CN111356468A - Composition for preventing or treating fibrotic disease comprising extract of Rhus toxicodendron - Google Patents

Abstract

The invention relates to a composition having anti-fibrotic activity comprising a Rhus succedanea extract as an active ingredient, and more particularly, to a Rhus succedanea extract having an activity capable of effectively inhibiting a signaling pathway of transforming growth factor- β 1 (TGF- β 1) that plays an important role in fibrosis, thereby being capable of inhibiting the expression and activity of extracellular matrices such as α smooth muscle actin (α -SMA), Connective Tissue Growth Factor (CTGF), fibronectin outer domain A (FN-EDA), collagen, etc., induced by transforming growth factor- β 1, and having high anti-oxidative activity, and thus the composition of the invention comprising the Rhus succedanea extract as an active ingredient has an effect of being effectively used for preparing a therapeutic agent for fibrotic diseases and a health functional food.

Description

Composition for preventing or treating fibrotic disease comprising extract of Rhus toxicodendron

Technical Field

The present invention relates to a composition for preventing or treating fibrotic diseases, comprising a rhus toxicodendron extract as an effective ingredient.

Background

Most organs undergo inflammation and healing processes following tissue injury, and if there is sustained injury, fibrosis of the tissue occurs during the healing process. During fibrosis, extracellular matrix (ECM) such as collagen or fibronectin accumulates in tissues and destroys normal tissues, resulting in dysfunction. In fibrosis, since extracellular matrix is derived from fibroblasts, activated fibroblasts and myofibroblasts (myofibroblasts) located at the center of fibroblasts have been actively studied in the research of fibrosis and anti-fibrosis.

Fibrosis is also found in various organs, tissues and cells, and fibrotic diseases are known to occur in skin (hyperproliferative scars, keloids, scleroderma), liver (fibrosis and cirrhosis), lung (fibrosis), heart (fibrosis), intestine (adhesions and strictures), joints (fibrosis of the joint membrane), bone marrow (fibrosis and dysplastic syndrome), and the like.

In particular, hepatic fibrosis is a precursor lesion of hepatic cirrhosis and is triggered by the action of various cytokines and growth factors due to severe liver injury causing chronic liver diseases in general, hepatic fibrosis is reversible and consists of fine fibers (thinfibrates), without nodule formation, in the case where the cause of hepatic injury is transient, extracellular matrix (ECM) increased by the action of apoptosis (apoptosis) process and Matrix Metalloproteinases (MMPs) is degraded and normally restored, however, if the hepatic fibrosis process is repeated, coarse fibers (thick fibers) are formed and progressed to hepatic cirrhosis with nodules, and hepatic cells are damaged due to various factors causing inflammation and induced by the process of accumulation of abnormal extracellular matrix proteins including collagen, thus, in order to regulate the expression of hepatic cirrhosis, the regulation of extracellular matrix accumulation is important in the case of inflammatory response, the regulation of extracellular matrix accumulation is induced by the action of various factors, the growth factors of collagen-derived extracellular matrix protein (TGF-5), the growth factor-activating factor is induced by the action of collagen-derived from collagen-derived growth factors (TGF-5), and the growth factor-stimulating the growth of liver fibrosis-derived from collagen-derived growth factor (TGF-5), and the growth factor-derived from collagen-derived collagen-activating factor-derived from collagen-activating collagen-producing collagen-activating factor (collagen-activating factor-collagen-producing collagen-activating factor-producing collagen-activating factor-collagen-activating collagen-producing collagen-activating factor-collagen-producing collagen-activating factor-collagen-producing collagen-activating factor-collagen-activating factor-collagen-producing collagen-activating factor-collagen-transforming the liver-activating factor-transforming the liver-activating factor-transforming the collagen-activating factor-transforming the collagen-activating factor-transforming the liver-fibroblast-activating factor-transforming the collagen-activating factor.

Pulmonary fibrosis (pulmonary fibrosis) is a disease in which a binding tissue, particularly collagen, excessively adheres to the alveolar wall to cause dyspnea, and pulmonary fibrosis is a dangerous condition in which a lung tissue becomes hard and an otherwise soft and elastic tissue cannot freely undergo contraction and expansion for breathing at a hardened site, so that the respiratory capacity is reduced, the remaining lung capacity is reduced, the lung function is decreased, and in the worst case, death may occur due to dyspnea or respiratory failure.

As a method for treating such fibrotic diseases such as hepatic fibrosis and pulmonary fibrosis, most of the therapeutic agents currently used are chemotherapeutic agents and have a problem of causing various side effects in the human body, and therefore, there is a need for development of a novel anti-fibrotic disease therapeutic agent having a more basic therapeutic effect and derived from a natural product stabilized in the body.

Further, a yellow sumac (Dendropanax morbifera Lev.) belonging to Araliaceae is an evergreen broad-leaved arbor growing in coastal areas of south Korea and Jizhou islands, which is a tree species that does not fall leaves even in winter, and a yellow sap flows out from a cut on the bark, and is called yellow paint.

Since the three kingdoms times of korea, yellow paint has been used as a rare paint, which emits golden yellow of the armour, helmet and other metal ornaments of emperor, the taking time, use and the like of yellow paint are recorded in "the section of the history of gorgeous", "the class of chicken" in china, "the forest of chicken", and "the history of the Shandong", written in the dynasty history of china, "the manyuan tortoise" in book and "the dictionary" as the former Baiji products. Also, it has been reported that the phase-transfer mahogany has effects of removing dysphoria with smothery sensation, treating eye diseases, treating jaundice, treating burn and leprosy, and is harmless to the human body.

To this end, the present inventors confirmed that the Rhus succedanea extract has the effect of regulating the expression and activity of transforming growth factor- β 1 and fibrosis-associated factors, which play an important role in fibrosis, and thus completed the present invention by confirming that the Rhus succedanea extract can be used for preventing and treating fibrotic diseases.

Disclosure of Invention

Technical problem

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating a fibrotic disease, comprising a rhus toxicodendron extract as an effective ingredient.

Also, another object of the present invention is to provide a health functional food for preventing or improving fibrotic diseases comprising extract of rhus toxicodendron.

Means for solving the problems

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating fibrotic diseases, comprising a rhus toxicodendron extract as an active ingredient.

In an embodiment of the present invention, the Rhus succedanea extract can be a crude Rhus succedanea extract, a polar solvent soluble extract or a non-polar solvent soluble extract.

In one embodiment of the present invention, the crude extract may be an extract obtained using a solvent selected from water including purified water, methanol, ethanol, butanol, or a mixed solvent thereof.

In an embodiment of the present invention, the polar solvent-soluble extract may be an extract obtained by using a solvent selected from water, ethanol, butanol or a mixture thereof

In an embodiment of the invention, the non-polar solvent soluble extract may be an extract obtained by using hexane, chloroform, dichloromethane or ethyl acetate.

In one embodiment of the present invention, the Rhus succedanea extract can inhibit the signal transduction pathway of TGF- β 1.

In an embodiment of the present invention, the fibrotic disease may be selected from the group consisting of proliferative scar, keloid, scleroderma, peritoneal adhesion, liver fibrosis, lung fibrosis, cardiac fibrosis, skeletal muscle fibrosis, synovial fibrosis, myelofibrosis, myelodysplastic syndrome and diabetic retinopathy.

Also, the present invention provides a health functional food for preventing or improving fibrotic diseases comprising the extract of Rhus toxicodendron.

In an embodiment of the present invention, the Rhus succedanea extract may be an extract obtained by using a solvent selected from the group consisting of water, methanol, ethanol, butanol, hexane, chloroform, dichloromethane, and ethyl acetate.

In an embodiment of the present invention, the fibrotic disease may be selected from the group consisting of proliferative scar, keloid, scleroderma, peritoneal adhesion, liver fibrosis, lung fibrosis, cardiac fibrosis, skeletal muscle fibrosis, synovial fibrosis, myelofibrosis, myelodysplastic syndrome and diabetic retinopathy.

ADVANTAGEOUS EFFECTS OF INVENTION

The Rhus succedanea extract of the present invention has an activity of effectively inhibiting a signal transduction pathway of TGF- β 1, which plays an important role in fibrosis, and thus can inhibit the expression and activity of extracellular matrices such as α smooth muscle actin (α -SMA), Connective Tissue Growth Factor (CTGF), fibronectin external domain A (FN-EDA), collagen, etc., induced by TGF- β 1, and has high antioxidant activity, so that the composition of the present invention comprising the Rhus succedanea extract as an active ingredient has an effect of being effectively used for the preparation of a therapeutic agent for fibrotic diseases and a health functional food.

Drawings

Fig. 1 shows a preparation process of the sumac extract of the present invention.

