KR20190003434A - Composition for preventing, improving or treating of fibrosis - Google Patents

Abstract

The present invention relates to a composition containing an extract of Phaseoli semen or a fraction of the same as an active component. The composition inhibits epithelial-mesenchymal transition, increases pulmonary volume reduced by pulmonary fibrosis and alleviates liver fibrosis and oxidative stress. Therefore, the composition can be usefully used for preventing, alleviating or treating fibrosis.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing, improving or treating fibrosis,

The present invention relates to a composition for preventing, ameliorating or treating fibrosis comprising an extract of Red Fox or a fraction thereof as an active ingredient.

Fibrosis refers to the loss of function of the tissue due to fibrosis, which is replaced with fibrous connective tissue as normal tissue is destroyed, and complete treatment is difficult because the fibrosis progresses chronically.

Idiopathic pulmonary fibrosis (IPF), one of the fibrosis, is an unidentified disease characterized by the chronic fibrosis of the lung. It is mainly localized in the lungs and is characterized by histologically distinctive interstitial pneumonia normal interstitial pneumonia, UIP). The prevalence of this disease is known to be 2 to 29 per 100 000 population and is designated as a rare disease of incurable disease in Korea. The clinical course of the disease is variable, and is generally known to be a fatal disease that progresses to respiratory failure due to a gradual onset of pulmonary dysfunction and an average survival time within 2 to 3 years after diagnosis.

Recently, in Korea, exposure to polyhexamethylene guanidine (PHMG) and oligo (2-) ethoxy ethoxyethyl guanidine chloride (PGH) in humidifier disinfectants has been associated with airway and lung injury , Respiratory distress, cough, and other symptoms are accompanied by pulmonary fibrosis patients, causing a great wave of social and economic. These wavelengths were measured using a similar material, 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one The product has been discontinued, and the cause of the onset of pulmonary fibrosis is unclear, making the national anxiety more intense.

In addition to these substances, the causes of pulmonary fibrosis include chemicals, smoking, micro dust, virus infection, and genetic factors. However, the definite etiologic factor has not yet been clarified, The lack of basic research on the past has not yet uncovered a clear therapeutic target.

Although glucocorticoids, immunosuppressive agents and anti-viral cytokines have been used for treatment of pulmonary fibrosis, according to the ATS / ERS guidelines for idiopathic pulmonary fibrosis in 2011, The combination therapy of the immunosuppressant azathioprine resulted in an increase in mortality.

Since the conventional method of treating pulmonary fibrosis using the immunity or inflammation-suppressing method as in the above guideline is limited, it is urgently required to develop drugs that directly inhibit the progress of fibrosis as well as suppress inflammation. Recently, a substance that directly inhibits the TGF-β signal transduction system, which is important for fibrosis progression, is in clinical trials. However, since TGF-β acts not only to promote fibrosis but also to suppress inflammation itself, The overall inhibition of -β can lead to many side effects.

Under these circumstances, the inventors of the present invention have studied to find substances for the prevention and treatment of fibrosis derived from natural products, which have little toxicity and side effects. As a result, the extracts of red beans and red beans inhibit pulmonary fibrosis and hepatic fibrosis To complete the present invention.

1. Korean Patent No. 10-1478109

Another object of the present invention is to provide a food composition for preventing or ameliorating pulmonary fibrosis or hepatic fibrosis comprising the red head extract or fraction thereof as an active ingredient.

An aspect of the present invention provides a food composition for preventing or ameliorating pulmonary fibrosis or hepatic fibrosis comprising a red head extract or a fraction thereof as an active ingredient.

As used herein, the term 'red bean' refers to the seed of the bean ( Phaseolus angularis Wight), which is round in the shape of a cylinder, and the seed coat is glossy in reddish brown. In terms of pharmacological activity, it has been used in folk remedies for the treatment of diuresis, inflammation, drainage, fever, systemic edema, cirrhosis, jaundice, swelling, purulent diseases, species, organs, gonorrhea and diarrhea.

According to one embodiment of the present invention, the toddler may be used to mean seeds of red bean ( Phaseoli angularis Wight) or red bean (or vine red beans, Phaseolus calciner Roxburgh), but the present invention is not limited thereto.

