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Title:
COMPOSITION COMPRISING PHENOLIC COMPOUND FOR PREVENTING AND TREATING LIVER CIRRHOSIS
Document Type and Number:
WIPO Patent Application WO/2004/002471
Kind Code:
A1
Abstract:
The present invention relates to a composition for treatment and prevention of liver cirrhosis comprising a phenolic compound. The composition comprises at least one selected from protocatechuic acid, butein, fustin, sulfuretin, fisetin and derivatives thereof as an effective ingredient and can be used as a medicament or health food for the purpose of prevention and treatment of liver cirrhosis.

Inventors:
NA CHUN-SOO (KR)
JUNG NAM-CHUL (KR)
DONG MI-SOOK (KR)
Application Number:
PCT/KR2003/001269
Publication Date:
January 08, 2004
Filing Date:
June 27, 2003
Export Citation:
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Assignee:
LIFETREE BIOTECH CO LTD (KR)
NA CHUN-SOO (KR)
JUNG NAM-CHUL (KR)
DONG MI-SOOK (KR)
International Classes:
A23L1/30; A23L2/52; A23L7/10; A23L11/00; A61K31/05; A61K31/343; A61K31/353; A61P1/16; (IPC1-7): A61K31/353; A61K31/05; A61K31/343; A61P1/16
Foreign References:
JPS54160740A1979-12-19
EP0586917A11994-03-16
Other References:
ZHANG MIN ET AL.: "Effect of six flavonoids on proliferation of hepatic stellate cells in vitro", ACTA PHARMACOL. SIN., vol. 21, no. 3, 2000, pages 253 - 256
SVEGLIATI-BARONI G. ET AL.: "Wine: risk factors for liver disease and antifibriotic compounds", DRUGS EXPTL. CLIN. RES. XXV, vol. 2/3, 1999, pages 143 - 145
TANAKA T. ET AL.: "Chemoprevention of diethylnitrosamine-induced hepatocarcinogenesis by a simple phenolic acid, protocatechuic acid, in rats", CANCER RES., vol. 53, no. 12, 1993, pages 2775 - 2779
Attorney, Agent or Firm:
Darae, Patent Firm (KIPS 647-9 Yeoksam-don, Kangnam-ku Seoul 135-980, KR)
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Claims:
What Is Claimed Is :
1. A pharmaceutical composition for prevention or treatment of liver cirrhosis comprising a phenolic compound as an effective ingredient.
2. The pharmaceutical composition according to claim 1, wherein the phenolic compound comprises at least one selected from protocatechuic acid, butein, fustin, sulfuretin, fisetin and derivatives thereof.
3. The pharmaceutical composition according to claim 1, wherein the phenolic compound comprises a mixture of two or more selected from protocatechuic acid, butein, fustin, sulfuretin, fisetin and derivatives thereof.
4. A food composition for prevention or treatment of liver cirrhosis comprising a phenolic compound as an effective ingredient.
5. The food composition according to claim 4, wherein the phenolic compound comprises at least one selected from protocatechuic acid, butein, fustin, sulfuretin, fisetin and derivatives thereof.
6. The food composition according to claim 4, wherein the phenolic compound comprises a mixture of two or more selected from protocatechuic acid, butein, fustin, sulfuretin, fisetin and derivatives thereof.
Description:
COMPOSITION COMPRISING PHENOLIC COMPOUND FOR PREVENTING AND TREATING LIVER CIRRHOSIS Technical Field and Background Art The present invention relates to a composition for treatment and prevention of liver cirrhosis comprising a phenolic compound as an effective ingredient.

Liver cirrhosis is the final stage of chronic liver disease. Main causes of liver cirrhosis are various, including hepatitis virus infection, alcoholism, defective bile acid secretion, medicinal poisoning, allergy, excessive iron deposition, etc.

Liver cells can be repaired by regeneration owing to their strong regenerating ability if they are destroyed to some degree. However, when the destruction exceeds a certain point, the destroyed cells are not regenerated but fibrosed, causing the liver to be hardened. Such circumstance, in which the liver is hardened by change of its structure and cannot go back to the original state, is designated liver cirrhosis.

The process of liver fibrosis leading to liver cirrhosis is a reaction occurring in response to continuous stimulation and involves three steps, similar to the wound healing process: 1) acute inflammation, 2) synthesis of components of extracellular matrix (ECM) including collagen, and 3) tissue reconstruction (scar formation) (Ramadori R, Knittel R, and Saile B. (1998) Fibrosis and Altered Matrix Synthesis Digestion 59: 372-375).

