TW201630598A - Flavonol skeleton-contained compound used in producing medicament for preventing and treating diseases related to activity of human chymosin or pharmaceutically acceptable salt thereof - Google Patents

Abstract

The present invention provides a novel medicament for preventing and treating diseases related to the activity of human chymosin. A human chymosin inhibitor containing a compound as shown in the following general formula (A) as an effective component shows selectivity for human chymosin and has an excellent enzyme activity inhibiting effect. Preferably, more than one component of this compound is selected from a group consisting of quercitrin, isoquercitrin, hyperoside, kaempferol and luteolin. A medicament containing the human chymosin inhibitor is a medicine used for preventing and treating various diseases related to the activity of human chymosin by means of a human chymosin activity inhibiting effect. (In the general formula (A), R1 to R5 respectively and independently represent hydroxyl or methoxyl, and X1 represents hydroxyl or -OG group. In addition, G represents a carbohydrate chain).

Description

Use of a flavonol skeleton-containing compound or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prophylaxis and treatment of a disease associated with activity of human chymosin

The present invention relates to a medicament for the prophylaxis and treatment of a human chymosin inhibitor and a disease associated with activity of human chymosin, and an application thereof.

The renin-angiotensin system is one of the endocrine mechanisms that maintain the constantity of systemic blood pressure, body fluid volume, and the like. In the renin-angiotensin system, angiotensin I is produced from angiotensinogen by renin, which is then converted to angiotensin II by angiotensin I. This conversion is caused by angiotensin-converting enzyme (ACE) in circulating blood.

Angiotensin II is known to be a major cause factor in hypertensive diseases due to its strong vasoconstriction. In addition, angiotensin II acts as a proliferation factor to promote the proliferation of fibroblasts, especially in cardiovascular diseases, involving hypertrophy of cardiomyocytes, migration/proliferation of smooth muscle cells, stimulation of intracellular matrix of fibroblasts, and cells. The regulation of a wide range of cellular functions, such as apoptosis induction, also plays an important role in the formation of fibrosis or nephrosclerosis and atherosclerotic plaque.

In this way, the final active peptide of the angiotensin II-based renin-angiotensin system can be expected to prevent or treat diseases caused by angiotensin II such as hypertension by inhibiting the production of angiotensin II.

As a pathway for producing angiotensin II, a pathway in which angiotensin II is produced from angiotensin I by angiotensin-converting enzyme (ACE) is known. Therefore, an ACE inhibitor which inhibits the activity of angiotensin-converting enzyme is a prophylactic and therapeutic agent for a disease caused by angiotensin II, and its effectiveness has been clarified in the clinic. However, it is also known that ACE inhibitors have side effects such as dry cough. In addition, although ACE inhibitors are considered to be useful for prevention or treatment of hypertension, there are cases in which ACE inhibitors are not recognized to be effective even if administered. Hypertension with antihypertensive effects (for example, hypertension caused by excessive salt intake).

On the other hand, human chymosin (hereinafter sometimes referred to as "chymosin") is an enzyme having an ability to produce angiotensin II other than ACE, and is produced in the production of angiotensin II in human tissues. It does not depend on ACE's "non-ACE dependency path" and is attracting attention.

The inventors of the present invention performed a single-strand extraction of an enzyme producing angiotensin II from angiotensin I from a human heart, and clarified that the human chymosin system is responsible for the production of angiotensin II by the chemical structure and gene selection of the enzyme ( Non-patent documents 1 to 3). In addition, the human chymosin system is thought to be produced in tissue-bound obese cells and stored in secretory granules, which are secreted by secretory granules and have physiological functions in the tissues. It is generally believed that in the blood vessel wall, when normal, human chymosin is more in the outer membrane, but instead is in the inner membrane. The ACE system is partially present, but when the blood vessel is impeded, the chymosin is involved in the remodeling of the outer membrane and the neointimal proliferation (Non-Patent Document 4). It has been reported that the non-ACE-dependent angiotensin II production pathway caused by this chymosin is higher in humans than in other animal species, and there is also angiotensin II in human heart. The production chymosin is responsible for about 80% of the reports (Non-Patent Document 5).

Human chymosin is mainly found in the secretory granules of human obese cells such as the heart, skin, blood vessel wall, and intestinal tube. If these tissues are disordered, they are secreted by secretory granules and released to the cells. In addition, angiotensin I in the tissue is converted into angiotensin II (Non-Patent Documents 3 and 6).

It is known that the angiotensin II system is associated with cardiovascular hypertrophy or remodeling depth, suggesting that human chymosin is closely related to cardiac/vascular lesions via the production of angiotensin II. Therefore, a human chymosin inhibitor which inhibits the activity of human chymosin can prevent or treat a disease caused by angiotensin II by specifically inhibiting the local production of angiotensin II in human tissues, in particular, various uses. A therapeutic agent for heart disease/vascular disease/kidney disease.

In addition, although the human chymosin differs in the amount of attached sugar chains due to its expression organization, the matrix specificity of the serine enzyme is consistent regardless of the tissue expression. For example, human chymosin extracted from the heart (see Non-Patent Document 1) and human chymosin extracted from skin or lung (see Non-Patent Documents 7 and 8) have enzyme-consistent properties. That is, produced by angiotensin I at approximately the same rate The vasoconstrictin II has the same isoelectric point or optimum pH.

To date, several human chymosin inhibitors have been reported. As a substance having an effect of inhibiting the activity of human chymosin, three have been reported Derivative (Patent Document 1), substituted aminopyrimidinylacetamide derivative (Patent Document 2), benzimidazole derivative (Patent Document 3), and the like.

In addition, the inventors of the present invention have reported that the chymosin inhibitor is used for the prevention or treatment of a disease accompanied by abnormal glucose tolerance (Patent Document 4) or a vascular dysfunction (Patent Document 5) by using curd together. The enzyme inhibitor and the ACE inhibitor are used for prevention or treatment of diseases of the circulatory system such as heart disease (Patent Document 6). Since the chymosin inhibitor inhibits the production of angiotensin II caused by the chymosin released from obese cells of various tissues by some stimulation, it is also effective for tissue lesions even by oral administration. The advantages of prevention/improvement of both systemic blood circulation dysfunctions (eg, hypertension).

Furthermore, the inventors of the present invention have reported that, by inhibition of chymosin activity, even the inhibition of the activity of angiotensin-converting enzyme (ACE) does not recognize the hypertension caused by excessive salt intake. It is also considered that there is a hypotensive effect (Non-Patent Document 5). In addition, hypertension caused by excessive salt intake is likely to become drug-resistant hypertension. Therefore, it is known that a hypertensive preventive agent (for example, an ACE inhibitor) of a known hypertensive disorder is rarely recognized as a blood pressure lowering effect or a blood pressure lowering effect is insufficient.

In addition, it has been suggested that the chymosin inhibitor is used as a preventive and therapeutic agent for a disease associated with the activity of human chymosin other than the above-mentioned diseases related to the production of angiotensin II. For example, since skin diseases such as atopic dermatitis, dryness, and urticaria are related to the activity of human chymosin, it is suggested that the chymosin inhibitor may become active by inhibiting the activity of human chymosin. A therapeutic drug for atopic dermatitis (see Non-Patent Document 9 and Non-Patent Document 10).

[Previous Technical Literature] [Patent Document]

[Patent Document 1] Japanese Patent Laid-Open No. Hei 8-208654

[Patent Document 2] Japanese Patent Laid-Open Publication No. 2003-104984

[Patent Document 3] Japanese Patent Laid-Open Publication No. 2001-199983

[Patent Document 4] International Publication No. 2005/018672

[Patent Document 5] International Publication No. 2001/032214

[Patent Document 6] International Publication No. 2003/018061

[Non-patent literature]

[Non-Patent Document 1] Urata H et al., The Journal of Biological Chemistry. 1990; 265: 222348-22357

[Non-Patent Document 2] Urata H et al., The Journal of Biological Chemistry. 1991; 266: 17173-17179

[Non-Patent Document 3] Urata H et al., The Journal of Clinical Investigation. 1993; 91: 1269-1281

[Non-Patent Document 4] Uehara Y et al., Hypertension. 2000; 35: 55-60

[Non-Patent Document 5] Urata H et al., Circulation Reaserch. 1990; 66: 883-890

[Non-Patent Document 6] Urata H et al., Journal of Hypertension. 1994; 12: S17-22

[Non-Patent Document 7] Reilly CF et al., J Biol Chem. 1982; 257: 8619-8622

[Non-Patent Document 8] Wintroub BU et al., J Clin Invest. 1986; 77: 196-201

[Non-Patent Document 9] Tani K et al., Journal of Leukocyte Biology, 2000, 67, 585-589

[Non-Patent Document 10] Tomimori Y et al., Biochemical Pharmacology, 2002, 64, 1187-1193

On the other hand, the compound which is an active ingredient of the chymosin inhibitor reported so far is not sufficient for the activity of human chymosin, or it is often difficult. In addition, the actual situation is the prevention or treatment effect on the above-mentioned diseases related to the activity of human chymosin against the component having the activity inhibition effect of human chymosin contained in the chymosin inhibitor, and the animal has been utilized so far. The empirical data of the experimental data or human clinical data is still not sufficient, for this component In fact, the verification of whether the disease has a preventive or therapeutic effect is not sufficient.

