阅读说明：本技术 用于预防、改善或治疗纤维化的chp的用途 (Use of CHP for the prevention, amelioration or treatment of fibrosis ) 是由 郑会润 李宪钟 全宗洙 李到炫 金演洙 梁承熹 金龙哲 文钟注 金智恩 于 2020-03-30 设计创作，主要内容包括：本发明涉及用于预防、改善或治疗纤维化的CHP的用途,更详细地,涉及包含CHP的用于预防或治疗纤维化的药学组合物、用于预防或改善纤维化的保健功能食品组合物、抗纤维化组合物、利用CHP的预防、改善或治疗纤维化的方法和/或制备用于预防或治疗纤维化的药学组合物时的CHP的用途。(The present invention relates to the use of CHP for preventing, improving or treating fibrosis, and more particularly, to a pharmaceutical composition for preventing or treating fibrosis, a health functional food composition for preventing or improving fibrosis, an anti-fibrotic composition, a method for preventing, improving or treating fibrosis using CHP, and/or the use of CHP in the preparation of a pharmaceutical composition for preventing or treating fibrosis, which comprises CHP.)

1. A pharmaceutical composition for preventing or treating fibrosis comprising CHP or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition for preventing or treating fibrosis according to claim 1, wherein said fibrosis occurs in at least one selected from the group consisting of kidney, liver, lung, skin, heart, pancreas, urinary system, reproductive system, sweat gland, nerve, brain, bone marrow, muscle and joint.

3. The pharmaceutical composition for preventing or treating fibrosis according to claim 1, wherein the fibrosis is one or more selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, radioactive lung injury or pulmonary fibrosis, pulmonary edema, cystic fibrosis, liver fibrosis, endomyocardial fibrosis, myocardial infarction, atrial fibrosis, glial scar, kidney fibrosis, bone marrow fibrosis, joint fibrosis, adipose fibrosis, skin fibrosis, nerve fibrosis and muscle fibrosis.

4. A health-care functional food composition for preventing or improving fibrosis, comprising CHP or a food-acceptable salt thereof.

5. The health functional food composition for preventing or improving fibrosis according to claim 4, wherein the fibrosis occurs in one or more selected from the group consisting of kidney, liver, lung, skin, heart, pancreas, urinary system, reproductive system, sweat gland, nerve, brain, bone marrow, muscle and joint.

6. The functional food composition for health use according to claim 4, wherein the fibrosis is one or more selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury or pulmonary fibrosis, pulmonary edema, cystic fibrosis, liver fibrosis, endomyocardial fibrosis, myocardial infarction, atrial fibrosis, glial scar, kidney fibrosis, bone marrow fibrosis, joint fibrosis, fatty fibrosis, skin fibrosis, nerve fibrosis and muscle fibrosis.

7. An anti-fibrotic composition comprising CHP or a pharmaceutically acceptable salt thereof.

8. A method of preventing or treating fibrosis comprising the step of administering an effective amount of CHP to a subject in need thereof.

9. The method of claim 8, wherein the fibrosis occurs in at least one selected from the group consisting of kidney, liver, lung, skin, heart, pancreas, urinary system, reproductive system, sweat gland, nerve, brain, bone marrow, muscle and joint.

10. The method according to claim 8, wherein the fibrosis is at least one selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury or pulmonary fibrosis, pulmonary edema, cystic fibrosis, liver fibrosis, endocardial myocardial fibrosis, myocardial infarction, atrial fibrosis, glial scar, renal fibrosis, bone marrow fibrosis, joint fibrosis, adipose fibrosis, skin fibrosis, nerve fibrosis and muscle fibrosis.

11. Use of CHP in the preparation of a pharmaceutical composition for the prevention or treatment of fibrosis.

12. The use of CHP according to claim 11, wherein the fibrosis occurs in one or more selected from the group consisting of kidney, liver, lung, skin, heart, pancreas, urinary system, reproductive system, sweat gland, nerve, brain, bone marrow, muscle and joint.

13. The use of CHP according to claim 11, wherein the fibrosis is one or more selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury or fibrosis, pulmonary edema, cystic fibrosis, liver fibrosis, endomyocardial fibrosis, myocardial infarction, atrial fibrosis, glial scar, renal fibrosis, bone marrow fibrosis, joint fibrosis, adipose fibrosis, skin fibrosis, nerve fibrosis and muscle fibrosis.

Technical Field

The present invention relates to the use of CHP for preventing, improving or treating fibrosis, and more particularly, to a pharmaceutical composition for preventing or treating fibrosis, a health functional food composition for preventing or improving fibrosis, an anti-fibrotic composition, a method for preventing or treating fibrosis using CHP, and/or the use of CHP in the preparation of a pharmaceutical composition for preventing or treating fibrosis, which comprises CHP.

Background

Fibrosis (fibrosis) is a disease in which abnormal production, accumulation and deposition of extracellular matrix occur in fibroblasts, and is caused by fibrosis of organs or tissues. Fibrosis is a very fatal disease that induces organ damage. As one example, Idiopathic Pulmonary Fibrosis (IPF) is the result of recurrent alveolar epithelial cell injury associated with fibroblast accumulation and myofibroblast differentiation, a chronic, progressive and fatal disease that results in the excessive accumulation of extracellular matrix (ECM) while causing irreversible destruction of lung parenchyma (lung) tissue.

In existing therapeutic studies, corticosteroids and immunosuppressive drugs are used, often targeting the inflammatory process of fibrosis. However, this preparation shows little effect in clinical experiments, and thus there is a need for a novel drug for treating fibrosis.

In addition, a composition for regulating blood glucose comprising a soybean hydrolysate containing CHP (cyclic (His-Pro)) at a high concentration is disclosed in korean laid-open patent No. 10-2013-0006170, but the anti-fibrotic effect of CHP is not yet clear.

Disclosure of Invention

Technical problem

The object of the present invention is to provide a pharmaceutical composition for preventing or treating fibrosis, comprising CHP.

It is still another object of the present invention to provide a health functional food composition for preventing or improving fibrosis, comprising CHP.

It is another object of the present invention to provide an anti-fibrotic composition comprising CHP.

It is still another object of the present invention to provide a method for preventing or treating fibrosis using CHP.

It is a further object of the present invention to provide the use of CHP in the preparation of a pharmaceutical composition for the prevention or treatment of fibrosis.

Technical scheme

In order to solve the problems as described above, the present invention provides a pharmaceutical composition for preventing or treating fibrosis, comprising CHP or a pharmaceutically acceptable salt thereof.

Also, the present invention provides a health functional food composition for preventing or improving fibrosis, comprising CHP or a food-acceptable salt thereof.

Also, the present invention provides a method for preventing or treating fibrosis comprising the step of administering an effective amount of CHP to a subject in need thereof.

Also, the present invention provides the use of CHP in the preparation of a pharmaceutical composition for the prevention or treatment of fibrosis.

According to a preferred embodiment of the present invention, the fibrosis may occur in one or more selected from the group consisting of kidney, liver, lung, skin, heart, pancreas, urinary system, reproductive system, sweat gland, nerve, brain, bone marrow, muscle and joint.

According to another preferred embodiment of the present invention, the Fibrosis is one or more selected from the group consisting of Pulmonary Fibrosis (Pulmonary Fibrosis), Idiopathic Pulmonary Fibrosis (Idiopathic Pulmonary Fibrosis), Radiation-induced lung injury (Radiation-induced lung injury) or Pulmonary Fibrosis, Pulmonary edema, Cystic Fibrosis (Cystic Fibrosis), hepatic Fibrosis, endocardial Fibrosis (Endomyocardial Fibrosis), Myocardial Infarction (Myocardial infarcation), atrial Fibrosis (atrial Fibrosis), Glial scar (Glial scar), Renal Fibrosis (Renal Fibrosis), Myelofibrosis (Myelofibrosis), joint Fibrosis (Arthrofibrosis), fatty Fibrosis, skin Fibrosis, nerve Fibrosis and muscle Fibrosis.

Also, the present invention provides an anti-fibrotic composition comprising CHP.

ADVANTAGEOUS EFFECTS OF INVENTION

The CHP-containing composition of the present invention inhibits fibrosis occurring in various tissues or organs such as kidney, liver, lung, skin, heart, pancreas, urinary system, reproductive system, sweat gland, nerve, brain, bone marrow, muscle and joint, thereby effectively preventing, improving or treating fibrosis.

Drawings

FIG. 1 shows the isolation of cells from human glomeruli and tubules and the primary culture process (left: glomeruli, right: tubules).

FIG. 2 shows morphological changes of cells according to CHP treatment concentrations (4. mu.g/ml, 20. mu.g/ml and 100. mu.g/ml, respectively) in a human-derived proximal tubular epithelial cell fibrosis model.

FIG. 3 is a graph showing the results of confirming the changes in the expression levels of E-cadherin (E-cadherin), fibronectin (fibronectin) and pSTAT3 proteins according to CHP treatment concentrations (4. mu.g/ml, 20. mu.g/ml and 100. mu.g/ml, respectively) in a human-derived proximal tubular epithelial cell fibrosis model by Western blotting.