The red head extract means an extract obtained by adding a solvent to red head, and can be prepared according to a conventional method known in the art. For example, a red toenite extract can be prepared by pulverizing a red head, adding a solvent conventionally used for extraction, and applying appropriate temperature and pressure. The red head doe extract includes all of the extract solution, the diluted solution or concentrate of the extract, the dried product produced by drying the extract, or the purified product thereof.

According to one embodiment of the present invention, the solvent is selected from the group consisting of purified water, hexane, 1,3-butylene glycol, an alcohol having 1 to 4 carbon atoms, ethyl acetate, diethyl ether, dichloromethane, acetone, And it is preferable to use purified water or an alcohol having 1 to 4 carbon atoms.

As used herein, the term " fraction " refers to a product obtained by a fractionation method that separates a particular component or a particular group from a mixture comprising various components. According to an embodiment of the present invention, the extract of Red Fox may be a fraction obtained by fractionating the solvent by a solvent fractionation method such as n-hexane or ethyl acetate, and it includes both a polar solvent fraction and a non-polar solvent fraction, Fractions, ethyl acetate fractions and water fractions can all be used.

As used herein, the term " fibrosis " refers to a symptom of loss of tissue function due to fibrosis, which is replaced with fibrous connective tissue when normal tissue is destroyed. For example, When fibrosis occurs, it is called "scar".

According to one embodiment of the present invention, the fibrosis may be selected from the group consisting of pulmonary fibrosis and hepatic fibrosis. Liver fibrosis is a symptom of hardening of liver tissue due to repeated damage and recovery of the liver.

Pulmonary fibrosis is a type of chronic interstitial lung disease in which pulmonary tissue cells are transformed into fibrous cells and cause diseases such as dyspnea, cough, cyanosis, and clubbing it means. Up to now, steroid therapy, interferon gamma, acetylcysteine, pyrennidone, and bosetan have been used as therapeutic agents, but no agents showing a specific therapeutic effect have yet been reported.

According to one embodiment of the present invention, the pulmonary fibrosis is selected from the group consisting of acute and chronic bronchitis, asthma, ssRNA and dsRNA viral infection, bacterium and fungal infection, pneumonia, hematemesis, chronic obstructive pulmonary disease, pulmonary tuberculosis, pulmonary nodule, emphysema, Idiopathic pulmonary fibrosis and interstitial lung disease.

According to one embodiment of the present invention, the composition is capable of inhibiting epithelial-mesenchymal transition, which is one of the main symptoms of fibrosis process. E-cadherin, N-cadherin, Vimentin, Snail, SMA, and Collagen I. < / RTI > In addition, the composition can inhibit the progression of fibrosis in an animal model of pulmonary fibrosis, and can be used for preventing or improving fibrosis because it can increase a reduced lung volume.

The red head extract or its fraction is derived from natural materials and has been proven to be safe and can therefore be used for food composition applications. The food composition may be added to foods, beverages, or the like to prevent or improve pulmonary fibrosis or hepatic fibrosis, or may be used as a raw material for foods and beverages. Examples of foods containing the food composition include various foods, beverages, gums, tea, vitamin complexes, health supplements, and the like, and they can be used as powder, granule, tablet, capsule or beverage. In addition to containing the food composition as an essential ingredient, the food comprising the food composition may further comprise a food-acceptable food-aid additive, such as natural carbohydrates and various flavors, as an additional ingredient.

The food composition may further include other additives appropriately selected according to the purpose and use of the food, and it may be mixed with other active ingredients such as additives used in common use, additives such as coloring matters, surfactants, preservatives, etc. . The additive may be prepared by pulverizing, granulating, tableting or liquefying according to the purpose and use of the additive. For commercialization, a conventional method may be used for packaging.

The red head extract or its fractions can improve the levels of hepatotoxicity indicators aspart aminotransferase (AST) and alanine aminotransferase (ALT), inhibit cellular necrosis by progression of hepatic fibrosis, and reduce oxidative stress of hepatocytes. And can be usefully used for prevention and improvement of fibrosis.

The composition comprising the red dragonfly extract or the fraction thereof as an active ingredient according to an embodiment of the present invention inhibits the epithelial-mesenchymal transition, thereby delaying the progress of fibrosis, increasing the lung volume reduced by pulmonary fibrosis, Oxidative stress can be improved.