Under such circumstances, an object of the present invention is to provide a use of a specific compound for the manufacture of a medicament for the prophylactic treatment of a disease associated with the activity of human chymosin.

In order to solve the above-mentioned problems, the inventors of the present invention have intensively studied and found a compound having a specific skeleton which has not been reported to the activity inhibition effect of human chymosin, and which exhibits excellent activity against human chymosin. Obstructing the effect, and completing the following invention.

Further, other aspects of the invention relate to the following invention.

<1> Use of a compound represented by the following general formula (A) or a pharmaceutically acceptable salt thereof for the production of a medicament for the prophylaxis and treatment of a disease associated with activity of human chymosin;

(In the general formula (A), R ¹ to R ⁵ each independently represent a hydrogen atom, a hydroxyl group or a methoxy group, X ¹ represents a hydrogen atom, a hydroxyl group or a -OG group, and G represents a sugar chain).

<2> The use according to <1>, wherein, in the compound represented by the general formula (A), X ¹ is a hydroxyl group, a rhamnose residue, a glucose residue, or a galactose residue.

<3> The use according to <1> or <2>, wherein the compound represented by the general formula (A) is from quercetin, isoquercitrin, hyperoside, kaempferol and luteolin. One or more selected from the group consisting of.

The use of the compound of the formula (A) is at least 1 selected from the group consisting of quercetin glycoside and quercetin derivatives, as described in any one of <1> to <3>. Kind.

<5> The use according to <1> to <4>, wherein the compound represented by the general formula (A) is hyperoside.

The use according to any one of <1> to <5>, wherein the disease associated with the activity of human chymosin is a disease associated with angiotensin II production.

<7> The use according to <6>, wherein the disease associated with the production of angiotensin II is hypertension and/or a disease caused by hypertension.

<8> The use according to <7>, wherein the hypertensive condition is hypertension caused by excessive salt intake.

<9> The use according to <7> or <8>, which comprises at least a hyperoside and is administered orally to a human in a manner of 0.1 mg/kg body weight/day or more as a method for A compound of the general formula (A).

The use according to any one of <1> to <5>, wherein the disease associated with the activity of human chymosin is a disease which is not associated with angiotensin II production.

<11> The use according to <10>, wherein the disease which is not related to angiotensin II production is atopic dermatitis, cognac and urticaria, and a group consisting of any combination of the above Selected skin diseases.

<12> The use according to <11>, wherein the skin disease is atopic dermatitis.

Further, the present invention relates to the following human chymosin inhibitor and an agent for the prophylactic treatment of a disease associated with activity of human chymosin containing the human chymosin inhibitor.

<1a> A human chymosin inhibitor which comprises a compound represented by the following general formula (A) as an active ingredient;

(In the general formula (A), R ¹ to R ⁵ each independently represent a hydrogen atom, a hydroxyl group or a methoxy group, X ¹ represents a hydrogen atom, a hydroxyl group or a -OG group, and G represents a sugar chain).

<2a> A medicament for the prophylaxis and treatment of a disease associated with the activity of human chymosin, which comprises the human chymosin inhibitor of <1a>.

The agent according to <2a>, wherein the disease associated with the activity of human chymosin is a disease associated with angiotensin II production.

<4a> The agent according to <3a>, wherein the disease associated with the production of angiotensin II is hypertension and/or a disease caused by hypertension.

<5a> The agent according to <4a>, wherein the hypertensive condition is hypertension caused by excessive salt intake.

<6a> The agent according to <4a> or <5a>, which contains at least a hyperoside and is administered orally to a human in a manner of 0.1 mg/kg body weight/day or more as a method for A compound of the general formula (A).

The agent according to <2a>, wherein the disease associated with the activity of human chymosin is a disease which is not associated with angiotensin II production.

<8a> The agent according to <7a>, wherein the disease which is not related to angiotensin II production is atopic dermatitis, cognac and urticaria, and a group consisting of any combination of the above Selected skin diseases.

According to the present invention, it is possible to provide a medicament for the prophylaxis and treatment of a disease associated with the activity of human chymosin, which exhibits an excellent enzyme activity inhibitory effect on human chymosin due to a compound contained as an active ingredient. Therefore, the use of the preventive therapeutic agent is prevention and treatment of a disease associated with activity of human chymosin.

Fig. 1 is a result of evaluation of the dependence of the amount of hyperoside of systolic blood pressure (SBP) in salt-dependent hypertensive mice (6 rats) (8 weeks).

Fig. 2 is a result of evaluation of the dependence of the amount of hyperoside of dilated blood pressure (DBP) in salt-dependent hypertensive mice (6 rats) (8 weeks).

Fig. 3 is a result of evaluation of the dependence of the amount of hyperoside on the heart rate of salt-dependent hypertensive mice (8 weeks).

Fig. 4 is a result of evaluation of the dependence of the amount of leeches of the systolic blood pressure (SBP) of the saline-dependent hypertensive mice (8 weeks).

Fig. 5 is a result of evaluation of the dependence of the amount of dried leech on the dilated blood pressure (DBP) of the salt-dependent hypertensive mice (8 weeks).

Fig. 6 is a result of evaluation of the dependence of the amount of dried leeches on the heart rate of salt-dependent hypertensive mice (8 weeks).

Figure 7 is an evaluation of the dependence of the amount of leeches of the systolic blood pressure (SBP) on humans.

Fig. 8 is a result of evaluation of the dependence of the amount of leech dry matter relative to human dilated blood pressure (DBP).

Fig. 9 is a result of evaluation of the dependence of the amount of leech dry matter relative to the number of human heart beats.

Figure 10 is a skin condition for the symptoms of atopic dermatitis A sample of the questionnaire (DLQI: Dermatology Life Quality Index).

Fig. 11 shows the results of a questionnaire on skin condition (DLQI) before and after administration of dried leech.

Fig. 12 shows the evaluation results of the eosinophil number before and after the leech dry product was taken.

In the following, the present invention is described in detail with reference to the embodiments of the present invention, and the present invention is not limited thereto, and may be arbitrarily changed and carried out without departing from the scope of the invention.

<1. Disease associated with human chymosin activity>

In the present specification, "a disease associated with the activity of human chymosin" means a disease in which symptoms are manifested, aggravated, and progressed due to an increase in the enzyme activity of human chymosin.

Diseases associated with the activity of human chymosin are roughly classified into diseases associated with angiotensin II production and diseases not associated with angiotensin II production.

The disease associated with the production of angiotensin II means a disease caused by angiotensin II, which causes symptoms, aggravation, and progression. Specific examples include hypertension, diseases caused by hypertension, diseases caused by abnormal sugar tolerance, and the like.

In this specification, "hypertension" is caused by The incidence of hypertensive disease such as excessive intake of salt, lack of exercise, smoking, drinking, and accumulation of stress, and those who are likely to be sick (slightly high blood pressure relative to healthy people). Further, specific examples of the diseases caused by hypertension include hypertensive arteriosclerotic diseases and hypertensive circulatory system diseases.

Examples of the hypertensive arteriosclerotic disease include a disease in which vascular function abnormality is caused by deposition of lipids into blood vessels, a disease in which symptoms are deteriorated due to abnormal vascular function due to lipid deposition of blood vessels, and lipid deposition due to blood vessels. For example, a disease that causes abnormal vascular function and delays healing. Specifically, for example, acute coronary artery syndrome such as unstable angina or acute myocardial infarction, restenosis after percutaneous coronary artery formation, occlusive atherosclerosis, occlusive thromboangiitis, porridge brain Arteriosclerosis, cerebral infarction, intermittent gangrene, gangrene of the lower extremities, renal vascular hypertension, renal aneurysm, and renal infarction are associated with lipid deposition of blood vessels, causing abnormal vascular function.

As a hypertensive circulatory system disease, it contains all hypertensive organ disorders. Specific examples include heart diseases such as coronary artery disease, cardiac insufficiency, and arteriosclerotic valvular disease; brain diseases such as cerebrovascular diseases; upper extremity/lower extremity occlusive arteriosclerosis, including hypertensive nephropathy and hypertensive diabetes For example, an arteriosclerotic disease of systemic blood vessels such as chronic kidney disease of renal disease.