FIG. 4 shows the morphological changes of cells according to CHP treatment concentrations (40. mu.g/ml and 100. mu.g/ml, respectively) in the human-derived glomerular endothelial cell fibrosis model.

FIG. 5a shows the results of the Western blotting to confirm the change in the expression level of fibronectin according to the CHP treatment concentration (62.5 ng/ml, 125ng/ml and 250ng/ml, respectively) in the human-derived hepatocyte fibrosis model, and FIG. 5b shows the results of quantifying the size of the band and plotting the results.

FIG. 6a shows the results of the Western blotting to confirm the change in the expression level of fibronectin according to the administration concentration of CHP (5 mg/kg and 35mg/kg, respectively) in an animal model of liver fibrosis, and FIG. 6b shows the results of quantifying the size of the band and plotting the same.

Fig. 7a shows the results of the western blotting to confirm the change in the expression level of fibronectin according to the CHP treatment concentration (62.5 ng/ml and 125ng/ml, respectively) in the human-derived lung cell fibrosis model, and fig. 7b shows the results of the quantitative and graphical visualization of the band sizes.

FIG. 8 shows the results of graphical representation of the changes in the expression levels of transforming growth factor beta (TGF β) and Collagen 3(Collagen 3) genes in accordance with the administration concentrations of CHP (5 mg/kg and 35mg/kg, respectively) in an animal model of pulmonary fibrosis.

FIG. 9a shows the results of the bands for confirming the change in the expression level of fibronectin according to the CHP treatment concentration (62.5 ng/ml, 125ng/ml, 250ng/ml and 500g/ml, respectively) in the human-derived skin cell fibrosis model by Western blotting, and FIG. 9b shows the results of the quantitative band size and the graphical representation.

FIG. 10 is a graph showing the results of the changes in the expression levels of genes for transforming growth factor beta, Fibronectin (Fibronectin), Collagen 1(Collagen 1), Collagen 2(Collagen 2), Collagen 3 and Collagen 4(Collagen 4) according to the administration concentrations (5 mg/kg and 35mg/kg, respectively) of the fibrosis markers in the animal model of cardiac fibrosis.

FIG. 11 shows the results of graphing the changes in the expression levels of the transforming growth factor beta, fibronectin, collagen 3 and Connective Tissue Growth Factor (CTGF) genes, which are fibrosis markers, according to the administration concentrations of CHP (5 mg/kg and 35mg/kg, respectively) in an animal model of adipose fibrosis.

Detailed Description

The present invention will be described in more detail below.

As described above, in the existing research on the treatment related to fibrosis, corticosteroids and immunosuppressive drugs are generally used to target the inflammatory process of fibrosis, but such a preparation shows little effect in clinical experiments, and thus a novel drug for treating fibrosis is required.

Accordingly, the present inventors confirmed that CHP inhibits fibrosis occurring in various tissues or organs and effectively prevents, improves or treats fibrosis, thereby completing the present invention.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating fibrosis comprising CHP or a pharmaceutically acceptable salt thereof and/or a health functional food composition for preventing or improving fibrosis comprising CHP or a food acceptable salt thereof.

Also, the present invention provides an anti-fibrotic composition comprising CHP.

In the present invention, "CHP (Cyclo-HisPro)" is a naturally occurring cyclic dipeptide (dipeptide) composed of histidine-proline, which is a metabolite of thyroid-stimulating hormone-releasing hormone (TRH), or a physiologically active dipeptide synthesized in vivo through thyroid-stimulating hormone-releasing hormone metabolic process and de novo, and refers to a substance widely distributed throughout the brain, spinal cord, gastrointestinal tract, and the like.

In the composition of the present invention, the CHP may be synthesized or commercially available. Also, it can be used by purifying CHP-containing substances such as prostate extract and soybean hydrolysate.

The term "purified" is used to refer to CHP in a concentrated form as compared to a form obtainable from a natural source such as prostate extract. The purified components may be concentrated from their natural sources or obtained by chemical synthesis.

In the present invention, the term "fibrosis" is used interchangeably with "fibrotic disorder", "fibroproliferative disorder", "fibrotic disease", "fibroproliferative disease", "fibrotic disorder" and "fibroproliferative disorder" to refer to a disorder, disease or disorder characterized by dysregulated proliferation or activity of fibroblasts, abnormal accumulation of fibronectin and/or pathological or excessive accumulation of collagen tissue. Generally, such conditions, diseases or disorders can be treated by administering compounds having anti-fibrotic activity.

For example, the fibrosis may occur in one or more selected from the group consisting of kidney, liver, lung, skin, heart, pancreas, urinary system, reproductive system, sweat gland, nerve, brain, bone marrow, muscle and joint.

For example, the fibrosis of the present invention may be one or more selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, radioactive lung injury or pulmonary fibrosis, pulmonary edema, cystic fibrosis, hepatic fibrosis, endocardial myocardial fibrosis, myocardial infarction, atrial fibrosis, glial scar, renal fibrosis, bone marrow fibrosis, joint fibrosis, fat fibrosis, skin fibrosis, nerve fibrosis and muscle fibrosis, but is not limited thereto.

Specifically, the CHP of the present invention has therapeutic effects on various fibrosis described in the present application.

In the prevention, amelioration, or treatment use of fibrosis of the composition of the present invention, the first embodiment relates to renal fibrosis, which is fibrosis occurring in the kidney.

Renal diseases are classified into acute renal failure and chronic renal failure according to their progression states, or into glomerulonephritis caused by deposition of vascular complexes, diabetic nephropathy accompanied with diabetes, hypertensive nephropathy accompanied with hypertension, toxic nephropathy caused by administration of drugs such as antibiotics and anticancer agents, bacterial infection, and the like. In renal diseases caused by any cause, when the glomerular filtration rate is reduced to 50% or less due to chronic renal insufficiency, the glomerular filtration rate is reduced continuously in most cases, and finally, end-stage renal failure is caused, and complications such as blood abnormality, nervous system complications, gastrointestinal complications, immune complications, infection, osteodystrophy, and the like are induced, and death is caused in severe cases.

Chronic renal failure refers to a state in which the renal function gradually decreases in one direction (irreversibility) and the homeostasis of the organism cannot be maintained. All renal diseases are accompanied by fibrosis of the kidneys, ultimately leading to end-stage renal failure. In particular, chronic renal failure is closely related to the progression of renal fibrosis, and therefore, when the progression of fibrosis is inhibited, the progression of chronic renal failure can be inhibited.

The term "renal fibrosis" in the present invention includes all diseases where fibrosis occurs in the kidney due to various causes, and the fibrosis may include one or more selected from the group consisting of catheter (cathter) installation, glomerulosclerosis, glomerulonephritis, nephritis, acute renal failure, chronic renal failure, end-stage renal disease, and metabolic diseases, but is not limited thereto.

The nephritis may be any interstitial nephritis, for example, streptococcal nephritis, staphylococcal nephritis, pneumococcal nephritis, viral nephritis accompanied by chicken pox, hepatitis B, hepatitis C, HIV, etc., nephritis caused by parasitic infection such as malaria, infectious interstitial nephritis accompanied by fungal nephritis, mycoplasma nephritis, etc., nephritis accompanied by systemic lupus erythematosus (lupus nephritis), systemic scleroderma (collagen-like kidney), collagen diseases such as Sjgren's syndrome, nephritis accompanied by purpura nephritis, polyarteritis, and autoimmune disease such as acute glomerulonephritis, interstitial nephritis accompanied by radiation irradiation, anticancer agents such as gold preparations, NSAIDs, penicillamine (penicillamine), bleomycin (bleomycin), antibiotics, drug-induced interstitial nephritis caused by paraquat, etc., interstitial nephritis, and the like, Allergic nephritis caused by insect sting, pollen, Anacardiaceae plants, etc., amyloidosis (amyloidosis) nephritis, diabetic nephropathy, chronic glomerulonephritis, malignant nephrosclerosis, polycystic kidney disease, etc., tubulointerstitial nephritis, pregnancy toxicity or cancer-associated nephritis, membranous proliferative glomerulonephritis, IgA nephropathy, mixed cryoglobulinemia (cryoglobulinemia) nephritis, Goodpasture's syndrome nephritis, Wegener granulomatosis nephritis, acute interstitial nephritis, etc., but is not limited thereto.

As demonstrated in fig. 2 to 4, CHP restores the expression of E-cadherin as a conjugation marker and reduces the expression of fibronectin and pSTAT3 proteins in fibrosis-inducing proximal tubular epithelial cells, thereby showing a fundamental anti-fibrotic effect, regardless of the cause of the induction of fibrosis, and is useful for treating various renal fibrosis.

In the use of the composition of the present invention for preventing, ameliorating or treating fibrosis, the second embodiment relates to liver fibrosis, which is fibrosis occurring in the liver.

The term "liver fibrosis (liver fibrosis)" in the present invention refers to a condition in which chronic damage of the liver causes proliferation of fibrous tissue, and may include, but is not limited to, symptoms caused by one or more selected from the group consisting of chronic liver disease, hepatitis B virus infection, hepatitis C virus infection, hepatitis D virus infection, schistosomiasis, alcoholic liver disease or non-alcoholic steatohepatitis, metabolic disease, protein deficiency, coronary artery disease, autoimmune hepatitis, cystic fibrosis, alpha-1 antitrypsin deficiency, primary biliary cirrhosis, drug reaction, and toxin.

