JPH0585934A - Anti-trypsin agent - Google Patents

Abstract

PURPOSE:To obtain an anti-trypsin agent useful for preventing and treating acute and chronic pancreatitis and thrombotic diseases and preventing transition to diabetes by pancreatitis because of strong inhibition of trypsin activity and no influence on thrombin. CONSTITUTION:An anti-trypsin agent comprising a flavonoid compound, preferably hydroxy and/or alkoxy derivatives of flavone or hydroxy derivative of flavanone such as quercetin, baicalein, luteolin, kaempferol, apigenin, oroxylin, scrucaflavone-II, wogonin or compounds of formula I and formula II (R<1> is H: R<2>=R<3>=OH; R is H or OH) as an active ingredient is obtained. The compound is obtained from a crude drug such as flower of Sophora japonica, root of Scutellaria baicalensis, flower of crysanthemum or cranesbill by extraction. A dose as the medicine is preferably about 60-600mg daily in the case of ordinary adult and is orally administered dividedly at least three times after a meal.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antitrypsin agent, and more particularly, it has an action of strongly inhibiting the protease trypsin and is useful for the prevention or treatment of acute or chronic pancreatic inflammation, thrombotic diseases and the like. It relates to antitrypsin agents.

[0002]

2. Description of the Related Art In recent years, due to westernized dietary habits, the number of patients suffering from acute and chronic pancreatitis due to excessive intake of meat, especially fat and excessive intake of alcoholic beverages has increased. In addition, this pancreatitis disease may cause diabetes, or may cause a state of preparation for thrombus formation due to an increase in blood trypsin due to pancreatic inflammation.

Several antitrypsin agents have been developed as therapeutic agents for improving the above-mentioned conditions (Japanese Patent Publication Nos. 57-35870, 60-49185, 61-41510, and 61-41510).
61-1063, Japanese Patent Publication No. 2-10822, Japanese Patent Publication No. 62-1389,
JP-A-60-163855 and JP-A-60-163815). These antitrypsin agents consist of guanidinobenzoic acid derivatives, amidine derivatives and arginine derivatives, all of which are synthetic compounds. As an antitrypsin agent obtained from a medicinal plant, heteragenine, which is a triterpene compound, has been developed (JP-A-1-42411 and JP-A-2-262510).

[0004]

The above-mentioned conventional techniques relating to antitrypsin agents have been focused on the development of synthetic compounds that are inhibitors of the protease trypsin that causes inflammation such as pancreatic inflammation. And, as a common action of those compounds, since it has an action of inhibiting proteolytic enzymes other than trypsin, for example, thrombin,
There is a problem in that it also impedes the physiological hemostatic action and causes a bleeding tendency. An object of the present invention is to provide an excellent antitrypsin agent which does not have such a problem.

[0005]

The present inventors have found that various flavonoid compounds contained in plants, in particular flavone, flavanone and their derivatives, strongly inhibit trypsin activity and further inhibit other serine proteases such as thrombin. The present invention has been completed based on the finding that it has no effect and is therefore effective in the prevention and treatment of pancreatitis, thrombotic diseases and the like. The gist of the present invention is an antitrypsin agent containing a flavonoid compound as an active ingredient.

The flavonoid compound used in the present invention is preferably a hydroxy and / or alkoxy derivative of flavone or a hydroxy derivative of flavanone. The hydroxy derivatives of flavone include quercetin, baicalen, luteolin, kaempferol and apigenin, while the hydroxy and alkoxy derivatives of flavone include 2 ', 5,
Examples include 5, ', 7-tetrahydroxy-6', 8-dimethoxyflavone, oroxyline-A, sukurucapflavone-II, and ogonin. Of these, derivatives having a hydroxy group at the 3-position of flavones, such as quercetin and kaempferol, are sometimes called flavonols. Examples of flavanone derivatives include 2 ', 5,6', 7-tetrahydroxyflavanone and 2 ', 3,5,6', 7-pentahydroxyflavanone.

Since flavonoid compounds are present in many kinds of plants throughout their entire tissues, they may be obtained by any method without limitation on materials, extraction methods and the like, but they are extracted from the following crude drugs. This is preferable from the viewpoint of convenience of material availability and safety, and the target compound can be easily obtained.

[0008] For example, quercetin, baicalein, luteolin and kaempferol are furanovoid compounds contained in Sophora, Huang Gong, Chrysanthemum and Ginkgo biloba, respectively, and can be obtained by extraction from each crude drug. There are the following extraction methods. First, each crude drug is extracted under reflux with ethyl acetate and concentrated under reduced pressure. The obtained concentrated extract is eluted with chloroform-methanol mixture (20: 1 to 5: 1 v / v) using silica gel column chromatography to obtain the target compound, quercetin, baicalein, luteolin and kaempferol.