Further, as a disease belonging to a disease associated with the production of angiotensin II, which is caused by an abnormal glucose tolerance, diabetes and/or diabetic complications may be mentioned. In diabetic complications, including diabetic nephropathy, Diabetic omental disease, diabetic peripheral neuropathy, hyperinsulinemia, insulin resistance syndrome, atherosclerosis, acute coronary artery syndrome, occlusive atherosclerosis, vasculitis, cerebral infarction, hypertension, renal insufficiency, kidney Symptoms, renal aneurysms, renal infarction, or obesity.

As a blood-related disease associated with the activity of human chymosin Tube contractile hormone II produces irrelevant diseases, including skin diseases such as atopic dermatitis, dryness, and urticaria; fibrosis related to organ diseases such as fatty liver, cirrhosis, and pulmonary fibrosis; Crohn's disease And ulcerative colitis and other diseases of the digestive system; etc. In addition, even in diseases related to the activity of human chymosin, skin diseases such as atopic dermatitis, dryness, and pigmented urticaria are highly related to the increase in the activity of human chymosin.

<2. Use of a compound represented by the general formula (A) for producing a medicament for prophylactic treatment of a disease associated with activity of human chymosin>

The present invention relates to a compound of the general formula (A) for use in the manufacture of a medicament for the prophylaxis and treatment of a disease associated with activity of human chymosin, or a pharmaceutically acceptable salt thereof (hereinafter, referred to as "the use of the present invention" ").

(In the general formula (A), R ¹ to R ⁵ each independently represent a hydrogen atom, a hydroxyl group or a methoxy group, X ¹ represents a hydrogen atom, a hydroxyl group or a -OG group, and G represents a sugar chain).

In the use of the present invention, a compound represented by the general formula (A) or a pharmaceutically acceptable salt thereof is used for the production of a medicament for the prophylaxis and treatment of a disease associated with the activity of human chymosin (hereinafter, it is described as "Prophylactic and therapeutic agent of the present invention" or "agent of the present invention"). In addition, in the present invention, the "prevention drug" means a drug which is useful for at least one of prevention, treatment, and improvement of symptoms of the target disease. In addition, in this specification, "pharmaceutical" means not only a pharmaceutical product but also a medical product.

The compound represented by the general formula (A) also includes a suitable aspect thereof, which is described later in <3. A compound represented by the general formula (A) and a human chymosin inhibitor containing the compound. Further, the compound represented by the general formula (A) has a flavonol skeleton or a flavonol-like skeleton, and is common in terms of substitution by a similar functional group. In addition, in the present specification, the compound represented by the general formula (A) may be referred to as "compound (A)" hereinafter.

The compound (A) according to the present invention has an action of inhibiting the activity of human chymosin. Therefore, the compound (A) can be used as an active ingredient of a human chymosin inhibitor. Hereinafter, the human chymosin inhibitor containing the compound (A) as an active ingredient may be described as "the human chymosin inhibitor of the present invention" or "the chymosin inhibitor of the present invention".

Thus, the prophylactic and therapeutic agent of the present invention is contained The compound (A) is a chymosin inhibitor of the present invention and has a prophylactic and therapeutic effect on a disease associated with the activity of human chymosin. In other words, the preventive therapeutic agent of the present invention may contain the compound (A) which is a substantial active ingredient. Therefore, the chymosin inhibitor of the present invention can be used as a prophylactic and therapeutic agent for diseases related to the activity of human chymosin, and in this case, the chymosin inhibitor of the present invention and the preventive treatment of the present invention The medicaments are not substantially different.

Since the prophylactic and therapeutic agent of the present invention comprising the compound (A) (including the chymosin inhibitor of the present invention) can inhibit the activity of human chymosin, it can be prevented from being treated by humans by administering it to humans. A disease associated with the activity of chymosin.

The details of the human chymosin inhibitor of the present invention and the preventive therapeutic agent of the present invention include the aspect and the method of use thereof, and are described later in <3. The compound represented by the general formula (A) and the compound containing the same. Human chymosin inhibitor>.

<3. A compound represented by the general formula (A) and a human chymosin inhibitor containing the same>

The human chymosin inhibitor of the present invention contains the compound represented by the above general formula (A) or a pharmacologically acceptable salt of the compound as an active ingredient.

R ¹ to R ⁵ in the general formula (A) each independently represent a hydrogen atom, a hydroxyl group or a methoxy group, and X ¹ represents a hydrogen atom, a hydroxyl group or an -OG group. Here, G is a sugar chain, and the details will be described later.

In the above general formula (A), the number of hydroxyl groups or methoxy groups constituting R ¹ to R ⁵ is not limited, but a suitable hydroxyl group is preferably 3 or more, and more preferably a hydroxyl group of 4 or more.

In the above general formula (A), X ¹ is a hydrogen atom, a hydroxyl group or a -OG group. In the case where X ¹ is an -OG group, the sugar chain is glycosidically bonded via an oxygen atom.

The structure of the sugar chain and the type of the sugar constituting the sugar chain are not particularly limited. The number of sugars constituting the sugar chain is not particularly limited, but is usually 1 to 4, preferably 1 or 2. Further, in the case where the number of sugar chains is 1, a so-called monosaccharide residue is used. The sugar constituting the sugar chain may be a five-carbon sugar or a six-carbon sugar.

The hydroxyl group contained in the above sugar chain may also be modified by other groups. For example, the above hydroxyl group may be esterified as described later or alkoxylated.

Specific examples of the sugar constituting the sugar chain include ribose, glucose, mannose, galactose, rhamnose, xylose, arabinose, glucuronic acid, rutinose, sophorose, and sophorose. Gentiobiose, sambubiose, lathyrose, laminaribiose, gentiotriose, robinobiose, and the like.

The sugar may also be a sugar (organic acid-bonded sugar) in which an organic acid is bonded alone or in multiple esterification. The bonding position of the organic acid is not particularly limited. Specific examples of the bonding position of the organic acid include For example, one or more of the 2, 3, and 6 positions of the sugar. The type and configuration of the above organic acid are not particularly limited. The above organic acid may be an aromatic organic acid or an aliphatic organic acid.

In terms of excellent human chymosin inhibitory activity, it is preferred that in the general formula (A), X ¹ is a hydrogen atom, a hydroxyl group, a rhamnose residue, a glucose residue or a galactose residue. In this case, the number of hydrogen atoms, hydroxyl groups or methoxy groups constituting R ¹ to R ⁵ is not limited, but a hydroxyl group is preferably 3 or more.

In the chymosin inhibitor of the present invention, the compound (compound (A)) represented by the general formula (A) preferably contains quercetin or quercetin which has the chemical formula shown below. Any one of the body and quercetin derivatives is an active ingredient, and particularly preferably a quercetin glycoside or quercetin derivative which is excellent in absorption in the body. Examples of the quercetin glycoside and the quercetin derivative include quercitrin, isoquercitrin, and hyperoside.

In addition, in the chymosin inhibitor of the present invention, examples of the compound (A) other than the quercetin, the quercetin glycoside, and the quercetin derivative include galangin and kaempferol. (kaempferol), myricetin, luteolin.

The compound (A) which is an active ingredient of the chymosin inhibitor of the present invention has an action of inhibiting the activity of human chymosin. In addition, The evaluation method is any as long as it can judge whether or not the enzyme activity of human chymosin is inhibited. A suitable example of the evaluation method for inhibiting the activity of human chymosin will be described later in the examples.

In the chymosin inhibitor of the present invention, the compound (A) may be contained alone or in combination of two or more. Even among the compound (A), it is selected from the group consisting of quercetin, isoquercitrin, hyperoside, kaempferol and luteolin which have excellent chymosin inhibitory activity. One or more of them are preferred as the active ingredient.

Further, since quercetin, isoquercitrin, and hyperoside are excellent in the inhibition of chymosin activity, and X ¹ has a functional group having a sugar chain, high bioabsorbability can be expected. Therefore, the chymosin inhibitor of the present invention preferably contains at least one of quercetin, isoquercitrin and hyperoside.

Specifically, since the hyperoside chymosin activity is particularly excellent and has high bioabsorbability, the chymosin inhibitor of the present invention preferably contains hyperoside.

The compound (A) may be a synthetic or a natural one. In the case where a natural product is used as a raw material, the extract obtained from the raw material animal and plant by a known extraction method may be used as it is, or may be further subjected to separation and purification.