Extracellular matrix is composed of collagen, matrix glycoproteins <BR> <BR> (fibronectin, laminin, etc. ), proteoglycan and the like, in which the main component is collagen. After liver cells are destroyed, a tissue of new liver cells is

reconstructed. At this time, if the cells fail to constitute perfect cellular structures of cytoplasm and extracellular membrane and only the extracellular membrane which supports the skeleton structure of a cell is developed, collagen which is a main component of extracellular matrix of liver cells is excessively deposited and by the fibrosed collagen, the liver tissue is hardened stiffly, leading to liver cirrhosis.

Therefore, in order to prevent liver fibrosis which induces liver cirrhosis, it is preferred to employ a method for inhibiting synthesis of collagen which is a main cause of liver fibrosis, or promoting degradation thereof.

Collagen is synthesized in hepatic stellate cells (HSC), among liver cells, of which proliferation is promoted when liver fibrosis is developed, and degraded by matrix metalloproteinase (MMP). The activity of MMP is controlled by gene expression, activation of proenzyme and MMP tissue inhibitor (TIMP) (Knittel T, Mehde M, Grundmann A, Saile B, Scharf JG, and Ramadori G. (2000) Expression of matrix metalloproteinases and their inhibitors during hepatic tissue repair in the rat Histochem Cell Biol 113 : 443-453.).

Therefore, preferred examples of drugs to inhibit liver cirrhosis include drugs to inhibit proliferation of stellate cells mainly taking charge of synthesis of collagen when liver fibrosis is developed, drugs to inhibit synthesis of components of extracellular matrix, including collagen, by other mechanisms, drugs to promote the activity of MMP, drugs to reduce the activity of TIMP, an MMP activity inhibitor, or drugs having two or more functions described above.

The present inventors have found that a group of compounds, among some phenolic compounds, can inhibit synthesis of components of extracellular matrix, including collagen, increase MMP activity, promotes inhibiting effect on TIMP activity and inhibit proliferation of stellate cells, and that these compounds or a

mixture thereof have effects to treat and prevent liver cirrhosis. Based on these discoveries, the present invention has been completed.

SUMMARY OF THE INVENTION Thus, it is an object of the present invention to provide a composition showing treating and preventing effects of liver cirrhosis.

It is another object of the present invention to provide a pharmaceutical composition or food composition showing treating and preventing effects of liver cirrhosis caused by liver fibrosis.

In accordance with the present invention, the above objects can be accomplished by the provision of a composition for treatment and prevention of liver cirrhosis comprising at least one phenolic compound as an effective ingredient The phenolic compound used in the composition according to the present invention may be any one selected from protocatechuic acid, butein, fustin, sulfuretin, fisetin or derivatives thereof.

According to the present invention, the phenolic compound can be used as a mixture of two or more selected from protocatechuic acid, butein, fustin, sulfuretin, fisetin or derivatives thereof.

The composition according to the present invention reduces synthesis of procollagen, TGF ß-1 and fibronectin which are representative proteins known to increase upon hepatic fibrosis, increase expression of MMP which degrades collagen, and reduce TIMP which is an MMP inhibitor.

Also, the composition according to the present invention can inhibit cell proliferation of HSC, whose proliferation is promoted upon hepatic fibrosis.

Further, the composition according to the present invention can treat or prevent fibrosis of liver cells and thus, prevent reduction of liver cells'ability to produce albumin and reduce GOT and GPT levels and bilirubin concentration in blood which are increased by hepatoxicity.

Therefore, the composition according to the present invention comprising a phenolic compound selected from protocatechuic acid, butein, fustin, sulfuretin, fisetin or a mixture of two or more thereof as an effective ingredient can used in treatment and prevention of liver cirrhosis by liver fibrosis.

The composition according to the present invention can provide a pharmaceutical composition or food composition useful for prevention and treatment of liver cirrhosis.

For administration, the composition according to the present invention can be prepared by combining at least one pharmaceutically acceptable or edible carrier in addition to the effective ingredient as described above.

The pharmaceutically acceptable or edible carrier includes saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol and a mixture of two or more thereof, and can be used in combination with other common additives such as an antioxidant, a <BR> <BR> buffer, a bacteriostatic agent, etc. , as needed. Also, the composition can be formulated into injection formulations, such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets by further adding a diluting agent, a dispersing agent, a surfactant, a binder and a lubricant. Further, the composition can be preferably formulated according to respective diseases or ingredients by a proper method known to the art or a method described in Remington's Pharmaceutical Science (the latest edition), Mack Publishing Company, Easton PA.