FIG. 2 shows the results of reverse transcription-polymerase chain reaction (RT-PCR) analysis of the expression level of α smooth muscle actin induced by solvent extract of Rhus succedanea treated with transforming growth factor- β 1 according to the present invention in different solvents, using HS27 and HHStec cell lines as targets.

FIG. 3 shows the results of analyzing the expression level of α smooth muscle actin induced by the transforming growth factor- β 1 treated with the hexane extract of Rhus succedanea according to the present invention by RT-PCR and immunoblotting using HS27 and HHStec cell lines as subjects.

FIG. 4 shows the results of analyzing the expression amount of messenger ribonucleic acid (mRNA) of the transforming growth factor- β 1 and the production amount of the transforming growth factor- β 1 of the xanthane extract-treated according to the present invention in the HS27 cell line.

FIG. 5 shows the results of analyzing the changes in expression of Connective Tissue Growth Factor (CTGF) induced by transforming growth factor- β 1 treated with the hexanes extract of Rhus succedanea according to the present invention in HS27 and HHStec cell lines by RT-PCR and immunoblotting.

FIG. 6 shows the results of analyzing the change in the expression of fibronectin external domain A (FN-EDA) induced by transforming growth factor- β 1 treated with the xanthane extract according to the present invention in HS27 and HHStec cell strains by RT-PCR and immunoblotting

FIG. 7 shows the results of analyzing the changes in the expression of type I collagen and type III collagen (ECM) in HS27 and HHStec cell lines treated with the hexane-fractionated extract of Rhus succedanea according to the present invention at various concentrations.

FIG. 8 shows the results of analyzing the amount of soluble collagen secreted from HS27 and HHStec cell lines by using the hexane-fractionated extract of Rhus succedanea according to the present invention at various concentrations.

Fig. 9 shows photographs of the degree of cell migration (recovery force) observed under a microscope and the results of analyzing the recovery degree after treating the scratched HS27 cell line with various concentrations of the hexane fraction extract of Rhus succedanea of the present invention and culturing for 24 hours.

Fig. 10 shows the results of analyzing the antioxidant activity of the Rhus succedanea extract according to the present invention according to various solvents.

FIG. 11 shows the results of analyzing the scavenging ability of 2 ', 7' -dichlorofluorescent xanthate diacetate (DCFH-DA) after treating the Rhus succedanea extract of the present invention in different solvents against the HS27 cell line.

FIG. 12 shows the results of analyzing the expression levels of mRNA of matrix metalloproteinase 1 (MMP 1), tumor necrosis factor α (TNF-a), and tissue inhibitor of metalloproteinase 1 (TIMP 1) as factors related to fibrosis, after treating various concentrations of the hexane fraction extract of Rhus toxicodendron according to the present invention against HS27 cell line.

FIG. 13 shows the results of analyzing the expression levels of mRNA of matrix metalloproteinase 2 (MMP 2) and matrix metalloproteinase 9 (MMP 9) which are factors associated with fibrosis, after treating various concentrations of the hexane fraction extract of Rhus succedanea according to the present invention with respect to HS27 cell line.

FIG. 14 shows the results of analyzing the expression level of mRNA of matrix metalloproteinase 1 and matrix metalloproteinase 2, which are factors associated with fibrosis, after treating various concentrations of the hexane fraction extract of Rhus toxicodendron according to the present invention against HHStec cell line.

FIG. 15 shows the results of measuring the expression levels of α smooth muscle actin and connective tissue growth factor expressed in the cells after treating ethanol extracts of Rhus toxicodendron (Dimethylaminoethoxyethanol (DMEE)) of the present invention at different concentrations against HHStec cell lines, and SM is a positive control group indicating a group treated with Silymarin (Silymarin).

FIG. 16 shows the results of measuring the distance of a scratch wound after scratching cells after pretreatment of various concentrations of the ethanol extract of Rhus succedanea (dimethylaminoethoxyethanol) of the present invention against HHStec cell line.

Fig. 17 shows the results of measurement of the expression levels of α smooth muscle actin and connective tissue growth factor expressed in the above cells after treating different concentrations of ethanol extract of toxicodendron amurense (dimethylaminoethoxyethanol) of the present invention against HS27 cell line as skin fibroblasts, and SM is a positive control group indicating a group treated with Silymarin (Silymarin).

Fig. 18 shows the results of measuring the distance of a scratch wound after scratching cells after treating various concentrations of the ethanol extract of Rhus succedanea (dimethylaminoethoxyethanol) against the HS27 cell line as skin fibroblasts.

FIG. 19 shows the results of analyzing the degree of the weight loss inhibitory effect of mice injected with carbon tetrachloride according to the treatment of the ethanol extract of Rhus succedanea as an animal model with respect to a white mouse (SD) animal model, CTL being a control group in which no treatment is performed, CC14 being a group injected with carbon tetrachloride, DM1 + CCl4 being a group injected with the ethanol extract of Rhus succedanea and carbon tetrachloride, SM + CCl₄Is the group of silymarin and carbon tetrachloride.

Fig. 20 shows the results of confirming whether or not the liver injury according to carbon tetrachloride injection in a white rat (SD) animal model can be improved when the ethanol extract of Rhus succedanea was treated by measuring the contents of glutamic acid Aminotransferase (ALT) and aspartic acid Aminotransferase (AST) as serum indices.

Fig. 21 shows the results of confirming whether or not the ethanol extract of Rhus succedanea can improve liver tissue damage (liver fibrosis symptoms) according to carbon tetrachloride injection in a white mouse (SD) animal model when treated by hematoxylin-eosin (HE) staining method.

Fig. 22 shows the results of confirming whether or not the hepatic tissue damage (hepatic fibrosis symptom) according to carbon tetrachloride injection from a white mouse (SD) animal model as a subject can be improved when the ethanol extract of toxicodendron amurense of the present invention is treated by analyzing the expression of Vascular Endothelial Growth Factor (VEGF).

Detailed Description

Best mode for carrying out the invention

The terms used in the present invention are defined as follows.

"extract" is characterized in that it is a crude extract, polar solvent-soluble extract or non-polar solvent-soluble extract of Rhus succedanea.

The "crude extract" includes a solvent soluble in a solvent selected from water including purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol, etc., or a mixed solvent thereof, preferably, an extract soluble in water and a mixed solvent of methanol, more preferably, an extract soluble in 50% to 100% methanol.

The "polar solvent-soluble extract" includes a solvent soluble in a solvent selected from water, methanol, butanol or a mixed solvent thereof, preferably, soluble in water or methanol, more preferably, soluble in methanol.

"non-polar solvent soluble extract" includes extracts soluble in hexane, chloroform, dichloromethane or ethyl acetate, preferably in hexane, dichloromethane or ethyl acetate, more preferably in hexane or ethyl acetate solvents.

"pharmaceutical composition" refers to a mixture of the Rhus succedanea extract of the present invention and other chemical ingredients, such as diluents or carriers.

"Carrier" is defined as a compound that facilitates the addition of the compound to cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the infiltration of large amounts of organic compounds into cells or tissues of an organism.

"diluent" is defined as a compound that not only stabilizes the biologically active form of the subject compound, but is also diluted in water in which the compound is dissolved. Salts dissolved in buffer solutions are used in the art as diluents. The buffer solution commonly used is phosphate buffered saline because it mimics the salt state of human solutions. Since buffer salts can control the pH of a solution at lower concentrations, buffer diluents rarely alter the biological activity of a compound.

By "subject" or "patient" is meant any individual in need of treatment, including humans, cows, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also, any subject who participated in a clinical study but did not show clinical findings of the disease or who participated in an epidemiological study or served as a control is included in the subject.

"tissue or cell sample" refers to a collection of similar cells obtained from within the tissue of a subject or patient. The source of the tissue or cell sample may be a fresh, frozen and/or preserved organ or tissue sample or solid tissue from biopsy or aspirate; blood or any blood component; cells at any point in time of pregnancy or postpartum in a subject. The tissue sample may also be primary cells or cultured cells or cell lines.

An "effective amount" is an appropriate amount that affects a beneficial or preferred clinical or biochemical outcome. The effective amount may be administered once or more than once. For the purposes of the present invention, an effective amount is an amount suitable for temporarily alleviating, ameliorating, stabilizing, reversing, slowing or delaying the progression of a disease state. A composition is said to be "pharmaceutically or physiologically acceptable" if, for example, the beneficiary animal is able to tolerate administration of the composition or administration of the composition to the animal is appropriate. In the case where the amount to be administered is physiologically important, the above-mentioned formulation can be said to be administered in a "therapeutically effective amount". An agent is physiologically significant if its presence brings about a physiologically detectable change in a subject patient.