Examples of the natural product of quercetin containing one of the compounds suitable as the compound (A) include citrus, onion, buckwheat, apple, green tea, broccoli, sorghum jute, and leeches. Further, as a natural product of a large amount of quercetin containing one of the same suitable compounds, fish may be mentioned Valerian, ginkgo, leeches, etc. Further, as a natural product of a large amount of isoquercitrin which contains one of the same suitable compounds, mango, dandelion, leeches and the like can be mentioned. Further, examples of the natural product containing a large amount of hyperoside (quercetin-3-O-galactoside) include daffodil, hypericum, camphor, and leeches. The site which can be used as such a raw material plant is not particularly limited, and examples thereof include an aerial part, a root portion, and a fruit. These raw material plants may contain one or more compounds (A) as a material, and may be used as an unprocessed sample, or as a dried product or an extract.

In the present specification, "dry matter" means a product obtained by drying a raw material plant. Specifically, the raw material plants are dried by a known drying method. From the viewpoint of usability, the dried product is usually pulverized and pulverized, and is used in the form of a dried pulverized material. The pulverization method is not particularly limited, and any conventionally known pulverizer may be used. The particle size of the powder is suitably determined depending on the intended use.

In the present specification, the term "extracted material" refers to a form in which the raw material of the raw material to be extracted is subjected to solvent extraction as it is, or if it is dried and finely cut as necessary, and the content of the active ingredient is increased. Concept of the person.

In addition, these compounds are also contained in the form of a pharmacologically acceptable salt, and the form of the salt is not particularly limited, and examples thereof include an alkali metal salt (sodium salt, potassium salt, lithium salt, etc.) and an alkaline earth metal. Salt (calcium salt, magnesium salt, etc.), inorganic salt (acetate, ammonium salt, etc.), organic amine salt (dibenzylamine salt, glucosamine salt, ethylenediamine salt, diethylamine salt, triethylamine) Salt, dicyclohexylamine salt, diethanolamine salt, tetramethylammonium salt Etc.), an amino acid salt (glycinate, a persalt, arginine, alanine, aspartate, etc.).

Since the chymosin inhibitor of the present invention can inhibit the activity of human chymosin, it can prevent the treatment of diseases associated with the activity of human chymosin. Since human chymosin is an enzyme that produces angiotensin II from angiotensin I independently of ACE, a medicament containing a chymosin inhibitor of the present invention capable of inhibiting the activity of human chymosin can be used as a so-called " A non-ACE-dependent angiotensin II production inhibitor, which inhibits the production of angiotensin II due to inhibition of human chymosin activity, and can be used as a prophylactic agent for the prevention and treatment of a disease caused by angiotensin II. .

Further, in the disease associated with the activity of human chymosin, a disease other than the disease caused by angiotensin II is also included, and the agent containing the chymosin inhibitor of the present invention can also be used as the cause of angiotensin II. A medicament for the prevention and treatment of diseases other than diseases.

The specific type of disease associated with the activity of human chymosin is as described above in <1. Disease associated with activity of human chymosin>.

In addition, animals with chymosin are also present in humans. For example, several chymosins have been reported in mice. Among them, mouse mast cell protease (MMCP) 4 has the same ability to produce angiotensin II as human chymosin and has no vasoconstrictin II decomposition ability (Science.1996;271 :502-505). That is, MMCP4 is angiotensin II producing lysine. Therefore, mice can be used to perform activities related to human chymosin activity. Disease model experiment.

On the other hand, there is no chymosin having the same properties as human chymosin in rats used in many animal experiments involving hypertension, and a model experiment on diseases related to human chymosin activity Not suitable. The foregoing refers to the fact that rat chymosin decomposes angiotensin I or II more rapidly than the production of angiotensin II (The Journal of investigative dermatology. 1984; 83: 336-339).

(form of chymosin inhibitor)

The chymosin inhibitor of the present invention may be formulated in an effective amount together with a pharmaceutically acceptable carrier, orally or parenterally, in the form of a solid preparation or a liquid preparation. Any dosage form may be used if it is usually administered orally or parenterally.

The dosage form used for oral administration or parenteral administration is specifically a powder preparation, a granule, a tablet, a capsule, an ingot or the like. Further, examples of the liquid preparation include an internal liquid preparation, an external liquid preparation, a suspension, an emulsion, a syrup, an injection, an infusion, and the like, and these dosage forms or other dosage forms are appropriately selected depending on the purpose.

In the solid preparation, an auxiliary agent such as an excipient, a binder, a disintegrant, a lubricant, a flavoring agent, or a stabilizer may be used as the main inhibitor of the chymosin inhibitor of the present invention. The ratio of the main agent to the adjuvant is suitably selected depending on the purpose.

As a suitable example of the excipient in the solid preparation, lactose, D-mannitol, starch, etc. are mentioned, for example. As a suitable example of the binder, it can be enumerated For example, crystalline cellulose, white sugar, D-mannitol, dextrin, hydroxypropyl cellulose, and the like. Suitable examples of the disintegrator include starch, carboxymethylcellulose, carboxymethylcellulose calcium, and the like. In addition, preparations such as preservatives, viscosity-enhancing stabilizers, coloring agents, and sweeteners may also be used as needed.

Further, when it is used in the form of a liquid preparation, as a solvent, the dispersibility of the above-mentioned compound as an active ingredient is selected, and it is biosafe. As a suitable example of a solvent, water for injection, ethanol, propylene glycol, etc. are mentioned, for example.

Further, the liquid preparation may further contain an auxiliary component such as a dissolution aid, a suspending agent, an isotonic agent, a buffering agent, a viscosity increasing stabilizer, etc. together with the chymosin inhibitor of the present invention as a main component.

Suitable examples of the dissolution aid include ethanol, polyethylene glycol, propylene glycol, benzyl benzoate, sodium carbonate, sodium citrate, and the like. Suitable examples of the suspending agent include sodium lauryl sulfate, lauryl alanine propionate, lecithin, benzalkonium chloride, benzethonium chloride, and single hard. Glycerylglycerol, polyvinyl alcohol, polyvinylpyrrolidone, hydroxymethylcellulose, and the like. A suitable example of the buffering agent is a buffer solution such as phosphate, acetate or carbonate. A suitable example of the viscosity-enhancing stabilizer is sulfite or ascorbate.

Further, in the chymosin inhibitor of the present invention, a known optional component may be contained within a range in which the activity inhibiting action is not inhibited. For example, a stabilizer, a wetting agent, or an emulsifier may be added, or a salt may be suitably used as an auxiliary in the form of an osmotic pressure adjusting agent or a pH adjusting agent. medicine.

The preparation method of the above solid preparation or liquid preparation can be applied to a general pharmaceutical product or a method of producing a pharmaceutical product.

The chymosin inhibitor of the present invention may be appropriately selected from a dosage form, an administration amount, and a administration method depending on the type or symptom of the disease associated with the activity of human chymosin.

In addition, the content of the above-mentioned compound (A) in the chymosin inhibitor of the present invention is an ingestible activity, such as an activity inhibitory action or a administration method necessary for the prevention, treatment, or symptom improvement of the target disease. It is appropriate to decide within the scope of the necessary amount. In addition, the method of administering the chymosin inhibitor of the present invention may be any one, and may include a choice of oral administration or parenteral administration, and is appropriately determined depending on the target disease.

Further, the compound (A) which is an active ingredient of the chymosin inhibitor of the present invention also contains a compound which lacks bioabsorbability. For example, although quercetin is water soluble, the bioabsorbability is not high. In the compound (A) which lacks bioabsorbability, since the above-mentioned pharmacological effects by the chymosin inhibitory action cannot be expected, it is preferable to use a bioabsorber for the purpose of improving bioabsorbability. The bioabsorption enhancer may be contained in the form of a component of the agent of the present invention, or may be used in combination with the agent of the present invention to use a bioabsorber.

The bioabsorber can be used as long as it has an effect of improving the bioabsorbability of the compound (A), and examples thereof include oils and fats and citrates. The oils and fats are not particularly limited, but may be obtained from cotton oil, soybean oil, palm oil, corn oil, sunflower oil, rapeseed oil, sesame oil, and the like. Vegetable oils; or animal fats obtained from fish such as sardines, mackerel, or livestock such as cattle and pigs. Examples of the citrate include sodium citrate, potassium citrate, and iron citrate. These bioabsorption enhancers may be used alone or in combination of two or more.

Further, the chymosin inhibitor of the present invention may contain an effective ingredient as another medicine in addition to the aforementioned active ingredient in the treatment of various diseases.