The composition according to the present invention can be administered orally or parenterally according to an intended method and the dose may vary depending on weight, age, sex and health condition of a patient, diet, administration time, administration method, excretion rate and severity of disease. The daily dose is in the range of 10 to 200 mg/kg, preferably in the range of 25 to 100 mg/kg, and this amount can be administered once or in portions several times per day.

The composition may be added to foods or drinks for the purpose of treatment and prevention of liver cirrhosis. Here, the amount of the phenolic compound added to a food and a drink is 0. 1 to 15 wt%, preferably 1 to 10 wt%, based on the total weight of the food, in case of a health food composition, or 1 to 30g, preferably 3 to lOg, per 100mE, in case of a health drink composition.

The health drink composition may contain additional ingredients including various flavors or natural carbohydrates like other common drinks. The natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin, cyclodextrin; and sugar alcohols such as xylitol, sorbitol and erythritol. As a sweetening agent, natural sweeteners such as thaumatin and stevia extracts or synthetic sweeteners such as saccharine and aspartame may be used. Generally, the natural carbohydrate may be added in the ratio of about 1 to 20g, preferably about 5 to 12g, per 1001ni of the composition.

Further, the composition may contain various nutrients, vitamins, electrolytes, flavors such as synthetic flavors and natural flavors, colorants, pectic acid and derivatives thereof, alginic acid and derivatives thereof, organic acids, a protective colloidal thickener, a pH controller, a stabilizer, a preservative, glycerin, alcohols, a carbonating agent for a carbonated drink and the like. In addition, the

composition may be combined with flesh of fruit to prepare natural fruit juices, fruit juice drinks and vegetable drinks. The above-described ingredients may be used alone or in combination with each other. The rate of these additives to the composition is generally selected from the range of 0 to 20 weight parts per 100 weight parts of the composition, though it is not critical.

For development of health foods, the composition may be added to various foods, including, for example, various food items, drinks, gums, vitamin complexes, health-aids, special nutrient supplements, functional foods and the like.

Formulation examples are described below. In the examples, the phenolic compound is any one selected from protocatechuic acid, butein, fustin, sulfuretin and fisetin or a mixture of two or more thereof.

Formulation 1: Preparation of powder Phenolic compound 2g Lactose Ig The above-described ingredients were mixed and charged in an air-tight package to prepare powder formulation.

Formulation 2: Preparation of tablet Phenolic compound 100nag Corn starch 100mg Lactose 200mg Magnesium stearate 2nig The above-described ingredients were mixed and tableted according to a conventional tableting method to prepare tablets.

Formulation 3: Preparation of capsule Phenolic compound 100mg Corn starch 100mg Lactose 1 00mg Magnesium stearate 2mg The above-described ingredients were mixed and sealed in gelatin capsules to prepare capsules.

Formulation 4: Preparation of Injection Phenolic compound 100mg Distilled water for injection appropriate amount pH controller appropriate amount The compounds were dissolved in distilled water for injection according to a conventional preparation method of an injection. The solution was adjusted to pH about 7.5 and charged into a 2mE ample with distilled water, followed by sterilization to prepare an injection.

Formulation 5: Preparation of powder of roasted grains, seeds, nuts, vegetables and mushrooms A health food was prepared as follows.

Brown rice, barley, glutinous rice and job's tears were made alpha and dried.

The dried product was roasted and milled into powder having a particle size of 60 mesh.

Black bean, black sesame and wild sesame were steamed and dried. The

dried product was roasted and milled into powder having a particle size of 60 mesh.

The powders of grains and seeds prepared above were combined with the phenolic compound and other additives in the ratio described below to prepare granules.

Grains: brown rice 30 wt%, job's tears 15 wt%, barley 20 wt%, Seeds: wild sesame 7 wt%, black bean 8 wt%, black sesame 7 wt%, Phenolic compound 3 wt%, Ten-thousand-year mushroom (Ganoderam Lucidum) 0.5 wt%, Chinese foxglove (Rehmannia glutinosa liboschitz) 0.5 wt% Formulation 6: Preparation of drink Drinks were prepared as follows.

