KR101810795B1 - Composition for preventing, improving or treating hepatic fibrosis or liver cirrhosis comprising Aster tataricus Linne fil extract - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating liver fibrosis or liver cirrhosis, which comprises an Aster tataricus Linne fil extract as an effective component, wherein the composition exhibits excellent hepatoprotective effects through increased apoptosis, collagen reduction, and ECM accumulation reduction in HSC-T6 cell lines and hepatic stellate cells. Additionally, the composition of the present invention exhibits excellent anti-fibroblastic effects by improving serum AST and ALT in an animal model of TAA-induced liver cirrhosis; restoring the total GSH level to a normal level; and down-regulating the expressions of TGF-, -SMA and Col1a2, which are genes related to hydroxyproline level reduction and liver fibrosis. Accordingly, the composition of the present invention may be beneficially used as an agent for improving liver functions or treating liver fibrosis or liver cirrhosis.

Description

TECHNICAL FIELD The present invention relates to a composition for prevention, improvement or treatment of hepatic fibrosis or liver cirrhosis,

The present invention relates to a composition for preventing, improving or treating hepatic fibrosis or cirrhosis, which contains as an active ingredient an extract of Zykylum. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating liver fibrosis or cirrhosis, / ≪ / RTI >

Liver disease is caused by a variety of causes such as alcohol, various drugs, toxic chemicals, hepatitis B and C viruses, cholestasis, autoimmunity, etc., and usually proceeds through liver to hepatitis, liver fibrosis and cirrhosis. Fatty liver is not a morbid condition in itself, but it is a reversible symptom that is recovered naturally when the causative substance is removed. However, if excess fat accumulation in the liver tissue is maintained constantly, hepatitis hepatitis occurs, resulting in repeated hepatocyte necrosis and regeneration. In this process, the increase of fibrous extracellular matrix (ECM) And fibrosis of the liver proceeds due to accumulation.

Liver fibrosis is still one of the major diseases of the world's population. Liver fibrosis is the result of chronic damage to the liver with the accumulation of extracellular matrix (ECM) proteins such as collagen, fibronectin and laminin, and the nature of most chronic diseases is also characterized by persistent inflammation, It is caused by repeated liver damage with change and organ failure. Hepatic stellate cells (HSCs) are noted to play a key role in liver fibrosis, which is a central source of fibrosis as a major source of fibrin and non-fibril maturation proteins. Hibernated HSCs produce less ECM protein than activated HSCs, but when repeated hurdles cause the hibernating HSCs to become active, they are transformed into myoblast-like phenotypes, a process that proliferates and is said to be activated.

Activated HSC function causes excessive accumulation of ECM and destroys the liver of normal structure, which causes pathophysiological damage to the liver. Overexpression of ECM proteins ultimately leads to cancer caused by liver failure, fibrosis, cirrhosis or HSC activation. Abnormal conditions of ECM proteins, including accumulation and overexpression in liver tissue, cause excessive liver maturation in late liver disease or disorder. This condition is the most important stage during progressive liver disease. Thus, the prevention and inhibition of liver fibrosis is very important in improving chronic liver disease.

For centuries, Western medicine, Asian countries and other countries have been using herbal medicines and natural products to treat liver disease. Many factors contribute to the appeal of herbal medicines, and supporters of medicinal herbs claim that medicinal herbs can cure and prevent disease.

On the other hand, Aster tataricus Linne The term "fil" refers to the roots of anthracnose, mainly distributed in China, Japan, and Korea. It has been reported that pharmacological effects include gadam, shinhae, antibacterial and anticancer activities. The major components are terpenoids, sterols, flavonoids, and cylclopeptides. Korean Patent Laid-Open No. 10-2004-0065498 discloses a natural composition having an effect of alleviating allergic dermatitis and skin itching, and Korean Patent No. 10-0516194 discloses a natural herbal extract having anti- And Korean Patent Laid-Open No. 10-2012-0117115 discloses a composition for promoting hair growth or preventing hair loss, which contains a hairy extract.

However, until recently, the anti-fibrosis effect of Jawan has not been revealed. Under these circumstances, the inventors of the present invention found that the anti-fibrosis effect of the in-vitro system using hepatic stellate cells and the in- vivo hepatic fibrosis rat model induced by thioacetamide (TAA) , The present inventors completed the present invention by confirming that the extract of Zaanbei shows inhibition of hepatic fibrosis and protection of liver.