Hepatic fibrosis is a pre-stage lesion of liver cirrhosis, is the result of severe liver injury leading to chronic liver disease, and is initiated by the action of various cytokines and growth factors. In general, liver fibrosis is reversible and consists of thin fibers (thi fibils), and in the case where nodules are not formed and the cause of liver injury is transient, increased extracellular matrix is decomposed by an apoptosis (apoptosis) process and Matrix Metalloproteinases (MMPs), thereby restoring normal, but if the liver fibrosis process is repeated, thick fibers (thick fibils) are formed and nodular liver cirrhosis is developed. Also, hepatic cells are damaged due to various inflammation-inducing factors and cirrhosis is induced by the process of hepatic fibrosis in which abnormal extracellular matrix proteins including collagen are accumulated, and in order to regulate the expression of cirrhosis, it is important to regulate the accumulation of extracellular matrix. The inflammatory response in the case of damaged hepatocytes activates resting hepatic stellate cells to secrete extracellular matrix and various cytokines (cytokines) and chemokines (chemokines), with transforming growth factor- β 1 acting as a potent growth inhibitor. A substance in which transforming growth factor- β 1 is 25kD is secreted in an inactive latent form by binding to latent transforming growth factor- β 1binding protein (latent TGF- β 1binding protein), exists in a state of binding to extracellular matrix such as type 1 collagen, type 4 collagen, laminin and decorin (decorin), and is activated by various stimuli. Transforming growth factor-beta 1 reduces the production of collagenase (collagenases) or increases the production of collagenase inhibitors to regulate collagen expression, increases the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), platelet-derived growth factor (PDGF), etc., in macrophages, and plays an important role in the fibrosis process. At present, transforming growth factor-beta 1 is expressed only at the site where fibrosis progresses, but not in normal liver tissues or inactive regions, and it is known that transforming growth factor-beta 1 plays an important role in hepatic fibrosis.

In addition, non-alcoholic fatty liver disease is induced by obesity, diabetes, hyperlipidemia, drugs, etc. regardless of drinking, and refers to a wide range of diseases including simple fatty liver (steatosis) which does not accompany inflammatory reaction according to the progression and non-alcoholic steatohepatitis (NASH) which accompanies inflammatory reaction of liver cells (hepatocellular inflammation), advanced fibrosis (advanced fibrosis) and cirrhosis (liver cirrhosis).

Non-alcoholic fatty liver disease (NAFLD) is an increase in adult diseases caused by high-fat and high-calorie diets in modern society, and it has been reported that 20% to 30% of the adult population, based on developed countries, shows non-alcoholic fatty liver disease, of which 2% to 3% are transferred to non-alcoholic steatohepatitis patients, and in particular, histologically, steatohepatitis showing fibrosis and accompanying inflammation has a very high risk of developing liver cirrhosis, liver failure, and liver cancer.

Thus, in one embodiment of the present invention, an animal model of liver fibrosis induced by a high fat diet was established, and it was confirmed that CHP showed a decrease in zonulin expression in the corresponding model.

Fatty liver is not itself a pathological state and is a reversible symptom that recovers spontaneously after removal of pathogens. However, if the state of excessive fat accumulation in the liver tissue is maintained, steatohepatitis occurs, and as a result, necrosis and regeneration of liver cells occur repeatedly, and in the process, fibrous extracellular matrix increases and fibrosis of the liver progresses. When the hepatic injury reaches a prescribed stage, the accumulation of extracellular matrix increases regardless of the kind of pathogen, and as hepatocytes are continuously destroyed and regenerated, regenerative nodules (regenerative nodules) are formed, thereby deteriorating into irreversible cirrhosis (liver cirrhosis).

Therefore, in the present invention, hepatic fibrosis is a disease clearly different from fatty liver, which is a reversible symptom, and refers to all diseases in which liver tissue is changed into fibrotic tissue such as regenerative nodules and the like, and liver function is decreased.

As shown in fig. 5, CHP reduced the expression level of fibronectin, which is a fibrotic protein, in hepatocytes induced by fibrosis, and as shown in fig. 6, CHP reduced the expression of fibronectin in animal models induced liver fibrosis, showed a fundamental anti-fibrotic effect, and was also applicable to liver failure and liver cancer that progress from cirrhosis, including various liver fibrosis, regardless of the cause of induced fibrosis.

In the fibrosis prevention, amelioration, or treatment use of the composition of the present invention, the third embodiment relates to pulmonary fibrosis, which is fibrosis occurring in the lung.

Among the fibrosis, Pulmonary fibrosis (Pulmonary fibrosis), in particular, refers to a disease in which chronic inflammatory cells infiltrate into the alveolar wall of lung tissue to induce tissue fibrosis and cause severe structural changes of the lung tissue. Once fibrosis is induced for any reason, the lung tissue becomes hard and the alveolar wall becomes thick, thereby reducing the amount of oxygen supplied from the blood, resulting in difficulty in breathing. There is currently no treatment that can fully restore lung tissue that has already developed fibrosis, and therefore patients die after 3 to 5 years when symptoms are usually present, in addition to early detection of fibrosis or lung transplantation.

Specifically, in the present invention, the term "pulmonary fibrosis" refers to development of scar (fibrotic) tissue due to excessive formation of fibrous associated tissue or development of fibrous associated tissue in the lung (fibrosis). In particular, pulmonary fibrosis refers to a chronic disease that induces swelling and scarring of the alveoli and the interstitial tissues of the lung. Scar tissue, as described above, replaces healthy tissue to induce inflammation, which can be identified as a precursor to fibrosis. Due to the damage of the lung tissue as described above, the lungs may become stiff and the individual may have difficulty breathing autonomously.

In the present invention, the pulmonary fibrosis may include one or more selected from the group consisting of idiopathic pulmonary fibrosis, radiation Lung injury, Nonspecific Interstitial Pneumonia (Nonspecific Interstitial pneumoniaa), Acute Interstitial Pneumonia (Acute Interstitial pneumoniaa), Cryptogenic organic Pneumonia (Cryptogenic Pneumonia), Respiratory bronchiolitis-associated Interstitial Lung (Respiratory Lung) disease, Desquamative Interstitial Pneumonia (Desquamative Interstitial pneumoniaa), Lymphoid Interstitial Pneumonia (Lymphoid Interstitial pneumoniaa), Interstitial pulmonary fibrosis and diffuse pulmonary fibrosis, pulmonary edema, cystic fibrosis and pulmonary fibrosis caused by metabolic disease, but is not limited thereto.

The pulmonary fibrosis may be caused by various causes, for example, fine damage to the lung by inhalation of fine particulate matter (asbestos, stone dust, metal dust, particles in cigarette smoke, silica dust, etc.). Pulmonary fibrosis occurs due to secondary effects of other diseases (autoimmune diseases, viral or bacterial infections, etc.), and can be induced by specific drugs such as cytotoxic agents (bleomycin, busulfan, methotrexate, etc.), antibiotics (nitrofurantoin, sulfasalazine, etc.), arrhythmia remedies (amiodarone, tocainide, etc.), anti-inflammatory agents (gold, penicillamine, etc.), illegal drugs (drugs, cocaine, heroin, etc.), and the above-mentioned idiopathic pulmonary fibrosis can be caused by unknown reasons other than the above-mentioned reasons. Also, chronic intake of high fat diet induces pulmonary fibrosis, which is caused by increased levels of inflammation after high fat intake.

As confirmed in fig. 7, CHP reduced the expression level of fibronectin, a fibrotic protein, in lung cells that induced fibrosis, and as confirmed in fig. 8, CHP reduced the expression of transforming growth factor β and collagen 3 genes, which are major fibrotic markers, in animal models that induced pulmonary fibrosis, showing a fundamental anti-fibrotic effect, and thus was applicable to the treatment of various pulmonary fibrosis regardless of the cause of induction of fibrosis.

In the fibrosis prevention, amelioration, or treatment use of the composition of the present invention, the fourth embodiment relates to skin fibrosis.

In the present invention, the term "dermal fibrosis" is excessive scarring of the skin and is the result of a pathological wound healing response. The range of fibrotic skin diseases is wide: scleroderma, renal fibrotic dermatoses, mixed connective tissue disease, sclerosing mucoedema, sclerosing edema, and eosinophilic fasciitis. Exposure to chemical substances or physical factors (mechanical trauma, burns) is also a potential cause of fibrotic skin diseases. Skin fibrosis may be dominated by immune, autoimmune and inflammatory mechanisms. The balance of collagen production and breakdown of fibroblasts plays an important role in the pathophysiological processes of skin fibrosis. Specific cytokines such as transforming growth factor-beta (TGB-beta) and interleukin-4 (IL-4) promote wound healing and fibrosis. Fibroblasts of normal skin are quiescent. They control the amount of desmin and have low proliferative activity. After skin damage, these cells are activated, i.e., they express alpha-smooth muscle actin (alpha-SMA) and synthesize a large amount of connective tissue protein. Activated cells are commonly referred to as muscle fibroblasts.

In the present invention, the term "skin fibrosis" is also intended to include "scleroderma".