Other flavonoid compounds can be obtained by the same method. 2 ', 5,5', 7-tetrahydroxy-6 ', 8-dimethoxyflavone, oroxyline-
A, Sukurucapflavone-II, Ogonin, 2 ', 5,6', 7
-Tetrahydroxyflavanone, 2 ', 3,5,6', 7-Pentahydroxyflavanone is a mixture of chloroform-methanol of a concentrated extract of yellow corn (Augon) with ethyl acetate extracted using silica gel column chromatography.
It can be obtained by eluting (20: 1 to 5: 1 v / v) and repeating silica gel column chromatography [Y. Kimura et al .: Chem. Pharm. Bull, 30, 1792.
(1982); Y. Kimura et al. Planta Medica, 50, 290 (198
Four)].

Apigenin is a mixture of chloroform-methanol mixture (20: 1 to 5: 1 v / v) of chrysanthemum flower ethyl acetate extract concentrated extract using silica gel column chromatography.
It can be obtained by eluting with and further repeating silica gel column chromatography. The method for extracting the flavonoid compound is not limited to the above method. Further, since the compound used in the present invention has been identified and the structure has been clarified, in addition to extraction from plants,
It may be produced by synthetic or plant biotechnology.

[0011]

The flavonoid compound used in the present invention has a strong antitrypsin activity and, therefore, has an effect of inhibiting tissue inflammation such as pancreatitis by a mechanism such as suppressing the production of kinin which is an inflammation-causing substance. .. It also has the effect of inhibiting the activation of blood coagulation factors, factor VII and factor XII, with the increase of blood trypsin due to pancreatic inflammation, and inhibiting thrombus formation.

Moreover, since this flavonoid compound does not inhibit the activity of other proteolytic enzymes such as thrombin, it is superior to other proteolytic enzyme inhibitors in that it does not affect the physiological hemostatic action. In addition, regarding flavonoid compounds, which are crude drug components, no toxicity was observed upon oral administration of 1 g / kg body weight (mouse, rat).

Therefore, the antitrypsin agent of the present invention containing a flavonoid compound as an active ingredient is a very excellent antitrypsin agent as a very excellent antitrypsin agent for the prevention and treatment of tissue inflammation such as pancreatitis, the prevention of transition to diabetes due to pancreatitis, It is also effective in preventing and treating thrombotic diseases.

The dose of the flavonoid compound administered as a medicine varies depending on the type of disease, the degree of symptoms, the age and weight of the patient, etc.
About 600 mg should be administered. It is desirable to administer this after meals at least 3 times a day. As a method of administration, oral administration is preferable in principle, but it is not limited to this.

When the flavonoid compound is administered, additives commonly used for formulation can be used, and the flavonoid compound can be formulated into various forms depending on the administration method. For example, dosage forms for oral administration include tablets, capsules, granules,
Either a powder or a fine granule may be used. Additives used for formulation include excipients, disintegrants, lubricants, binders, dispersants, plasticizers and the like. Any of these may be used with commonly used additives. Hereinafter, the action of the antitrypsin agent of the present invention will be described with reference to experimental examples.

(Experimental example) 1. Extraction and isolation of flavonoid compounds from each crude drug (1) Extraction and isolation of quercetin from Sophora cabbage 500 g of Sophora reflex extraction with ethyl acetate (1 liter x 3 times) Then, it was concentrated under reduced pressure. The obtained concentrated extract was subjected to silica gel (Merck) column chromatography to obtain a chloroform-methanol mixture (20: 1 to 5: 1 v / v).
Elution was performed with and the silica gel column chromatography was repeated to obtain 5 g of quercetin. The chloroform-methanol mixture used here is used by gradually changing the ratio while checking the necessary components by silica gel thin layer chromatography.

(2) Extraction and Isolation of Flavonoids from Yellow Sesame (Ougon) The following flavonoid compounds were obtained from 10 kg of yellow rice (Ougon) by the same operation method as in (1). That is, baicalein 31.2 g, 2 ', 5,5', 7-tetrahydroxy-6 ', 8
-3.5 g of dimethoxyflavone, 1.1 g of oroxylin-A, 6.3 g of sucurcapflavone-II, 10.5 g of ogonine, 2 ',
3.8 g of 5,6 ', 7-tetrahydroxyflavanone and
4.2 g of 2 ', 3,5,6', 7-pentahydroxyflavanone was obtained.

(3) Extraction and isolation of flavonoids from chrysanthemum flowers 1 kg of chrysanthemum flowers and 1 g of luteolin and 0.5 g of apigenin were obtained in the same manner as in (1).

(4) Extraction and isolation of kaempferol from ginger ginger Pepper geranium 2 g was obtained from 1 kg gem ginger by the same operation method as in (1).