Further, in the case where the chymosin inhibitor of the present invention is to be ingested by a daily diet, it may be contained in the food or drink in the form of a functional food. The term "functional food" as used herein means foods and/or beverages that are ingested for health purposes, such as health foods, nutritional supplements, nutritional functional foods, and nutraceutical foods, in addition to general foods. In addition, in the case of productized in the form of functional foods, various additives for foods may be added, specifically, coloring materials, preservatives, viscosity increasing stabilizers, antioxidants, bleaching agents, and antibacterial agents. Mold inhibitors, sour materials, seasonings, emulsifiers, fortifiers, manufacturing agents, perfumes, and the like.

(Application of chymosin inhibitor)

The agent for the prophylactic treatment of a disease associated with the activity of human chymosin (hereinafter referred to as "the agent for prophylactic treatment of the present invention" or "the agent of the present invention") may contain the above-described chymosin of the present invention. Obstruction agent.

In the present specification, the "prophylactic drug for therapeutic use" means a drug which is useful for at least one of prevention, treatment, and improvement of symptoms of a target disease. In addition, in this specification, "pharmaceutical" means It not only contains pharmaceuticals, but also contains foreign products from the Ministry of Medicine.

The agent of the present invention may contain other agents or pharmacologically acceptable optional components in addition to the chymosin inhibitor of the present invention within a range that does not impair its efficacy. Further, the agent of the present invention may contain the compound (A) which is a substantial active ingredient, and the chymosin inhibitor of the present invention may be used as the agent of the present invention.

As described above, the chymosin inhibitor of the present invention contains the above compound (A) as an active ingredient, and the compound (A) has an action of inhibiting the activity of human chymosin. Therefore, the agent for prophylactic treatment of the present invention is useful for at least one of prevention, treatment, and improvement of symptoms of a disease associated with activity of human chymosin. In addition, the disease related to the activity of human chymosin which is a target of the prophylactic and therapeutic agent of the present invention is as described above, which is related to the activity of human chymosin.

The compound (A) contained in the agent of the present invention inhibits the activity of human chymosin. Thus, human chymosin produces angiotensin II from angiotensin I independently of ACE. Therefore, the agent of the present invention is a non-ACE-dependent angiotensin II production inhibitor, inhibits the production of angiotensin II, and can prevent the treatment of diseases associated with angiotensin II. Further, examples of the diseases associated with angiotensin II include hypertension, diseases caused by hypertension, diseases caused by abnormal glucose tolerance, and the like, and the details are as follows: <1. Related to the activity of human chymosin Disease > above.

The agent of the present invention can be used as a blood having excellent blood pressure caused by hypertensive disorder, particularly excessive salt intake. A blood pressure lowering agent that acts under pressure drop. In addition, the "blood pressure lowering action" in the present specification means a blood pressure lowering effect on the onset of hypertension and the person who is likely to have a disease (a person with mild hypertension as compared with a healthy person). That is, for the onset of hypertension and the person who is likely to suffer from the disease, it is restored to a near normal blood pressure by the action of blood pressure lowering.

In addition, hypertensive disorder caused by excessive salt intake is likely to become drug-resistant hypertension. If long-term continuous use of ACE inhibitors or blood pressure inhibitors such as angiotensin-receptor antagonist (ARB), the blood pressure lowering effect is mostly weakened. .

Since the agent of the present invention does not inhibit the production of angiotensin II by the ACE pathway, but inhibits the production of angiotensin II produced by the non-ACE pathway of human chymosin by hindering the activity of human chymosin As a mechanism, even if it is a hypertensive disorder caused by excessive salt intake, an effective blood pressure lowering effect can be recognized.

Therefore, the agent of the present invention is particularly suitable as a prophylactic or therapeutic agent for a disease caused by hypertensive disease caused by excessive intake of salt or a disease caused by hypertension.

Further, when the agent of the present invention is used as a blood pressure lowering agent, it may be used in combination with an ACE inhibitor or an angiotensin II receptor antagonist in the case where a synergistic preventive/therapeutic effect can be expected.

Although the agent of the present invention is a non-ACE-dependent angiotensin II production inhibitor by the inhibition of chymosin activity, it is used in combination with an ACE inhibitor, and the ACE inhibitor is used alone. Or a chymosin inhibitor, which completely inhibits the production of angiotensin II in the tissue, maintains the inhibition of vasopressin breakdown by the ACE inhibitor, and inhibits the various types of intermediate chymosin. Physiological effects, and the synergistic therapeutic effect, is effective.

Further, as an agent which is frequently used as a conventional blood pressure lowering agent, there is a calcium antagonist. This drug system is often accompanied by a reflexive frequency pulse due to its strong vasodilatability. Japanese epidemiological studies have shown that if the absolute number or variation of the heartbeat increases, the average remaining life is shortened (for example, Tianzhu Pill Research or Daqiang Research).

Therefore, in consideration of the long-term prognosis of the general nationals, it is desired to develop a blood pressure lowering agent that reduces the number of heartbeats. In the blood pressure lowering agent currently used, an ACE inhibitor or an angiotensin II receptor antagonist is known as a drug which does not increase the number of heart beats even after the blood pressure reduction, but the effect of reducing the number of heartbeats has not been affected. prove. Due to this background, it is desirable to develop antihypertensive drugs that reduce the number of heartbeats while sufficiently reducing blood pressure. As the only agent that is accompanied by blood pressure reduction and the number of heartbeats is reduced, there is a beta blocker, but the actual situation is that the side effects involve many aspects (excessive Xumai/asthmatic induction/crown contracture stenosis/insulin secretion reduction/lipid metabolism) Deterioration, etc.) has limited practical use in clinical practice.

When the dose of the agent of the present invention is used as a blood pressure lowering agent, it may be selected as long as it exhibits a blood pressure lowering action. However, a typical example is given based on the compound (A). A method of orally administering to humans in an amount of 0.01 mg/kg body weight per day or more.

Further, the drug of the present invention exhibits a heartbeat number lowering action together with a blood pressure lowering action by administering a specific amount or more of the compound (A).

In the present specification, the "heartbeat number reduction effect" refers to an effect of lowering the number of heartbeats, and specifically, an effect of lowering the number of heartbeats by increasing the activity of the sympathetic nervous system.

In addition, the cause of hypertension caused by excessive salt intake and the possibility that the person suffering from the disease may have an increase in the activity of the sympathetic nervous system and increase the number of original heartbeats, but the agent of the present invention has such an increase. The effect of the number of heartbeats is reduced. As shown in the examples described later, in addition to the significant blood pressure lowering effect by the oral administration of the agent of the present invention, a significant decrease in the number of heart beats was observed as a result of the clinical experiment. In the case of a conventional antihypertensive drug, the number of heartbeats generally increases or does not change with a decrease in blood pressure. Therefore, the "heartbeat number reduction effect" is specific to the agent of the present invention.

The hypertensive disease which is a target of the agent of the present invention is not particularly limited, but even in hypertensive patients, hypertensive disorder caused by excessive salt intake is one of the particularly effective hypertensive agents of the present invention. Hypertension caused by excessive salt intake is likely to become drug-resistant hypertension. Therefore, in the case of a hypertensive disorder caused by excessive salt intake, a conventional blood pressure prophylactic agent (for example, an ACE inhibitor) is rarely recognized as a blood pressure lowering effect, or a blood pressure lowering effect is insufficient. On the other hand, the agent of the present invention can be considered to have a significant blood pressure lowering effect on hypertension caused by excessive salt intake. Here is "significant blood The effect of the pressure drop means that a statistically significant (risk ratio P < 0.05) low value is obtained when the blood pressure in the placebo period is compared with the blood pressure in the intake period of the agent of the present invention.

Further, the agent of the present invention does not necessarily need to be used for the purpose of lowering blood pressure, and may be used as a heartbeat number lowering agent which reduces the number of heart beats caused by administration of a specific amount of the compound (A). In this case, in addition to hypertension, it is also suitable for use in a case where the number of heartbeats in the disease in which the number of heartbeats increases is increased (inhibiting the increase in the number of heartbeats). Then, in addition to the compound (A), other pharmaceutical ingredients which are effective as a treatment for the target disease can be used in combination.

In addition, in the case where the target disease is hypertensive, the preventive/therapeutic effect of the multiplication can be expected, and it can also be used together with a well-known blood pressure lowering agent. Examples of known blood pressure lowering agents include ACE inhibitors, angiotensin II receptor antagonists, calcium channel antagonists, and diuretics (including thiophenes). A blood pressure lowering agent such as a diuretic/loop diuretic/potassium-retaining diuretic), an alpha blocker, a beta blocker, and an alpha beta blocker.