1) Preparation of carbonated drink 5 to 10% of sugar, 0.05 to 0.3% of citric acid, 0.005 to 0.02% of caramel, 0.1 to 1% of vitamin C and 0.1 to 10% of the phenolic compound were mixed and 10 to 30% of purified water was added thereto to form syrup. The syrup was sterilized at 85 to 98 C for 20 to 180 seconds and mixed with cooling water in the ratio of 1: 4.

To the resulting solution, 0.5 to 0.82% of carbonic acid gas was injected to prepare a carbonated drink containing the phenolic compound according to the present invention.

2) Preparation of health drink The phenolic compound was combined with sub-ingredients such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), table salt (0.5%) and water (75%), instantaneously sterilized and packed in a small size container such as glass bottles,

PET bottles, etc. to prepare a health drink.

BRIEF DESCRIPTION OF THE DRAWINGS The above objects, features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings, in which: Fig. 1 shows the result of RT-PCR to confirm the effect of the phenolic compounds according to the present invention on expression of proteins related to liver cirrhosis (C: control, Sil: positive control, Ra: treatment group receiving a mixture of 5 different phenolic compounds, Bu: treatment group receiving butein alone, Fu: treatment group receiving fustin alone, Sul: treatment group receiving sulfuretin alone, Fi: treatment group receiving fisetin alone); Fig. 2 shows graphs obtained by scanning bands to quantify each of the proteins shown in Fig. 1; Fig. 3 shows the inhibiting effects of the phenolic compound according to the present invention on cell proliferation of the stellate cell (C: control, Sil: positive control, Ra: treatment group receiving a mixture of 5 different phenolic compounds, Bu: treatment group receiving butein alone, Fu: treatment group receiving fustin alone, Sul: treatment group receiving sulfuretin alone, Fi: treatment group receiving fisetin alone); and Fig. 4 shows the results of serological tests to confirm the inhibiting effect of the phenolic compound according to the present invention on liver cirrhosis [A: albumin level, B: bilirubin level, C: AST (GOT) level, D: ALT (GPT) level] (C: control, DMN : treatment group receiving dimethylsulphoxide alone, DMN+RA :

treatment group receiving dimethylsulphoxide and the phenolic compound).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Now, constructions and effects of the present invention will be described using the following examples. However, the present invention is not limited thereto.

Example 1: Inhibiting effect on liver fibrosis All the phenolic compounds used as reagents, protocatechuic acid, butein, fustin, sulfuretin, fisetin and sulfuretin were products of Indofinechemical Co. , Ltd., (USA).

As a cell line, HSC-T6, a rat stellate cell line was distributed from Dr.

Friedman (Mount Sinai Medical Center, NY).

The cell culture was performed by passage culturing once per 2 to 3 days in Dubellcos'modified Eagles Medium (DMEM) (Gibco BRL, Gaithersberg, MD, USA) containing 10% fetal calf serum and antibiotics (100U/m, penicillin, 100 g/m streptomycin).

Samples were each of the five phenolic compounds according to the present invention and a mixture thereof and the final treated concentration was set to 50//g/) n.

At 24 hours after treatment with each sample, expressions of procollagen, fibronectin, TGF P-1, MMP-13, TIMP-1 and TIMP-2 were examined by RT-PCR.

As a positive control, a cell line was injected with silybin.

The proteins were quantified by RT-PCR using a primer set described below.

Easy blue solution (Intronbiotec) was used to separate whole RNA, and the first cDNA was synthesized using oligo-dT primer, an RNase inhibitor, MMLV reverse transcriptase. The PCR product was relatively quantified on the basis of the amount of P-actin. Gene Primer Sequence Product (bp) MMP-13 (f) 5'AAA GAA CAT GGT GAC TTC TAC C 3' (SEQ ID NO : 1) 282 bp (rev) 5'ACT GGA TTC CTT GAA CGT C 3' (SEQ ID NO : 2) TIMP-1 (f) 5'ACA GCT TTC TGC AAC TCG 3' (SEQ ID NO : 3) 334 bp (rev) 5'CTA TAG GTC TTT ACG AAG GCC 3' (SEQ ID NO : 4) TIMP-2 (f) 5'ATT TAT CTA CAC GGC CCC 3' (SEQ ID NO : 5) 341 bp (rev) 5'CAA GAA CCA TCA CTT CTC TTG 3' (SEQ ID NO : 6) Fibronectin (f) 5-GGCCTGAACCAGCCTACG (SEQ ID NO : 7) 255 bp (rev) 5-CATTCCCGAGACATGTGC (SEQ ID NO : 8) TGFp-1 (f) 5-GCTTTGTACAACAGCACC (SEQ ID NO : 9) 882 bp (rev) 5-CTGCTCCACCTTGGGCTT (SEQ ID NO : 10) Procollagen 1 (f) 5-AACGATGGTGCCAAGGGT (SEQ ID NO : 11) 974 bp (rev) 5-AGCACCATCTCTTCCAGG (SEQ ID NO : 12) P-actin (f) 5-CCATGGAGAGCCTCTGTGGATAT (SEQ ID NO : 13) 422 bp (rev) 5-TGAGGATCTGATCTGTCCACAAG (SEQ ID NO : 14)