1를 처리하여 EMT를 유도한 후 적소두 추출물 또는 OAA 처리에 의한 E-cadherin, N-cadherin, Vimentin 및 α-SMA의 발현 변화를 확인한 결과를 보여준다.
도 8은 블레오마이신을 투여하여 폐섬유화증을 유도한 동물모델에서 OAA 투여에 의한 폐섬유화 억제 효과 및 폐용적 변화를 측정한 결과를 보여준다.
도 9는 사염화탄소를 투여하여 급성 간섬유화증을 유도한 동물모델에서 체중, 간 무게, AST(aspartate aminotransferase) 및 ALT(alanine aminotransferase)를 측정한 결과를 보여준다.
도 10은 사염화탄소를 투여하여 간섬유화증을 유도한 동물모델에서 체중, 간 무게, AST(aspartate aminotransferase) 및 ALT(alanine aminotransferase)를 측정한 결과를 보여준다.
도 11은 간섬유화증 동물모델의 간조직을 헤마토자일린&에오신으로 염색한 결과를 보여준다.
도 12는 간섬유화증 동물모델의 간조직에서 마손 삼색 염색(Masson trichrome stain)을 수행한 결과를 보여준다.
도 13은 간섬유화증 동물모델의 간조직에서 유도성 산화질소 합성효소(inducible NO synthase) 및 글루타티온 과산화효소(glutathione peroxidase)의 mRNA 발현량을 측정한 결과를 보여준다.FIG. 1 shows the cell survival rate after treatment of non-small cell lung cancer (NSCLC) cell line with oleanolic acid acetate (OAA) or red head extract.
FIG. 2 shows the results of confirming the cell proliferation change induced by OAA after inducing an epithelial-mesenchymal transition (EMT) in A549 cells.
FIG. 3 shows the results of confirming the degree of cell migration by inducing EMT in A549 cells and then treating the cells with red toenne extract or OAA.
FIG. 4 shows the results of confirming the inhibitory effect on the infiltration of red toenite and OAA by inducing EMT in A549 cells and using Matrigel.
FIG. 5 shows the results of confirming the changes in protein expression by treatment with TGF-β1 and A549 cells after induction of EMT and treatment with red cells or OAA.
FIG. 6 shows the results of confirming the expression of protein by A549 cells treated with IL-6 after induction of EMT and treatment with Red Soybean extract or OAA.
Figure 7 shows that A549 cells express TGF-

1, and the expression of E-cadherin, N-cadherin, Vimentin and α-SMA by OAA treatment was examined after EMT induction.
FIG. 8 shows the results of measurement of lung fibrosis inhibitory effect and lung volume change by OAA administration in an animal model in which pulomycin was administered to induce pulmonary fibrosis.
FIG. 9 shows the results of measurement of body weight, liver weight, aspartate aminotransferase (ALT) and alanine aminotransferase (ALT) in an animal model in which acute liver fibrosis was induced by administration of carbon tetrachloride.
FIG. 10 shows the results of measurement of body weight, liver weight, aspartate aminotransferase (ALT) and alanine aminotransferase (ALT) in an animal model in which hepatic fibrosis was induced by administration of carbon tetrachloride.
Fig. 11 shows the result of staining liver tissue of hepatic fibrosis animal model with hematoxylin and eosin.
Figure 12 shows the results of performing masson trichrome staining in liver tissue of an animal model of hepatic fibrosis.
FIG. 13 shows the results of measurement of mRNA expression levels of inducible NO synthase and glutathione peroxidase in liver tissue of an animal model of hepatic fibrosis.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example 1: Isolation of red head extract, fractions and active ingredients

1-1: Manufacture of Red Pepper Extract

Red bean (red bean or red bean) was thoroughly washed with water, dried in the shade, and pulverized with a waring blender to prepare powder. 100 L of methanol was added to 20 kg of the red powder, and the mixture was cold-extracted at room temperature for 3 days. After 3 days, the extract was filtered under reduced pressure through a filter paper (Whatman, USA), and methanol was removed by vacuum rotary evaporator to obtain 450 g of red head extract.

1-2: Preparation of fractions

To separate the active fractions from the obtained red yeast extract, the red head extract was suspended in 1 liter of water, and the same amount of n-hexane was added thereto, followed by mixing and fractionation. This process was repeated four times to obtain 1 liter of water soluble fraction and 4 liter of n-hexane soluble fraction. Thereafter, the n-hexane soluble fraction was concentrated under reduced pressure to obtain 50 g of the n-hexane soluble extract.