The oral administration amount for exhibiting a decrease in the number of heart beats together with the blood pressure lowering effect depends on the kind of the compound (A), and is 0.1 mg/kg body weight in terms of the amount of the hyperoside in humans. More preferably, it is 0.12 to 0.35 mg/kg body weight/day in terms of the weight of the hyperoside.

Here, "the hyperoside conversion weight" means that in the case of hyperoside, the weight of the hyperin itself is equivalent to the converted weight, and in the gold wire The case of the compound (A) other than the amyloside is the converted weight calculated from the relative value of the human chymosin inhibition rate caused by 0.5 mM hyperoside.

Further, as described above, in the case of using the compound (A) derived from a natural product, a plurality of compounds (A) may be contained in the raw material plant. For example, in detail, in the examples, Persicaria hydropiper contains hyperoside as a main component, and in addition to hyperoside, a small amount of quercetin, quercetin and the like are equivalent to the compound (A). The ingredients. In the case of using the raw material plant as the compound (A) contained in the agent of the present invention, the amount of the drug to be converted into a hyperoside conversion weight may be determined.

In addition, the agent of the present invention can be used as a drug which is useful for at least one of prevention, treatment, and improvement of symptoms of a disease which is not associated with angiotensin II production in a disease associated with activity of human chymosin. The disease which is not related to the production of angiotensin II is, for example, the disease associated with the activity of human chymosin, and the skin diseases such as atopic dermatitis, dryness, and measles; fatty liver, Hepatic cirrhosis and pulmonary fibrosis and other fibrosis related to organ disorders; Crohn's disease and ulcerative colitis and other diseases of the digestive system;

In addition, the dose of the agent of the present invention in the case of a disease to be treated for the prevention and treatment of the present invention is appropriately selected in consideration of not only the type of the disease but also individual differences such as the age and sex of the subject.

The agent of the present invention, even in the above, and angiotensin II Among the diseases that are not related, it is particularly useful for at least one of prevention, treatment, and improvement of symptoms of skin diseases such as atopic dermatitis. When the agent of the present invention is used as a medicament for the prophylactic treatment of skin diseases, any of the dosage forms may be used if it is usually administered orally or parenterally. In the oral administration, the agent of the present invention is used for the prevention, treatment, and symptom improvement of atopic dermatitis, and has little toxicity or irritation to the human body, and has few side effects. Therefore, in the case of the agent of the present invention, a dosage form for oral administration which can be taken daily is one of suitable forms. The specific dosage form is described in the above (form of the chymosin inhibitor), and thus the description thereof will be omitted.

In addition, when it is applied to a skin-type skin external preparation as a dosage form for parenteral administration, it is generally in the form of a liquid or a cream. In this case, the pharmaceutical composition may contain any components such as an oil component, a solubilizing agent, a moisturizing agent, a coloring matter, an emulsifier, a tackifier, and a fragrance which are usually formulated in a pharmaceutical product or a pharmaceutical product.

Further, the compound (A) which is an active ingredient of the agent of the present invention may be formulated into a product other than a pharmaceutical product or a pharmaceutical product. The product is not particularly limited, and examples thereof include a bathing composition, a body soap, and a shampoo.

The agent of the present invention may be appropriately selected from a dosage form, an administration amount, and a administration method depending on the type or symptom of the skin disease associated with the activity of human chymosin.

In addition, the content of the above compound (A) in the chymosin inhibitor of the present invention is determined according to the prevention, treatment or symptom of the target disease. It is necessary to improve the activity inhibition effect, the administration method, and the like, and it is appropriately determined within the range in which the necessary amount can be ingested. In addition, the method of administering the chymosin inhibitor of the present invention may be any one, and may include a choice of oral administration or parenteral administration, and is appropriately determined depending on the target disease.

Further, as described above, in the case of using the compound (A) derived from a natural product, a plurality of compounds (A) may be contained in the raw material plant. For example, in detail, in the examples, Persicaria hydropiper contains hyperoside as a main component, and in addition to hyperoside, a small amount of quercetin, quercetin and the like are equivalent to the compound (A). The ingredients. In the case of using the raw material plant as the compound (A) contained in the agent of the present invention, the amount of the drug to be converted into a hyperoside conversion weight may be determined.

[Examples]

Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to the following examples without departing from the spirit of the invention.

<Evaluation 1: Evaluation of inhibition of chymosin activity (in vitro)>

The compound (A) used for the evaluation of the inhibition of chymosin activity is as follows.

"Compound (A)"

. Quercetin (Wuguang Pure Pharmaceutical Industry Co., Ltd.)

. Quercitrin (SIGMA)

. Isoquercitrin (SIGMA)

. Hyperoside (Wako Pure Chemical Industries Co., Ltd.)

. Galangin (Wako Pure Chemical Industries Co., Ltd.)

. Kaempferol (Wako Pure Chemical Industries Co., Ltd.)

. Myricetin (Wako Pure Chemical Industries Co., Ltd.)

. Luteolin (Funakoshi)

Further, for the compound having a flavanone skeleton and a similar skeleton and a flavanol-like skeleton shown below as a comparative example, evaluation of the inhibition effect of rennet activity was also performed.

. Eriodictyol (Funakoshi)

. (+)-Yellowwood ((+)-taxifolin) (SIGMA)

. (±)-Yellow Taxol ((±)-taxifolin hydrate) (SIGMA)

. Catechin ((+)-catechin) (SIGMA)

. Epicatechin (Wako Pure Chemical Industries Co., Ltd.)

The evaluation of the chymosin inhibitory activity (in vitro) was carried out by the method described below which was modified by the method described in Eur J Biochem 268(22), 5885-93 (2001). This evaluation method utilizes the use of angiotensin I as a substrate for human chymosin for Dnp/Nma A modified matrix, chymosin cleaves the matrix to produce angiotensin II; and an easy method for performing fluorescent color development. The following is a summary of the evaluation method.

The culture buffer was 20 mM phosphate buffer containing 100 mM NaCl, and the total culture solution amount was 100 μL. First, a compound to be measured was added to a final concentration of 0.1 mM, 0.2 mM, 0.5 mM, and 1 mM, and adjusted to contain 0.0012 units of standard human chymosin (SIGMA), and cultured for 30 minutes before being administered at room temperature. Dnp/Nma-modified angiotensin I (developed as a protein) was added as a matrix at a final concentration of 200 μM, and cultured at 37 ° C for 30 minutes. The culture was terminated by adding 25 μL of 0.5 M NaOH. The luminescence fluorescence (460 nm) of the produced Dnp angiotensin II was measured, and the amount produced was calculated from a standard curve prepared using standard Dnp angiotensin II. The human chymosin inhibitory activity and the obstruction rate of the test sample were determined by using a control group to which no inhibitor was added as a control.

As a compound (A), quercetin, quercetin, isoquercitrin, hyperoside, galangin, kaempferol, myricetin and luteolin are added so as to have the concentrations shown in Table 1, respectively. The human chymosin inhibitory activity rate was evaluated by the above method.

Further, as a comparative example, erioconiferin, (+)-cabinin, (±)-caesar, catechin, and epicatechin were added as the concentrations shown in Table 1, and the above method was used. To assess the rate of human chymosin inhibition activity.

The results are shown in Table 1.

From the above results, it was confirmed that the compound (A) has a chymosin activity inhibitory action. Specifically, it is understood that quercetin, isoquercitrin, hyperoside, kaempferol, and luteolin as the compound (A) have excellent chymosin activity inhibitory action.

<Evaluation 2: Evaluation of the improvement effect of hypertension in mice>

The chymosin inhibitor of the present invention (the agent of the present invention) was used to evaluate the improvement effect of hypertension caused by excessive salt intake as a disease caused by angiotensin II. Further, as an active ingredient of the chymosin inhibitor, hyperoside is used.

In the following five groups (control group, test group 1 to 4), evaluation of systolic blood pressure (SBP) and diastolic blood pressure (DBP) was performed.

As an experimental animal, wild type mice obtained from SLC, Japan (C57BL/6J) was bred at a temperature of 24 ± 1 ° C, humidity of 50-60%, and the illumination was adjusted in a 12-hour light-dark cycle in an experimental animal breeding room. CE-2 (Japan CLEA) was used as a feed, and 2% by weight of saline was used as drinking water to allow free feeding. Experiments were started with N=6 in each of the examples, and mice that died in the middle of the experiment were not identified. In addition, in each group, no difference was found in body weight, amount of feed, and amount of water intake.

[Control group (salt saline only)]

Six mice were fed with CE-2 (Japan CLEA) as a feed, and 2% by weight of saline was used as drinking water to prepare salt-dependent hypertensive mice that were subjected to a 2% saline solution for 3 months. For salt-dependent hypertensive mice, subsequent breeding was performed under the same conditions, and the above evaluation was performed every week for 8 weeks.