The term "treating … …" means, unless otherwise indicated, reversing, alleviating, preventing or inhibiting the progression of the condition or disease for which the term is applied or the symptoms of one or more of the conditions or diseases. As used herein, the term "treatment" when defined as "treating … …" refers to the act of treating.

The "functional food" refers to a food in which the Rhus succedanea extract of the present invention is added to a general food to improve the functionality of the general food. The functionality can be roughly classified into physical properties and physiological functionalities, and when the extract of the present invention is added to a general food, the physical properties and physiological functionalities of the general food are improved.

The present invention will be described in detail below.

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In addition, although preferred methods and samples are described in the present specification, methods and samples similar or equivalent to these are also included in the scope of the present invention. The contents of all publications cited in this specification as references are incorporated herein by reference.

The invention relates to an anti-fibrosis application of a Rhus succedanea extract, and relates to the exertion of specific physiological activity and function contained in the Rhus succedanea extract.

Yellow lacquer wood (Dendropanox morbiferaLEV.) is an evergreen broad-leaved tree belonging to araliaceae, and is a korean unique tree species grown on south and west coast of korea, such as jizhou island, skimmia, fujima, and hainan. The clove component of Rhus succedanea contains a small amount of polyterpenes (Terpene) and a large amount of sesquiterpenes (Sesquiterpene), depending on the time and place takenDifferent from the original ones, the composition comprises d-germacrene-d, β -celeriarene (β -selinene), α -amorphene (α -amorphene), α -celeriarene (α -selinene), delta-cadinene (delta-cadinene), gamma-cadinene (gamma-cadinene), T-ylanisole (T-muuronol), β -elemene (β -elemene), bicyclo [4, 4, 0]Decyl-1-dilute-2-isopropyl-5-methyl-9-methylene (bicyclo [4, 4, 0 ]]dec-1-en-2-isoproyl-5-methyl-9-methyl ene, β -cadinene (β -cadinene), myrcene B (germacrene-B), β 0-pinene (β 1-copaene), β 2-humulene (β 3-humulene), α -cadinene (α -cadinene) and small amounts of L-linalool (linalool L), α -terpinene (α -pinene), α -cubene (α -cubene), α -ylanene (α -ylanene), (+) -dihydromyrcene () -calamenene ((+) -calarene), 3, 7-guaiadine, (-) -isobornylene, β 4-bergene (β 5-cubenee), limonene (1-limonene), limonene (1-cadinene), etc.

The part of the sumac usable in the present invention is not limited to leaves, branches, barks, etc., but preferably, leaves can be used.

The yellow sumac wood can be prepared and used by methods well known in the art, methods of modification thereof, or methods according to the present invention. As an example, it can be prepared by the following method.

The yellow sumac wood extract or crude extract of the present invention can be obtained by adding a solvent selected from the group consisting of water including purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol, etc., or a mixed solvent thereof in a volume amount of about 1 to 30 times, preferably 2 to 15 times (w/v%) by weight of the yellow sumac wood, preferably, a mixed solvent of water and methanol, more preferably, 50 to 100% methanol, at a temperature of 0 to 100 ℃, preferably, at room temperature for 10 to 60 hours, preferably, at a temperature of 30 to 50 hours, by an extraction method such as cold immersion extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, or heating extraction, to obtain a yellow sumac wood crude extract.

Also, the polar solvent-soluble extract or the non-polar solvent-soluble extract of the present invention may be obtained by preferably dispersing the above-obtained crude extract in about 1 to 150 times, preferably, 5 to 100 times volume (w/v%) of water based on the weight of 50 to 100% of the methanol crude extract, and then sequentially adding about 1 to 10 times, preferably, 1 to 5 times volume of hexane, ethyl acetate and butane to the water, and, preferably, may be fractionated 1 to 5 times, preferably, 2 to 4 times to obtain the polar solvent-soluble extract or the non-polar solvent-soluble extract of the present invention. Preferably, it can be used by obtaining a hexane extract.

Also, in the present invention, the Rhus succedanea extract having anti-fiber activity may use not only hexane extract but also ethanol extract.

The concentrated solution of the above extract can also be obtained in powder state by freeze drying at-80 deg.C or vacuum decompression at 50 deg.C.

The invention includes a method of preparing the above-described Rhus succedanea extract. The above preparation method is only an exemplary method thereof, and may be appropriately modified and used according to various methods based on the techniques in the art. For example, extraction methods not exemplified according to the present invention can be successfully carried out by modifications apparent to those skilled in the art.

The detailed explanation of the reaction conditions and the like for preparing the Rhus succedanea extract of the present invention will be omitted by those of ordinary skill in the art to which the present invention pertains since they can be confirmed by the examples described later.

The present invention is characterized by initially elucidating a novel use of the Rhus succedanea extract, i.e., having an activity of preventing, ameliorating or treating a fibrotic disease.

Since it is known that a factor which plays a central role in the progress of fibrosis (fibrosis) is transforming growth factor- β 1, fibrosis can be suppressed as long as a signal transduction system based on transforming growth factor- β 1 can be suppressed.

In addition, stellate cells are activated by cytokines derived from Kupffer cells (Kupffer cells) or infiltrating cells and then converted into activated cells, thereby producing extracellular matrices with high clarity during the fibrosis process.

In order to confirm whether the Rhus succedanea extract of the present invention can prevent, improve and treat fibrotic diseases, the present inventors first analyzed the effect of Rhus succedanea extract extracted by various solvents on the expression changes of α smooth muscle actin, connective tissue growth factor and collagen induced by transforming growth factor- β 1.

As a result, it was confirmed that the Rhus succedanea extract of the present invention can effectively inhibit the expression levels of α smooth muscle actin, connective tissue growth factor and collagen, which are increased by transforming growth factor- β 1.

In another example of the present invention, it was confirmed whether the Rhus succedanea extract not only stimulates the production of prefibrosis cytokines by the kupffer cells of the liver, but also inhibits the activity of Reactive Oxygen Species (ROS), which is an early-inducing factor of fibrosis, by activating hepatic stellate cells, and it was revealed that the Rhus succedanea extract of the present invention has an effective activity of inhibiting Reactive Oxygen Species (ROS).

The connective tissue growth factor is a substance mainly secreted from hepatic stellate cells, and it is known that hepatic fibrosis is induced by stimulating proliferation and survival of hepatic stellate cells and production of collagen, whereas α smooth muscle actin and collagen increase when fibrosis is induced.

Among extracellular matrix components, fibronectin (FN, fibronectin) and fibronectin outer domain a (FN-EDA, fibronectin extra domain a) as a part thereof are produced in abnormal states such as wound healing, and their expression is also increased by transforming growth factor- β 1, and it is a factor inducing fibrosis.

Therefore, the Rhus succedanea extract has the activity of effectively inhibiting the fibrosis induction cause and the factor expression, and can be finally effectively prepared into medicines and health-care functional foods for preventing, improving and treating fibrosis diseases.

Accordingly, the present invention can provide a pharmaceutical composition for preventing or treating fibrotic diseases, comprising the extract of Rhus toxicodendron.

The fibrotic diseases that can be prevented, improved or treated by the Rhus succedanea extract of the present invention may be, but are not limited to, proliferative scars, keloids, scleroderma, peritoneal adhesions, hepatic fibrosis, pulmonary fibrosis, cardiac fibrosis, skeletal muscle fibrosis, synovial fibrosis, myelofibrosis and myelodysplastic syndrome and diabetic retinopathy.

In skin fibrosis (skin fibrosis), the treatment process of a wound is sequentially divided into three steps of 1) an inflammation step, 2) a proliferation step, 3) a remodeling step and the like, wherein the skin fibrosis comprises physiological scars, proliferative scars (hypertrophicscars), keloids (keloids) and scleroderma (scleroderma).

Furthermore, hepatic fibrosis is a disease affecting a high mortality rate in one hundred million people worldwide and can be caused by viruses, alcoholism, radiation, bile retention, oxidative stress, CCl₄And toxic chemical substances such as nitrosamine, and chronic liver injury induced by drugs such as diethylstilbestrol (stilbestrol), methyldopa (methyldopa) and choline-deficient diet therapy can lead to liver fibrosis, and if fibrosis is not regulated, late cirrhosis or hepatocellular carcinoma may develop. During chronic liver injury, hepatic stellate cells (HSC, hepatic adipocytes, adipose storage cells, lto cells, peri-sinusoidal cells (perisininoudal)) are activated and proliferate, leading to induction of collagen andextracellular matrix (ECM) such as fibronectin is excessively accumulated, thereby causing hepatic fibrosis. Recent research results have found that hepatic fibrosis (Liver fibrosis) is reversible when the cause of the disease is weakened or eliminated, and therefore, studies are being attempted to screen medicinal plants as anti-fibrotic plants, and as pharmacological effects, assays for activity of inhibiting hepatic stellate cell activity and inhibiting extracellular matrix deposition have been conducted.