Further, the same amount of ethyl acetate (C ₄ H ₈ O ₂ ) was added to 1 liter of the water-soluble fraction, followed by mixing and fractionation. This procedure was repeated three times to obtain 1 liter of the water-soluble fraction and 3 liter of the ethyl acetate-soluble fraction . The obtained ethyl acetate soluble fraction was concentrated under reduced pressure to obtain 35 g of ethyl acetate soluble extract. The remaining water-soluble fraction was concentrated under reduced pressure to obtain 35 g of a water fraction.

1-3: HPLC analysis of red head extract and fractions

HPLC analysis was performed on each of the red head extracts and fractions obtained in Examples 1-1 and 1-2.

Agilent Technologies 1200 series was used for HPLC, and J'sphere ODSH80 (YMC, 4 μm, 4.6 mm ID x 150 mm) column of YMC (Japan) was used for the analysis column. The analytical solvent was analyzed at 210 nm while flowing 5% to 90% acetonitrile (CH ₃ CN) at a flow rate of 1 ml / min, and 10 μl of the sample was injected. The HLPC analysis conditions are shown in Table 1 below.

Time (minutes) menstruum(%) CH ₃ CN H ₂ O 0 5 95 15 5 95 25 30 70 30 30 70 50 90 10 70 90 10

HPLC analysis showed that the peaks of catechin-7-glucopyranoside, catechin-7-glu, rutin, oleanolic acid acetate (OAA) and stigmasterol 5.5, 24.5, 35.5, and 35.5, respectively. HPLC chromatograms of red bean and red bean showed similar patterns.

1-4: Purification of active ingredients

80 g of the n-hexane fraction obtained in Example 1-2 was purified by silica gel column chromatography using a step gradient solvent system consisting of hexane: ethyl acetate (100: 1 to 1: 1) column chromatography to obtain five active fractions (Fr.1 to 5). Among the above active fractions, methanol was added to fractions 3 and 4 to carry out a recrystallization process, thereby purifying the two compounds exhibiting a white powder phase.

The two purified compounds were analyzed by instrumental analysis ( ¹ H-, ¹³ C-NMR, MS) and literature values (Voutquenne L. et al. Phytochemistry 2003, 64, 781-789; Kongduang D. et al. Tetrahedron letters 2008 , 49, 4067-4072). As a result, it was confirmed that oleanolic acid acetate (Formula 1) was used.

[Formula 1] < EMI ID =

10a, 12aR, 14bS-10-hydroxy-2,2,6a, 6b, 9,9,12a-heptamethyl-1,3,4,5,6,6a, 7a, 6a, 6aR, 6aS, 6bR, 8aR, 8,8a, 10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid acetate

Example 2: Determination of cell survival rate by treatment with oleanolin acid acetate

The cell survival rate was determined by MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] in order to examine the effect of the red cells extract and the oleanolin acetate obtained in Example 1 on cell viability Respectively.

Non-small cell lung cancer (NSCLC) cell lines were suspended in the medium at a concentration of 1 x ¹⁰ < ⁴ > / uL and 200 [mu] l was added to 96-well plates for 24 hours. After 24 hours, medium containing oleanolin acetate (hereinafter referred to as OAA; 0, 1, 3, 6, 10 and 30 uM) or red head extract (0, 5, 10, 30 and 60 ng / Were added to each well and cultured for 24 hours or 48 hours, respectively.

After the incubation, MTT was diluted (0.5 ㎎ / ㎖) in PBS (phosphate buffered saline), and 20 ㎕ was added to each well and cultured in a CO ₂ incubator for 4 hours. After 4 hours, the MTT solution was removed and 200 μl of DMSO (dimethyl sulfoxide) was added to each well. The formazan precipitate was dissolved for 5 minutes and the absorbance was measured at 595 nm.

As a result, the cell survival rate was not affected in the group treated with Red Pepper Extract, but the cell survival rate was decreased with increasing OAA concentration in the OAA treated group.

FIG. 1 is a graph showing cell survival rate after culturing non-small cell lung cancer cells for 24 hours or 48 hours after treatment with OAA or Red Pepper Extract. FIG.

Example 3 Confirmation of EMT Inhibitory Effect by OAA Administration on TGF-β1-Induced Epithelial-mesenchymal Transition (EMT)

The epithelial-mesenchymal transition (hereinafter referred to as EMT) is known to play an important role in the pathogenesis of pulmonary fibrosis, as epithelial cells are transformed into mesenchymal cells . As the proliferation and migration of epithelial cells increase with the progress of EMT, the efficacy of EMR suppression of the red head extract and OAA was confirmed as follows.