Scleroderma is a chronic autoimmune connective tissue disease of unknown cause characterized by sclerosing changes and abnormalities in the vascular system that accumulate excess collagen in the dermis, causing stiffening and thickening of a portion of the skin or the whole body of the skin. The Collagen (Collagen) constitutes connective tissue to support and connect body tissues. Scleroderma has several forms, with forms that exhibit symptoms only in specific parts of the body, and also forms that exhibit symptoms throughout the body, including internal organs. That is, there are limited scleroderma (localized scleroderma) in which only a part of the skin is hardened and systemic scleroderma (systemic scleroderma) in which internal organs including the skin, such as the lung, digestive system, kidney, and heart, show fibrosis due to increased gliosis. Systemic scleroderma is classified into a limited type and a wide-spread type according to the degree of invasion of skin and internal organs, prognosis, and immunological examination results.

In the preventing, ameliorating or treating use of skin fibrosis of the present invention, the skin fibrosis may include one or more selected from the group consisting of scars, hypertrophic scars, keloids, localized scleroderma, and systemic scleroderma, but is not limited thereto.

The cause of scleroderma is not clear, but fibrosis of the tissue is thought to play an important role.

The early symptoms of scleroderma are very diverse and, in particular, the skin symptoms become evident in the later stages. Common symptoms of scleroderma are joint pain, morning stiffness, fatigue and weight loss. Also, when exposed to cold, blood supply to the fingers, toes, nose, and ears is temporarily restricted. The symptom is an early symptom of scleroderma patients and is one of common symptoms. Patients with scleroderma have hardened skin. This skin stiffening phenomenon is commonly occurring on both sides of the body while spreading widely. Eventually, the tissue is damaged, and the skin is pigmented.

The incidence of localized scleroderma in women is 2.6 times that in men. The age of 75% of patients with localized scleroderma is between 20 and 50 years, and linear scleroderma shows a tendency to develop in the earlier ages. Localized scleroderma is classified as localized scleroderma, systemic localized scleroderma, linear localized scleroderma, or subcutaneous localized scleroderma.

Localized scleroderma usually starts with erythema or purplish red spots, with a clear boundary with the surrounding normal skin, and the skin at the prescribed site loses elasticity and becomes hard. The part is brown, and in many cases, it is discolored to be white. Most of them occur in one place, but there are cases where a plurality of them occur, and the sizes of them are different from each other, and vary from the size of a coin to the size of an adult palm. Although causing cosmetic problems, it does not progress to systemic sclerosis. In some patients, hard skin softens and recovers itself even without treatment.

Systemic localized scleroderma is a severe form of localized scleroderma, a condition where large areas of skin become hardened and hyperpigmentation is observed. The skin of the trunk, buttocks and legs becomes widely hard. However, it is distinguished from systemic sclerosis because of the absence of Raynaud's (Raynaud's) phenomenon, a symptom of visceral organ invasion.

Linear limited scleroderma is a condition in which the skin hardens linearly and long, often occurs in the order of legs, arms, forehead, and chest, and particularly, a condition in which a recess occurs in a vertical line on the forehead is called sword-injured scleroderma (en coup de sabre), which induces cosmetic problems. Unlike localized scleroderma, linear scleroderma does not only invade the skin, but also the muscle, periosteal tissue beneath the skin to anchor the underlying tissue. It is more common in children and can sometimes induce severe dysplasia in the extremities and face.

Subcutaneous localized scleroderma shows hardening of the adipose layer, fascia, muscle, and bone, and joint movement may be limited. Since the lesions occurred deep, no characteristic skin pigmentation change of scleroderma was observed.

Systemic scleroderma (systemic scleroderma) is 4 times more prevalent in women than in men, and can occur at any age, but is most commonly between the ages of 30 and 50. The wide-ranging onset is younger than the localized type. Raynaud's phenomenon is the first symptom of systemic scleroderma, after which, hardening symptoms begin to develop in the skin and internal organs. Systemic scleroderma is divided into cutaneous symptoms and hidden organ symptoms.

In systemic sclerosis, the initial skin symptoms begin in the fingers and hands. Initially, only reynolds phenomenon was exhibited, after which the fingers and hands became swollen, stiff, red. The skin gradually hardens and spreads to the arms and face, and in the wide range of cases, it continues to harden throughout the body including the trunk. When the face is hardened, wrinkles disappear as a whole and expression is difficult to make. The nose looks sharp, the mouth is difficult to open, the lips are thin, radial wrinkles appear around the mouth, and the nose becomes like the mouth of an elderly person. Finger-toe sclerosis, in which the movement of joints is limited, the hand cannot be held all the way, fingers are bent and become sharp, is exhibited. Painful and difficult to heal ulcers on the fingertips and joints.

It can show extensive hyperpigmentation and local hypopigmentation, hair loss and decreased sweat secretion at the affected site. Telangiectasia appears as a circular spot on the face and upper torso, or is observed around nail wrinkles. Systemic sclerosis may then occur if the capillaries of the nail wrinkles of patients with raynaud's disease are abnormal, and changes in capillaries are important in determining prognosis. Skin calcification can be observed, relatively common around finger joints.

Visceral organ symptoms occur in the gastrointestinal tract, lungs, heart or kidneys. In the gastrointestinal tract, the esophagus is the most commonly affected site, and is affected in more than 90% of cases. Impaired esophageal motility, dysphagia, and reflux esophagitis often occur. Constipation, diarrhea, malabsorption, etc. may occur due to the reduced intestinal motility. The most important cause of death in patients with systemic sclerosis is pulmonary symptoms, which occur in about 70% of patients. When moving, dyspnea, cough are complained of due to fibrosis of the lungs. Alveolitis often occurs, so that lung function continues to decline. In the heart, symptoms of cardiac conduction disorder, heart failure, and pericarditis may occur, and myocardial sclerosis may occur in 50% to 70% of patients. Renal symptoms occur in about 45% of patients with systemic sclerosis. Acute renal failure, hypertension, etc. may be manifested suddenly, and uremia may also occur slowly.

Therefore, in the prophylactic, ameliorating or therapeutic use of skin fibrosis of the present invention, skin fibrosis includes, without limitation, organs such as blood vessels and veins, submandibular ducts (ducts), gall bladder, thyroid follicular, sweat duct, ovary, kidney, or the lumen of gland (gland); epithelial cells of the gums, tongue, palate, nose, throat, esophagus, stomach, small intestine, rectum, anus, and vagina; fibrosis of the dermis, scar, skin and any skin tissue and epithelial cells in the scalp.

As confirmed in fig. 9, CHP exhibits a fundamental anti-fibrotic effect by reducing the expression level of fibronectin, which is a fibrotic protein, in skin cells that induce fibrosis, and therefore can be applied to the treatment of various skin fibrosis (skin sclerosis) regardless of the cause of the induction of fibrosis.

In the prevention, amelioration, or treatment use of fibrosis in the composition of the present invention, the fifth embodiment relates to cardiac fibrosis, which is fibrosis occurring in the heart.

In the present invention, the term "cardiac fibrosis" refers to a phenomenon in which matrix protein is excessively deposited between heart cells to harden the heart, mainly occurs in the heart of a patient with myocardial infarction, refers to a phenomenon that is a major cause of reduced function of the heart, and may include one or more selected from the group consisting of endocardial myocardial fibrosis, atrial fibrosis, heart failure, myocardial infarction, and cardiac fibrosis caused by metabolic diseases, but is not limited thereto.

Fibrosis is characterized by an unbalanced accumulation of fibrotic collagen, inflammation, increased workload, hypertrophy, and stimulation by various hormones, cytokines, and growth factors that occur after cardiac muscle cell death.

Cardiac fibrosis may also be abnormal thickening of a heart valve caused by uneven proliferation of cardiac fibroblasts, and further, refers to proliferation of fibroblasts of cardiac muscle. Fibroblasts (fibrocytes) are generally referred to as collagen, and function to provide structural support to the heart. If too active, the process becomes a cause of thickening and fibrosis of the valve.

Fibrosis of the heart is the leading cause of heart failure and myocardial infarction, and thus the term "heart fibrosis" may be interpreted to include the meaning of heart failure and/or myocardial infarction induced by heart fibrosis.

As confirmed in fig. 10, CHP reduces the expression of fibronectin, collagen 1, collagen 2, collagen 3, and collagen 4 genes, which are major fibrosis markers in an animal model for inducing cardiac fibrosis, and exhibits a fundamental anti-fibrotic effect, and thus can be used for the treatment of various cardiac fibrosis regardless of the cause.

In the fibrosis prevention, amelioration, or treatment use of the composition of the present invention, the sixth embodiment relates to fat fibrosis.

In the present invention, the term "fat fibrosis" is meant to include all diseases in which fibrosis occurs in fat tissue.

It is reported that fibrosis of adipose tissue occurs when normal mice are fed a high fat diet containing 60% fat for 16-24 weeks (Hu, M et al, evolution-Based comparative and Alternative Medicine,1-12,2018; Kwon, e.y., & Choi, m.s., nutriments, 10(10),1415,2018; Nakazeki, F et al, Scientific Reports,8(1), 2018; muniappon, L et al, Scientific Reports,7(1), 2017; Lancha, a., PLoS ONE,9(5), e98398,2014; Vel α zquez, K.T et al, physiologics, 5(18 e13412,2017; long, L, Ye, hue, wash, y, 20127, biological report, 121).