Systemic sclerosis patients have a incidence of invading the lungs of up to 90% and interstitial lung disease associated with systemic sclerosis is another leading cause of death. Like hepatic fibrosis, lung fibroblasts are activated and then differentiated into myofibroblasts capable of producing extracellular matrices such as collagen in pulmonary fibrosis. Also, Idiopathic Pulmonary Fibrosis (IPF) is a chronic progressive lung disease with a poor prognosis characterized by death due to respiratory failure within a few years after onset of symptoms, inhibition of lung transplantation and fibrosis being used as a method for treating idiopathic pulmonary fibrosis.

Renal fibrosis (Renal fibrosis) is classified into glomerulosclerosis and Renal Interstitial Fibrosis (RIF) which are induced by various pathogenic stimuli such as inflammation, immune response, trauma, hypoxia, and low blood circulation, and it is found that the Renal interstitial fibrosis is involved in the progression of life-threatening chronic kidney diseases. Once renal fibrosis occurs, renal interstitial fibroblast-like cells (renal interstitial fibroblast-like cells) are activated into myofibroblasts, and it is known that sufficient expression of red blood cell-producing factors is lost due to induced anemia.

Cardiac fibrosis (Cardiac fibrosis) naturally leads to myocardial infarction, the progression of which leads to impaired Cardiac function and ultimately to cardioplegia. Therefore, inhibition of cardiac fibrosis is also important for the prevention of heart failure.

In addition, complications such as abdominal pain, intestinal occlusion, and infertility after abdominal surgery are also induced, and in order to prevent adhesion between the intestine and the peritoneum, tissue adhesion barriers such as films, membranes, knits, sprays, and hydrogels have been developed to prevent adhesion between the intestine and the peritoneum, and studies have been underway to treat adhesion-related diseases such as peritoneal adhesion by suppressing fibrosis.

Also, mucocapsulitis is characterized by fibrosis and flexion of the joint capsule, thereby causing a problem of pain in the movement. Myelofibrosis is found in various blood diseases such as myeloproliferative diseases and acute osteomalacia leukemia, and it is known that re-adhesion occurs in up to 50% of patients with coronary artery disease who undergo Percutaneous Transluminal Coronary Angioplasty (PTCA) and stenting, and neointimal proliferation and fibrosis are known to be associated with adhesion of damaged arteries and vascular stents.

On the other hand, although death caused by fibrotic diseases accounts for about one third of natural death, no effective therapeutic method for regulating fibrotic disorders has been developed yet, and recently, studies on medicinal plants having effects of inhibiting hepatic stellate cell activation and inhibiting extracellular matrix deposition while being advantageous in terms of safety and cost have been conducted as a scheme for effectively treating hepatic fibrosis.

As described above, fibrosis is associated with the onset of many diseases, and it is clarified in the present invention that the Rhus succedanea extract of the present invention can be effectively used for the preventive improvement or treatment of diseases associated with such fibrosis.

Accordingly, the present invention can provide a method for treating and preventing fibrotic diseases using Rhus succedanea extract by providing a method for blocking the signal transduction of TGF- β 1 by administering an effective amount of Rhus succedanea extract to a subject in need thereof.

As used herein, the term "treating … …" is intended to, unless otherwise indicated, reverse, alleviate, prevent or inhibit the progression of the condition or disease for which the term is applied or one or more symptoms of the condition or disease. As used herein, the term "treatment" when defined as "treating … …" refers to the act of treating.

The composition for preventing or treating a fibrotic disease comprising the extract of Rhus toxicodendron comprises the above extract in an amount of 0.1 to 50% by weight, relative to the weight of the composition.

The pharmaceutical composition comprising the Rhus succedanea extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used for the preparation of pharmaceutical compositions.

The pharmaceutical composition of the present invention comprising the Rhus succedanea extract can be formulated or combined with an already used pharmaceutical agent such as a steroid drug, an antihistamine, an anti-inflammatory analgesic, and an antibiotic.

The pharmaceutical composition of the present invention comprising the Rhus succedanea extract can be formulated into oral dosage forms such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, etc., external preparations, adjuvants, and sterile injection solutions by various conventional methods. In particular, wound healing can be used in the form of a skin preparation for external use.

Examples of the carrier, excipient and diluent that can be included in the composition containing an extract of the present invention include vaseline, lactose (lactose), glucose, sucrose (sucrose), sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

When the preparation is carried out, the preparation is carried out by using a diluent or excipient such as a commonly used filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant and the like.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and are prepared by mixing at least one of the above-mentioned excipients, for example, starch, calcium carbonate (calcium carbonate), sucrose or lactose, gelatin and the like. Besides simple excipients, lubricants such as magnesium stearate and talc are used. Liquid phase preparations for oral administration include suspensions, solutions for internal use, emulsions, syrups and the like, and may include various excipients such as wetting agents, sweeteners, aromatics, preservatives and the like, in addition to water and liquid paraffin which are commonly used as simple diluents.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, oils, freeze-dried preparations, adjuvants. Examples of the nonaqueous solvent and the suspending agent include propylene glycol (propylene glycol), polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the adjuvant, witepsol, polyethylene glycol, Tween (tween), cacao butter, trilaurin, glycerogelatin, etc. can be used.

The amount of the extract of the present invention to be used may vary depending on the age, sex, and body weight of the patient, and may be administered once or several times daily in an amount of 0.0001mg/kg to 100mg/kg, preferably 0.001mg/kg to 10 mg/kg. The dose can be increased or decreased depending on the administration route, the degree of disease, sex, body weight, age, etc. Accordingly, the above-mentioned administration dose does not limit the scope of the present invention in any respect.

The pharmaceutical composition can be administered to mammals such as mice, rats, domestic animals, and humans by various routes. All modes of administration are contemplated, for example, transdermal, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine, epidural or intracerebral vascular, intra-articular and intra-abdominal administration.

The pharmaceutical administration form of the composition of the present invention can be used in the form of a pharmaceutically acceptable salt thereof, and can be used not only alone or in combination with other pharmaceutically active compounds but also in an appropriate combination.

On the other hand, the present invention relates to a functional composition for preventing and improving fibrotic diseases, comprising the Rhus succedanea extract as an active ingredient from other viewpoints. For example, such a functional composition includes a health functional food composition.

The functionality can be roughly classified into physical properties and physiological functionality, and when the Rhus succedanea extract of the present invention is added, the physical properties and physiological functionality of the composition may be improved.

For example, the molecular mechanism of the Rhus succedanea extract of the present invention can be utilized to prepare a functional composition for preventing and improving fibrotic diseases.

The Rhus succedanea extract can be used as a main component or an additive and an auxiliary agent when preparing various functional foods. As a specific example, the amount of the above extract of the functional composition is usually 0.01 to 15 weight percent based on the total composition weight, and may be appropriately selected according to the method of a general food composition or cosmetic composition, except for containing the above extract.

Further, various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and the like may be appropriately contained.

Since the composition of the present invention containing the Rhus succedanea extract is a natural plant ingredient, it has little toxicity and side effects, and thus can be used with confidence even if it is used for a long period of time for preventive purposes.

Detailed description of the invention

The present invention will be described in more detail below with reference to examples. These examples are merely illustrative of the present invention, and it is obvious to those of ordinary skill in the art that the scope of the present invention should not be construed as being limited by these examples.

Raw materials and Experimental methods

1. Preparation of Rhus succedanea extract

Leaves of Rhus succedanea were collected from a grain farm located in Han Guo An and then dried. The dried leaves were extracted with methanol at room temperature. The Rhus succedanea extract extracted using methanol was filtered through a Whatman number 1filter paper (Whatman number 1filter paper), and then concentrated under vacuum using a rotary evaporator concentrator. The methanol extract was then suspended with water and then fractionated with n-hexane, ethyl acetate, n-butanol and water in order according to polarity. Fig. 1 shows the steps for obtaining the Rhus succedanea extract using each solvent and the yields obtained by fractional distillation using each solvent.

2. Cell culture

HS27 cells, which are a normal human keratinocyte cell line, were cultured using 10% heat-inactivated Fetal Bovine Serum (FBS) and 100. mu.g/ml DMEM medium (Dulbecco modified Eagles media) containing antibiotics at 5% carbon dioxide and a temperature of 37 ℃. These cells were maintained in monolayer culture and subcultured when the cultured cells reached saturation.

