JP4822291B2 - Liver fibrosis inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hepatic fibrosis inhibitory agent which can inhibit pathological progress from chronic hepatitis to cirrhosis, exhibits excellent pharmacological activity, and can be used for a long time with a small side effect. <P>SOLUTION: The hepatic fibrosis inhibitory agent contains a proanthocyanidin composition as an active constituent. The proanthocyanidin composition has a specific configuration. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a liver fibrosis inhibitor containing proanthocyanidins (hereinafter referred to as “PAC”) as an active ingredient.

  Liver fibrosis is a condition caused by numerous factors such as liver inflammation, damage to hepatocytes by toxins, changes in hepatic blood flow, or viral, bacterial, fungal or parasitic infections. Also, inborn errors of metabolism are often associated with fibrosis, abnormal lipid metabolism (Gaucher's disease), glycogenosis, alpha antitrypsin deficiency, iron overaccumulation syndrome (hemochromatosis), copper accumulation disease (Wilson's disease). Accumulation of toxic metabolites (tyrosinemia, fructoseemia, galactosemia), peroxisome abnormalities (Zelweger syndrome), etc. are known. In addition, chemical substances and drugs such as alcohol and methotrexate, or hepatic circulatory disturbance (chronic heart failure, Bad-Chiari syndrome, venous occlusion, portal thrombosis, etc.) and chronic biliary outflow tract obstruction Cause.

In the process of progression from chronic hepatitis such as viral hepatitis to cirrhosis, liver fibrosis is a major pathological condition, and suppressing liver fibrosis is extremely important in preventing cirrhosis. In particular, in cases where interferon is ineffective in the treatment of hepatitis C, development of liver fibrosis suppression therapy is urgent. The main cause of liver fibrosis is abnormal production of extracellular matrix (collagen, laminin, proteoglycan, etc.) from hepatic stellate cells, one of the non-parenchymal cells of the liver. The function of hepatic stellate cells is regulated by cytokines and growth factors produced by Kupffer cells, which are hepatic macrophages. Platelet-derived growth factor (PDGF) is one of the activation promoting factors of hepatic stellate cells, and causes liver fibrosis by promoting the proliferation of hepatic stellate cells. Therefore, if a drug that suppresses the proliferation of hepatic stellate cells or activation by PDGF is developed, it is possible to suppress hepatic fibrosis and suppress the progression from chronic liver damage such as viral hepatitis to cirrhosis. It is.

  As natural product compounds that suppress the activation of hepatic stellate cells, catechins derived from green tea, epicatechins, and the like are known. Green tea catechin therapy has been reported to reduce oxidative stress and suppress liver fibrosis in patients after biliary atresia.

  In Non-Patent Documents 1, 2 and 3, epigallocatechin-3-O-gallate (hereinafter referred to as “EGCG”) is a recombinant human platelet-derived growth factor-BB; hereinafter referred to as “PDGF-BB”. It is reported that the amount of hepatic stellate cells synthesized by treatment is suppressed.

  Patent Document 1 states that high-molecular polyphenols containing PAC extracted from fermented tea such as oolong tea and black tea are effective for the prevention and treatment of fatty liver. In Patent Document 2, a composition containing polyphenols containing procyanidins and derivatives thereof is effective for treatment of tumors such as breast cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma or colon cancer. Has been suggested.

JP 2007-320958 A Special table 2007-519852 gazette Sakata R.M. et al. , J. et al. Hepatol, 40, 52-59 (2004) Chen A. et al. , J. et al. Biol. Chem. , 278, 23381-23389 (2003) Higashi N. et al. , J. et al. Lab. Clin. Med. , 145, 316-322 (2005)

  An object of the present invention is to provide a liver fibrosis inhibitor capable of suppressing the progression of the pathology from chronic hepatitis to cirrhosis, which exhibits excellent pharmacological activity, has few side effects and can be used for a long period of time. There is.

  In order to achieve the above object, the present inventors have conducted various studies on components derived from natural products using the inhibitory effect of activation of cultured human hepatic stellate cells LI90 by PDGF-BB as an index. The polymer was found to have an effect of suppressing liver fibrosis equivalent to EGCG, leading to the present invention.

  That is, this invention relates to the liver fibrosis inhibitor which uses a proanthocyanidin composition as an active ingredient.

  The hepatic fibrosis inhibitor of the present invention has an excellent pharmacological activity by having the above-described configuration, and can be used for a long time with few side effects. According to such a liver fibrosis inhibitor, liver fibrosis in chronic liver disorders such as viral hepatitis and alcoholic liver disorder can be effectively suppressed. Moreover, the onset or progress of diseases such as cirrhosis or hepatocellular carcinoma caused by liver fibrosis can be effectively prevented or suppressed.

The inhibitor of hepatic fibrosis according to the present invention is such that the proanthocyanidin composition has a flavan-3-ol skeleton represented by the following general formula (1), and the binding mode is the following (i), (ii) or (iii): It is preferable that two or more have a structure bonded to each other.
(I) 4-position and 8-position carbon bonds (ii) 4-position and 6-position carbon bonds (iii) 4-position and 8-position carbon bonds and 2-position and 7-position oxygen bonds

In formula (1), R ₁ and R ₃ each independently represent a hydrogen atom or a hydroxyl group, R ₂ represents a hydroxyl group, and R ₄ represents a hydrogen atom or a monovalent organic group.

  The liver fibrosis inhibitor as described above is more excellent in pharmacological activity, has few side effects, and can be used over a long period of time.

  In the hepatic fibrosis inhibitor of the present invention, the proanthocyanidins are preferably derived from natural products. Such an inhibitor of liver fibrosis exhibits excellent pharmacological activity, has few side effects, can be used for a long time, and can be easily processed from natural products.

  Moreover, this invention relates to the hepatic stellate cell growth inhibitor which uses a proanthocyanidin composition as an active ingredient.

  The hepatic stellate cell growth inhibitor of the present invention is a hepatic stellate cell growth inhibitor capable of suppressing the proliferation or activation of hepatic stellate cells, and exhibits excellent pharmacological activity and side effects by having the above-described configuration. There are few and can be used for a long time.

In the hepatic stellate cell growth inhibitor of the present invention, the proanthocyanidin composition has a flavan-3-ol skeleton represented by the following general formula (1), which is a bond of the following (i), (ii) or (iii): It is preferable to have a structure in which two or more are bonded to each other in a manner.
(I) 4-position and 8-position carbon bonds (ii) 4-position and 6-position carbon bonds (iii) 4-position and 8-position carbon bonds and 2-position and 7-position oxygen bonds

In formula (1), R ₁ and R ₃ each independently represent a hydrogen atom or a hydroxyl group, R ₂ represents a hydroxyl group, and R ₄ represents a hydrogen atom or a monovalent organic group.