[Test group 1 (Hyperin administration: 20 mg/kg body weight/day)]

In the same manner as the control group, salt-dependent hypertensive mice (6 rats) administered with 2% saline solution for 3 months were prepared. For the salt-dependent hypertensive mice (6 rats), hyperoside was administered at 20 mg/kg body weight/day together with the above-mentioned feed and saline, and the above evaluation was performed for 8 weeks each time.

[Test group 2 (hyperin administration: 10 mg/kg body weight/day)]

In the same manner as the control group, salt-dependent hypertensive mice (6 rats) administered with 2% saline solution for 3 months were prepared. For the salt dependence Hypertensive mice (6 rats), together with the above-mentioned feed and saline, were administered with hyperoside at 10 mg/kg body weight/day, and the above evaluation was performed for 8 weeks each time.

[Test group 3 (hyperin administration: 5 mg/kg body weight/day)]

In the same manner as the control group, salt-dependent hypertensive mice (6 rats) administered with 2% saline solution for 3 months were prepared. For the salt-dependent hypertensive mice (6 rats), hyperoside was administered at 5 mg/kg body weight/day together with the above-mentioned feed and saline, and the above evaluation was performed for 8 weeks each time.

[Test group 4 (hyperin administration: 2.5 mg/kg body weight/day)]

In the same manner as the control group, salt-dependent hypertensive mice (6 rats) administered with 2% saline solution for 3 months were prepared. For the salt-dependent hypertensive mice (6 rats), hyperoside was administered at 2.5 mg/kg body weight/day together with the above-mentioned feed and saline, and the above evaluation was performed for 8 weeks each time.

Figures 1 to 3 show the results of systolic blood pressure (SBP) and diastolic blood pressure (DBP) in the control group and the test group 1 to 4. In addition, a distributed analysis of the binary configuration is performed, and the Bonferroni method is performed in the post-mortem test. The significant level was set to P < 0.05.

As shown in Fig. 1 to Fig. 3, in the control group, during the administration of saline for 3 months, the systolic blood pressure, the diastolic blood pressure and the number of heartbeats were confirmed to increase with the passage of the administration period. Continued during the test. In other words, it is considered that in the control group in which only 2% by weight of saline is administered, the symptoms of hypertension caused by excessive salt intake occur, and the number of heartbeats increases due to the hypertension. As a result, systolic blood pressure and an increase in blood pressure during dilatation were also identified during the subsequent trial period.

On the other hand, it can be understood that after administration of saline for 3 months (after the onset of symptoms of hypertension), in the test group 1 to 4 administered with hyperoside, the first week of administration from hyperoside As a result, both the systolic blood pressure and the increase in blood pressure during the dilated phase began to be inhibited compared with the control group, and were significantly inhibited individually after the second week. Therefore, it can be confirmed that the more the amount of hyperoside administered, the greater the antihypertensive effect of systolic blood pressure and blood pressure during dilatation. That is, it has been shown that the administration of hyperoside contributes to the improvement of salt-dependent hypertension.

In addition, salt-dependent hypertensive disorder may become drug-resistant hypertension, and it is not possible to determine that there is a hypotensive effect by obstructing the ACE system. Therefore, it is considered that the production of non-ACE-dependent angiotensin II is inhibited by the inhibition of chymosin activity caused by administration of hyperoside, thereby exhibiting a hypotensive effect.

<Evaluation 3: Evaluation of the ingredients contained in the leech>

As a raw material plant containing the compound (A), a perhydrate (Persicaria hydropiper) was used, and the dried product was used in the test described later.

The shoots of the leeches (purchased from Seishin Co., Ltd.) were dried in a shed dryer to obtain a dried mash. Drying obtained The material was sufficiently pulverized by a jet mill. The water content of the obtained pulverized dried leeches (5 samples) obtained by the heat reduction method was 1.5% by weight (average value), and was 2% by weight or less in all the samples.

As an evaluation of the components contained in the leeches, the components of the dried matter (pulverized product) containing the mash portion were evaluated by the following methods.

First, 10 parts by volume (by weight) of 50% by volume of ethanol/water was added to a predetermined amount of dried leech (n=3), and the mixture was stirred at room temperature for 2 hours to extract components contained in the leech. Next, the supernatant was separated by centrifugation (3000 rpm, 10 minutes), diluted 10-fold with 50% by volume of ethanol/water, filtered through a filter (0.45 μm), and then subjected to an Alliance HPLC system (Waters). analysis.

The analysis results of the solvent-extracted components of the dried leech were shown in Table 2. As shown in Table 2, the main component of the dried product of the mink bud portion was hyperoside. Therefore, in the following evaluation, the compound (A) contained in the dried leech was tested as hyperoside and tested.

<Evaluation 4: Evaluation of the improvement effect of hypertension in mice>

The septic blood pressure (SBP) of the dried leeches of the salt-dependent hypertensive mice was administered to the rats in the following five groups (control group, test group 1A to 4A) using the dried mash (pulverized material), Assessment of dilated blood pressure (DBP) and heart rate (Heart Rate).

As experimental animals, wild type mice (C57BL/6J) obtained from SLC, Japan, were bred in a laboratory animal breeding room adjusted at a temperature of 24 ± 1 ° C, a humidity of 50-60%, and a lighting system with a 12-hour light-dark cycle. CE-2 (Japan CLEA) was used as a feed, and 2% by weight of saline was used as drinking water to allow free feeding. Experiments were started with N=6 in each of the examples, and mice that died in the middle of the experiment were not identified. In addition, in each group, no difference was found in body weight, amount of feed, and amount of water intake.

[Control group (salt saline only)]

Six mice were fed with CE-2 (Japan CLEA) as a feed, and 2% by weight of saline was used as drinking water to prepare salt-dependent hypertensive mice that were subjected to a 2% saline solution for 3 months. For salt-dependent hypertensive mice, subsequent breeding was performed under the same conditions, and the above evaluation was performed every week for 8 weeks.

[Test group 1A (dried leech dry product: 500 mg/kg body weight/day)]

The salt-dependent hypertensive mice (6 rats) prepared in the same manner as the control group, together with the above feed and saline, were 500 mg/kg body weight/ The mash was dried and pulverized daily, and the above evaluation was performed every week for 8 weeks.

[Test group 2A (dried leech dry matter: 250 mg/kg body weight/day)]

The salt-dependent hypertensive mice (6 rats) prepared in the same manner as the control group, together with the above-mentioned feed and saline, were given dry pulverized material at 250 mg/kg body weight/day, and the above evaluation was performed every week. A total of 8 weeks.

[Test group 3A (dried leech dry matter: 125 mg/kg body weight/day)]

The salt-dependent hypertensive mice (6 rats) prepared in the same manner as the control group were administered with leeches and dried pulverized materials at 125 mg/kg body weight/day together with the above-mentioned feed and saline, and the above evaluation was performed every week. A total of 8 weeks.

[Test group 4A (water-dried dry matter administration: 62.5 mg/kg body weight/day)]

The salt-dependent hypertensive mice (6 rats) prepared in the same manner as the control group were given the above-mentioned feed and saline, and the mash was dried at 62.5 mg/kg body weight/day, and the above-mentioned per week was performed. Evaluation for a total of 8 weeks.

The evaluation results of systolic blood pressure (SBP), dilated blood pressure (DBP) and heart rate in the control group and the test group 1A to 4A are shown in Figs. 4 to 6. In addition, a distributed analysis of the binary configuration is performed, and the Bonferroni method is performed in the post-mortem test. The significant level was set to P < 0.05.

As shown in Fig. 4 to 6, in the control group, during the administration of saline for 3 months, with the passage of time, it was confirmed that there was an increase in systolic blood pressure, dilated blood pressure, and heart rate, which was during the test period. continued. On the other hand, after the administration of the saline solution for 3 months, in the test group 1A to 3A to which the dried leech was administered, the heartbeat from the first week of the leech dry matter administration was compared with the control group. The increase in the number, the systolic blood pressure, and the increase in the blood pressure during the dilatation phase were all inhibited, and were significantly suppressed after the second week. In the test group 4A, although the systolic blood pressure was not affected, the increase in the blood pressure during the expansion period was significantly suppressed from the fourth week. The suppression of the increase in the number of heartbeats has little effect.

According to the above results, similarly to the oral administration of hyperoside in the above evaluation 2, it was confirmed that the blood pressure lowering effect in the mice caused by the oral administration of the dried leech was confirmed. Further, it can be confirmed that when the dose is increased, the effect of lowering the heart rate is exhibited together with the blood pressure lowering action.