3. Real-time quantitative Polymerase Chain Reaction (PCR)

Total ribonucleic acid was obtained using MagExtractor of MFX-2100 (Osaka Toyobo Co., Japan) as an automatic nucleic acid extraction system and according to the method of the product specification. Subsequently, ribonucleic acid (1. mu.g) was subjected to reverse transcription reaction using 200 units (units) of Moloney murine leukemia virus reverse transcriptase (Invitrogen, USA) and oligonucleotide (oligo (dT)) primers at 37 ℃ for 1 hour. The real-time polymerase chain reaction was carried out using Light Cycler 2.0 equipment (Mannheim Roche, Germany) of the fluorescent probe method (Taqman Master Mix), using a total of 20. mu.l of a reaction solution containing 4. mu.l of 5-fold diluted complementary deoxyribonucleic acid (cDNA), 10pmol of each primer and probe, 4. mu.l of a buffer solution containing a premix, base triphosphate deoxynucleotides (dNTPs), MgCl₂And Tag polymerase (Tag polymerase), and data analysis was performed using Light cyclesoft version 4.0 (roche).

Total cytoplasmic ribonucleic acid was extracted using TRI reagent (RNAiSo Plu from Protoyoho, Japan), and the extracted ribonucleic acid was synthesized into complementary deoxyribonucleic acid using reverse transcription polymerase chain reaction (reverse transcription PCR) (Invitrogen, USA). The above reaction was carried out by using a mixture of 4. mu.l of a mixture of 1: 5 diluted complementary deoxyribonucleic acid, 4mM MgCl₂10pmol of each primer, and 4. mu.l of Fast Start Mix buffer (dNTPs, SYBR green dye and Tag polymerase). Reversing ribonucleic acid in samples Using Moloney murine leukemia VirusA reverse transcription reaction was performed with a transcriptase (molonejeukemia virus reverse transcriptase) (invitrogen, usa) and an oligonucleotide (dT) primer at 37 ℃ for 1 hour, and the synthesized complementary deoxyribonucleic acid was diluted with water to 1: after 5, amplification was carried out by a GeneAmp PCR9600 (Perkin-Elmer-Cetus, USA) apparatus using 2.5 units of Tag polymerase (Promega, USA) and 10pmol of each primer. The primers, probes and conditions for polymerase chain reaction used in the above-described polymerase chain reaction of this experiment are shown in tables 1 and 2 below.

TABLE 1

Primers for use in polymerase chain reactions

TABLE 2

Conditions for polymerase chain reaction

4. Western blotting method

The concentration of the protein was determined using the Coomassie Brilliant blue method (Bradford) (Schleicher and Wieland, 1978). A standard curve was prepared using serial dilutions of bovine serum albumin (BSA, bovine serum albumin), and bole (BioRad) protein assay staining reagents were run at 1: 4 in water. Mu.l of the standard and sample were added to 1ml of the diluted staining reagent, and absorbance measurement was performed at 595 nm. The concentration of the sample protein was determined from a standard curve prepared with bovine serum albumin.

After washing the cells with phosphate buffer solution, the cells were lysed with 50mM Tris (hydroxymethyl) aminomethane (Tris-Cl) (ph 7.4), 250mM sodium chloride, 0.5% polyethylene glycol octylphenyl ether (Triton X100), 10% glycerol, 1mM Dithiothreitol (DTT), 1mM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (protein inhibitor cocktail biotechnology, usa), after centrifugation of the cell lysate, electrophoresis protein blotting was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-santa) and protein-attached membrane (membrane) was subjected to coupling reaction with anti- α smooth muscle actin (ACAM corporation, usa), connective tissue growth factor (ACAM corporation, usa), fibronectin outer domain a (sarsan charis corporation), and peroxidase dehydrogenase (garbanseki-3-phosphate dehydrogenase) (pdh 2 h) after reaction with horseradish peroxidase clone (sepharose peroxidase, usa), and subsequent detection was performed at room temperature using a first time after electrophoresis, and subsequent detection using a horseradish peroxidase cloning, 2 h.

5. Cell migration analysis

HS27 cell line was transformed into 1 × 10⁵Cells/well were seeded into 6-well culture plates and cultured until the plates were filled with cells. Subsequently, 20 μ l of Erlotib was used for scraping, and the culture plate was washed with phosphate buffer solution to remove the cells ruptured by the scraping. Subsequently, the cells were cultured in a medium containing 50 μ g of the Rhus succedanea extract of the present invention for 24 hours, and the rate of migration of the cells to the cell-free site was confirmed.

6.1 analysis of 1, 1-Diphenyl-2-trinitrophenylhydrazine (DPPH) scavenging Capacity

The radical scavenging ability was analyzed using 1, 1-diphenyl-2-trinitrophenylhydrazine. Each of the Rhus succedanea extracts of the present invention was dissolved in methanol, 5ml of a reaction solution containing 3.98ml of methanol, 20. mu.l of each extract and 1ml of 1, 1-diphenyl-2-trinitrophenylhydrazine (dissolved in methanol to a concentration of 0.15 mM) was left at room temperature for 30 minutes, and then the degree of decrease in absorbance was measured at 517nm using a spectrophotometer (spectrophotometer) (Perkin Elder, USA). Each experiment was repeated three times and the semi-Inhibitory Concentration (IC) was calculated from the radical scavenging ability of each extract at different treatment concentrations₅₀Inhibition of 50% of 1, 1-diphenyl-2-trinitrophenylhydrazine free radical (50% in)hibition of DPPH radics)). In this case, silybum marianum and ascorbic acid known to have antioxidant activity were used as a positive control group.

7.2 ', 7' -Dichlorofluoroxanthate (DCFH-DA) assay

Suspension of AML-2/DX100 cell line and HS27 cell line (1 × 10) in phosphate buffer solution⁵Cells/ml), each sample (Rhus succedanea extract) at different concentrations was treated for 30 minutes post-cell reaction. Subsequently, after adding 1. mu.M of 2 ', 7' -dichlorofluoroxanthate and 4mM of hydrogen peroxide to react for 2 hours, 2 ', 7' -Dichlorofluorescein (DCF) was measured at 485nm to 530nm using a spectrophotometer (Perkin El, USA).

8. Preparation of ethanol extract (ethoxyethanol) of Rhus succedanea

Leaves of Rhus succedanea (Dendropanax morbifera) were collected from a grain farm located in Han Guo Ming and dried, and then ethanol (30%, 50% and 70%) was added to the dried leaves at various concentrations, followed by heating to 90 ℃ to obtain an ethanol extract. The ethanol extract of Rhus succedanea was filtered through Woltmann No. 1filter paper, and then concentrated under vacuum using a rotary evaporator concentrator, and used in the following examples.

9. Using CCL₄Preparation of mouse model for inducing hepatic fibrosis

To prepare a murine model for inducing hepatic fibrosis, male white rats (SpragueDawley, SD) aged 4 weeks were acclimated for 1 week and divided into 4 groups (solvent-only group (control group), carbon tetrachloride-treated group, ethanol extract of Rhus toxicodendron (ethoxyethanol) + CCl of the present invention)₄Treated group, positive control group (SM + carbon tetrachloride treated group)), 6 carbon tetrachloride-treated groups were used alone, while 5 carbon tetrachloride-treated groups were used in each of the other groups, and CCl was added₄All samples were dosed orally (PO) 30 minutes before Intraperitoneal (IP) injection for 6 weeks twice weekly. These experiments were carried out by the University of Korea Animal Experimental ethics Committee (Chossun University institute Industrial Care and Use Committee) on the IRB program andafter approval, all experiments were performed.

10. Sirius red (sirius red stain) staining of collagen of liver tissue

After obtaining liver tissues from liver injury-inducing mice, sirius red staining was performed according to the experimental method guidelines of the ScyTek Laboratories kit, specifically, formalin-fixed liver tissues were embedded in paraffin in a conventional manner, cut into 4 μm sections using a microtome and attached to a glass slide, paraffin was removed, and a hematoxylin reagent was treated and reacted for 5 minutes. Subsequently, the tissue sections were washed twice with distilled water and reacted for 15 seconds after the treatment bluing reagent completely covered the tissue sections. Subsequently, it was washed twice with distilled water. The tissue slices were immersed in an ethanol solution to terminate the reaction. Thereafter, eosin Y solution treatment was treated and allowed to react for 3 minutes, and then the slides containing the tissue sections were washed with ethanol solution. Then, dehydration treatment was performed using an ethanol solution and fixation was performed using Histomount (National Diagnostics, usa) to complete all dyeing processes. Type I and type III collagen appear red under the light microscope if sirius red staining is used.