A549 cells and NIH-3T3 fibroblasts, which are lung cancer-derived cells, were suspended in 1 × 10 ⁴ / ml of the culture medium, and then 200 μl of each was added to a 96-well plate and cultured for 24 hours. After 24 hours, each well was treated with TGF-? 1 and cultured for 48 hours with 200 占 퐇 of medium containing OAA (30 uM) or Pirfenidone (0.3 mg / ml). After the incubation, 20 μl of the Cell Counting Kit-8 (CCK-8 kit; Dojindo, Japan) solution was dispensed into each well and cultured in a CO ₂ incubator for 1 hour. The absorbance was then measured at 450 nm.

1 투여에 의하여 증가된 EMT 관련 세포 증식이 OAA 투여에 의하여 A549 세포 및 NIH-3T3 세포 모두에서 유의적으로 감소된 것을 확인할 수 있었다.As a result,

1-induced EMT-related cell proliferation was significantly reduced in both A549 cells and NIH-3T3 cells by OAA administration.

FIG. 2 is a graph showing the results of confirming cell proliferation changes by OAA after induction of EMT by administering TGF-β1 to A549 cells. In Fig. 2, Pir means pypenidone, A in Fig. 2 shows A549 cells, and Fig. 2 shows results in NIH-3T3 cells.

Example 4: Determination of the effect of red head extract and OAA on TGF-? 1 induced EMT

4-1. Confirmation of cell movement inhibition effect of red head extract and OAA by scratch wound healing method

A549 cells were plated on a 6-well plate at 1 x ¹⁰ < ⁵ > / ml and cultured for 24 hours. After 24 hours, the single cell layer was scratched with 200 [mu] l tip and the medium containing OAA (10 and 30 uM) or red head extract (30 and 60 [mu] g / ml) was added to each well in an amount of 1 ml . After incubation for 48 hours, the degree of migration of the cells was confirmed by a microscope and images were taken.

As a result, it was found that the cell migration was increased in the TGF-β1-treated cells compared with the control (Control), and the cell migration was reduced when the red cells or OAA was treated. Especially, treatment with high concentration of OAA significantly decreased cell migration.

FIG. 3 A is a photograph showing the degree of cell migration by administering TGF-β1 to A549 cells and inducing EMT, followed by treatment with reddish-purine extract or OAA.

4-2. Confirmation of cell movement inhibition effect of red head extract and OAA by ECIS (Electrical Cell-Substrate Impedance Sensing) method

ECIS is a device that monitors the morphological changes of cells and responses to various drugs in real time. As the cell migration increases by TGF-β1 treatment, the resistance value increases. Therefore, the cell migration by OAA or Reduced Toxin It can be measured indirectly.

A549 cells were cultured in a well plate equipped with an electrode and treated with OAA (10 and 30 uM) or red head extract (30 and 60 uM) in the same manner as in Example 3-1. The change of the resistance value (Impedance) against the current generated in this process was measured.

As a result of the measurement, the resistance value was increased and the cell migration was increased in the case of treatment with TGF-β1 compared to the control (Con A549). When 30 μM of OAA was treated, the resistance value was remarkably decreased, .

FIG. 3B is a graph showing the results of ECIS analysis of cell migration by treating TGF-β1 with A549 cells to induce EMT, and then treating the cells with ODS or O7A.

Example 5: Confirmatory effect of TGF-beta1 induced EMT on the infiltration of red toenne extract and OAA

A trans-well insert (Corning, USA) was coated with 2 mg / ml of matrigel (Corning). A549 cells were diluted to about 2 x 10 ⁴ in 100 μl of serum-free media and OAA (10 and 30 μM) or red head extract (30 and 60 μg / mg) was added thereto and added to the trans-well insert Respectively. Receive chambers were seeded with medium containing 10% serum and the cells were cultured for 48 hours. After the incubation, the cells remaining in the insert were removed, and the cells infiltrated into the receive chamber were stained with crystal violet and confirmed by a microscope.

As a result, it was found that the cells treated with TGF-β1 increased the number of cells infiltrated into the receive chamber as compared with the control (control). On the other hand, in the case of cells treated with red cells or OAA, Of the total number of patients.

FIG. 4 is a photograph showing the results of confirming the inhibition of cell infiltration of red papilla extract and OAA by using Matrigel after inducing EMT by treating TGF-β1 with A549 cells.