2. Effect of various flavonoid compounds on trypsin activity 100 μl of proteolytic enzyme trypsin (45 μg / ml) in 100 ml of 100 mM phosphate buffer (pH 7.4) and various flavonoid solutions prepared to several concentrations Add 100 μl, 25 ℃, 10
I pre-incubated for a minute. Next, the synthetic substrate N-
Benzoyl-Phe-Val-Arg-p-nitroanilide
(Sigma, 1 mg / ml) 200 μl was added to a final volume of 2.0 m
It was further reacted at 25 ° C. for 15 minutes as l. 50% in reaction solution
Stop the enzymatic reaction by adding 100 μl of trichloroacetic acid,
After leaving it in ice water for 30 minutes, it was centrifuged at 4 ° C. and 10,000 rpm for 20 minutes, and the amount of p-nitroaniline released in the supernatant was quantified. The 50% inhibitory concentration was determined from the correlation between the flavonoid compound concentration and the amount of released p-nitroaniline, and is shown in Table 1.

3. Effects of various flavonoid compounds on thrombin activity 100 μl of proteolytic enzyme thrombin (5 IU / ml) in 0.6 ml of 100 mM phosphate buffer (pH 7.4) and various flavonoid solutions prepared to several concentrations 100 μl was added, and preincubation was performed at 25 ° C. for 10 minutes. Next, 200 μl of the synthetic substrate N-benzoyl-Phe-Val-Arg-p-nitroanilide (manufactured by Sigma, 1 mg / ml) was added to make a final volume of 1.0 ml and further reacted at 25 ° C. for 15 minutes. The enzymatic reaction was stopped by adding 100 μl of 50% trichloroacetic acid to the reaction solution, allowed to stand in ice water for 30 minutes and then centrifuged at 10,000 rpm for 20 minutes at 4 ° C. to quantify the released p-nitroaniline. ..
The 50% inhibitory concentration was determined from the correlation between the flavonoid compound concentration and the amount of released p-nitroaniline, and is shown in Table 1.
As is clear from Table 1, each flavonoid compound exhibits strong antitrypsin activity, but not antithrombin activity.

[0022]

[Table 1]

Next, formulation examples of each flavonoid compound will be shown.

[0024]

【Example】

Example 1 Tablets Corn starch 65 g Crystalline cellulose 20 g Carboxymethylcellulose calcium 3.5 g Light anhydrous silicic acid 0.5 g Magnesium stearate 1 g Flavonoid compound (quercetin) 10 g Total 100 g According to the above formulation, It was compression-molded by a machine to obtain 200 mg tablets. Each tablet contains 20 mg of a flavonoid compound, and an adult should take 3 to 30 tablets daily in 3 divided doses after eating.

Example 2 Granules Corn starch 84 g Carboxymethyl cellulose calcium 5 g Light anhydrous silicic acid 0.5 g Magnesium stearate 0.5 g Flavonoid compound (baicalein) 10 g Total 100 g According to the above formulation, the After compression molding with a machine, it was crushed with a crusher and sieved to obtain granules. 1 g of this granule contains 10 flavonoid compounds.
0 mg is contained, and for adults, take 1 to 6 g daily in 3 divided doses after meals.

Example 3 Capsule Corn starch 89.5 g Light anhydrous silicic acid 0.5 g Flavonoid compound (luteolin) 10 g Total 100 g According to the above-mentioned prescription, to were uniformly mixed to give 200 mg.
No. 2 capsule was filled. This capsule contains 20 mg of a flavonoid compound, and an adult takes 6 to 30 capsules daily in 3 divided doses after meals.

Example 4 Powder corn starch 65 g Crystalline cellulose 20 g Carboxymethyl cellulose calcium 3.5 g Light anhydrous silicic acid 0.5 g Magnesium stearate 1 g Flavonoid compound (Kemperol 10 g Total 100 g) According to the above formulation, are uniformly mixed with No. 18 sieve and 30 The powder passed through a No. Sieve was obtained as a powder, and 1 g of this powder contains 100 mg of flavonoid compound, and 1 to 6 g per day is taken by an adult in 3 divided doses after meals.

[0028]

INDUSTRIAL APPLICABILITY As described above in detail, flavonoid compounds contained in plants, particularly flavone, flavanone and their derivatives, have a strong trypsin activity inhibitory action,
Moreover, since it does not affect thrombin, it is useful as an antitrypsin agent for the prevention and treatment of acute and chronic pancreatitis, the prevention of transition to diabetes due to pancreatitis disease, and the prevention and treatment of thrombotic diseases. Can be used as a drug.

Claims (2)

[Claims] 1. An antitrypsin agent containing a flavonoid compound as an active ingredient. 2. The antitrypsin agent according to claim 1, wherein the flavonoid compound is a hydroxy and / or alkoxy derivative of flavone or a hydroxy derivative of flavanone.