  The hepatic stellate cell growth inhibitor as described above is more excellent in pharmacological activity, has few side effects, and can be used over a long period of time.

  In the hepatic stellate cell growth inhibitor of the present invention, the proanthocyanidins are preferably derived from natural products. Such an inhibitor of liver fibrosis exhibits excellent pharmacological activity, has few side effects, can be used for a long time, and can be easily processed from natural products.

  In one aspect, the hepatic stellate cell growth inhibitor of the present invention inhibits the growth or activation of hepatic stellate cells based on hepatic stellate cell apoptosis.

  The present invention also relates to an agent for inhibiting the onset / progress of liver disease caused by liver fibrosis, including the liver fibrosis inhibitor or the hepatic stellate cell growth inhibitor.

  Further, the present invention includes a liver fibrosis inhibitor, a hepatic stellate cell proliferation inhibitor, a hepatic stellate cell apoptosis induction, or a liver disease caused by liver fibrosis. The present invention relates to a functional auxiliary food or drink for suppressing the onset or suppressing the progression of liver disease caused by liver fibrosis.

  According to the present invention, there is provided a liver fibrosis inhibitor capable of suppressing the progression of pathology from chronic hepatitis to cirrhosis, which exhibits excellent pharmacological activity, has few side effects and can be used for a long time. be able to. Further, according to the present invention, a hepatic stellate cell growth inhibitor capable of suppressing the proliferation or activation of hepatic stellate cells, which exhibits excellent pharmacological activity, has few side effects and can be used for a long period of time. Can be provided. Furthermore, according to the present invention, it is possible to provide a liver disease onset / progress inhibitor and a functional auxiliary food or drink characterized by containing the above-mentioned liver fibrosis inhibitor or the above-mentioned hepatic stellate cell growth inhibitor.

  Hereinafter, the best mode for carrying out the present invention will be described in detail with reference to the drawings as necessary. However, the present invention is not limited to the following embodiments.

  The hepatic fibrosis inhibitor and hepatic stellate cell growth inhibitor according to this embodiment comprise a proanthocyanidin composition (hereinafter referred to as “PAC composition”) as an active ingredient. Here, the PAC composition is a composition comprising one or more proanthocyanidins (hereinafter referred to as “PAC”), and the liver fibrosis inhibitor and the hepatic stellate cell proliferation inhibitor according to the embodiment are: In many cases, a composition comprising a plurality of PACs is used as an active ingredient. The PAC composition may also contain a derivative of PAC, a physiologically acceptable salt thereof, a hydrate thereof, a solvate thereof, and the like.

  PAC is a general term for a group of polymer compounds formed by polymerizing monomers having hydroxyl groups at various sites based on a flavan skeleton. Among the structural units of such a polymer, the base end portion is called a terminal unit, and the others are called extension units. In addition, the degree of polymerization of the polymer ranges from dimers and trimers to polymers of 100 or more.

  In this embodiment, from the viewpoint of pharmacological activity, the degree of polymerization of PAC is preferably 3 or more, more preferably 5 or more, and further preferably 5 to 20. However, as long as the main component is a polymer having a polymerization degree of 3 or more, the PAC composition may contain monomers and other fragments. The average degree of polymerization of PAC contained in the PAC composition is preferably 3 or more, more preferably 5 or more, and further preferably 5 to 20. In the present invention, the average degree of polymerization refers to the average number of the above-mentioned monomers bonded, and can be measured, for example, by the method shown in the examples described later.

Examples of the PAC include a polyphenol component mainly composed of a catechin, that is, a polymer having a degree of polymerization of 2 or more having flavan-3-ol as a structural unit, as shown in the chemical formula of FIG. Note that R in the chemical formula of FIG. 1 corresponds to OR ₄ in the general formula (1). In addition, gallic acid is often ester-bonded to the 3-position hydroxyl group of the flavan ring.

  Common names are given to PACs in which a specific monomer (skeleton) is polymerized, and typical PACs are propellers defined and classified according to the number of hydroxyl groups in the B ring (see FIG. 1). Examples include nidin (4'-OH), procyanidin (3'-OH, 4'-OH) and prodelphinidin (3'-OH, 4'-OH, 5'-OH). For example, polymers having a component lacking a hydroxyl group at the 5-position of the A ring include progaiballinidine, proficetinidine, and prolovinetinidine, and depending on the presence or absence of hydroxyl groups at other specific sites. There are common names such as proteracacidin, promelacacidin, proapigenidin, and prolteolinidin.

  In the present invention, PAC is preferably procyanidin, propelargonidin or prodelphinidin. The structural units (skeletons) of “procyanidins” may be the same or different and can be selected from catechin, epicatechin, catechin gallate and epicatechin gallate. The structural unit (skeleton) of “prodelphinidin” may be the same or different, and can be selected from gallocatechin, epigallocatechin, gallocatechin gallate and epigallocatechin gallate.

  Moreover, as PAC, the condensed tannin widely contained in various plants is mentioned, for example. Such a PAC can obtain cyanidin, delphinidin, pelargonidin, aurantidine, luteolinidine, peonidin, malvidin, petunidin, europeinidine, rosinidine, hirastidine, apigenidin and the like by acid treatment.

  Furthermore, various compounds are known for PACs, such as the difference in the bonding position on each ring, the conformation of substituents in the structural unit, and the bonding order in the polymer of different structural units. The chemical names of some of the PACs included in the present invention are exemplified below. In addition, the molecular formula (molecular formula) is in parentheses. In the present invention, not only the following PACs but also PACs having a higher degree of polymerization in which these PACs are further condensed with structural units such as catechin and epicatechin or other PACs are included.