The present invention has been made in view of the above-mentioned needs, and an object of the present invention is to provide a composition for prevention, improvement or treatment of hepatic fibrosis or cirrhosis using a human milk extract. However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for prevention or treatment of liver fibrosis or cirrhosis, which comprises an asparagine extract as an active ingredient.

The present invention also provides a health functional food composition for prevention or improvement of liver fibrosis or liver cirrhosis, which comprises an asparagine extract as an active ingredient.

According to a preferred embodiment of the present invention, the human milk extract may be extracted from at least one solvent selected from the group consisting of C ₁ to C ₄ alcohols and water.

According to another preferred embodiment of the present invention, the extraction solvent may be 70% ethanol.

According to another preferred embodiment of the present invention, the chrysanthemum extract may inhibit hepatic stellate cell activation and proliferation.

The composition containing the human milk extract of the present invention as an active ingredient exhibits excellent liver protecting effect through increased cell death, collagen reduction and reduction of ECM accumulation in the HSC-T6 cell line and hepatic stellate cells. In addition, TAA-induced liver fibrosis animal models have improved serum AST and ALT, restored total GSH levels to normal levels, decreased levels of hydroxyproline and genes associated with liver fibrosis, TGF-β, α-SMA, The expression of Col1a2 is down-regulated and exhibits excellent antifibrotic effect. Therefore, the composition of the present invention can be usefully used as a therapeutic agent for improving liver function or liver fibrosis or liver cirrhosis.

In addition, the human milk extract of the present invention is safe as a natural material and has no side effects, and the raw material cost is low, which is very economical.

FIG. 1 is a graph showing cell viability of Chang cells and HSC-T6 cells according to the treatment of human milk ethanol extract. FIG.
FIG. 2 is a photograph showing microscopic observation of morphological changes of activated HSCs according to the treatment with a human milk extract. In the picture, arrows indicate HSC.
FIG. 3 shows the results of analysis of the cell cycle of HSC-T6 cells treated with the extract of chrysanthemum extract, wherein (A) is a histogram and (B) is a graph.
FIG. 4 shows the results of analysis of cell death of HSC-T6 cells treated with the human milk extract, wherein (A) is a histogram and (B) is a graph. Data values were expressed as mean ± SEM (* P <0.05 compared with control, ** P <0.01).
Fig. 5 is a schematic diagram showing the process of establishing an animal model of liver fibrosis.
FIG. 6 is a photograph of a liver taken from an animal model of liver fibrosis of FIG. 5, wherein A is a normal group, B is a TAA treated group, C is a positive control group treated with silymarin, D is a treated group of 0.5 mg / ATE 500 group, and E represents the ATE 100 group treated with 0.1 mg / kg of human milk extract.
FIG. 7 is a graph showing changes in serum AST (A) and ALT (B) levels in TAA-induced liver fibrosis rats following treatment with humanized extract.
FIG. 8 is a graph showing the change in total GSH content in rat liver tissues of TAA-induced liver fibrosis according to the treatment with humanized extract.
FIG. 9 graphically illustrates the change in total hydroxyproline levels in the liver of TAA-induced liver fibrosis in rats treated with humanized extract.
FIG. 10 is a photograph showing the liver of a rat stained with H & E staining. In FIG. 10, A shows a normal group, B shows a TAE treated group, C shows a positive control group treated with silymarin, D shows an ATE 500 treated with a 0.5 mg / And E represents the ATE 100 group treated with 0.1 mg / kg of human milk extract.
Fig. 11 (A) is a photograph showing the liver of a rat stained with Mason's trichrome staining method, wherein A is a normal group, B is a TAA treated group, C is a positive control group treated with silymarin, D is a 0.5 mg / , And ATE 100 group treated with 0.1 mg / kg of human milk extract, and FIG. 10 (B) is a graph showing the ratio of fibrosis area.
FIG. 12 is a graph showing measurement results of fibrosis-related gene expression in liver tissue using real-time PCR. Data values were expressed as normalized fold values for the control.

Hereinafter, the present invention will be described in more detail.

As described above, until recently, the anti-fibrosis effect of Jawan has not been revealed. Under these circumstances, the inventors of the present invention were studying the anti-fibrosis effect of an in vitro system using hepatic stellate cells and an anti-fibrosis effect of TAA-induced hepatic fibrosis rat model in vivo , Liver fibrosis inhibition and liver protection effect, and the present invention has been completed.

