters of the above-mentioned extraction solvent, for example, water is added to 1 kg of the powder, followed by extraction. This extraction may be performed at room temperature,
It may be performed by heating to about 00 ° C. At this time, in order to effectively perform the solvent extraction, it is advantageous to stir the mixture appropriately so that the extraction solvent sufficiently penetrates into the inside.

After the completion of the extraction, solid-liquid separation is carried out by using known means such as filtration, centrifugation and decantation. The obtained extract is concentrated under reduced pressure if necessary, and then n-
After adding a hydrocarbon solvent such as hexane and stirring or shaking sufficiently, the mixture is separated into a hydrocarbon solvent layer and an aqueous layer.
Next, the aqueous layer is subjected to column chromatography to separate into a methanol elution part and a water elution part. By evaporating the methanol from the methanol elution part, a desired cinnamon extract can be obtained. The means for distilling off methanol is not particularly limited, and a conventionally used method can be used.

The extraction process can be carried out efficiently by subjecting the cinnamon powder to a steam distillation or a de-essential oil treatment with a hydrocarbon solvent such as n-hexane before the extraction process.

The urease activity inhibitor of Helicobacter pylori according to the present invention contains the thus obtained cauliflower extract as an active ingredient, and the cauliflower extract may be used as it is, or may be diluted or concentrated. Alternatively, after lyophilization, it can be prepared in powder or paste form and optionally formulated as appropriate, and used as a prophylactic or therapeutic agent for gastritis, gastric ulcer or duodenal ulcer.

The content of cinnamon extract in the preparation is as follows:
The dry weight is usually in the range of 0.0001 to 50% by weight, preferably 0.001 to 30% by weight. The dosage form of the preparation is not particularly limited, and may be any of tablets, capsules, pills, granules, liquids and the like.
Further, additives such as excipients can be appropriately used according to the dosage form. The urease activity inhibitor of the present invention may optionally contain other active ingredients such as an antacid and a gastric mucosal repair agent.

Further, the urease activity inhibitor of the present invention can be prepared in the form of a liquid or solid food, for example, according to a conventional method, and other well-known food materials,
Flavors and the like can also be used as appropriate. Such other food materials include those conventionally used in the food field, such as flour, starch, proteins and their degradation products, saccharides, lipids, vitamins, and minerals.

[0021]

Industrial Applicability The urease activity inhibitor of the present invention has an activity of inhibiting the activity of urease produced by H. pylori, is safe and can be administered for a long period of time, and prevents and treats gastritis, gastric ulcer, duodenal ulcer and the like. It is effective as an agent. Further, the urease activity inhibitor of the present invention can be provided in any of a pharmaceutical form and a food form.

[0022]

Next, the present invention will be described in more detail with reference to examples and test examples, but the present invention is not limited to these examples.

Example 1 2.0 kg of Guangnan Cahigium powder was put into 15 liters of water, subjected to an extraction treatment at 40 ° C. for 5 hours, and then centrifuged to separate into a solid and an extract. . The extract was then concentrated to a total volume of 2.2 liters. This concentrated liquid was divided into two equal parts, and 1.1 liter of one concentrated liquid was freeze-dried to obtain 61.1 g of a solid substance.
The yield of the extract with water was 6.11% by weight. Next, the remaining 1.1 L of the concentrated solution was subjected to extraction with 1.1 L of n-hexane three times.
The n-hexane soluble fraction was 460 mg, and the yield was 0.046% by weight. On the other hand, the aqueous layer after the extraction treatment with n-hexane was subjected to column chromatography (column: diameter 8.2 cm, length 31 cm, filler: MCI-g).
el. CHP-20), eluting with 2 liters of water and 1 liter of methanol, separating into a water elution portion and a methanol elution portion, and concentrating and drying the methanol elution portion. 77 g (yield 1.78% by weight) were obtained. Further, 6 g of cinnamon extract (a) was dissolved in 100 ml of methanol, and column chromatography (column: diameter 4 cm, length 35 cm, filler: Se)
phadex LH-20) and eluted sequentially with ethanol and methanol to obtain 9 fractions. By concentrating and drying each fraction, cinnamon extracts (b) to (nu) were obtained. On the other hand, when the water-eluted portion was freeze-dried, 31.43 g (yield 3.14% by weight) of cinnamon extract (lu) was obtained.

Example 2 2.0 kg of Vietnamese calyx powder was poured into 15 liters of water, subjected to an extraction treatment at 40 ° C. for 5 hours, and then centrifuged to remove solids from the extract. Then, the extract is concentrated under reduced pressure to 1.5 liter, and then freeze-dried to obtain 105.98 g of a solid product (yield: 10.10 g).
59%). 55.93 g of the solid product thus obtained was dissolved in 1 liter of water and subjected to column chromatography (column: diameter 8.2 cm, length 31 cm, filler: MCI-gel CHP-20). , 4 liters of water and 3 liters of methanol. The eluate was freeze-dried to obtain 40.04 g (yield: 3.79%) of the extract of cabbage extract (ヲ) from the portion eluted with water and 14.16 g (yield 1.79 g) of cabbage extract (wa) from the portion eluted with methanol.
34%).