Proanthocyanidin PZ5 (C75 H62 O31),
Proanthocyanidin BP 1 (Unspecified),
Proanthocyanidin RP 4 (C129 H106 O67),
Proanthocyanidin RP 3 (C136 H120 O70),
Proanthocyanidin CS 4 (C136 H120 O70),
Proanthocyanidin CS 3 (C127 H128 O69),
Proanthocyanidin CS 2 (C113 H110 O62),
Proanthocyanidin CS1 (C121 H118 O65),
Proanthocyanidin RP 2 (C120 H114 O64),
Proanthocyanidin RP 1 (C125 H130 O69),
Proanthocyanidin T4 (C128 H122 O65),
Proanthocyanidin T3 (C105 H102 O59),
Proanthocyanidin T2 (C67 H54 O29),
Proanthocyanidin T1 (C87 H72 O43),
4- (Benzylthio) proanthocyanidin A2 (C37 H30 O12 S),
Proanthocyanidin A7 (C30 H24 O12),
Proanthocyanidin (C30 H24 O12),
Proanthocyanidin A5 ′ (C30 H24 O12),
Proanthocyanidin A4 (C30 H24 O12),
Proanthocyanidin B6 (C37 H30 O16),
Proanthocyanidin B2 3,3′-O-gallate (C44 H34 O20),
Proanthocyanidin B4 (C30 H26 O12),
Proanthocyanidin A2 4α-benzylthioether (C37 H30 O12 S),
Proanthocyanidin A2 (C30 H24 O12),
Proanthocyanidin C1 (C45 H38 O18),
Proanthocyanidin B2 (C30 H26 O12),
Proanthocyanidin B (C31 H28 O12),
Methylated polymer proanthocyanidin {(C29 H32 O10) x},
Proanthocyanidin B1 (C30 H26 O12),
Proanthocyanidin A (C31 H28 O12),
Proanthocyanidin C (C30 H26 O12),
Proanthocyanidin B5 (C30 H26 O12),
Proanthocyanidin P-1 (Unspecified).

  In this embodiment, in the PAC composition, the flavan-3-ol skeleton represented by the following general formula (1) has (i) a bond between the 4-position carbon and the 8-position carbon, and (ii) a 4-position carbon and a 6-position. It is preferable to include those having a structure in which two or more carbon atoms are bonded to each other in any one of carbon bonds, (iii) 4-position carbon and 8-position carbon bonds, and 2-position carbon and 7-position oxygen bonds.

In formula (1), R ₁ and R ₃ each independently represent a hydrogen atom or a hydroxyl group, R ₂ represents a hydroxyl group, and R ₄ represents a hydrogen atom or a monovalent organic group.

Examples of the monovalent organic group represented by R ₄ in the general formula (1) include a gallate group, an organic acid residue, a sugar residue, and the like (wherein the residue is an atom or group in the molecule). Means a monovalent group formed by removing. Examples of organic acids that form organic acid residues include p-coumaric acid, caffeic acid, ferulic acid, sinapinic acid, p-hydroxybenzoic acid, gallic acid, acetic acid, oxalic acid, malonic acid, succinic acid, malic acid Etc. The sugar that forms the sugar residue is modified at any position, for example, via a phenol hydroxyl group and an ester bond, as necessary. Examples of the glycoside-forming residue include a pyranose type hexose residue and a furanose type pentose residue. Specifically, examples of sugar forms include disaccharides and trisaccharides in addition to monosaccharides such as glucose, galactose, rhamnose, xylose, and arabinose.

R ₄ may be a glycoside-forming residue in which a sugar is bonded to a gallate group or an organic acid group. In this case, for example, the sugar is bonded to a phenolic hydroxyl group of a gallate group or an organic acid group at an arbitrary position via an ester bond. Examples of the glycoside-forming residue include a pyranose type hexose residue and a furanose type pentose residue. Examples of sugar forms include disaccharides and trisaccharides in addition to monosaccharides such as glucose, galactose, rhamnose, xylose, and arabinose.

Among them, R ₄ preferably exhibits good gallate group which may have a substituent. The “gallate group optionally having substituent (s)” includes “gallate group” and “gallate group having substituent (s)”, and “gallate group having substituent (s)” includes “gallate to which sugar is bound. Group "and the like.

  In formula (1), the hydroxyl group at the 5-position and / or 7-position of the A ring may be a modifying group to which a gallate group, a sugar, an organic acid or the like is bonded.

  Examples of proanthocyanidins having a structure in which the flavan-3-ol skeletons are bonded to each other by the bonding mode (i) above include those represented by the following general formula (2).

Examples of the proanthocyanidins having a structure in which the flavan-3-ol skeletons are bonded to each other by the bonding mode (iii) include those represented by the following general formula (3).

  The main binding sites between PAC structural units include (i), (ii) or (iii) as described above. Specific examples include, for example, an A-type bond; 2 positions of oxygen and 2-position and 7-position, for example, B-type bond; any one position of 4-position and 6-position or 4-position and 8-position is mentioned. There are also PACs having various configurations consisting of these bonds.

  PAC can be derived from natural products or synthetic products, but PACs derived from natural products are preferred, and plant-derived PACs are more preferred. As starting materials for obtaining such PACs, raw materials from plant materials derived from plants, such as vegetables, nuts, bark, and other plant materials including polyphenol compounds can be used. For example, most colored fruits, strawberries, and vegetables are known to contain polyphenolic compounds. Examples of plants, fruits, strawberries, and vegetables that contain polyphenol compounds include, for example, blueberry leaves, grape seeds, taro, bilberries, elderberries, plums, blackberries, strawberries, red currants, black currants, cranberries, Examples include, but are not limited to, cherry, raspberry, grapefruit, currant, hibiscus flower, pepper, beans, peas, soybean hulls, red cabbage, purple corn, purple sweet potato. Of these, the use of PAC compositions such as those derived from blueberry leaves, grape seeds, and croton seed sap is practical.

  Table 1 shows proanthocyanidin compositions obtained from blueberry leaves, grape seeds, and croton seed sap as specific examples of the PAC composition according to this embodiment.

  In the starting material of the present invention, the parts of the plant to be used are leaves, petals, sepals, flowers, petiole, shoots, roots, stems, seeds, pods, tubers, bark, formation layers, timbers, fungi, fruits, vegetables, Herbs, ferns, sap, resin, grapes, apples, skins such as onions and avocados, citrus peels, skins including fruit hulls, apples, wine pomace, grain hulls, straw, hay, olives, rapes or A lump of oil seed derived from canola and other oil crop extracts can be used arbitrarily.

A preferred starting material in the present invention is blueberry. Blueberry is a Ericaceae (Ericaceae) the genus Vaccinium (Vaccinium) Saianokokasu clause of the American native deciduous or evergreen belonging to the (Cyanococcus) shrub or semi-tall fruit trees. There are about six types of blueberries, but the following three are important for fruit gardening. In the present invention, the type, origin, and place of origin of the blueberries to be used are not particularly limited.

(1) Highbush blueberry (Highbush blueberry, Vaccinium corymbosum L.): O'neill, Sharp blue, Georgia gem, Flowable, Leveley, Spartan, Darrow, Duke, Berkeley, Harrison, etc.

(2) rabbit eye blueberries (Rabbiteye blueberry, V. ashei Reade, V. virgatum Aiton): Woodard, Garden Blue, Tiff Blue, home Bell, Meyer and the like.