Accordingly, the present invention sought to solve the above-mentioned problems by providing a composition for prevention, improvement or treatment of liver fibrosis or cirrhosis, which comprises an extract of Zygomycetes as an active ingredient. The composition containing the human milk extract of the present invention as an active ingredient exhibits excellent liver protecting effect through increased cell death, collagen reduction and reduction of ECM accumulation in the HSC-T6 cell line and hepatic stellate cells. In addition, TAA-induced liver fibrosis animal models have improved serum AST and ALT, restored total GSH levels to normal levels, decreased levels of hydroxyproline and genes associated with liver fibrosis, TGF-β, α-SMA, The expression of Col1a2 is down-regulated and exhibits excellent antifibrotic effect. Therefore, the composition of the present invention can be usefully used as a therapeutic agent for improving liver function or liver fibrosis or liver cirrhosis.

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

The term &quot; pharmaceutical composition &quot; refers to a mixture of other chemical components, such as a diluent or carrier, in the subject-matter extract of the present invention.

The term &quot; carrier &quot; is defined as a compound that facilitates the addition of a compound into a cell or tissue. For example, dimethylsulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic compounds into cells or tissues of an organism.

The term &quot; diluent &quot; is defined as a compound that not only stabilizes the biologically active form of the compound of interest, but also dilutes in water to which the compound is dissolved. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline, since it mimics the salt state of the human solution. Since buffer salts can control the pH of the solution at low concentrations, buffer diluents rarely modify the biological activity of the compounds.

The term &quot; treatment &quot; means an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction Also, &quot; treatment &quot; may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. &Quot; Treatment &quot; refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented. "Alleviating" a disease may result in a reduction in the extent of the disease state and / or undesirable clinical symptoms and / or a slower or longer time course of the progression, It means to lose.

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention.

The present invention provides a pharmaceutical composition for prevention or treatment of hepatic fibrosis or cirrhosis, which comprises a humanized extract as an active ingredient.

The above-mentioned Jia-Yuan extract is preferably prepared by a manufacturing method including, but not limited to, the following steps:

1) extracting the human milk with an extraction solvent;

2) filtering the extract of step 1); And

3) Concentrating the filtered extract of step 2) at a reduced pressure, and then drying the extract to prepare a chrysanthemum extract.

The extraction solvent is preferably water, alcohol or a mixture thereof. The alcohol may be a C ₁ to C ₄ lower alcohol, preferably a C ₁ to C ₂ lower alcohol, but it is not limited thereto. Any solvent that can effectively extract the active ingredient from a plant may be used without limitation Can be used. As the C ₁ to C ₂ lower alcohol, 30% to 100% methanol or ethanol is preferably used. In one embodiment of the present invention, a human milk extract was prepared using 70% ethanol as a solvent.

As the extraction method, it is preferable to use but not limited to stirring extraction, low-pressure high-temperature extraction, hot water extraction, reflux extraction, hot water extraction, cold extraction, room temperature extraction, ultrasonic extraction or steam extraction. It is preferable that the extraction solvent is added by 1 to 10 times the weight of the mother liquor. The extraction temperature is preferably 20 to 120 DEG C, and the temperature can be appropriately changed according to the extraction method. The extraction time is preferably from 2 hours to 72 hours, but is not limited thereto. In addition, the number of times of extraction is preferably 2 to 5 times, more preferably 3 to 4 times, but is not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator in the vacuum concentration step 3), but it is not limited thereto, and it is preferable that drying is carried out under reduced pressure, vacuum drying, boiling, spray drying or freeze drying It is not limited.

The present invention provides a method of inhibiting and inhibiting HSC-T6 and primary HSC (FIGS. 2 and 4) in a TAA-induced liver fibrosis animal model (FIG. 8), decreased levels of hydroxyproline (FIG. 9) and genes associated with hepatic fibrosis, TGF-beta, alpha-SMA And Col1a2 expression, thereby exhibiting excellent antifibrotic effect (Fig. 9). Therefore, the composition of the present invention can be usefully used as a therapeutic agent for improving liver function or liver fibrosis or liver cirrhosis.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The pharmaceutical composition according to the present invention can be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository and sterilized injection solution according to a conventional method Can be used.