Therefore, the present inventors established an animal model of adipose fibrosis induced by high fat diet, and confirmed the expression changes of transforming growth factor β, fibronectin, collagen 3 and connective tissue growth factor genes as fibrosis markers by administration of CHP. As a result, as confirmed in fig. 11, CHP exhibits an anti-fibrotic effect by significantly reducing the expression of the above-mentioned fibrosis marker, and thus is useful for treating fatty fibrosis.

As described above, it was confirmed in the present invention that CHP exhibits an anti-fibrosis activity by reducing the expression of various fibrosis marker genes or proteins in various tissue cells and animal models in which fibrosis is induced, and is thus expected to be effectively applied to the prevention, improvement, or treatment of fibrosis occurring in various tissues and/or organs in the body including kidney, liver, lung, skin, heart, pancreas, urinary system, reproductive system, sweat gland, nerve, brain, bone marrow, muscle, and joint.

In the compositions of the invention for the prevention, amelioration, or treatment of fibrosis, the terms "prevention", "amelioration", and/or "treatment" refer to all acts of inhibiting or delaying the progression of a disease or disorder, all acts of ameliorating or beneficially altering the state of a disease or disorder, and all acts of delaying, halting, reversing the progression of a disease or disorder.

In the present application, the term "pharmaceutically acceptable" means physiologically acceptable and does not generally cause allergic reactions or the like when administered to a human body, and preferably, the above salts are acid addition salts formed by pharmaceutically acceptable free acids (free acids).

The pharmaceutically acceptable salt may be an acid addition salt formed using an organic acid or an inorganic acid, and the organic acid includes, for example, formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid, malonic acid, fumaric acid, succinic acid monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminoxyacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or methanesulfonic acid. Inorganic acids include, for example, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, or boric acid. The acid addition salts are preferably in the form of chlorate or acetate, more preferably chlorate.

Other examples of acceptable salts include, among others, gamma-aminobutyric acid (GABA) salt, gabapentin salt, pregabalin salt, nicotinate, adipate, hemi-malonate, cysteine salt, acetylcysteine salt, methionine salt, arginine salt, lysine salt, ornithine salt, or aspartate salt, and the like.

Also, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Carriers for parenteral administration include water, suitable oils, saline solutions, aqueous dextrose, glycols and the like. In addition, the above-mentioned carrier may further include a stabilizer and a preservative. Suitable stabilizers are antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl or propyl p-hydroxybenzoate and chlorobutanol. Other pharmaceutically acceptable carriers can be found in the following references (Remington's Pharmaceutical Sciences,19th ed., Mack Publishing Company, Easton, Pa, 1995).

The pharmaceutical compositions of the present invention may be administered to a mammal, including a human, by any method. For example, it can be administered orally or parenterally, and the parenteral administration can be intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, rectal administration, but is not limited thereto.

The pharmaceutical composition of the present invention may be formulated into preparations for oral administration or parenteral administration according to the administration routes as described above. In the case of dosage formulation, it may be formulated by using one or more buffers (e.g., saline solution or Phosphate Buffered Saline (PBS)), carbohydrates (e.g., glucose, mannose, sucrose, or dextran, etc.), antioxidants, bacteriostats, chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA) or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants, surfactants, diluents, or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions, capsules and the like, which can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient, examples of which include starch (including corn starch, wheat starch, rice starch, potato starch and the like), Calcium carbonate (Calcium carbonate), Sucrose (Sucrose), Lactose (Lactose), glucose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, gelatin and the like. For example, tablets or sugar-coated tablets can be obtained by compounding the active ingredient with a solid excipient and pulverizing the same, followed by adding an appropriate adjuvant thereto and processing the resulting mixture into a granulated mixture.

In addition to simple excipients, lubricants such as magnesium stearate and talc may be used. Liquid preparations for oral administration correspond to suspensions, liquids for internal use, emulsions, syrups and the like, and may include several excipients, such as wetting agents, sweeteners, aromatics or preservatives and the like, in addition to water or liquid paraffin, which are frequently used as simple diluents.

In addition, as disintegrating agent, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate, etc. may be added, and anticoagulant, lubricant, wetting agent, perfume, emulsifier and antiseptic may also be added.

In the case of parenteral administration, the pharmaceutical composition of the present invention can be formulated into injection dosage forms, transdermal administration agents and intranasal inhalants by methods known in the art together with suitable carriers for parenteral administration. The above injection must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections may include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), mixtures thereof, and/or solvents or dispersants including vegetable oils. More preferably, Hanks 'solution, ringer's solution, Phosphate Buffered Saline (PBS) containing triethanolamine, or sterile water for injection, or an isotonic solution (e.g., 10% ethanol, 40% propylene glycol, or 5% glucose) may be used as a suitable carrier. In order to protect the above injection from microbial contamination, the injection may further comprise various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Also, in most cases, the above injection may further comprise an isotonic agent such as sugar or sodium chloride.

The transdermal administration preparation forms include ointments, creams, lotions, gels, external solutions, plasters, packs, aerosols and the like. The term "transdermal administration" as used above refers to the topical administration of a pharmaceutical composition to the skin to deliver an effective amount of the active ingredient contained in the pharmaceutical composition into the skin.

In the case of an inhaler, the compounds for use according to the present invention may conveniently be delivered from pressurized packs or a nebulizer, using a suitable propellant, e.g. dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas, in the form of an aerosol spray. In the case of a pressurized aerosol, the unit of administration may be determined by providing a valve that delivers a measured amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder material such as lactose or starch. Dosage forms for parenteral administration are described in the formulary generally known in Pharmaceutical chemistry (Remington's Pharmaceutical Science,15th Edition,1975.Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour).

When the pharmaceutical composition of the present invention includes an effective amount of CHP, it can provide a preferable effect of preventing, ameliorating or treating fibrosis. In the present application, the term "effective amount" refers to an amount that indicates a greater response compared to a control group, preferably an amount sufficient to prevent, ameliorate or treat fibrosis. The pharmaceutical compositions of the invention may contain from 0.01% to 99.9% CHP with the remainder being a pharmaceutically acceptable carrier. The effective amount of CHP included in the pharmaceutical composition of the present invention may vary depending on the form in which the composition is to be produced.

The total effective amount of the pharmaceutical composition of the present invention can be administered to a patient as a single dose (single dose) or as multiple doses (multiple dose) by a fractionated treatment method for long-term administration (fractionated treatment protocol). The pharmaceutical composition of the present invention may vary the content of the effective ingredient depending on the degree of the disease. For example, based on CHP, 0.001mg to 100mg per 1kg body weight is administered once a day or more, more preferably 0.01mg to 10mg per 1kg body weight is administered once a day or more. However, the dose of CHP is determined in consideration of various factors such as the route of administration and the number of treatments of the pharmaceutical composition, the age, body weight, health status, sex, severity of disease, diet and excretion rate of the patient, and thus, a person of ordinary skill in the art can determine an appropriate effective dose of CHP for a specific use for preventing, treating or improving fibrosis. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, administration route and administration method as long as it exhibits the effects of the present invention.

The pharmaceutical composition for preventing or treating fibrosis of the present invention can be used alone or in combination with a method employing surgery, radiation therapy, hormone therapy, chemotherapy, or a biological response controlling agent.

The pharmaceutical composition for preventing or treating fibrosis according to the present invention can also be provided in the form of a topical preparation or the like containing CHP. In this aspect, the composition of the present invention may be a quasi-drug composition for preventing or improving fibrosis and a quasi-drug comprising the above composition.

The external preparation can be directly applied to the skin or the oral cavity. In the case where the pharmaceutical composition for preventing or treating fibrosis according to the present invention is used as an external preparation, any ingredient generally used in external preparations for skin, such as a fatty material, an organic solvent, a solubilizing agent, a concentrating agent, a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent (formulation), a fragrance, a surfactant, water, an ionic emulsifier, a nonionic emulsifier, a filler, a sequestering agent, a chelating agent, a preservative, a vitamin, a blocking agent, a wetting agent, an essential oil, a dye, a pigment, a hydrophilic active agent, a lipophilic active agent, or a lipid vesicle, may be further included. The above ingredients can be introduced in amounts generally used in the field of skin science.

When the composition of the present invention is provided as a preparation for external use, the composition may be in the form of a liquid preparation, an ointment, a patch, a gel, a cream, an aerosol, or the like, but is not limited thereto. According to an example of the present invention, the quasi-drug of the present invention may be an oral care product including toothpaste, mouthwash and oral spray, ointment, mask, poultice, patch, transdermal absorbent, and the like.

In the case where the composition of the present invention is used as a quasi-drug composition, CHP may be added directly or used appropriately together with other quasi-drug ingredients according to a conventional method. The mixing amount of the effective ingredients can be appropriately determined depending on the purpose of use (prevention, health care or treatment).

The contents of the pharmaceutical composition and the health-care functional food composition of the present invention can be applied to the quasi-drug composition and the quasi-drug of the present invention.

In the present invention, the term "health functional food" means "functional food" and "health food".