Example 6: Regulation of TGF-β1 Inducible EMT by Expression of Red Piglet Extract and OAA

A549 cells were plated on a 6-well plate at 1 x ¹⁰ < ⁵ > / ml and cultured for 24 hours. The medium containing OAA (10 and 30 uM) or red head extract (30 and 60 占 퐂 / mg) was then added to each well in an amount of 1 ml, followed by incubation for another 48 hours. After incubation, the wells were incubated with RIPA lysis buffer [50 mM Tris-Cl (pH 7.4), 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg / ml aprotinin, / ㎖ leupeptin, and 1 mM Na ₃ VO _4] was added to each 500 ㎕ dissolve the cells for 15 minutes at 4 ℃. Next, the supernatant was collected by centrifugation (14,000 rpm and 10 minutes) at 4 ° C, and proteins were separated by SDS-PAGE (SDS-polyacrylamide gel electrophoresis). The separated proteins were transferred to a PVDF membrane (polyvinylidene difluoride membrane) and reacted with the primary antibody at 4 ° C for 12 hours. After 12 hours, the cells were further reacted with the secondary antibody for each primary antibody, and the protein bands were confirmed using an ECL kit (Thermo Fisher Scientific, USA) according to the manufacturer's protocol.

Expression of E-cadherin, N-cadherin, Vimentin, Snail, α-SMA, and Collagen I was increased by TGF-β1 treatment and decreased by OAA treatment.

In addition, when IL-6 was treated, the level of phosphorylated STAT3 was increased in addition to the expression of the protein, but the phosphorylation of STAT3 was decreased by treatment with Reduced Toxin extract or OAA.

FIG. 5 is a graph showing the results of confirming the changes in protein expression by treatment with TGF-β1 and EMR-induced red cells or OAA in A549 cells.

FIG. 6 is a graph showing the results of confirming the expression of protein by A549 cells treated with IL-6 and EMT, followed by treatment with Pfu extract or OAA.

Example 7: Regulation of TGF-beta1 induced EMT by morphological change of cells by treatment with Reddorp extract and OAA

A549 cells were plated on a 4-well plate at 1 × 10 ⁵ / ml and cultured for 24 hours. The medium containing OAA (10 and 30 uM) or red head extract (30 and 60 占 퐂 / mg) was then added to each well in an amount of 1 ml, followed by incubation for another 48 hours. After incubation, the cells were washed with PBS (phosphate buffered saline) and fixed with 4% paraformaldehyde for 20 min. TritonX-100 (0.2% in PBS) was added to the fixed cells, reacted at room temperature, and blocked with 5% normal goat serum at room temperature for 1 hour. The primary antibody was diluted 1: 200 in a blocking buffer, reacted at 4 ° C for 12 hours, and further reacted with the secondary antibody for each primary antibody for 90 minutes. After completion of the reaction, a mounting solution containing DAPI was dispensed and observed under a microscope, and images were taken.

As a result, it was confirmed that the expression of E-cadherin, N-cadherin, Vimentin and α-SMA increased by TGF-β1 treatment was decreased by the treatment with Reduced tofu extract or OAA treatment.

FIG. 7 is a photograph showing the results of confirming the expression of E-cadherin, N-cadherin, Vimentin and α-SMA by treatment with TGF-β1 and EMT and treatment with OEA extract of A549 cells.

Example 8: Confirmatory effect of OAA on lung fibrosis

8-1. Production of an animal model of pulmonary fibrosis

Mice were intubated and bleomycin (2 mg / kg) was injected intraperitoneally in a volume of 50 ul. After injection, the incision site was sutured with 5-0 nylon, and OAA was administered 24 hours after injection of bleomycin. Mice were sacrificed at 3 days, 7 days, 10 days, 13 days, 17 days, 20 days, and 21 days after bleomycin injection, and BALF (broncho-alveolar lavage fluid) Respectively.

8-2. Confirming the inhibitory effect of OAA on pulmonary fibrosis

CT images were taken using volume CT (computed tomography) based on a cone-beam type flat-panel detector (flat-panel detector). At this time, the tube voltage and the tube current of the x-ray generator were 50 kV and 120 각각, respectively. CT reconstructed a total of 720 images at 360 ° rotation by 0.5 ° rotation per projection. The reconstructed images consisted of 1024x1,024 pixels (pixels) of the smallest units composing the image, and a total of 512 reconstructed tomographic images were obtained.