(3) Low bush blueberry ( V. angustifolium Aiton; V. mytilloides Michaux): signature, bronzewick, bromidone, etc.

  The natural product-derived PAC composition which is the active ingredient of the present invention can be obtained by appropriately combining the following steps according to the purpose.

(1) Pretreatment processing :
Depending on the whole plant parts such as leaves, skin, fruits, stems, roots, etc., impurities are physically removed in advance by washing with water or filtering. Alternatively, components other than the PAC of the present invention, such as chlorophyll and fibrin, can be distilled off with a solvent. For this solvent, chloroform, hexane, acetone or the like can be used, although it varies depending on the object to be distilled off. The raw material may be pulverized as it is, or may be pulverized after drying, and may be used in the powder form for the next step, or the juice or extract from the raw material may be used for the next step. The squeezed juice or extract may be concentrated or dried and used in the next step in powder form.

  The drying means is not particularly limited as long as the pharmacological effect of the present invention is not impaired, and vacuum freeze drying, hot air drying, far-infrared drying, vacuum drying, microwave vacuum drying, superheated steam drying, and the like can be widely used. Among these, vacuum freeze-drying with little component change can be preferably used. The vacuum freeze-drying conditions can be appropriately selected depending on the type of raw material and the state of the part. For example, the freezing temperature when drying fresh leaves as it is is −30 to −20 ° C., the drying temperature is −30 to 30 ° C., and the drying time is A range of 15 to 24 hours is preferred.

(2) Extraction process :
The processed product obtained in the previous step can then be solvent extracted. For solvent extraction, the following solvents are combined as appropriate, and multistage extraction is performed as necessary. The extraction solvent to be used is not particularly limited, but it is preferable to use water or a polar solvent compatible with water. Examples of polar solvents that are compatible with water include lower alkyl alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as ethylene glycol, butylene glycol, propylene glycol, and glycerin. it can. As the alcohol, it is practical to use a lower alcohol, particularly methanol or ethanol, from the viewpoint of safety. Examples of other organic solvents include acetone, diethyl ether, dioxane, acetonitrile, acetic acid ethyl ester, xylene, chloroform, toluene, hexane, and the like. These solvents including combinations of water and polar solvents may be used alone or in combination of two or more. For example, it is possible to use a mixed solvent of acetone and ethyl ether, preferably a 1: 1 (v / v) mixture of acetone and ethyl ether. In general, it is desirable to use a mixed solvent of a polar solvent and water. These water-containing solvents are water-containing alkyl alcohols, more preferably water-containing methanol and water-containing ethanol. The alcohol concentration in the hydrous alcohol is 5 to 90% by volume, preferably 80 to 90% by volume, more preferably 50 to 90% by volume. As a preferable mixed solvent other than alcohol, a mixed solution of water and acetone can be exemplified.

  Examples of the extraction operation include immersion treatment such as cold immersion and digestion of a processed product in a solvent. Usually, extraction is performed with stirring under heating, followed by filtration to obtain an extract. For example, in stirring extraction with an 80% by volume ethanol aqueous solution, the solution temperature is desirably room temperature, and the immersion time varies depending on the temperature, but is preferably in the range of 30 seconds to 1 hour. It can also be based on the percolation method.

  From the obtained extract, solids are removed by filtration or centrifugation as necessary. The filtrate may be used as it is depending on the requirements of the next step, or may be used after partially concentrating or drying by distilling off the solvent. If necessary, these extracts and concentrates are purified. The purification method is not particularly limited, and examples thereof include a column method and a resolution method using a solvent (see WO 00/64883).

  The PAC composition can be used in various forms such as an extract, a concentrated extract, a paste, a dried product, and a semi-dried product. Since the purified PAC composition is concentrated, it generally has a higher pharmacological activity (liver fibrosis inhibiting activity) than a simple extract.

  The obtained PAC composition can be used as it is or as a prodrug or a drug. The hepatic fibrosis inhibitor or hepatic stellate cell growth inhibitor according to this embodiment is a drug comprising a PAC composition, a physiologically acceptable salt thereof, a hydrate thereof and / or a solvate thereof. However, it is generally desirable to prepare a pharmaceutical composition containing the above substances and pharmacologically and pharmaceutically acceptable additives.

  Examples of pharmacologically and pharmaceutically acceptable additives include excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, dyes, diluents, bases, solubilizers, and dissolution agents. Adjuvants, tonicity agents, pH adjusters, stabilizers, propellants, adhesives, and the like can be used.

  When the hepatic fibrosis inhibitor or hepatic stellate cell growth inhibitor according to this embodiment is used as a pharmaceutical, it is preferably administered in a dosage unit form, and is administered orally, intra-tissue (subcutaneous, intramuscular, intravenous Administration), local administration (transdermal administration, etc.), or rectal administration.

  The oral dosage form may be appropriately selected from tablets, pills, capsules, fine granules, granules and the like. Examples of liquid pharmaceutical preparations for oral administration include emulsions, solutions, suspensions, and syrups. In the preparation, excipients, binders, disintegrants, lubricants, colorants, flavoring agents, pH adjusters and the like according to various preparations can be appropriately blended. For example, in the case of a tablet, if necessary, it can be made into a tablet with a normal coating, such as a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric tablet, a film-coated tablet, a double tablet, or a multilayer tablet. When preparing into tablet form, as a carrier, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, etc .; water, ethanol, propanol, simple syrup , Glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone and other binders; dried starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene Disintegrants such as sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose; disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil; quaternary ammonium salts, sodium lauryl sulfate Humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; and lubricants such as purified talc, stearate, boric acid powder and polyethylene glycol .

  An example of the intra-tissue dosage form is an injection. For preparation as an injection, it is desirable to use forms such as non-toxic solutions, emulsions and suspensions. They are preferably sterilized and isotonic with blood. In preparing these solutions, emulsions and suspensions, for example, water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. are used as diluents. be able to. A sufficient amount of sodium chloride, glucose, or glycerin to prepare an isotonic solution, and usual solubilizers, buffers, pH adjusters, soothing agents, and the like can also be blended. These injections can be administered subcutaneously, intramuscularly or intravenously.

  Examples of the topical dosage form include external preparations such as topical solutions, creams, powders, pastes, gels and ointments. These include a certain amount of a PAC composition, a prodrug thereof, a pharmaceutically acceptable salt thereof, and the like according to the present embodiment, a fragrance, a colorant, a filler, a surfactant, a moisturizing agent that are suitable for the purpose of external preparation. It can be prepared by combining with one or more of agents, emollients, gelling agents, carriers, preservatives, stabilizers and the like.