In the present invention, the term "functional food" is the same term as a food for special health use (FoSHU) and refers to a food which is processed in such a manner as to effectively exert a bioregulatory function in addition to supplying nutrients and has a good medical and medical effect.

In the present invention, the term "health food" refers to a food having a positive health-care or health-promoting effect as compared with a general food, and a health supplement food (health supplement food) refers to a food for the purpose of health supplement. The terms functional food, health supplementary food are used interchangeably depending on the situation. The above food can be prepared into various forms of tablets, capsules, powders, granules, liquid preparations, pills, etc. in order to obtain useful effects of improving and restoring liver function.

As a specific example of such a functional food, a processed food having good storability while utilizing the characteristics of agricultural products, livestock products, or aquatic products can be produced by using the above composition.

The health functional food composition of the present invention may also be prepared in the form of nutritional supplements (nutritional supplements), food additives (food additives), feeds, etc., for animals including humans or livestock.

The above-mentioned types of food compositions can be prepared in various forms according to conventional methods well known in the art. Can be prepared by adding CHP to general foods including, but not limited to, beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruits, bottled foods, jams, etc.), fish, meats and their processed products (e.g., ham, sausage, salted beef, etc.), bread, noodles (e.g., udon, buckwheat, ramen, pasta, macaroni, etc.), fruit juices, various beverages, cookies, syrups, dairy products (e.g., butter, milk, etc.), edible vegetable oils, margarine, vegetable proteins, steamed foods, frozen foods, and various seasonings (e.g., soybean paste, soy sauce, etc.).

Also, the nutritional supplement may be prepared by adding CHP to capsules, tablets, pills, etc., but is not limited thereto.

The health functional food may be ingested by making the CHP into a liquid phase, a granule, an encapsulation, or a powder, and may be prepared as tea, fruit juice, or a beverage (health drink), for example, but not limited thereto. In order to use the CHP as a food additive, the CHP can be prepared and used in the form of a powder or a concentrated solution. The CHP may be mixed with an active ingredient known to have an effect of preventing or improving fibrosis to prepare a composition.

In the case where the food composition of the present invention is used as a health drink composition, the above health drink composition may contain some flavoring agents or natural carbohydrates, etc. as additional ingredients as in ordinary drinks. The above-mentioned carbohydrate may be: monosaccharides such as glucose and fructose; disaccharides, such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; sugar alcohols, such as xylitol, sorbitol and erythritol. The sweetener may be: natural sweeteners such as thaumatin, stevia extract; and synthetic sweeteners such as saccharin and aspartame. The proportion of the above natural carbohydrates is generally from about 0.01g to 0.04g, preferably from about 0.02g to 0.03g, per 100mL of the composition of the invention.

The CHP may be contained as an effective ingredient in the food composition for preventing or improving fibrosis in an amount effective to obtain the above-mentioned preventing or improving effect, for example, preferably 0.01 to 100 weight percent relative to the total weight of the entire composition, but is not limited thereto. The food composition of the present invention can be prepared by mixing other active ingredients known to have an effect of preventing or improving fibrosis together with CHP.

In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acids and salts thereof, alginic acids and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, or carbonating agents, and the like. In addition, the health food of the present invention may contain pulp for use in the manufacture of natural fruit juice, fruit juice beverages and vegetable beverages. Such ingredients may be used alone or in admixture. The proportion of such additives is not critical, but is generally selected from the range of 0.01 to 0.1 parts by weight, relative to 100 parts by weight of the composition of the invention.

Also, the present invention relates to a method for preventing or treating fibrosis comprising the step of administering an effective amount of CHP to a subject in need thereof.

In the methods of the present invention, the term "subject" includes any animal (e.g., human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent), but is not limited thereto. Such terms do not denote a particular age or gender. Thus, female/female, male/male, adult/adult and newborn subjects, as well as embryos are included. A patient refers to a subject suffering from a disease or disorder. The term "patient" includes human and veterinary subjects.

In the method of the present invention, the effects of CHP and the explanation of the administration route, the number of administrations, the dose, etc. are the same as those described above, and therefore, the description thereof will be omitted.

Also, the present invention provides the use of CHP in the preparation of a pharmaceutical composition for the prevention or treatment of fibrosis.

The present invention will be described in more detail below with reference to examples. However, the present invention may be modified in various ways and may have various embodiments, and the specific examples and the description described below are only for the purpose of understanding the present invention, and the present invention is not limited to the specifically disclosed embodiments. It should be understood that the scope of the present invention includes all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention.

Modes for carrying out the invention

Preparation example

The CHP used in the examples described below was purchased from BaHenry (BACHEM) of Switzerland and used.

Example 1

Confirmation of anti-fibrotic Effect in human-derived proximal tubular epithelial cells treated with CHP

1-1, isolation and culture of human proximal tubular epithelial cells

As shown in fig. 1, pure human glomerular endothelial cells and human proximal tubular epithelial cells that can be used for cell experiments were isolated and cultured by isolating glomerular and tubular interstitial tissues from normal adult renal tissues and primary culturing the same.

1-2 observation of cytological morphology following CHP treatment in a model of human proximal tubular epithelial cell fibrosis

The recombinant transforming growth factor β (2ng/ml) was treated in the human-derived proximal tubular epithelial cells isolated and primary-cultured in the above example 1-1 to induce cell fibrosis, whereby cytoskeletal remodelling and morphological changes (elongated and structurally disappeared) occurred, confirming that fibrosis was properly induced (fig. 2).

After that, when CHP (4. mu.g/ml, 20. mu.g/ml and 100. mu.g/ml) was treated at various concentrations, improvement of fibrosis was confirmed by cell morphology (FIG. 2).

1-3, confirmation of E-cadherin, fibronectin after treatment of CHP in a model of human proximal tubular epithelial cell fibrosis Zonulin and pSTAT3 protein expression

Under the same conditions as in example 1-2, the expression levels of proteins of E-cadherin as a junction marker (junction marker), fibronectin as a fibrosis marker, and pSTAT3 as a fibrosis transcription factor were confirmed by Western blotting after administration of CHP.

As a result, as shown in FIG. 3, it was confirmed that the protein expression of E-cadherin was restored and the protein expression of fibronectin and pSTAT3 was decreased with the administration of CHP.

Example 2

Confirmation of anti-fibrotic Effect in human-derived glomerular endothelial cells according to CHP

Observation of cell morphology after treatment of CHP in a human-derived glomerular endothelial cell fibrosis model

As in the human-derived proximal tubular epithelial cell fibrosis model of example 1-1 above, cytoskeletal remodelling and morphological changes occurred by treating r-transforming growth factor β (2ng/ml) to induce cell fibrosis in glomerular endothelial cells, confirming that fibrosis was properly induced (fig. 4).

After that, when CHP (40. mu.g/ml and 100. mu.g/ml) was treated at various concentrations, improvement of fibrosis was confirmed by cell morphology (FIG. 4).

From the above results, it was confirmed that CHP has an excellent therapeutic effect on renal fibrosis.

Example 3

Confirmation of anti-fibrotic Effect in human hepatocytes treated with CHP

3-1, culturing human liver cells, inducing fibrosis and post-treating CHP

To culture Huh7 liver thinCell lines, purchased from Hyclone Laboratories Inc. in USA in DMEM medium and Fetal Bovine Serum (FBS), and R from USA&The company D systems bought transforming growth factor beta for inducing fibrosis. DMEM medium supplemented with 10% fetal bovine serum was used at 37 ℃ with 5% CO₂The culture chamber of (1) was cultured, and the transforming growth factor beta was treated so that the concentration thereof became 2ng/ml, and CHP was treated so that the concentration thereof became 0ng/ml, 62.5ng/ml, 125ng/ml, and 250ng/ml, and the cells were recovered 48 hours after the treatment.

3-2 confirmation of fibronectin expression as a fibrotic marker after treatment of CHP in a model of human-derived hepatocyte fibrosis

For analysis of protein expression of fibronectin as a fibrotic protein, RIPA lysis buffer (lyses buffer) and Halt from Thermo Fisher Scientific Inc. were purchased from Sammer Feishel technologies, USA^TMFor protein quantification, a BCA protein quantification kit from siemer feishel technologies, usa was purchased as a mixture of Protease (Protease) and Phosphatase (Phosphatase) inhibitors. For the Western blotting, Bolt of Saimer Feishale scientific Inc. in USA was prepared^TMProtein gel electrophoresis systems and wet transfer systems from Burley (Bio-Rad Laboratories, Inc.) Inc., USA. Antibodies to fibronectin as a marker of hepatic fibrosis and GAPDH antibodies as loading controls were purchased from Abcam, usa. To the recovered Huh7 cells, 100ul of RIPA buffer supplemented with protease and phosphatase inhibitors was added, and the cells were lysed by pipetting. After leaving on ice for 10 minutes, it was centrifuged at 15000rpm at a temperature of 4 ℃ for 10 minutes. The supernatant was recovered and the protein concentration was determined by BCA quantification using Bolt^TMThe same amount of protein of the sample was separated by a protein gel electrophoresis system and transferred to a Nitrocellulose membrane (Nitrocellulose membrane). The membrane was blocked (blocking) with 5% skim milk (ski) at room temperature for 1 hour, and then reacted with fibronectin and GAPDH antibodies as primary antibodies at 4 ℃ overnight (overlap). The reaction mixture was washed 3 times with TBST for 10 minutes, and then reacted with a secondary antibody at ordinary temperature for 1 hour. Washing with TBST for 3 min, 10 min eachAnd reacted with ECL to determine the extent of expression. The indicated band sizes were quantified using the ImageJ program and corrected by dividing the fibronectin band size value by the GAPDH band size value. Statistical significance was analyzed with Student's t-test statistics of the control group. # p<0.05 (negative control group: transforming growth factor beta treated control group).

