JP2005053818A - Trypsin inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a safe trypsin inhibitor having excellent trypsin inhibitory activity. <P>SOLUTION: This trypsin inhibitor comprises at least one kind of a plant selected from the group consisting of Hamamelis virginiana, Rosmarinus officinalis, Forsythiae fructus, Hibiscus syriacus, Syzygium aromaticum, Lagerstroemia speciosa Liquidambaris fructus, Melissa officinalis and Granati pericarpium or its extract. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a trypsin inhibitor, and more particularly to a trypsin inhibitor that has an action of strongly inhibiting the protease protease trypsin and is useful for the prevention or treatment of acute, chronic pancreatitis, thrombosis and the like.

In recent years, the Westernized diet has increased the number of patients suffering from acute and chronic pancreatitis due to excessive intake of meat, particularly fat and alcoholic beverages. Moreover, this pancreatitis may cause diabetes, or thrombosis associated with an increase in blood trypsin due to pancreatitis may occur. Several trypsin inhibitors have been developed so far for the prevention or treatment of these diseases (see Patent Documents 1 to 8). These trypsin inhibitors are composed of guanidinobenzoic acid derivatives, amidine derivatives and arginine derivatives, all of which are synthetic compounds. Examples of the medicinal plant origin include hederagenin (see Patent Documents 9 and 10), acacia genus, Eucalyptus genus (refer to Patent Document 11), sage, mulberry genus (see Patent Document 12), and white beans (refer to Patent Document 13). , Bitter gourd (see Patent Documents 14 and 15) and the like have been reported as having trypsin inhibitory activity, but a sufficient effect has not yet been obtained.
Japanese Patent Publication No.57-35870 Japanese Patent Publication No. 60-49185 Japanese Patent Publication No. 61-41510 Japanese Patent Publication No.61-1063 Japanese Patent Publication No. 2-10822 Japanese Examined Patent Publication No. 62-1389 JP-A-60-163855 JP-A-60-163815 JP-A-1-42411 JP-A-2-262510 Japanese Patent Laid-Open No. 2002-322076 JP-A-11-147834 JP 2000-86530 A JP-A-8-143467 JP-A-8-140477

  An object of the present invention is to provide a trypsin inhibitor exhibiting excellent trypsin inhibitory activity and high safety. Another object of the present invention is to provide a preventive / therapeutic agent for acute and chronic pancreatitis and a preventive / therapeutic agent for thrombosis.

  Therefore, the present inventor has searched for plant-derived trypsin inhibitors in consideration of safety. As a result, certain types of plants or extracts thereof have strong trypsin inhibitory activity, and preventive and therapeutic agents for pancreatitis and thrombus And the present invention was completed.

  That is, the present invention relates to witch hazel (Hamamelis virginiana), rosemary (Rosmarinus officinalis), forsythiae fructus, muskweed (Hibiscus syriacus), clove (Syzygium aromaticum), banaba (Lagerstroemia speciosa), fu (Liquidambaris) Trypsin inhibitor containing at least one plant selected from the group consisting of Melissa officinalis and pomegranate (Granati pericarpium) or an extract thereof, and acute and chronic pancreatitis preventive and therapeutic agents containing them, thrombosis Prophylactic and therapeutic agents are provided.

  Since the plant of the present invention or an extract thereof strongly inhibits trypsin activity, it is effective in preventing and treating acute and chronic pancreatitis, diabetes and thrombosis.

  Plants used in the present invention include witch hazel (Hamamelis virginiana), rosemary (Rosmarinus officinalis), forsythiae fructus, Hibiscus syriacus, clove (Syzygium aromaticum), banaba (Lagerstroemia speciosa), fus (Liquis) fructus), Melissa officinalis and Pomegranate (Granati pericarpium). The plant in the present invention can use all parts such as leaves, stems, buds, flowers, woody parts, bark parts (bark) and the like, and underground parts such as roots and tubers, seeds, fruits and resins. The plant in the present invention can be used as a dried product obtained by drying the plant itself, a pulverized product thereof, a juice obtained by compressing and extracting the plant itself, or an extract, but is preferably used as an extract. .