  Suppositories can be mentioned as a rectal dosage form. In preparing the suppository form, the base material is a low-melting-point base material such as higher esters such as myristyl palmitate, higher alcohols, polyethylene glycol, cacao butter, gelatin, semi-synthetic glycerides, and mixtures thereof. Can be mentioned. The suppository can be prepared by mixing and molding the PAC composition according to the present embodiment, a prodrug thereof, a pharmaceutically acceptable salt thereof, and the like into the base material as described above.

  When the hepatic fibrosis inhibitor or hepatic stellate cell growth inhibitor according to this embodiment is administered as a pharmaceutical, the effective dose of the PAC composition (or its prodrug, or a pharmaceutically acceptable salt thereof) is: Although it cannot be specified because it varies depending on the patient's age, weight, nature of disease, progress, administration route, etc., it is usually in the range of 1 to 2000 mg per day in terms of 60 kg body weight for an adult. Depending on the patient's condition and the like, a dose below or above the above range can also be administered. When administering a large amount, it is desirable to divide the dose into several times a day. In the case of pharmaceuticals, the effective content of PAC cannot be generally specified in relation to the dose or dose, but it can be selected in the range of 0.1 to 99.5% by weight, preferably 0.5 to 90% by weight. Good.

  Examples of the use of the pharmaceutical using the PAC composition according to the present embodiment include a liver fibrosis inhibitor, a hepatic stellate cell growth inhibitor, a hepatic stellate cell DNA synthesis activation inhibitor, a hepatic stellate cell apoptosis inducer, or a liver Examples include inhibitors of the onset or progression of liver diseases (for example, cirrhosis and liver cancer) caused by fibrosis.

  The PAC composition according to the present embodiment can also be used as a functional supplement food or drink for preventing the onset and progression of the above diseases, including suppression of liver fibrosis. The functional food and drink preparation can be in the form of a solid, liquid, emulsion or the like, but a pharmaceutically or food-acceptable carrier or additive can be blended as necessary. In addition to general dosage form adjuvants and the like, the additives may include active ingredients such as other drugs, functional ingredients such as vitamins and other trace ingredients. The dosage form is not particularly limited, but is preferably a form suitable for oral administration. Moreover, the form is not particularly limited, but it can be prepared in the form of tablets, pills, capsules, granules, powders, powders, troches, or solutions (drinks).

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples. In the following examples, “%” means “w / w% (mass%)” unless otherwise specified.

1. Preparation of PAC from each material As described above, PAC is widely found in the plant kingdom. The materials listed below (Samples 1 to 3) reported to contain PAC were obtained, and PAC fractions were prepared.

Sample 1: Blueberry leaf Sample 2: Grape seed extract (Gravinol ^TM : Kikkoman Corporation)
Sample 3: Croton sap (Sangre de Drago; Raintree Nutrition, Inc.)

(Sample 1: Preparation of PAC fraction derived from blueberry leaves)
Freezing dry leaves of rabbit eye blueberry seeds ( Vaccinium virgatum Aiton) by freeze-drying (FTS System, Dura-Top MP & Dura-Dry MP) and grinding with ultracentrifuge grinder (MRK & RETSCH, EM-1) A dry powder was obtained. 100 g of hexane was added to 10 g of this lyophilized powder, and the mixture was extracted by shaking at room temperature for 30 minutes, and the residue was collected by decantation. This extraction operation was repeated three times to obtain a washed product of blueberry leaf hexane.

  Furthermore, 100 ml of ethyl acetate was added to the resulting washed product of blueberry leaf hexane, and similarly extracted by shaking for 30 minutes, and the residue was recovered by decantation. This was similarly repeated three times to obtain a washed product of hexane-ethyl acetate.

  Next, 100 ml of methanol was added to the washed product of hexane-ethyl acetate, extracted by shaking for 30 minutes, the extract was collected by filtration, 100 ml of methanol was added again to the residue, and the extracts were combined three times in the same manner.

The extract was concentrated under reduced pressure using an evaporator and then freeze-dried to obtain about 3.5 g of a blueberry leaf methanol extract. Furthermore, about 500 mg of this extract was dissolved in 60% methanol and applied to a Sephadex ^™ LH-20 (volume 100 ml; manufactured by GE Healthcare Bioscience) column equilibrated with 60% methanol. Washed and then washed with 400 ml of 100% methanol. Thereafter, the residue was eluted with 400 ml of 70% acetone, concentrated under reduced pressure, and lyophilized to obtain about 100 mg of a blueberry leaf-derived PAC fraction (sample 1). Similarly, the following samples were prepared.

(Preparation of PAC fractions derived from samples 2-3)
Samples 2 to 3 were obtained directly, extracted with 100% methanol, concentrated under reduced pressure, and then lyophilized. Thereafter, as in the above preparation, a PAC fraction was prepared with Sephadex LH-20.

Composition analysis of PAC preparation PAC purity, composition and average degree of polymerization were examined for samples 1 to 3 extracted from each source. The analysis methods and results used are shown below.

2. Butanol-hydrochloric acid hydrolysis method (Porter method)
PAC was analyzed using the porter method according to the method of the following literature.
-Porter L. J. et al. et al. "Phytochemistry", 1986, Vol. 25, p. 223-230
Shoji T. et al. "J. Agric. Food Chem.", 2006, Vol. 54, p. 884-892

  PAC breaks the bond between flavans by heating under acidic conditions to give carbenium ions and catechins. The former is further oxidized to red anthocyanidins. The coloration method using this principle is the Porter method, and the PAC in the sample can be detected sharply by increasing the absorbance at 540 nm.

(1) Qualitative and quantitative determination of PAC by Porter method The purified fraction is dissolved in methanol, and 200 μl of 1-butanol containing 5% (v / v) hydrochloric acid and 750 μl of 1% (w / v) ammonium iron sulfate are contained. 50 μl of 2 mol / L hydrochloric acid was added and reacted at 105 ° C. for 40 minutes. The reaction solution was returned to room temperature, and the absorbance at 540 nm was measured. Procyanidin B2 (manufactured by Sigma) was used as a control.