As a result of the experiment, as shown in fig. 5a and 5b, it was confirmed that when transforming growth factor β and CHP of 250ng/ml were treated, the expression amount of fibronectin, which is a fibrotic protein, increased with the treatment of transforming growth factor β decreased. Thus, it was confirmed that CHP can improve fibrosis in Huh7 cells as a liver cell line.

Example 4

Confirmation of anti-fibrotic Effect in hepatic fibrosis animal models based on administration of CHP

Long-term intake of high-fat diets is well known as a risk factor for inducing liver fibrosis. Due to insulin resistance and increased blood fatty acids, fatty factors, cytokines, which occur when a fat diet is ingested for a long period of time, an inflammatory environment is induced in liver tissues, and the interaction of immune cells accumulated in liver tissues promotes fibrosis, in particular, interleukin-13 cytokine secreted from Th2CD4+ T cells produces a large amount of transforming growth factor β, which is a key cause of fibrosis, from macrophages. Signalling through transforming growth factor beta causes transformation of cells within the liver tissue and promotes progression of fibrosis (Rosselii, M et al, Current Pharmaceutical Design,20(31), 5010-.

When normal mice were fed a high fat diet containing 60% fat for 10-16 weeks, liver fibrosis was observed and could be confirmed by expression of fibronectin, which is a marker protein for fibrosis (Kim, I.H et al, AGE,38(4), 291-.

4-1, designing experimental animal and inducing hepatic fibrosis

For the experiment of hepatic fibrosis in animals, 12-week-old C57BL/6 mice ingested with a high fat diet containing 60% fat for 6 weeks were purchased from Jackson in the united states, Research diets D12492 as a high fat diet feed containing 60% fat were purchased from Saeronbio in korea, and the mice were ingested for 17 weeks. Mice were free to eat and drink water at 24 ± 3 ℃. C57BL/6 mice on a 6-week high fat diet were acclimatized to a 1-week feeding environment and weighed and evenly divided into 3 groups as shown in table 1. CHP was administered once a day at a concentration of 5mg/kg or 35mg/k to each group orally for 16 weeks, and the same amount of distilled water was orally administered by the same method for the control group.

TABLE 1

4-2 confirmation of fibronectin expression as a fibrotic marker in CHP-administered animal models of liver fibrosis

Isoflurane (Isoflurane) required for Anesthesia and dissection of mice was purchased from Hana Pharm corporation, and an RC2 Rodent Circuit Controller Anesthesia System of Witpur, USA was prepared. Phosphate buffer was purchased from sea clone, usa. To isolate liver tissue of mice, mice were anesthetized by respiratory anesthesia using 3% to 3.5% isoflurane. Immediately after blood collection from the heart of an anesthetized mouse, liver tissue was harvested, washed with phosphate buffer, 50mg of liver tissue was excised, 500ul of RIPA buffer to which protease and phosphatase inhibitors were added was placed, and the liver tissue was pulverized by a T10 homogenizer (homogenizer) of IKA, germany. After being left on ice for 15 minutes, it was centrifuged at 15000rpm at a temperature of 4 ℃. In order to analyze the expression of fibronectin, which is a fibrotic protein, western blotting was performed by the same method as in example 3-2. Statistical significance was analyzed by Student's t-test statistics with HFD control groups (. p <0.05,. p < 0.01).

As a result of the experiment, as shown in fig. 6a and 6b, it was confirmed that the expression level of fibronectin, which is a representative fibrosis marker, was significantly decreased in the CHP-administered group, and the effect was enhanced depending on the concentration of CHP. This indicates that CHP ameliorates liver fibrosis, a clear evidence that can be applied to the treatment of liver fibrosis.

Example 5

Confirmation of anti-fibrotic Effect in human Lung cells treated with CHP

5-1, culturing human lung cells and post-treatment of CHP after induction of fibrosis

For culturing L-132 lung cell line, MEM medium and fetal bovine serum were purchased from Hai clone, USA, and R&Transforming growth factor beta for inducing fibrosis was purchased from D systems. The culture medium was supplemented with 10% fetal bovine serum in MEM at 37 ℃ with 5% CO₂The culture chamber of (1) was cultured, and the transforming growth factor beta was treated so that the concentration thereof became 2ng/ml, and CHP was treated so that the concentration thereof became 0ng/ml, 62.5ng/ml or 125ng/ml, and the cells were recovered after 48 hours of treatment.

5-2 confirmation of the protein of the fibrosis marker fibronectin after treatment of CHP in a model of human derived Lung cell fibrosis Expression of the plasmid

In order to analyze fibronectin expression as a fibrotic protein, western blotting was performed by the same method as in example 3-2. Statistical significance was analyzed by Student's t-test statistics with control (# p <0.05 (negative control: transforming growth factor beta treated control); p <0.01 (transforming growth factor beta treated control: CHP 125ng/ml treated).

As a result of the experiment, as shown in fig. 7a and 7b, it was confirmed that when transforming growth factor β and CHP of 125ng/ml were treated, the expression amount of fibronectin, which is a fibrotic protein, increased with the treatment of transforming growth factor β was significantly reduced. It was confirmed that CHP has an excellent therapeutic effect on pulmonary fibrosis as a result of improvement of fibrosis in L-132 cells as a lung cell line.

Example 6

Confirmation of anti-fibrotic Effect in animal models of pulmonary fibrosis based on administration of CHP

Pulmonary fibrosis occurs due to long-term intake of high-fat diet. This appears to be caused by increased levels of inflammation due to high fat intake, and it has been reported that pulmonary fibrosis occurs when normal mice are allowed to take a high fat diet containing 60% fat for 15 weeks (Ge, X.N et al, Experimental Lung Research,39(9), 365-. In particular, chronic ingestion of high fat diets induces obesity, hypertrophic adipose tissue and adipose and cytokines secreted by immune cells accumulated therein, interfering with the migration of immune cells from bone marrow to lung, thereby causing chronic inflammatory responses (de Vries A et al, Clin Exp Allergy,39: 731) -739, 2009). Moreover, interleukin-13 cytokine secreted from Th2CD4+ T cells abundantly produces transforming growth factor beta as a key inducer of fibrosis, causing transformation of lung tissue and promoting fibrosis phenomenon by signaling of transforming growth factor beta (Fichtner-Feigl S et al, Nat Med,12:99-106,2006; Lee CG et al, J Exp Med,194:809-21, 2001). Fibrosis of the Lung can be confirmed by increasing the expression level of transforming growth factor beta gene or the like relative to the normal level (Ge, X.N et al, Experimental Lung Research,39(9), 365-.

For pulmonary fibrosis experiments in animals, an animal model of pulmonary fibrosis was established by the same method as in example 4-1.

6-2 confirmation of fibrotic markers, i.e., transforming growth factor beta and collagen, in CHP-administered animal models of pulmonary fibrosis Expression of the Pro 3 Gene

Isoflurane required for Anesthesia and dissection of mice was purchased from Hana Pharm, Inc. (HANA PHARM CO., LTD.) of Korea, and an RC2 Rodent Circuit Controller Anesthesia System of Witt Pop, USA was prepared. Phosphate buffer was purchased from sea clone, usa. To isolate lung tissue from mice, mice were anesthetized by respiratory anesthesia using 3% to 3.5% isoflurane. Immediately after blood collection from the heart of an anesthetized mouse, lung tissue was taken out, 50mg of the lung was cut out and put into 500. mu.L of nucleoZOL, which was pulverized by a T10 homogenizer from Aika, Germany. Next, RNA was extracted according to the total RNA isolation protocol of NucleoZOL, and 1. mu.g of RNA was synthesized into cDNA by Reverse Transcription Polymerase Chain Reaction (Reverse Transcription Polymerase Chain Reaction) using the iScript cDNA synthesis kit. The synthesized cDNA was analyzed by real-time polymerase chain reaction by iQ SYBR Green Supermix using the forward/reverse primer sets corresponding to the respective genes. The expression value of each gene was corrected by dividing by the expression value of GAPDH as a housekeeping gene. The primers used for the real-time polymerase chain reaction were the base sequences as shown in table 2, synthesized and used by the korean Bioneer company.

TABLE 2

Statistical significance was analyzed using analysis of variance (One-way ANOVA) statistical analysis, comparing significance to control groups by Dunnett's post-hoc test (. p <0.05,. p < 0.001).

As a result of the experiment, as shown in fig. 8, it was confirmed that the expression of transforming growth factor β and collagen 3 genes, which are major fibrosis markers, in the lung was more significantly reduced depending on the administration concentration of CHP. This means that CHP improves pulmonary fibrosis, and it can be confirmed that CHP has an excellent therapeutic effect for the treatment of pulmonary fibrosis.