EXAMPLE 3 After extracting 1.0 kg of Guangnan Cahi powder in 1.5 liters of water at 60 ° C. for 3 hours each for 3 hours, the respective extracts are combined until the volume becomes 1.5 liters. Concentrated. The aqueous extract was then diluted with 1.5 liters of water-saturated n-butanol
After extraction twice, the n-butanol extract was concentrated and dried to obtain 16.3 g of an n-butanol extract. This n
300 ml of n-hexane was added to the -butanol extract, and the mixture was refluxed and extracted at 60 ° C. for 1 hour. The hexane extract was concentrated and dried to obtain 0.21 g of a hexane extract. The residue was subjected to reflux extraction with 300 milliliters of acetone for 30 minutes, and the operation was repeated three times. Then, the acetone extract was concentrated and dried to obtain 9.98 g of an acetone extract.
The residue was dried to obtain 6.0 g of a cinnamon extract (f).

Example 4 An operation of extracting 1.0 kg of Vietnamese cabbage powder with 1.5 liters of water at 60 ° C. for 3 hours was repeated three times, and the water extracts were combined and concentrated to 1.5 liters. The aqueous extract was then diluted with 1.5 liters of water-saturated n-butanol for 2 hours.
After extraction twice, the n-butanol extract was concentrated and dried to obtain 15.3 g of an n-butanol extract. To this butanol extract, add 300 ml of hexane,
The mixture was refluxed and extracted at 60 ° C. for 1 hour, and the hexane extract was concentrated and dried to obtain 0.83 g of a hexane extract. The operation of reflux extraction of the extract with 300 ml of acetone for 30 minutes was repeated three times, and then the acetone extract was concentrated.
After drying to dryness, 3.07 g of an acetone extract was obtained. The residue was dried to obtain 1.22 g of a cinnamon extract (Y).

Test Example Inhibition of urease activity for each of the fractions (a) to (nu) obtained in Example 1 and (ヲ), (wa) obtained in Example 2 and (f) obtained in Example 3 The measurement test was carried out by the following method using urease derived from nata beans (jack bean) which is structurally similar to urease derived from H. pylori and is generally used for measurement of activity inhibition of urease derived from H. pylori. . 1 mg of ¹³ C-labeled urea (99 atomic% ¹³ C, manufactured by Tracer Technology) is placed in an NMR tube having an outer diameter of 5 mm, and 580 μl of a sodium phosphate buffer (pH 7) at a concentration of 1/15 mol / liter is added. ^{After 13} C-urea was dissolved and ¹³ C-NMR measurement was performed to confirm the signal of ¹³ C-urea, the NMR tube was ice-cooled for 10 minutes. TSP (sodium 3-trimethylsilylpropionate-d ₄ ) was used as a reference for ¹³ C-NMR, and the TSP signal was set to 0 ppm.

On the other hand, a urease solution containing 30 mg of urease [derived from nata beans, manufactured by Wako Pure Chemical Industries, Ltd.] in 20 ml of a sodium phosphate buffer (pH 7) at a concentration of 1/15 mol / liter was cooled on ice for 10 minutes. Thereafter, 20 μl of the urease solution was added into the above-mentioned NMR tube, and ¹³ C-NMR measurement was immediately started. From 1 minute to 15 minutes, the number of integration was 20 times per minute (measurement time: 1 minute). The spectrum was measured.

FIG. 1 shows an NMR spectrum measured every minute from 1 minute to 15 minutes after the reference (TSP).
Are superimposed so that the signal height is constant. One minute later, the spectrum of urea (16
5.2 ppm) and a signal (162.6 ppm) of carbon dioxide (carbonate) derived from ¹³ C-labeled carbon dioxide generated from ¹³ C-labeled urea.

It was confirmed that the urea signal decreased with time, while the carbon dioxide signal increased with time. The pH of the reaction solution after the measurement was completed
Was 8. Moreover, when the interior of the NMR tube after the measurement sampling gas, were GC-MS measurement, ¹³
The presence of C-labeled carbon dioxide (m / z: 45) was confirmed.

According to the above method, a urease solution was placed in an NMR tube, and the cabbage extracts (a) to (f) obtained in Examples as test drugs, other crude drugs of the type shown in Table 1, and ¹³ C
¹³ C-NMR measurement was performed by adding -labeled urea, and the disappearance time of ¹³ C-labeled urea was compared and examined using the control without the test drug as a control. Table 1 shows the results. For reference, the estimated disappearance time of ¹³ C-labeled urea of hydroxamic acid and disulfide, which are conventionally known urease activity inhibitors, is also shown.

[0032]

[Table 1]

As is clear from Table 1, it was found that Guangnan cabbage extracts (nu) and (f) have strong urease activity. About this Guangnan Cahi extract (mosquito), ¹³
FIG. 1 shows NMR spectra obtained by observing the decomposition of C-labeled urea over time from 1 minute to 27 minutes. In this figure, the signal at 165.2 ppm is derived from urea, and the signal at 162.6 ppm is derived from ¹³ C-carbon dioxide generated from ¹³ C-urea.

[Brief description of the drawings]

FIG. 1 is a ¹³ C-NMR spectrum showing an example of a change with time of the degradation of ¹³ C-labeled urea by urease.

Claims (3)

[Claims] 1. Helicobacter pylori (Helicobacter) comprising a cinnamon extract as an active ingredient.
ter pylori) urease activity inhibitor. 2. The inhibitor of urease activity of Helicobacter pylori according to claim 1, wherein the extract of cinnamon bark is an extract with an aqueous solvent. 3. The urease activity inhibitor of Helicobacter pylori according to claim 1, which is used as an agent for preventing and treating gastritis, gastric ulcer or duodenal ulcer.