  Examples of the solvent to be extracted include water; lower alcohols such as ethanol and isopropanol; ethers such as ethyl ether and isopropyl ether; polar solvents such as acetone. These may be used alone or in combination of two or more. It may be used as Among these, water or a lower alcohol is particularly preferable. Examples of the extraction method include a method of extraction by dipping in a room temperature, cooled and heated state. The ratio of the plant and the solvent during extraction is preferably 1 to 100 times by weight, particularly 2 to 20 times by weight of the solvent relative to the plant. Moreover, although extraction time changes also with extraction temperature, when using water, it is preferable to set it as the range for 1-120 minutes, and 5-30 minutes are especially preferable. On the other hand, when a lower alcohol is used, the range is preferably 1 hour to 7 days, and particularly preferably 1 hour to 48 hours. The extract thus obtained can be used as it is, but can also be used as an extract fraction by using various chromatographies such as column chromatography.

  Particularly preferred extracts are water extracts of mugwort, banaba, pokeweed and pomegranate; and lower alcohol extracts of witch hazel, rosemary, forsythia, clove, fus and mugwort.

  Moreover, you may use these plants or its extract 1 type or in mixture of 2 or more types.

  It has not been known at all that the dry matter or extract of these specific plants used in the present invention has trypsin inhibitory activity, and is a new finding obtained by the present invention. The plant according to the present invention has a trypsin inhibitory activity superior to that of an antitrypsin agent obtained from a conventional medicinal plant, and is highly safe and prevents acute, chronic pancreatitis, thrombosis, etc. It is useful as a therapeutic agent and as a food for these purposes.

  As a method for administering the trypsin inhibitor of the present invention, a prophylactic / therapeutic agent for acute and chronic pancreatitis, thrombosis and the like, either oral administration or parenteral administration can be employed. Upon administration, these plants or extracts thereof are mixed with solid or liquid non-toxic pharmaceutical carriers suitable for administration methods such as oral administration, rectal administration, and injection, and administered in the form of conventional pharmaceutical preparations. be able to. Examples of such preparations include solid preparations such as tablets, granules, powders and capsules; liquid preparations such as solutions, suspensions and emulsions; lyophilized preparations and the like. These preparations can be prepared by usual means. Examples of the non-toxic pharmaceutical carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin , Water, physiological saline and the like. Further, if necessary, conventional additives such as a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent can be appropriately added.

  When the trypsin inhibitor of the present invention is used as a food, the above-mentioned plant is used as it is, added with various nutritional components, or contained in food and drink, to treat acute and chronic pancreatitis or thrombosis and Used as a health food or food material useful for prevention. For example, after adding the above-mentioned appropriate auxiliary agent to the above-mentioned plant, using conventional means, it is formed into an edible form, for example, granular, granular, tablet, capsule, paste, etc. It may also be used, and various foods such as processed foods such as ham and sausage; processed fish foods such as kamaboko and chikuwa; used in addition to bread, confectionery, butter, powdered milk, fermented milk products, water It may be used by adding to beverages such as fruit juice, milk and soft drinks. The effective dose of the plant or extract of the present invention when used as a pharmaceutical product depends on the patient's age, weight, symptoms, patient grade, route of administration, administration schedule, formulation, strength of the inhibitory activity of the material, etc. For example, in the case of oral administration, generally about 0.001 to 1000 mg / kg body weight per day as a dried product of a plant or an extract is desirable. In the case of hot water extract of Mukuge, it is about 5 to 300 mg / kg body weight per day, and may be divided into several times a day.

  Since the plant used in the present invention is natural, its toxicity is low. For example, even if a hot water extract of banaba is orally administered to a rat for a long period of 1000 mg / kg daily for 100 days, no death is observed. No weight change was observed. Therefore, it can be said that the trypsin inhibitor of the present invention has a highly safe and excellent trypsin inhibitory activity.

  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

Example 1
(Plant extraction) 200 g of distilled water was added to 10 g of each of Hibiscus cortex, Lagerstroemia speciosa, Melissa officinalis, and pomegranate (Granati pericarpium), boiled at 100 ° C. for 30 minutes, and then freeze-dried By doing this, each hot water extraction dried material was obtained.

(Trypsin inhibitory activity test) Trypsin activity was obtained by adding porcine pancreatic trypsin (5207 USP unit / mg, Wako Pure Chemical Industries) to 0.7 mL of 0.2 mol / L triethanolamine buffer (pH 7.8) containing 20 mmol / L of calcium chloride. (Trade name) solution (100 μg / mL) 0.05 mL and extract-containing sample sample solution 0.05 mL (in the case of blind, 0.05 mL of water) are added, and the mixture is incubated at 25 ° C. for 15 minutes, and the substrate is 4 mmol / After adding 0.2 mL of LBz-L-Arg-4-NA (N-α-benzoyl-L-arginine-4-nitroanilide hydrochloride) and further incubating at 25 ° C. for 10 minutes, 1 mL of 0.1N hydrochloric acid solution was added. The reaction was stopped by addition, and evaluation was made by measuring the absorbance at 405 nm. Trypsin inhibitory activity was calculated using the following formula.