(2) Composition analysis; LC / MS method The LC / MS was used to analyze the Porter method reaction decomposition product. If the PAC was purified from blueberry leaves, it would detect anthocyanidins. The apparatus and conditions used for LC / MS are as follows.
・ Device: Shimadzu LC / MS-IT-TOF
Column: Atlantis T3, 2.1 mm I.D. D. × 100 mm, 3 μm (manufactured by Waters), 40 ° C.
Mobile phase (eluent): (A) 0.5% (v / v) formic acid containing 5 mM ammonium formate
(B) Acetonitrile gradient: eluent B 10% (0 min) → 40% (15 min) → 100% (15 min) → 100% (22.5 min)
-Mobile phase flow rate: 0.25 ml / min-Detector 1: Photodiode array 540 nm
Detector 2: ESI-MS
(Positive-Ion Mode, interface voltage: 4.5 kV,
CDL temperature 200 ° C., nebulization gas flow rate: 1.5 L / min,
(Dry gas pressure: 200 kPa, heat block temperature: 200 ° C.)

  The samples of the plant-derived PAC preparations prepared above showed a red color by this method, which revealed the presence of PAC. Further, as a result of LC / MS analysis, the elution time of the main peak of the decomposed product was the same as that of the cyanidin sample, and the MS / MS spectrum was also matched, so that it was identified as PAC (FIG. 2). Furthermore, as a result of quantifying procyanidin B2 as a sample, the purity of PAC was 55.91 to 88.12% (Table 1).

  Moreover, as a result of analyzing the anthocyanidin composition by LC / MS, the preparation derived from blueberry leaves and grape seeds was 100% procyanidin, but the one derived from croton seed sap contains about 40% prodelphinidin. (Table 1).

3. Analysis of average polymerization degree and structural unit composition of PAC by thiol cleavage From the above results, it was shown that the main component of each plant-derived preparation was PAC. Next, in order to examine the structural unit, the binding mode and the degree of polymerization of PAC, the method of Guyot et al. (Guyot S. et al., “J. Agric. Food Chem.”, 2001, vol. 49, p. 14-20), thiol cleavage analysis was performed.

FIG. 3 shows the basic reaction of thiol cleavage. In the basic reaction of thiol opening, by reacting PAC with toluene-α-thiol under acidic conditions, the extension unit becomes a benzylthioether adduct and the terminal unit becomes free flavan-3-ol. By analyzing these thiol cleavage reaction products by reverse phase HPLC, it is possible to obtain information on each composition and average degree of polymerization (mDP) of the terminal unit and the extension unit. The average degree of polymerization (mDP) can be determined by the following equation.
Average degree of polymerization (mDP) = [terminal unit + extension unit] / [terminal unit]

  Catechin and epicatechin were used as preparations and corrections were made taking into account the isomerization of free flavan-3-ols produced during the thiol cleavage reaction (Gu L. et al., “J. Agric. Food. Chem. ", 2002, 50, pp. 4852-4860).

(1) Thiol cleavage, high performance liquid chromatography (HPLC) conditions Blueberry leaf-derived active compound PAC is prepared to 1 mg / ml with methanol, 50 μl is taken, and 50% of 3.3% (v / v) hydrochloric acid methanol is added, After adding 100 μl of 5% (v / v) toluene-α-thiol and reacting at 50 ° C. for 30 minutes, the reaction solution was diluted 5-fold with methanol and subjected to HPLC. The HPLC conditions are shown below.
-Equipment: Shimadzu Prominence LC-20A
Column: Atlantis T3, 4.6 mm I.D. D. × 150 mm, 3 μm (Waters), 40 ° C.
Mobile phase (eluent): (A) 0.05% (v / v) trifluoroacetic acid
(B) Acetonitrile gradient: Eluent B 15% (0 min) → 25% (10 min) → 60% (40 min) → 100% (40 min) → 100% (50 min)
-Mobile phase flow rate: 0.70 ml / min-Detector: UV280nm

(2) Identification of reactants; liquid chromatograph mass spectrometry (LC / MS)
The reaction product was similarly subjected to LC / MS to identify each detection peak. The analysis conditions are shown below.
・ Device: Shimadzu LC / MS-IT-TOF
Column: Atlantis T3, 2.1 mm D. × 100mm (made by Waters)
Mobile phase (eluent): (A) 0.05% (v / v) trifluoroacetic acid
(B) Acetonitrile gradient: Eluent B 15% (0 min) → 25% (10 min) → 60% (40 min) → 100% (40 min)
-Mobile phase flow rate: 0.25 ml / min-Detector 1: Photodiode array 280 nm
Detector 2: ESI-MS
(Positive-Ion Mode, interface voltage: 4.5 kV, CDL temperature 200 ° C., nebulization gas flow rate: 1.5 L / min, dry gas pressure: 200 kPa, heat block temperature: 200 ° C.)

  When PAC prepared from blueberry leaves was cleaved by thiol and separated by HPLC, 8 main peaks were mainly confirmed. An HPLC chromatogram is shown in FIG. Catechin, epicatechin and toluene-α-thiol were confirmed from the elution time of the sample, and peak A was found to be catechin, peak C was epicatechin, and peak H was found to be toluene-α-thiol. Other peaks were analyzed again by LC / MS, and the MS spectrum of each peak was confirmed.

  Peak E detected m / z = 411.0892, and this composition was estimated to be C22H20O6S (Error = −3.8 ppm) by composition estimation. In addition, m / z = 287.0510 was detected in the MS / MS spectrum, and the difference between the parent MS and MS / MS was 124.0382. Therefore, it was estimated that peak E was a benzylthioether adduct. . In addition, subtraction of the toluene-α-thiol composition formula C7H8S from the composition formula C22H20O6S estimated from the parent ion yields C15H14O6, confirming that peak E is a benzylthioether adduct of catechin or epicatechin. did it. Furthermore, when the procyanidin B2 preparation is subjected to thiol cleavage, epicatechin and epicatechin benzylthioether are produced. When this decomposition product was analyzed, the elution time of epicatechin benzyl thioether and peak E was the same, so peak E was epicatechin benzyl thioether.

  Peak G detects m / z = 697.1385 as the parent ion and m / z = 573.0987 in MS / MS, and the difference is 124.0398. This peak is also a benzylthioether adduct. I understood that. From this exact mass, it was estimated as C37H30O12S. When the benzylthioether adduct is removed from this composition, it becomes C30H24O12, and therefore peak G was estimated to be an A-type dimer benzylthioether adduct composed of catechin and epicatechin.

According to Thompson et al. (See Thompson R. S. et al., “J. Chem. Soc. Perkin Trans. I”, 1972, p. 1387-1399), the A-type interflavan linkage is against thiol cleavage. It is said that it is resistant. After the thiol cleavage reaction, if A-type is present in the terminal unit, it is released as the corresponding A-type dimer, and if it is in the extension unit, the corresponding benzylthioether adduct is detected (Foo L. Y). Et al., “J. Nat. Prod.”, 2000, 63, pp. 125-1228).