The reconstructed image was three-dimensionally analyzed using a 3D-rendering software program Xelis (Infinitt Technology, Seoul, Korea). The image analysis was performed using a sagittal plane, an axial plane, and a coronal plane. .

Observation showed that pulmonary fibrosis progressed when the bleomycin was injected, but when OAA was administered after bleomycin injection, it was confirmed that OAA concentration-dependent pulmonary fibrosis was suppressed, and that pulmonary fibrosis did not progress even after the lapse of time there was.

FIG. 8 shows the inhibitory effect of OAA on pulmonary fibrosis in an animal model in which pulmomycin was induced to induce pulmonary fibrosis, and the second and fourth panels show reconstructed tomographic images.

8-3. Determination of lung volume change by OAA administration

Since pulmonary volume decreases when pulmonary fibrosis progresses, lung volume was measured after OAA administration in an animal model of pulmonary fibrosis. As a result, bleomycin administration decreased lung volume, but OAA administration showed a decrease in lung volume.

FIG. 8 shows the results of measurement of lung volume change by OAA administration in an animal model in which pulmomycin was induced to induce pulmonary fibrosis. The first and third panels show the results of lung volume measurement.

Example 9: Confirmation of OAA inhibitory effect on acute liver fibrosis

9-1. Production of an animal model of acute liver fibrosis

(23 ± 3 ° C), humidity (55 ± 15%) and irradiation dose (7: 1) in an SPF animal breeding room by purchasing a 4-week-old male SD rat (Oriata Bio, Seoul, Korea) 00 to 19:00). Rats were stabilized for one week and used in the experiment and were randomly divided into five groups: control (CON, Control), carbon tetrachloride (CCl ₄ ), carbon tetrachloride & oleanolic acid acid, OA) combined administration group _{(CCl 4 + OA 50 ㎎ /} ㎏), carbon tetrachloride & olreahnol acid acetate combined administration group _{(CCl 4 + OAA 10 ㎎ /} ㎏) and carbon tetrachloride & olreahnol acid acetate combined administration group (CCl ₄ + OAA 50 ㎎ / Kg). OA and OAA were orally administered daily for three days at a defined dose, and 24 hours after the intraperitoneal injection of carbon tetrachloride (100 μg / g) on the third day of oral administration, the rats were sacrificed.

9-2. Identification of body weight, liver weight and liver function indicators in an animal model of acute liver fibrosis

The rats were sacrificed, their weights were measured, and the liver was weighed and weighed. Blood was collected from the heart and centrifuged at 3000 rpm for 15 minutes at 4 ° C. The serum was separated and analyzed with an automatic analyzer (Fuji Dry-Chem NX500i, Japan) using a biochemical indicator AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were measured. AST and ALT are enzymes present in hepatocytes, which leak into the serum upon acute liver injury, and elevated levels of AST and ALT in the blood usually signify liver damage.

As a result, as shown in FIG. 9, in the CCl ₄ administration group, weight and weight of the liver were decreased compared to the control group, and it was found that acute liver fibrosis was induced. In addition, AST and ALT levels were significantly increased in CCl ₄ treated group, whereas AST and ALT levels were decreased in CCl ₄ + OA treated group or CCl ₄ + OAA treated group. In particular, CCl ₄ + and OA in CCl ₄ + OAA combination group than the combination group AST and ALT levels decreased effect is more noticeable it was found that fiberization treatment effect between the OAA is better than OA.

Example 10: Inhibitory effect of OAA on hepatic fibrosis

10-1. Production of liver fibrosis animal model

The rats were randomly divided into four groups after the rats were raised under the same conditions as in Example 9-1. Control group (Control, CON), carbon tetrachloride (CCl ₄ ), carbon tetrachloride & oleanolin combined administration group _{(CCl 4 + OA 50 ㎎ /} ㎏) and carbon tetrachloride & olreahnol acetate acid combination group _{(CCl 4 + OAA 50 ㎎ /} ㎏). OA and OAA were orally administered daily for three weeks at a defined dose, and carbon tetrachloride (100 μl / 100 g) was intraperitoneally administered three times per week for 3 weeks to sacrifice the rat on day 22.

10-2. Determination of body weight, liver weight and liver function indicators in liver fibrosis animal models

After sacrificing the rats, body weight, liver weight, ALT and AST levels were measured in the same manner as in Example 9-2.