11. Immunochemical staining of liver tissue

Liver tissues obtained from rats induced hepatic fibrosis were subjected to immunochemical staining, and the liver tissues fixed in 10% formalin were embedded in paraffin according to a conventional method, and then cut into 4 μm sections using a microtome and attached to a glass slide, and the paraffin was removed using xylene, thereby completing the soaking process. The antigen retrieval process was performed in Citrate buffer (Citrate buffer) (pH 6.0) in a constant temperature bath for 40 minutes. After washing with distilled water, a Peroxidase blocking buffer (Peroxidase blocking buffer) was treated at room temperature for 10 minutes to remove endogenous Peroxidase. Subsequently, anti-SMA mouse monoclonal antibodies (ebo, uk) and anti-vegf mouse monoclonal antibodies were expressed as 1: after diluting at a ratio of 100, 300. mu.l was added, and the reaction was carried out overnight at 4 ℃ after meal. After washing with TBS Buffer (TBST) containing 0.1% Tween 20, the mixture was reacted at room temperature for 30 minutes with 300. mu.l of mouse enhancer (mousnenhancer) in Polink 2 Plus kit from GBI. After washing with TBST buffer, the resultant was developed with Diaminobenzidine (DAB). At this time, the development time was 5 minutes, and after washing, it was reacted with Mayer's hematoxylin for 1 minute, and then soaked in TBST buffer and subjected to indigo dyeing (bluring). The washed slides were dehydrated in ethanol and xylene and then sealed with Histomount.

All experimental results analyzed in the experiments of the present invention were obtained by repeating the experiments three times, with the resulting values expressed as mean ± standard deviation (means ± SE), statistical significance determined by paired t-tests (paired Student's ttest), and P-values (P values) < 0.05 considered significant.

Example 1

Analysis of the Effect of Rhus succedanea extract on the overexpression of α smooth muscle actin mRNA caused by TGF- β 1

In the process of fibrosis, if transforming growth factor- β 1 is activated, intracellular collagen production and accumulation increase, leading to fibrosis, thereby disabling tissues and organs, and, when cells or tissues are damaged by fibrosis, fat and retinoid components in cells are lost, expression of α smooth muscle actin increases and causes cell changes, and thus, researchers have been conducting research for inhibiting the expression and activity of transforming growth factor- β 1 to prevent hepatic fibrosis.

To this end, the inventors analyzed whether the Rhus succedanea extract of the present invention affects the expression of α smooth muscle actin caused by transforming growth factor- β 1, which plays a role in inducing fibrosis, and for this purpose, after treating HS27, which is a human dermal fibroblast cell line, and HHStec (human hepatic stellatecell), which is a human hepatic stellate cell line, with transforming growth factor- β 1, the Rhus succedanea extract was treated separately, and then the degree of expression of intracellular α smooth muscle actin was analyzed by polymerase chain reaction and Western blotting.

As a result, as shown in fig. 2, the expression level of α smooth muscle actin, which was significantly increased in both HS27 cell line and HHStec cell line when transforming growth factor- β 1 was treated, was effectively suppressed by the treatment of the astragalus membranaceus tree extract of the present invention, and particularly, the hexane extract had more excellent effects than ethyl acetate, n-butanol and water extracts (fractions).

The present inventors treated HS27 cell line and HHStec, which is a human hepatic stellate cell line, with varying concentrations of the hexane extract from toxicodendron amurense, and analyzed how much the inhibitory effect on the expression of α smooth muscle actin increased by tgf- β 1, and further analyzed whether the inhibitory effect on the expression of tgf- β 1 itself, by the polymerase chain reaction and western blotting.

As a result, as shown in FIG. 3, the expression of α smooth muscle actin increased by TGF- β 1 was significantly inhibited in both cell lines in a concentration-dependent manner, and the expression of TGF- β 1 was also inhibited in a concentration-dependent manner (see FIG. 4).

From these results, the present inventors have found that the Rhus succedanea extract of the present invention can inhibit the expression and activity of transforming growth factor- β 1, which is a fibrosis-promoting cytokine, and transforming growth factor- β 1-promoting factors involved in fibrosis, and particularly, hexane fraction of Rhus succedanea has the strongest anti-fibrotic activity.

Example 2

Analysis of Effect of Rhus chinensis extract on connective tissue growth factor and fibronectin external Domain A

Among the components of extracellular matrix, fibronectin and the various fibronectin external domains a (FN-EDA, fibronectin extra domain a) as its isomers are produced in abnormal states such as wound healing, and their expression is also increased by transforming growth factor- β.

Therefore, the present inventors examined whether or not the effect of inhibiting the expression of connective tissue growth factor and fibronectin ectodomain a, which were increased by transforming growth factor- β 1, was exhibited when the extract of Rhus succedanea was treated with different concentrations of Rhus succedanea extract in HS27 cell line and HHStec, which is a human hepatic stellate cell line, and then analyzed the expression levels of connective tissue growth factor and fibronectin ectodomain a by polymerase chain reaction and Western blotting.

As shown in fig. 5 and 6, the sumac extract of the present invention exhibited an activity effective in inhibiting the expression of connective tissue growth factor and fibronectin ectodomain a increased by transforming growth factor- β 1, and in particular, in the case of using the hexane extract, the inhibitory effect of connective tissue growth factor was 5-fold at a concentration of 2 μ g/ml in the HS27 cell line, 7-fold at a concentration of 10 μ g/ml, 14-fold at a concentration of 50 μ g/ml, 9-fold at a concentration of 2 μ g/ml in the HS27 cell line, and 18-fold at a concentration of 10 μ g/ml.

Furthermore, the hexane extract of Rhus succedanea of the present invention was found to have an activity of effectively inhibiting the fibronectin outer domain A in both HS27 and HHStec cell lines.

Example 3

Analysis of the Effect of Rhus succedanea extract on the expression of collagen

It is known that transforming growth factor- β 1, which is a major stimulator for the production of extracellular matrix, inhibits the activity of fibroblasts and inhibits the accumulation and production of collagen in hepatocytes in the case of using transforming growth factor- β 1 small interfering ribonucleic acid (siRNA) to inhibit the expression of transforming growth factor- β 1.

Therefore, in order to confirm whether the Rhus succedanea extract of the present invention can inhibit the expression of type I collagen A1 and type I collagen 3A, the present inventors treated the transforming growth factor- β 1 and Rhus succedanea extracts of different concentrations in HS27 cell line and HHStec cell line to analyze the expression levels of type I collagen and type III collagen mRNA expressed in the cells and the amount of collagen produced.

As a result, as shown in fig. 7 and 8, the hexanes extract of Rhus succedanea according to the present invention was effective in inhibiting the expression of type I collagen and type III collagen in a treatment concentration-dependent manner, and also showed inhibition of the production and secretion of soluble collagen induced by transforming growth factor- β 1.

Example 4

Analysis of the Effect of Rhus succedanea extract on cell migration

To confirm whether the Rhus succedanea extract of the present invention affects cell migration, experiments were performed according to the cell migration assay described above.

Analysis results as shown in fig. 9, the Rhus succedanea extract of the present invention showed that wounds caused by scraping were restored by cell migration, and when hexane extract at a concentration of 50 μ g/ml was treated, it showed about 50% restoring force.

Example 5

Inhibitory effect of Rhus succedanea extract on early onset of hepatic fibrosis

Inhibition of Reactive Oxygen Species (ROS) not only stimulates kupffer cells of the liver to produce profibrotic cytokines, but also induces early onset of fibrosis by activating hepatic stellate cells. Thus, antioxidant activity may inhibit early onset and development of liver fibrosis.

Therefore, in order to confirm whether the Rhus succedanea extract of the present invention has the function of inhibiting active oxygen species, the present inventors examined whether the Rhus succedanea extract of the present invention has antioxidant activity by the analysis of <6>1, 1-diphenyl-2-trinitrophenylhydrazine scavenging ability and the analysis of <7>2 ', 7' -dichlorofluoroxanthate.

As shown in fig. 10 and 11, the sumac extract of the present invention has excellent antioxidant activity, and in particular, it was confirmed that the ethyl acetate fraction and the butanol fraction have very strong 1, 1-diphenyl-2-trinitrophenylhydrazine scavenging ability, and the ethyl acetate fraction has the strongest 2 ', 7' -dichlorofluorescent xanthate diacetate scavenging ability, which is significantly superior to vitamin C and silybum marianum. Silybum marianum is known as a therapeutic agent for liver diseases such as alcoholic liver diseases, liver cirrhosis, hepatotoxicity, etc., and has antioxidant, antifibrotic, anti-inflammatory, and liver regeneration effects, but the Rhus succedanea extract of the present invention shows more excellent antioxidant activity than this type of silybum marianum, and has more excellent effects of preventing, treating, and improving hepatic fibrosis than the existing drugs.