  The parent ion of peak B was m / z = 8633.1822, and the composition was estimated from this exact mass, resulting in C45H36O18 (Error = −0.86 ppm). Since the molecular formula of the B-type trimer is C45H38O18 and the A-type is C45H34O18, this peak B was assumed to be a trimer in which A-type and B-type are mixed.

  The peak D was m / z = 985.2009, and when the benzylthioether adduct was removed, C45H36O18 was obtained, and this compound was presumed to be an AB-type mixed trimer benzylthioether.

  Peak F detected m / z = 6055.1449, and m / z = 481.1109 was detected by MS / MS, so this peak was considered to be a benzylthioether adduct, but when the adduct was removed, C25H24O10 Since no further information was obtained, “Unknown” is displayed in Table 2.

  As a result of thiol cleavage of the PAC prepared from the above, as a result of decomposition of the PAC, a free trimer, an A-type dimer and a trimer other than a flavan-3-ol monomer and a benzylthioether adduct of a flavan-3-ol monomer Since the benzylthioether adduct was detected and its composition ratio was relatively high, the calculation method of mDP was revised.

That is, assuming that the degree of polymerization of the detected flavan-3-ol is n (including the benzylthioether adduct), mDP was determined by the following equation.
mDP = [sum of (benzylthioether adduct × n) + sum of (free flavan-3-ols × n)] / [sum of free flavan-3-ols]

  In addition, the area ratio of the detected flavan-3-ol and its benzylthioether adduct was calculated from a thiol cleavage reaction product of a known procyanidin B2 preparation, and each was corrected. As a result, as shown in Table 2, the mDP of PAC prepared from blueberry leaves was 11.2. In addition, the composition was generally high in epicatechin ratio, 42.6% for the terminal unit and 69.6% for the extension unit, but the detected A-type dimer and AB- Since the composition of the type-mixed trimer is unknown, it was found that 95% or more was composed of epicatechin when the entire composition was viewed with only the detected monomer.

Tables 2 and 3 show the average degree of polymerization (mDP) and composition of the PAC used in the present invention.

From the results in Table 2, the PAC preparations of each origin were characterized as follows:
(1) Blueberry leaf-derived PAC had an average degree of polymerization of 11.2, a high epicatechin composition ratio, and was composed entirely of procyanidins. In addition, it has an A type bond, and unidentified side chains were also confirmed.
(2) The grape seed-derived one had an average degree of polymerization of 14.4, which was longer than that of blueberries, the terminal unit had a higher catechin composition ratio, and the extension was about 1: 1 for epicatechin and epicatechin gallate. Side chains other than A-type binding mode and gallate were not confirmed.
(3) Those derived from croton seed sap had an average degree of polymerization of 8.3 and confirmed gallocatechin and epigallocatechin. Its prodelphinidin content was about 40%.

4). Functional analysis of PAC preparation As a hepatic stellate cell, a human cultured hepatic stellate cell subculture strain (LI90; purchased from Human Science Foundation, Catalog No., JCRB0160) was used to evaluate the liver fibrosis suppression function.

As test samples, BB-PAC purified by the method of Preparation Example 1, GS-PAC purified by the method of Preparation Example 2, and CL-PAC purified by the method of Preparation Example 3 were used. PAC or EGCG [(−)-epigallocatechin-O-gallate; Kurita High Purity Reagent, Kurita Analysis Center Co., Ltd., Lot No. 038311], catechin [(±) -catechin; Kurita high purity reagent, Kurita Analysis Center Co., Ltd., Lot No. C012002J15], epicatechin (SIGMA, E1753-1G, Lot No. 026K2611) and procyanidin B2 (Bio Chemika, 42157, 1 mg) are all serum-free DMEM so that the final concentration is 0.1 to 10 μg / ml. Diluted with medium and added. The final concentration of DMSO, which is the solvent for the reagent, was unified to 0.1% for all groups. After 1 hour of pre-culture, PDGF-BB (recombinant human plate growth factor, R & D SYSTEMS, No. 220-BB) was added to LI90 to a final concentration of 10 ng / ml and further cultured. After completion of the culture, the growth of LI90 was evaluated by MTT method and the amount of DNA synthesis was evaluated using BrdU labeling & detection kit (Roche Diagnostics Co., USA). Moreover, the relative amount of DNA fragmentation caused by apoptosis was measured by Cell Death Detection ELISA ^PLUS (Roche Diagnostics Co., USA). The phosphorylation of ERK (p42 and p44) and Akt (Ser473) was detected by Western blot method, and the intensity of the obtained band was quantified and digitized. The antibodies used in the Western blot method were Phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (D13.14.4E) rabbit monoclonal antibody (Cell Signaling TECHNOLOGY, # 9910), p44 / 42 (Erk1 / 2) (137F5) rabbit monoclonal antibody (Cell Signaling TECHNOLOGY, # 9926), Phospho-Akt (Ser473) (D9E) rabbit monoclonal antibody (Cell Signaling TECHNOLOGY, # 9916) and Akt (pan) (C67E7) rabbit monoclonal antibody (Cell) Signaling TECHNOLOGY, # 9916).

  The test of statistical significance in each experimental result was based on a multiple comparison test (Tukey method and Dunnet method).

  FIG. 5 shows the growth inhibitory effect on the number of LI90 cells by BB-PAC. As shown in the figure, BB-PAC suppressed the proliferation of the number of LI90 cells and the increase in the number of LI90 cells treated with PDGF-BB in the concentration range of 1 to 10 μg / ml.

  FIG. 6 shows the DNA synthesis activation inhibitory effect of LI90 cells treated with PDGF-BB by BB-PAC. As shown in the figure, BB-PAC suppressed the increase in the amount of DNA synthesis to almost the control level at 1 μg / ml.

  FIG. 7 shows the ERK and Akt phosphorylation inhibitory effect of LI90 cells treated with PDGF-BB by BB-PAC. As shown in the figure, BB-PAC completely suppressed ERK phosphorylation by PDGF-BB treatment at 1 μg / ml. In addition, phosphorylation of Akt (Ser473) by PDGF-BB treatment was partially suppressed.

  FIG. 8 shows the effect of inhibiting DNA synthesis activation of LI90 cells treated with PDGF-BB by PAC other than BB-PAC. As shown in the figure, grape seed-derived GS-PAC and croton seed sap-derived CL-PAC have the same concentration as GS-PAC and, like BB-PAC, increased the amount of LI90 cell DNA synthesis by PDGF-BB. Was strongly suppressed.