As a result, as shown in FIG. 10, in the CCl ₄ administration group, weight and liver weight were decreased compared to the control group, and liver fibrosis was induced. In addition, AST and ALT levels were significantly increased in the CCl ₄ treated group compared to the control group, but the AST and ALT levels were decreased in the CCl ₄ + OA combination group or the CCl ₄ + OAA combination group. In particular, CCl ₄ + and OA in CCl ₄ + OAA combination group than the combination group AST and ALT levels decreased effect is more noticeable it was found that fiberization treatment effect between the OAA is better than OA.

10-3. Liver histopathology analysis of liver fibrosis animal model

After sacrificing the rats, the liver was separated and fixed with 4% formaldehyde and embedded in paraffin to make a paraffin block. The paraffin blocks were sectioned to produce liver tissue sections. Hematoxylin & eosin (H & E) staining or masson trichrome staining was performed on the sections and observed with an optical microscope.

As a result of hematoxylin & eosin staining, cell necrosis around the portal vein was observed in the CCl ₄ administration group as shown in FIG. 11, but in the group treated with CCl ₄ + OA or CCl ₄ + OAA, .

In addition, as a result of staining of collagen in hepatic tissue with Maeson's tricolor dyeing, it was confirmed that hepatocyte fibrosis was severe in the CCl ₄ administration group as shown in Fig. On the other hand, the degree of fibrosis was mitigated in the combination of CCl ₄ + OA and CCl ₄ + OAA.

10-4. Analysis of antioxidant enzyme expression in liver tissue of liver fibrosis animal model

After sacrificing the rats, the liver was separated and homogenized by addition of Trizol reagent (Invitrogen; USA). The RNA was isolated by adding chloroform thereto, and was precipitated by addition of isopropanol. The precipitated RNA was washed with 75% ethanol and the RNA concentration and purity were measured with a 2100 Bioanalyzer system (Agilent Technologies, USA). CDNA was synthesized with the Taqman reverse transcription reagent kit (Applied Biosystems, Foster City, CA, USA) using the separated RNA as a template. The expression level of inflammatory factor was confirmed by Real-time PCR using SYBR Green PCR master mix kit (Applied Biosystem, Foster City, CA, USA).

As a result of measuring the expression level of inducible NO synthase (iNOS) in the hepatocyte in order to examine the oxidative stress, the amount of iNOS expression increased in the CCl ₄ administration group as shown in FIG. 13 was higher in the CCl ₄ + OA combination administration group And CCl ₄ + OAA, respectively.

The excess oxygen produced by oxidative stress is removed by glutathione peroxidase (GPx). As a result of measurement of GPx mRNA expression level, it was confirmed that the expression amount of GPx mRNA was decreased by oxidative stress and hepatocyte injury in the CCl ₄ administration group as compared with the control group as shown in FIG. However, in the group treated with CCl ₄ + OA and the group treated with CCl ₄ + OAA, the amount of GPx mRNA decreased by CCl ₄ was increased again.

From the above results, oleanolin acetate can be effectively used for the treatment of hepatic fibrosis because it can improve hepatic fibrosis induced by carbon tetrachloride, suppress the expression of oxidative stress-related genes, and effectively remove active oxygen Able to know.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (5)

In a food composition for preventing or improving fibrosis,
Wherein the composition comprises a red head extract or a fraction thereof as an active ingredient,
Wherein the fibrosis is lung fibrosis or liver fibrosis.
The method of claim 1, wherein the red head extract is selected from the group consisting of purified water, hexane, 1,3-butylene glycol, an alcohol having 1 to 4 carbon atoms, ethyl acetate, diethyl ether, dichloromethane, acetone, Lt; / RTI > solvent.
The composition of claim 1, wherein the fraction is fractionated with a solvent selected from the group consisting of ethanol, hexane, ethyl acetate, water, and mixtures thereof.
The method of claim 1, wherein the pulmonary fibrosis is selected from the group consisting of acute and chronic bronchitis, asthma, ssRNA and dsRNA viral infections, bacterial and fungal infections, pneumonia, hemorrhage, chronic obstructive pulmonary disease, pulmonary tuberculosis, pulmonary nodules, emphysema, Fibrosis, and interstitial lung disease.
2. The composition of claim 1, wherein the composition inhibits epithelial-mesenchymal transition (EMT).

Images