Example 6

Analysis of influence of Rhus succedanea extract on expression of fibrosis-associated factor

6-1. influence analysis of Rhus succedanea extract on human dermal fibroblast (HS 27) metalloprotease 1, metalloprotease 2, metalloprotease 9, tumor necrosis factor α, and tissue inhibitor of metalloprotease 1

After transforming growth factor- β 1 was treated in HS27 cell line, the Rhus succedanea extract of the present invention was treated, and then the expression levels of metalloproteinase 1, metalloproteinase 2, metalloproteinase 9, tumor necrosis factor α, and metalloproteinase tissue inhibitor 1 expressed in the above cell lines were analyzed by reverse transcription polymerase chain reaction, and at this time, metalloproteinase 2 and metalloproteinase 9 were analyzed by zymography.

It is known that the above-mentioned metalloprotease 1 is a target gene of transforming growth factor- β 1, and that a factor which is down-regulated by transforming growth factor- β 1 is a molecule which is a target of a therapeutic agent for liver fibrosis, and it is known that tumor necrosis factor α causes apoptosis by a factor which participates in the decomposition (resolution) of fibrotic tissue, and the expression thereof is inhibited by transforming growth factor- β 1. therefore, if the expression and activity of these factors are promoted, the progress of fibrosis caused by transforming growth factor- β 1 is inhibited, in the case of inhibiting the expression of tissue inhibitor of metalloprotease 1 (TIMP 1) which affects the activation of hepatic stellate cells, the progress of liver fibrosis is inhibited, and the influence of the Rhus succedanea extract of the present invention on tissue inhibitor of metalloprotease 1 is also analyzed.

As a result, as shown in fig. 12, the hexane fraction extract of xanthane wood of the present invention showed an effect of increasing the expression of metalloproteinase 1 and tumor necrosis factor α decreased by tgf- β 1, and thus inhibiting the expression of cathepsin tissue inhibitor 1 increased by tgf- β 1.

The present inventors also analyzed the activity of the Rhus succedanea extract on the expression of metalloprotease 2 and metalloprotease 9, which are fibrosis-promoting factors, and as a result, it was found that the expression of metalloprotease 2 and metalloprotease 9 is increased by transforming growth factor- β 1 in a concentration-dependent manner, as shown in FIG. 13.

6-2 analysis of the Effect of Rhus succedanea extract on metalloprotease 1 and metalloprotease 2 of human hepatic stellate cells (HHstec)

In the above 6-1 experiment, human hepatic stellate cells (HHstec) were used as the subject cell line, and the experiment was performed in the same manner.

As a result, as shown in FIG. 14, the expression of metalloprotease 1 increased dependently with the treatment concentration of the E.xanthanane fractionated extract of the present invention, and the degree of increase was more superior to that of the silybum marianum treated group as the positive control group, and, in the analysis of metalloprotease 2, the E.xanthanane fractionated extract of the present invention suppressed the expression of metalloprotease 2 increased by transforming growth factor- β 1, and particularly, the metalloprotease 2 treated at a concentration of 10. mu.g/ml had the most effective inhibition rate.

Example 7 analysis of anti-fibrotic efficacy of ethanol extract of Rhus succedanea

7-1. analysis of the ability of Rhus succedanea extract to scavenge free radicals of 1, 1-diphenyl-2-trinitrophenylhydrazine and 2 ', 7' -dichlorofluoroxanthate according to the concentration of ethanol

The ethanol extract of Rhus succedanea prepared by varying the ethanol concentration (treating 30%, 50%, 70% ethanol) of the present invention was analyzed for its ability to scavenge 1, 1-diphenyl-2-trinitrophenylhydrazine free radical and 2 ', 7' -dichlorofluorescent xanthodiacetate free radical.

TABLE 3

As shown in table 3, in the case of the sumac extract obtained by changing the ethanol concentration, the scavenging ability of the 1, 1-diphenyl-2-trinitrophenylhydrazine radical and the 2 ', 7' -dichlorofluoroxanthylacetate radical was better than that of the 50% and 70% ethanol extracts in the 30% ethanol extract. In addition, the inhibitory effect of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) was more excellent in the 30% ethanol extract-treated group than in the other groups.

Therefore, in the case where the present inventors obtained a Rhus succedanea extract using ethanol, it was found that in order to obtain excellent anti-fibrosis efficacy, an extract using 30% ethanol was effective.

7-2 Activity analysis of ethanol extract of Rhus chinensis Mill in hepatic stellate cells for the inhibition of α smooth muscle actin and connective tissue growth factor expression

To analyze the anti-fibrotic activity of the ethanol extract of Rhus succedanea, it was analyzed whether the expression levels of α smooth muscle actin and connective tissue growth factor, which are known to increase when fibrosis occurs due to liver cell tissue damage, change according to the ethanol extract of Rhus succedanea.

As shown in FIG. 15, the ethanol extract of Rhus toxicodendron (30% ethanol extract) treated according to the present invention significantly inhibited the expression level of α smooth muscle actin and connective tissue growth factor compared to the Control (CTL) not treated with the above extract, and it was confirmed that the ethanol extract of 30% was most effective when treated at a concentration of 40. mu.g/ml, indicating that the inhibitory effect was 2 times or more at a concentration of 40. mu.g/ml compared to that at a concentration of 20. mu.g/ml, whereas the effect was similar between the group treated at a concentration of 80. mu.g/ml and the group treated at a concentration of 40. mu.g/ml.

7-3. test of the recovery of scraping of the ethanol extract of Rhus succedanea on hepatic stellate cells

The ethanol extract of Rhus succedanea was subjected to a cell scraping recovery test. For this, when hepatic stellate cells were cultured, the ethanol extract of Rhus succedanea (30% ethanol extract) of the present invention was added to a cell culture medium and cultured for 12 hours, and the cells were scraped and the distance of the cell wound was measured.

Results as shown in figure 16, the distance to scratch the wound increased dependently depending on the concentration of the ethanolic extract of Rhus succedanea, with the same results from two replicates. The above results indicate that the ethanol extract of Rhus succedanea can inhibit proliferation and/or migration of hepatic stellate cells.

7-4 Activity analysis of ethanol extract of Rhus chinensis Mill in dermal fibroblasts on the inhibition of α smooth muscle actin and connective tissue growth factor expression

After the ethanol extract of toxicodendron amurense (ethoxyethanol) of the present invention was treated with HS27 cells, which are skin fibroblasts, the effect of the ethanol extract of toxicodendron amurense on α smooth muscle actin messenger ribonucleic acid expression was measured, and as a result, it was confirmed that α smooth muscle actin messenger ribonucleic acid expression was inhibited at the treatment concentrations of the extract of 20 μ g/ml, 40 μ g/ml and 80 μ g/ml, and the effect of the ethanol extract of toxicodendron amurense on inhibition of connective tissue growth factor messenger ribonucleic acid expression was measured, and as a result, it was shown that the extract of toxicodendron amurense had the activity of inhibiting connective tissue growth factor messenger ribonucleic acid expression at the treatment concentrations of 20 μ g/ml and 40 μ g/ml, but had no effect at the higher concentration of 80 μ g/ml (refer to fig. 17).

7-5. test of the recovery of the ethanol extract of Rhus succedanea from scratching skin fibroblasts

After 24 hours of treatment of the ethanol extract of toxicodendron amurense (ethoxyethanol) on HS27 cells as skin fibroblasts, whether scraping (scratch) was recovered or not was tested, and the result showed that the distance of the scraped wound increased in a concentration-dependent manner (see fig. 18). The above results indicate that the ethanol extract of Rhus succedanea inhibits the proliferation and/or migration of dermal fibroblasts.

Example 8 analysis of ethanol extract of Rhus succedanea by animal experiments for anti-fibrotic Activity

Further, the present inventors performed experiments for confirming whether the treatment with the ethanol extract of Rhus succedanea of the present invention has an improvement or therapeutic effect on fibrotic diseases, with respect to a white mouse animal model that induces fibrotic diseases. For this purpose, experimental groups for mice were prepared and the following experiments were performed as shown in table 4 below.

TABLE 4

Classes of Experimental Components

Fruit of Chinese wolfberry Test (experiment) Group of	Processing content	Remarks for note
			Group
1	Carboxymethyl cellulose (CMC) (4 ml/kg BW, PO) + Vehide (olive oil 2ml/kg BW, IP) treatment group	Control group treated with solvent only
			2 groups of	Carboxymethyl cellulose (CMC) (4 ml/kg BW, PO) + CCL4 (2 ml/kg BW, IP, 30% carbon tetrachloride in olive oil)	CCL4 Treatment group
Group
	3	Pretreatment of 0 day yellow sumac ethanol extract (2 ml/kg content) BW + carbon tetrachloride treatment at 100mg/kg	The invention of the yellow sumac wood B for pretreatment After alcohol extraction, CCL4Treatment group
4 groups of				Silybum marianum pretreatment (2 ml/kg BW harbouring 101 mg/kg) CCL4Treatment)	And CCL4Treated together as a positive pair Group of silybum marianum according to group medicine

8-1, measuring body weight

As shown above, the body weights of the white mice of 4 groups were measured and averaged to analyze the body weight of each group. As a reference, when an anti-fibrotic disease is induced, a phenomenon of weight loss occurs, and thus it was confirmed whether the extract of the present invention can inhibit the anti-fibrotic disease by the phenomenon of weight loss.