Example 7

Confirmation of anti-fibrotic Effect in human skin cells treated with CHP

7-1, culturing human skin cells and post-treatment of CHP after induction of fibrosis

For culturing cell lines derived from HS68 dermal fibroblasts, DMEM medium and fetal bovine serum were purchased from Haihong, USA, and R was obtained from USA&Transforming growth factor beta for inducing fibrosis was purchased from D systems. DMEM medium supplemented with 10% fetal bovine serum was used at 37 ℃ with 5% CO₂The culture chamber of (4) is cultured, the transforming growth factor beta is treated to a concentration of 2ng/ml, and CHP is treated to a concentration of 2ng/mlThe cells were recovered 48 hours after treatment at a concentration of 0ng/ml, 62.5ng/ml, 125ng/ml, 250ng/ml or 500 ng/ml.

7-2 confirmation of fibronectin expression as a fibrotic marker after treatment of CHP in a model of human skin cell fibrosis

In order to analyze fibronectin expression as a fibrotic protein, western blotting was performed by the same method as in example 3-2. Statistical significance was analyzed by Student's t-test statistics with control (# p <0.05 (negative control: transforming growth factor beta treated control); p <0.05 (transforming growth factor beta treated control: CHP 500ng/ml treated).

As a result of the experiment, as shown in fig. 9a and 9b, it was confirmed that when transforming growth factor β and CHP of 500ng/ml were treated, the expression amount of fibronectin, which is a fibrotic protein, increased with the treatment of transforming growth factor β was significantly reduced. It was confirmed that CHP can be used for treating skin fibrosis as a result of improvement of fibrosis by CHP in HS68 cells as a skin cell line.

Example 8

Confirmation of anti-fibrotic Effect in an animal model of cardiac fibrosis based on administration of CHP

Well known risk factors for cardiac fibrosis include poor dietary habits and obesity, high cholesterol levels, and the like. When a high-fat diet is taken for a long time, hypervolemia caused by obesity occurs, so that the heart is overloaded and the systolic and diastolic functions are deformed. The symptoms of hyperglycemia, insulin resistance, hyperlipidemia, chronic inflammation, etc., which occur at the same time, are known to play a decisive role in cardiac fibrosis (Kaltman AJ, gold RM, Am J Med,60:645-653, 1976; Xia Y et al, Histochem Cell Biol,131:471-481, 2009; Ulasova E et al, J Mol Cell Cardiol, 50:147-156, 2011; Lo CS et al, J Cell biochem, 103:1999-2009, 2008; Cavalera, M et al, trans Research,164(4),323-335, 2014). In particular, obesity-induced increases in Cardiac adipocyte cells (Cardiac mast cells) and their degranulation provide fibrosis-inducing factors such as transforming growth factor β to Cardiac tissue and further promote Cardiac fibrosis (Kong P, Christia P, Frangoganninis NG, Cell Mol Life Sci.,71: 549-.

Also, it is reported that cardiac fibrosis occurs when normal mice ingest a high-fat diet for a long period of time. It has been reported that when normal mice were allowed to take a high-fat diet containing 60% fat for 14 to 22 weeks, cardiac fibrosis was induced and increased expression of transforming growth factor beta, collagen gene, etc. was confirmed as compared with normal (Li, W et al, Nutrition & Metabolism,14 (1); 68,2017; Ternac, J et al, European Heart Journal,18(11), 1283-.

8-1, design of experimental animal and Induction of cardiac fibrosis

For the experiment of cardiac fibrosis in animals, an animal model of cardiac fibrosis was established by the same method as in example 4-1.

8-2, confirming the fibrosis markers, i.e. transforming growth factor beta, fibrosis, in the CHP-administered heart fibrosis animal model Zonulin, collagen 1, collagen 2, collagen 3 and collagen 4 expression

Isoflurane required for Anesthesia and dissection of mice was purchased from Hana Pharm, Korea, and RC2 Rodent Circuit Controller Anesthesia System, Witt Pop, USA, was prepared. Phosphate buffer was purchased from sea clone, usa. To isolate the heart tissue of the mice, the mice were anesthetized by respiratory anesthesia using 3% to 3.5% isoflurane. Immediately after blood collection from the heart of the anesthetized mouse, heart tissue was harvested, 50mg of the heart was excised, and RNA extraction and gene expression analysis were performed by the same method as in example 6-2. The primers used for the real-time polymerase chain reaction were the base sequences as shown in table 3, synthesized and used by Bioneer corporation, korea. The expression value of each gene was corrected by dividing the expression value of β -actin (β -actin) of the housekeeping gene.

TABLE 3

Statistical significance was analyzed using analysis of variance statistical analysis, comparing significance to HFD control groups by Dunnett post hoc testing (. p <0.05,. p <0.01,. p < 0.0001).

As a result of the experiment, as shown in fig. 10, it was confirmed that the expression of fibronectin, transforming growth factor β, collagen 1, collagen 3, and collagen 4 genes, which are major fibrosis markers shown in the heart, was significantly reduced by administration of CHP. However, the effect of further decreasing the concentration of CHP was not confirmed, and it was considered that sufficient effect of CHP occurred only at a concentration of 5 mg/kg. The results that CHP inhibits the progression of cardiac fibrosis and improves cardiac fibrosis suggest that CHP may be applied in the treatment of cardiac fibrosis.

Example 9

Confirmation of anti-fibrotic Effect in an animal model of adipose fibrosis based on administration of CHP

The best known method to prevent fibrosis is to block the inflammatory response. In particular, adipose tissue secretes inflammatory substances to cause the malignant cycle of inflammation-induced lipofibrosis. Fibrosis of adipose tissue has been reported to occur when normal mice are allowed to ingest a high fat diet containing 60% fat for 16 to 24 weeks (Hu, M et al, evolution-Based comparative and Alternative Medicine,1-12,2018; Kwon, e.y., & Choi, m.s., nutriments, 10(10),1415,2018; Nakazeki, F et al, Scientific Reports,8(1), 2018; muniappon, L et al, Scientific Reports,7(1), 2017; Lancha, a et al, PLoS ONE,9(5), e98398,2014; vei α zquez, K.T et al, physical Reports,5(18 e13412,2017; Wang, L., she, wash, hux., huey, biological # 105, biological 121, 121). When a high-fat diet is taken for a long time, if swelling of fat cells occurs due to overnutrition, neutral fat accumulation, fat cell apoptosis, production of fat factors and cytokines, endoplasmic reticulum stress, fat tissue hypoxia, etc. occur, so that immune cells infiltrate into fat tissues and chronic inflammation occurs (Schenk S et al, J Clin Invest,118: 2992-. The interaction between various inflammatory immune cells accumulated in adipose tissue and macrophage-adipocytes generates transforming growth factor beta which is a key cause of fibrosis, whereby the deformation of adipose tissue occurs, thereby inducing the adipose fibrosis (Lee CG et al, J Exp Med,194:809-21, 2001; Fichtner-Feigl S et al, Nat Med,12:99-106,2006; Pessin, J.E., & Kwon, H, Journal of Investigative Medicine,60(8),1147-1150, 2012). Fibrosis of adipose tissue can be confirmed by increased expression of markers representative of the fibrosis gene relative to normal adipose tissue.

9-1. design of experimental animals and Induction of fat fibrosis

For the experiment of fat fibrosis in animals, an animal model of fat fibrosis was established by the same method as in example 4-1.

9-2, confirming the fibrosis markers, i.e. transforming growth factor beta, fibrosis, in the CHP-administered animal model of fatty fibrosis Zonulin, collagen 3 and connective tissue growth factor gene expression

Isoflurane required for Anesthesia and dissection of mice was purchased from Hana Pharm, Korea, and RC2 Rodent Circuit Controller Anesthesia System, Witt Pop, USA, was prepared. Phosphate buffer was purchased from sea clone, usa. To isolate the adipose tissues of the mice, the mice were anesthetized by respiratory anesthesia using 3% to 3.5% isoflurane. Immediately after blood collection from the heart of the anesthetized mouse, Epididymal Adipose Tissue (EAT) was harvested, and 100mg of fat was excised, and RNA extraction and gene expression analysis were performed in the same manner as in example 6-2. The primers used for the real-time polymerase chain reaction were the base sequences as shown in Table 4, synthesized by Bioneer and used. The expression value of each gene was corrected by dividing by the expression value of GAPDH as a housekeeping gene.

TABLE 4

Statistical significance was analyzed using analysis of variance statistical analysis, and significance was compared to control groups by Dunnett's post hoc test (. p <0.05,. p < 0.01).

As a result of the experiment, as shown in fig. 11, it was confirmed that fibronectin, transforming growth factor β, collagen 3, and connective tissue growth factor gene expression, which are major fibrosis markers shown in fat, were significantly reduced by administration of CHP. This means that CHP improves the fatty fibrosis, and it was confirmed that CHP has an excellent therapeutic effect on the treatment of fatty fibrosis.

Sequence listing

<110> Nomota pharmaceuticals, Inc

SEOUL NATIONAL University Hospital

Seoul National University, Industry-University Cooperation

<120> use of CHP for preventing, ameliorating or treating fibrosis

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<151> 2019-03-28

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