<Evaluation 5: Evaluation of the improvement effect of human hypertension>

With the approval of the ethics committee of the Fukuoka University Chitose Hospital, the testee who has symptoms of hypertension is used for the purpose of confirming the effectiveness and safety caused by the administration of dried leech (orally administered). Eleven people (average weight: 65 kg, standard deviation: 12.4) were subjected to human trials.

Indigestible dextrin 400 mg/person/day was taken as a placebo for 2 weeks, and then dried with leeches 200 mg/person/day for 2 weeks. In addition, the weight of the test subject was set to 65 kg, and the dried mink 200 mg/person/day. It corresponds to a dried leech product of 3.1 mg/kg body weight/day, which corresponds to a total of 0.03 mg/kg body weight/day of hyperoside.

Next, each was taken for 2 weeks in the order of 400 mg/person/day, 800 mg/person/day, 2000 mg/person/day of dried leech. In addition, the timing of taking placebo and dried leech on the 1st day was set as the test subject's breakfast. The relationship between the amount of the leech dry product administered and the amount of hyperoside administered is shown in Table 3.

Blood pressure measurement and heart rate measurement were performed using an automatic sphygmomanometer (HEM-7251G) manufactured by Omron Healthcare Co., Ltd. Further, in this automatic sphygmomanometer, after the measurement, the individual blood pressure data and the pulse data are automatically transmitted to the server via the Internet, and thus are not subjectively subject to the subject. As the blood pressure of the subject, the systolic blood pressure (SBP) and the diastolic blood pressure (DBP) of the day after taking the dried leech were used. In addition, as the heart rate, the pulse data of the morning of the morning when the leech was dried was used.

The evaluation results for systolic blood pressure (SBP) are shown in Fig. 7, and the results of evaluation for dilated blood pressure (DBP) are shown in Fig. 8. this In addition, the result of the heartbeat number is shown in FIG. In addition, the values in Figures 7 to 9 are the average of the placebo period (2 weeks) of 11 subjects and the average of the dry period of each leech (2 weeks each). The amount of hyperoside contained in the dried leech.

As shown in Fig. 7, if the placebo period and the dry season of leech are compared, it can be determined that the amount of hyperoside taken is 0.03 mg/kg body weight/day (200 mg/person/day of dried leech) The systolic blood pressure system is significantly reduced. In addition, as shown in Fig. 8, the blood pressure during the dilatation period also has the same result.

On the other hand, as shown in Fig. 9, it can be confirmed that the number of heartbeats in the amount of hyperoside taken 0.03 mg / kg body weight / day (water sputum dry product 200 mg / person / day), 0.06 mg / kg body weight / day (water leeches The dry matter was 400 mg/person/day, which was the same as the placebo period, but the amount of hyperoside administered was 0.12 mg/kg body weight/day (water sputum dry product 800 mg/person/day), 0.30 mg/kg body weight. The number of heartbeats decreased when the day/day (water drips 2000 mg/person/day).

From the above results, it was confirmed that the blood pressure lowering effect caused by the oral administration of the dried leeches containing the compound (A) such as hyperoside to humans can be confirmed, and it can be clarified that if the amount of dried leeches is increased, The effect of blood pressure lowering shows a reduction in the number of heartbeats.

Furthermore, as can be seen from the comparison between Assessment 2 (mouse) and Assessment 5 (human), it was confirmed that humans were taking a small amount of hyperoside on a body weight basis compared to mice. It shows the effect of lowering blood pressure and lowering the number of heartbeats.

<Evaluation 6: Evaluation of the effectiveness of atopic dermatitis>

Affected by the ethics committee of the Fukuoka University Chitose Hospital, in order to confirm the effectiveness of atopic dermatitis caused by the administration of dried leech (orally administered), atopic dermatitis occurs. For the test, 11 subjects (average weight: 65 kg, standard deviation: 12.4) were subjected to human trials.

For the effectiveness of atopic dermatitis, the following methods were used for evaluation.

First, before the start of the clinical trial, a questionnaire on the skin condition (DLQI: Dermatology Life Quality Index) and atopic skin were measured for the subject's symptoms of atopic dermatitis. The number of eosinophils in the indicator of inflammation.

Next, each of the dried lycopene 800 mg/person/day (desorubicin 0.12 mg/kg body weight/day), 2000 mg/person/day (the amount of hyperoside taken 0.30 mg/kg body weight/day) was taken. 2 weeks. In addition, the timing of taking placebo and dried leech on the 1st day was set as the test subject's breakfast.

(1) Evaluation using the DLQI questionnaire

A sample of the DLQI questionnaire is shown in Fig. 10. The DLQI is an indicator of the inconvenience caused by the skin condition, and the higher the value is, the more the symptoms of atopic dermatitis are improved.

The evaluation results of the questionnaire on skin condition (DLQI) before and after the administration of the dried leech were shown in Fig. 11 . In addition, the amount of administration is expressed as the amount of hyperoside taken in the dried leech.

As shown in Figure 11, if the test is taken before the start of the clinical trial The DLQI of the tester is used as a reference value, and the dried lycopene 800 mg/person/day (desperate 0.12 mg/kg body weight/day), 2000 mg/person/day (the amount of hyperoside taken 0.30 mg/kg body weight) is taken. After the day/day, the measured value showed a high value, and the symptoms of atopic dermatitis were improved.

(2) Evaluation of the number of eosinophils

In addition, an evaluation of the eosinophil number before and after administration of the dried leech was performed. A blood sample of a certain amount is taken from the subject, and the number of white blood cells (/mm ³ ) and white blood cells (%) in the blood sample are calculated, and the number of blood eosinophils per mm ^{3 is} calculated. In addition, the number of eosinophilic globules is used as an indicator for the pathological state of atopic dermatitis, and is increased when the symptoms are deteriorated.

As shown in Fig. 12, the number of eosinophilic spheres after taking the dried leeches 2000 mg (the amount of hyperoside taken 0.30 mg/kg body weight/day) showed a low value compared to before taking the dried leeches. .

From the above results, it is suggested that by taking the dried leech, the effect of preventing/improving the symptoms of atopic dermatitis and the hyperoside which inhibits the activity of human chymosin are effective components.

(3) Confirmation of safety

For the safety, a single administration test of lycopene dry matter 4000 mg/person/day (the amount of hyperoside taken 0.60 mg/kg body weight/day) was performed. The test subject is safely caused by the use of his or her name and consent to participate in the administration of dried leech after receiving the instructions or the description of the risk of the clinical trial. Sex test. As a result, among the 11 subjects who were tested, even if a single-dose test of squid dry matter of 4000 mg/person/day was carried out, there was no conscious feeling such as abdominal pain/nausea/vomiting, such as digestive tract stagnation or laxative abnormality. symptom. In addition, in blood tests, urine tests, and the like, metabolic abnormalities caused by the use of dried leech are not recognized at all. From the above results, the safety of the dried leech can be confirmed.

Claims (12)

Use of a compound represented by the following general formula (A) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prophylaxis and treatment of a disease associated with activity of human chymosin; (In the general formula (A), R ¹ to R ⁵ each independently represent a hydrogen atom, a hydroxyl group or a methoxy group, X ¹ represents a hydrogen atom, a hydroxyl group or a -OG group, and G represents a sugar chain). The use of claim 1, wherein, in the compound of the general formula (A), X ¹ is a hydroxyl group, a rhamnose residue, a glucose residue, or a galactose residue. The use of claim 1, wherein the compound of the general formula (A) is derived from quercitrin, isoquercitrin, hyperoside, kaempferol, and rhinoceros. One or more selected from the group consisting of luteolin. The use of the formula (1), wherein the compound represented by the general formula (A) is at least one selected from the group consisting of a quercetin glycoside and a quercetin derivative. The use of claim 1, wherein the compound represented by the general formula (A) is hyperoside. The use according to any one of claims 1 to 5, wherein the aforementioned disease associated with the activity of human chymosin is a disease associated with angiotensin II production. The use of claim 6, wherein the aforementioned disease associated with angiotensin II production is hypertension and/or a disease caused by hypertension. The use of claim 7, wherein the hypertensive condition is hypertension caused by excessive salt intake. The use of claim 7, which comprises at least a hyperoside and is administered orally to a human in a manner of 0.1 mg/kg body weight/day or more as a compound represented by the general formula (A). . The use according to any one of claims 1 to 5, wherein the aforementioned disease associated with the activity of human chymosin is a disease unrelated to angiotensin II production. The use of claim 10, wherein the aforementioned diseases not associated with angiotensin II production are atopic dermatitis, cognac and urticaria, and skin diseases selected from the group consisting of any combination of the above. The use of claim 11, wherein the aforementioned skin disease is atopic dermatitis.