The results are shown in Figs. 1 and 2.

As shown in Fig. 1, the cells administered with the phenolic compounds according to the present invention (Bu: butein, Fu: fustin, Sul: sulfuretin, Fi: fisetin) and the mixture thereof (Ra) showed significant reduction in expression of procollagen, as compared with control (C) with nothing administered. Also, expression of hepatic MMP-13 was increased but expression of TIMP-1 was reduced.

In the positive control (Sil), it was observed that synthesis of procollagen was inhibited. From these results, it was noted that the composition according to the present invention affects various proteins, as compared to silyvin used in the positive control and inhibits many stages of the liver fibrosis process.

Fig. 2 shows graphs obtained by scanning bands to quantify each of the proteins shown in Fig. 1 Example 2: Inhibiting effect on cell proliferation of HSC The stellate cell is characterized in that, when liver fibrosis is developed, its proliferation is promoted and synthesis of extracellular matrix is activated.

Therefore, the inhibiting effect of the phenolic compounds according to the present invention on liver cirrhosis was confirmed by observing their inhibiting effect on cell proliferation of HSC.

Similar to Example 1, HSC-T6 cell line was used. The cells were treated with each of the five phenolic compounds according to the present invention, prepared in Example 1 and a mixture thereof at various concentrations in the range of 0 to 100//g/m. 60 hours later, the cells were examined by the MTS assay using CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay Kit (Promega).

Similar to Example 1, a cell line treated with silyvin was used as the positive control.

The results are shown in Fig. 3.

As shown in Fig. 3, the treatment groups, in which the cells had been treated with each of the five phenolic compounds according to the present invention. (F1 : protocatechuic acid, Bu: butein, Fu: fustin. Sul: sulfuretin, Fi: fisetin) and the treatment group, in which the cells were treated with a mixture thereof, (RA) had excellent inhibiting effects on cell proliferation, as compared to the control group and the positive control also had inhibiting effect on cell proliferation in a level similar thereto.

Example 3: Confirmation of inhibiting effect on liver cirrhosis by animal test Through animal tests, the composition was confirmed to have inhibiting effects on liver cirrhosis.

7 weeks-old Wister male rats were used for this test. The animals could freely access to feed (Samtaco) and water and the breeding room was maintained at a temperature of 23 2 C and a relative humidity of 55 10% and the lighting cycle in the farm was set at a 12/12 h light-dark cycle.

The animals were randomly divided into three groups: a control group, a treatment and a comparison group. The control group was orally administered with 2m. /kg of PEG400 once per day, continuously for 5 weeks, while being intraperitoneally administered with I in/kg of physiological saline for injection once per day, for continuous three days per week, over 5 weeks.

The treatment group and the comparison group were intrabperitoneally administered with 1 mE/kgof DMN (dimethylnitrosamine), a liver cirrhosis inducing substance, once per day, for continuous three days per week, over 5 weeks. The treatment group was also intraperitoneally injected with the mixture of the phenolic compounds prepared in Example 1 in an amount of 40mg/kg, once per day, continuously over 5 weeks..

For 24 hours after the final administration, the animals fasted and were sacrificed with ether. Blood was taken from the abdominal artery and measured for an albumin level, a bilirubin level, an AST (GOT) level and an ALT (GPT) level in blood.

The results are shown in Fig. 4.

As shown in Fig. 4, when the phenolic compounds according to the present

invention were administered, the albumin level (A) in blood was increased, as compared to the comparison group which had been administered with DMN alone.

Also, when the phenolic compounds were administered, the bilirubin level (B), indicating jaundice level, and the GOT (C) and GPT (D) levels, which are increased in case of hepatotoxicosis, were reduced, as compared to the comparison group which had been administered with DMN alone.

The composition according to the present invention can be used in medicaments or health foods for treatment and prevention of liver cirrhosis.