Examples of carriers, excipients and diluents that may be contained in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose , Gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

In addition, the pharmaceutical composition of the present invention may further contain other components effective for liver disease. Examples of such active ingredients include silymarin derived from natural substances and bile acids such as ursodesoxycholic acid (UDCA). However, the inclusion of silymarin or UDCA is merely an example of a pharmaceutical composition containing a chrysanthemum extract as a main component, and does not limit the scope of the present invention.

The amount of the composition of the present invention to be used may vary depending on the age, sex, and body weight of the patient and may vary depending on the condition of the subject to be treated, the specific category or type of the subject disease, Lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt;

The pharmaceutical composition of the present invention can be administered individually as a prophylactic or therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

In particular, it can be administered by any medically appropriate method, for example by injection into normal cerebrospinal fluid, such as normal intravenous or intraarterial administration. In some cases, intradermal administration, intracavity administration, intrathecal administration, tumor or direct administration to the arteries supplying the tumor is advantageous. When the tumor or a portion thereof has previously been removed by surgical operation, the therapeutic agent can be delivered directly to the tumor site (and, in particular, the enclosed cavity or "ablation "Quot; resection cavity ").

The composition of the present invention is appropriately selected depending on the degree of absorption of the active ingredient in the body, the excretion rate, the age and weight of the patient, the sex and condition of the patient, the severity of the disease to be treated and the like, but is generally administered to the adult at 1 to 500 mg / . The unit dosage formulations thus formulated may be administered several times at predetermined time intervals as necessary.

The present invention also provides a health functional food composition for prevention or improvement of liver fibrosis or liver cirrhosis, which comprises an asparagine extract as an active ingredient.

The self-supporting extract contained in the health functional food composition can be prepared in the same manner as the self-supporting extract contained in the pharmaceutical composition of the present invention, and the effect of the extracted extract is also the same, so a description thereof will be omitted.

The health functional food may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, Organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. It can also contain natural fruit juices and pulp for the production of fruit juices and vegetable drinks. These components may be used independently or in combination. The health functional food may be any one of meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol and vitamin .

The health beverage composition of the present invention is not particularly limited to a liquid ingredient other than the one containing a spiritual extract, and may contain various flavoring agents or natural carbohydrates as an additional ingredient such as ordinary beverages.

In addition, the health functional food may further include food additives, and the suitability of the food functional food as a &quot; food additive &quot; is not limited to the corresponding items in general rules and general test methods approved by the Food and Drug Administration Shall be determined according to the relevant standards and standards.

Examples of the products that have been used in the above-mentioned "food additives" include natural products such as ketones, chemical products such as glycine, potassium citrate, nicotinic acid and cinnamic acid, sensory coloring matter, licorice extract, crystalline cellulose, high- - Mixed preparations such as a sodium glutamate preparation, a noodle-added alkaline agent, a preservative preparation, a tar coloring agent and the like.

In addition to the above, the health functional food composition of the present invention can be used as a nutritional supplement, a vitamin, a mineral (electrolyte), a flavoring agent such as a synthetic flavoring agent and a natural flavoring agent, a coloring agent and a thickening agent (cheese, chocolate, Alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. In addition, it may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination.

At this time, the content of the self-extract according to the present invention added to the food during the production of the health functional food can be appropriately increased or decreased as needed. The effective dose of the self- The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use such as preventive or therapeutic treatment. In the case of long-term consumption intended for health and hygiene purposes or for health control purposes, it may be below the above range.

The present invention will now be described more specifically with reference to the following examples. However, the following examples should not be construed as limiting the scope of the present invention, but should be construed to facilitate understanding of the present invention.

1-1. Wisdom  Preparation of extract

Commercially available humanoids were pulverized into powder form using a blender and extracted with a 70% ethanol (1000 ml of ethanol + 500 g of human milk) for 3 days (72 hours). After the extraction, After concentration, the mixture was freeze-dried at -70 ° C for 7 days using a drier. The lyophilized chrysanthemum extract was randomly used in the experiment. The lyophilized mother-in-law was 31.51 g and the extraction yield was 15.7%.

1-2. Reagents and compounds

Thioacetamide (TAA), and silymarin, hydroxyl-proline, p-dimethylaminobenzaldehyde, 1,1,3,3-tetraethoxypropane (TEP), chloramine-T, 5,5-dithiobis- (DTNB), reduced glutathione, glutathione reductase (GSH-red), glutathione peroxidase (GSH-px), beta -nicotinamide adenine dinucleotide phosphate and its reduced form (? -NADPH) Missouri, USA) and perchloric acid was purchased from GFS Chemical Co. (Columbus, Ohio, USA). All plants were purchased from Jecheon Herb Medicine (Jecheon, Korea).