Trypsin inhibitory activity = 100− (AT−AB _T ) / (AC−AB _C ) × 100 (%)
AT: Absorbance of sample sample AB _T : Blank absorbance of sample sample AC: Absorbance of blind sample AB _C : Blank absorbance of blind sample As a comparative example, Acacia that has already been reported to have trypsin inhibitory activity Similar tests were conducted on eucalyptus, sage, and mulberry. The results are also shown in Table 1.

  As can be seen from Table 1, it can be seen that the water extract of Mucuge, Banaba, Atlantic mint, and pomegranate of the present invention has high trypsin inhibitory activity.

Example 2
(Plant extraction) Witch hazel bark (Hamamelis virginiana), rosemary (Rosmarinus officinalis), forsythiae fructus, clove (Syzygium aromaticum), fu (Liquidambaris fructus), 2.5g ethanol (Hibiscus syriacus) And extracted for 1 week at room temperature. The obtained extraction solution was subjected to vacuum concentration to completely remove the solvent, and each alcohol extraction dried product was obtained.

(Trypsin inhibitory activity test) The trypsin inhibitory activity was measured by the method described in Example 1. However, 50 μL of ethanol was used for the blind test. As a comparative example, a similar test was performed for acacia, eucalyptus, sage, and mulberry, which have already been reported to have trypsin inhibitory activity. The results are also shown in Table 2. As can be seen from Table 2, it can be seen that the lower alcohol extract of the present invention witch hazel bark, rosemary, forsythia, clove, fu and mukuge has high trypsin inhibitory activity.

Formulation Example 1 (tablet)
Various ingredients were mixed according to the following formulation, granulated, dried and sized, and then tableted to produce tablets.
(Prescription) (mg)
Witch hazel extract 40
Microcrystalline cellulose 100
Lactose 80
Magnesium stearate 0.5
Methylcellulose 12

Formulation example 2 (soft drink)
According to the following formulation, each component was blended according to a conventional method and homogenized to obtain a soft drink. The obtained soft drink was filled in a brown bottle, sealed with an aluminum cap, and heat-treated. These had good appearance and flavor.
(Prescription) (g)
Mukuge extract 0.8
Fragrance 0.8
Water 100
Reduced starch saccharified product 24
Fructose 18

Claims (9)

  A trypsin inhibitor comprising at least one plant selected from the group consisting of witch hazel, rosemary, forsythia, mugweed, clove, banaba, fu, wild mint and pomegranate, or an extract thereof.   The trypsin inhibitor according to claim 1, wherein the plant extract is an aqueous extract of at least one plant selected from plants consisting of mugwort, banaba, Atlantic mint and pomegranate.   The trypsin inhibitor according to claim 1, wherein the plant extract is a lower alcohol extract of at least one plant selected from the group consisting of witch hazel, rosemary, forsythia, clove, fu and mukuge.   An agent for preventing or treating pancreatitis, comprising at least one plant selected from the group consisting of witch hazel, rosemary, forsythia, mugweed, clove, banaba, fu, wild mint, and pomegranate.   The pancreatitis preventive / therapeutic agent according to claim 4, wherein the plant extract is an aqueous extract of at least one plant selected from the plant consisting of mugwort, banaba, pokeweed and pomegranate.   The agent for preventing and treating pancreatitis according to claim 4, wherein the extract of the plant is a lower alcohol extract of at least one plant selected from the group consisting of witch hazel, rosemary, forsythia, clove, fu and mukuge.   An agent for preventing or treating thrombosis comprising at least one kind of plant selected from the group consisting of witch hazel, rosemary, forsythia, mugweed, clove, banaba, fu, wild mint, and pomegranate.   The thrombosis preventive or therapeutic agent according to claim 7, wherein the plant extract is an aqueous extract of at least one kind of plant selected from plants consisting of mugwort, banaba, wild mint, and pomegranate.   The thrombosis preventive or therapeutic agent according to claim 7, wherein the plant extract is a lower alcohol extract of at least one plant selected from the group consisting of witch hazel, rosemary, forsythia, clove, fu and mukuge.