  FIG. 9 shows the DNA synthesis activation inhibitory effect of LI90 cells treated with PDGF-BB depending on the degree of polymerization of PAC. As shown in the figure, catechin composed of PAC monomer, epicatechin and EGCG, and procyanidin B2 composed of epicatechin dimer have the same concentration as BB-PAC (average degree of polymerization 11.2) of positive control. In the range (3 and 10 μg / ml), an inhibitory effect on the increase in the amount of DNA synthesis comparable to BB-PAC and EGCG (control) was not observed.

  FIG. 10 shows the growth-suppressing and apoptosis-inducing effects of LI90 cells treated with PDGF-BB by BB-PAC. As shown in the figure, BB-PAC suppressed the proliferation of LI90 cells treated with PDGF-BB in a concentration-dependent manner in the concentration range of 3-10 μg / ml and significantly promoted DNA fragmentation. The latter suggests that BB-PAC is active in inducing apoptosis of PDGF-BB treated LI90 cells.

From these, the following can be concluded as a conclusion.
(1) Blueberry leaf-derived BB-PAC strongly suppresses the proliferation of LI90 cells and the DNA synthesis activation of LI90 cells by PDGF-BB treatment, similar to EGCG.
(2) Grape seed-derived GL-PAC and croton sap-derived CL-PAC have the same inhibitory effect as blueberry leaf-derived BB-PAC.
(3) Monomers and dimers such as catechin, epicatechin and procyanidin B2 have almost no similar inhibitory effect.
(4) Blueberry leaf-derived BB-PAC suppresses phosphorylation of ERK and Akt by PDGF-BB treatment.
(5) BB-PAC induces apoptosis of PDGF-BB treated LI90 cells.

  An inhibitor of liver fibrosis, an inhibitor of hepatic stellate cell proliferation, an inhibitor of hepatic stellate cell DNA synthesis activation, an hepatic stellate cell apoptosis inducer, or a liver disease caused by liver fibrosis, comprising a PAC composition as an active ingredient, For example, an inhibitor of the onset or progression of cirrhosis or liver cancer has an excellent effect in preventing or treating liver disease. In addition, since it can be taken over a long period of time, it can also be used as a functional auxiliary food for these improvements.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a structural formula diagram of epicatechin and catechin, which are representative components of a flavan compound constituting PAC, and gallic acid that often has an ester bond to a 3-position hydroxyl group of a flavan ring. (A) is an MS / MS spectrum of a cyanidin preparation, and (B) is a graph showing a porter-degraded product MS / MS spectrum of a blueberry leaf-derived PAC. It is the schematic diagram which showed thiol cleavage which is reaction which heats PAC with benzyl mercaptan on acidic conditions. It is a graph which shows the reverse phase chromatogram of the thiol cleavage reaction product of PAC derived from a blueberry leaf. It is a graph which shows the effect | action of BB-PAC with respect to the cell growth of LI90 cell by PDGF-BB processing or a non-processing. It is a graph which shows the effect | action of BB-PAC with respect to the DNA synthesis | combination of LI90 cell by PDGF-BB processing or non-processing. The photograph and graph which show the effect | action of BB-PAC with respect to ERK and Akt phosphorylation of LI90 cell by PDGF-BB or an untreated, (A) and (C) are a photograph, (B) and (D) are a graph. It is a graph which shows the effect | action of BB-PAC, GS-PAC, and CL-PAC with respect to the DNA synthesis | combination of LI90 cell by PDGF-BB processing or non-processing. It is a graph which shows the effect | action of BB-PAC with respect to the DNA synthesis | combination of LI90 cell by PDGF-BB treatment or non-processing, and the effect | action of EGCG, catechin, epicatechin, and procyanidin B2. It is a graph which shows the effect | action of BB-PAC with respect to the cell proliferation and DNA fragmentation of LI90 cell by PDGF-BB treatment.

Claims (12)

A liver fibrosis inhibitor comprising a composition comprising one or more proanthocyanidins as an active ingredient ,
The proanthocyanidins have the following general formula (1)

[In Formula (1), R ₁ and R ₃ each independently represent a hydrogen atom or a hydroxyl group, R ₂ represents a hydroxyl group, and R ₄ represents a hydrogen atom or a gallate group. ]
A hepatic fibrosis inhibitor having a structure in which three or more of the flavan-3-ol skeletons represented by (i), (ii), or (iii) are bonded to each other.
(I) Bond at the 4-position carbon and the 8-position carbon
(Ii) 4-position carbon and 6-position carbon bond
(Iii) 4-position and 8-position carbon bonds and 2-position and 7-position oxygen bonds The liver fibrosis inhibitor of Claim 1 whose content of the composition which consists of the said proanthocyanidins is 55.91-88.12%. The hepatic fibrosis inhibitor according to claim 1 or 2, wherein the composition comprising proanthocyanidins is derived from blueberry leaves. The hepatic fibrosis inhibitor according to claim 1 or 2, wherein the composition comprising proanthocyanidins is derived from grape seeds. The hepatic fibrosis inhibitor according to claim 1 or 2, wherein the composition comprising proanthocyanidins is derived from croton seed sap. A hepatic stellate cell growth inhibitor comprising a composition comprising one or more proanthocyanidins as an active ingredient ,
The proanthocyanidins have the following general formula (1)

[In Formula (1), R ₁ and R ₃ each independently represent a hydrogen atom or a hydroxyl group, R ₂ represents a hydroxyl group, and R ₄ represents a hydrogen atom or a gallate group. ]
A hepatic stellate cell growth inhibitor having a structure in which three or more of the flavan-3-ol skeletons represented by the formula (i), (ii) or (iii) are bonded to each other .
(I) Bond at the 4-position carbon and the 8-position carbon
(Ii) 4-position carbon and 6-position carbon bond
(Iii) 4-position and 8-position carbon bonds and 2-position and 7-position oxygen bonds The hepatic stellate cell growth inhibitor according to claim 6, wherein the content of the composition comprising proanthocyanidins is 55.91 to 88.12%. The hepatic stellate cell growth inhibitor according to claim 6 or 7, wherein the composition comprising proanthocyanidins is derived from blueberry leaves. The hepatic stellate cell growth inhibitor according to claim 6 or 7, wherein the composition comprising proanthocyanidins is derived from grape seeds. The hepatic stellate cell growth inhibitor according to claim 6 or 7, wherein the composition comprising proanthocyanidins is derived from croton seed sap. The hepatic stellate cell growth inhibitor according to any one of claims 6 to 10 , based on hepatic stellate cell apoptosis. A liver disease caused by liver fibrosis, comprising the liver fibrosis inhibitor according to any one of claims 1 to 5 or the hepatic stellate cell growth inhibitor according to any one of claims 6 to 11. Onset / proliferation inhibitor.