The results are shown in FIG. 19, in which CCL as an anti-fibrosis-inducing tissue was treated as compared with the control group₄The weight loss phenomenon of the group (b) is prominent, and on the contrary, the group treated with the ethanol extract of Rhus succedanea (ethoxyethanol) of the present invention has the effect of effectively inhibiting the absorption of CCL₄The activity of inducing the weight loss phenomenon was more excellent than that of the group of pretreated silybum marianum as a positive control group in the effect of inhibiting weight loss.

8-2, analyzing liver index of ethanol extract of Rhus chinensis in animal model of liver tissue injury caused by carbon tetrachloride

Blood was collected from the white mouse animal test group used in 8-1, and glutamate Aminotransferase (ALT) and aspartate Aminotransferase (AST) contained in serum as liver indices were measured. Glutamate aminotransferase and aspartate aminotransferase, which are known as indicators of liver damage, are free from blood and have high activity as liver tissue is destroyed, and particularly glutamate aminotransferase is present mostly in the liver and aspartate aminotransferase is present mostly in the heart, liver, skeletal muscle, and thus their specificity has been recognized.

The results of the analysis are shown in FIG. 20, in comparison with the control group, in the case of carbon tetrachloride peroxide (CCl)₄) In the group of glutamic acid aminotransferase and aspartic acid aminotransferaseHigh level of transferase activity free in blood, in contrast to the pretreatment of carbon tetrachloride (CCl) after pretreatment of the ethanol extract of Rhus succedanea according to the invention₄) Significantly less increased levels of glutamate and aspartate aminotransferases due to carbon tetrachloride, which is known to improve and prevent liver damage caused by carbon tetrachloride.

8-3 analysis of improving Effect of hepatic tissue by administering ethanol extract of Rhus chinensis to hepatic tissue injury animal model caused by carbon tetrachloride

After liver tissues were collected from the white mouse animal experimental group used in 8-1, the expression of hepatic collagen was analyzed by sirius red (sirius red) staining.

As shown in fig. 21, in the hepatocytes in the group treated with carbon tetrachloride (carboxymethylcellulose (CMC) + carbon tetrachloride), severe cystic fibrosis occurred, and the continuous collagen fiber membrane connected between the central vein and the central vein, and the central vein and the portal vein were connected to each other, compared to the group untreated with carbon tetrachloride (carboxymethylcellulose + oil), resulting in the occurrence of hepatic fibrosis. On the other hand, with carbon tetrachloride (CCl)₄) In comparison with the group treated with ethanol extract of Rhus succedanea (ethoxyethanol + carbon tetrachloride), it was observed that the change of vesiculation of hepatocytes was reduced, collagen septa irradiated around blood vessels were weak and discontinuous, and the phenomenon of hepatic fibrosis caused by carbon tetrachloride was improved, which had similar effect to the group treated with Silybum marianum as a positive drug.

Further, it was found that the expression of vascular endothelial growth factor increased when liver injury was induced by immunohistochemical staining for vascular endothelial growth factor in the liver tissue obtained from the experimental animal group. To this end, the present inventors analyzed whether the extract of the present invention has an activity of inhibiting the expression of vascular endothelial growth factor in hepatocytes or liver tissues.

As a result, as shown in fig. 22, the expression of vascular endothelial growth factor in liver tissue was increased in the group treated with carbon tetrachloride compared to the control group untreated with carbon tetrachloride (carboxymethylcellulose + oil), whereas the expression of vascular endothelial growth factor caused by carbon tetrachloride was increased in the positive control group (the group treated with silybum marianum) although the ethanol extract of toxicodendron amurense of the present invention slightly decreased the expression of vascular endothelial growth factor. This fact indicates that the ethanol extracts of Rhus succedanea have a stronger effect than Silybum marianum, or that they have different mechanisms in the variation of the expression of vascular endothelial growth factor.

Therefore, through the experimental results as described above, the present inventors have found that the toxicodendron amurense extract of the present invention can effectively prevent, improve, inhibit and treat fibrosis by modulating the expression and secretion of extracellular matrix such as collagen, which is a fibrosis-related factor, and in particular, the ethanol extract of the leaves of the toxicodendron amurense has more effective anti-fibrotic activity than silybum marianum.

The invention has thus been described with the focus on preferred embodiments. It will be appreciated by those skilled in the art that the present invention can be embodied in many alternate forms without departing from the essential characteristics thereof. Accordingly, the embodiments disclosed herein are to be considered in a descriptive sense only and not for purposes of limitation. The scope of the invention is indicated in the appended claims, rather than in the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

<110> university school labor cooperative group of Korean university

<120> composition for preventing or treating fibrotic disease comprising extract of Rhus yunnanensis Bunge as active ingredient

<130>NPNC63238

<160>14

<170>KoPatentIn 3.0

<210>1

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> a-SMA F primer

<400>1

tagctgagcg tggctatt 18

<210>2

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> a-SMA R primer

<400>2

cccatcaggc actcgta 17

<210>3

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> CTGF F primer

<400>3

cgaggagtgg gtgtgtga 18

<210>4

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> CTGF R primer

<400>4

cccacaggtc ttggaac 17

<210>5

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> FN-EDA F primer

<400>5

tccaagcgga gagagt 16

<210>6

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> FN-EDA R primer

<400>6

gtgggtgtga cctgag 16

<210>7

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> collagen 1A 1F primer

<400>7

gcttcaccta cagcgtcact 20

<210>8

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> collagen 1A 1R primer

<400>8

agaggagttt acaggaagca 20

<210>9

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> collagen 3A 1F primer

<400>9

aggtcctgcg ggtaacactg tcg 23

<210>10

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> collagen 3A 1R primer

<400>10

actttcaccc ttgacaccct g 21

<210>11

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> TGF-beta 1F primers

<400>11

acatcaacgg gttcactac 19

<210>12

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> TGF-beta 1R primers

<400>12

ctgaagcaat agttggtgtc c 21

<210>13

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> GAPDH F primer

<400>13

cgagatccct ccaaaatcaa 20

<210>14

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> GAPDH R primer

<400>14

tgtggtcatg agtccttcca 20

Claims (10)

1. A pharmaceutical composition for preventing or treating fibrotic diseases, comprising Rhus toxicodendron extract as an active ingredient.

2. The pharmaceutical composition for preventing or treating fibrotic diseases according to claim 1, wherein the Rhus succedanea extract is a crude Rhus succedanea extract, a polar solvent-soluble extract or a non-polar solvent-soluble extract.

3. The pharmaceutical composition for preventing or treating fibrotic diseases according to claim 2, wherein the crude extract is an extract obtained using a solvent selected from water including purified water, methanol, ethanol, butanol or a mixed solvent thereof.

4. The pharmaceutical composition for preventing or treating a fibrotic disease according to claim 2, wherein the polar solvent-soluble extract is an extract obtained using a solvent selected from water, ethanol, butanol, or a mixed solvent thereof.

5. The pharmaceutical composition for preventing or treating fibrotic diseases according to claim 2, wherein the non-polar solvent-soluble extract is an extract obtained using hexane, chloroform, dichloromethane, or ethyl acetate.

6. The pharmaceutical composition for preventing or treating fibrotic diseases according to claim 1, wherein the Rhus succedanea extract inhibits the signal transduction pathway of transforming growth factor β 1.

7. The pharmaceutical composition according to claim 1, wherein the fibrotic disease is selected from the group consisting of hyperproliferative scars, keloids, scleroderma, peritoneal adhesions, liver fibrosis, lung fibrosis, cardiac fibrosis, skeletal muscle fibrosis, synovial fibrosis, myelofibrosis and myelodysplastic syndrome, and diabetic retinopathy.

8. A health functional food for preventing or improving fibrotic diseases, comprising Rhus toxicodendron extract as an active ingredient.

9. The functional health food for preventing or ameliorating fibrotic diseases according to claim 8, wherein the Rhus succedanea extract is an extract obtained using a solvent selected from the group consisting of water, methanol, ethanol, butanol, hexane, chloroform, dichloromethane, and ethyl acetate.

10. The health functional food for preventing or ameliorating fibrotic diseases according to claim 8, wherein the fibrotic diseases are selected from the group consisting of hyperproliferative scars, keloids, scleroderma, peritoneal adhesions, liver fibrosis, lung fibrosis, cardiac fibrosis, skeletal muscle fibrosis, synovial fibrosis, myelofibrosis, myelodysplastic syndrome and diabetic retinopathy.

Images