1-3. Cell line

The hepatic details pimp (HSC-T6) immortalization has received offers from sonchanggyu professor of Oriental Medicine Hospital, Daejeon University, cell line Chang liver cell line normal liver was purchased from ATCC (ATCC ^® CCL-13 ™ meomae Manassas, Virginia, United States).

2-1. Cell culture

Chang hepatocytes were used as normal human cell lines derived from normal hepatocyte tissue. The cells 37 ℃, 10% in the wet atmosphere of 5% CO ₂ fetal bovine serum (FBS, GIBCO, USA), 1% antibiotic-antifungal drug (Invitrogen, USA), the minimum essential medium (EMEM, Gibco, USA) supplemented with Lt; / RTI &gt;

Immortalized hepatic stellate cells (HSC-T6) were cultured in DMEM supplemented with 5% FBS, 1% antibiotic-antifungal drug in a humidified atmosphere of 5% CO ₂ at 37 ° C.

2-2. Cell survival rate Assay

Cell viability assays were evaluated by 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) method. In 96-well plates, it was cultured Chang cells (7x10 ⁵ cells / ㎖) with HSC-T6 (6x10 ⁵ cells / ㎖) in a DMEM medium supplemented as described above. The sample materials were evaluated at various concentrations (0, 0.1, 0.5 and 1 mg / ml) for 24 hours at 37 ° C in an atmosphere of 5% CO ₂ and 95% humidity. The cells were then incubated with 0.5 mg / ml MTT (Sigma, USA) for 3 hours and the reaction was stopped by the addition of dimethylsulfoxide (DMSO, JUNSEL, Japan). Results were obtained at 540 nm using an ELISA reader. The survival rate of control cells was used as a control value of 100%.

As a result, as shown in Fig. 1, the hepatic ethanol extract was toxic at a concentration of 1 mg / ml or more and the toxicity was not confirmed in the normal liver cell line. From the results above, it was confirmed that the hepatocyte ethanol extract showed almost no toxicity in the normal hepatocytes and toxicity in the hepatic detailed pimples. Therefore, in the subsequent experiments, experiments were carried out using 0.5 mg / ml of human milk ethanol extract.

Primary HSC  Check the shape

HSCs were isolated from seven-week-old male SD rats in situ using protease, collagenase, dienergic perfusion, and a single-step Histogenz gradient.

Specifically, the anesthetized rats using the CO ₂ and after laparotomy for abdominal, 18 Gaussian support catheter to cannulation to the portal vein Ca ^{2 +} / Mg ^{2 +} that do not contain HBSS (Hank's balanced salt solution) and then flowing through the 200 ㎖ , 60 ml of a solution containing HBSS containing Ca ²⁺ / Mg ²⁺ and 0.015% (W / V) collagenase, followed by 10 ml of HBSS without Ca ²⁺ / Mg ²⁺ Ml tube and the extracted liver was washed.

The washed liver was placed in a 50 ml tube containing 6 ml of plasma, 6 ml of collagenase and 6 ml of DNase and stored for 10 minutes in a water bath for activation. The digested liver was put into 36 mL of 10 mg DNase TYPE II-containing solution in HBSS containing Ca ²⁺ / Mg ²⁺ , and the liver membrane was peeled off. Two dishes of 100 pi size were used and 17 ml each was used. The hepatocytes were filtered from the free solution and filtered again using a 0.45 nylon mesh. The filtered solution was placed in a 50 ml tube and centrifuged at 50 g for 1 minute. The residue was collected using 10 ml of DNase Type II solution in a 100 π size dish, and centrifuged again at 50 g for 1 minute Respectively.

After centrifugation, the supernatant was removed to a new tube, centrifuged at 450 g for 10 minutes, the supernatant was removed, and the collected hepatocytes were resuspended in 10 ml of HBSS containing cold Ca ^{2 +} / Mg ^{2 +} containing 0.3% BSA Suspended and centrifuged at 450 g for 10 minutes. After removing the supernatant, 0.3% BSA (that does not contain ^{Ca 2+ / Mg 2+ HBSS) 9.5} ㎖ and 28.7% Nycodenz (that does not contain Ca ²⁺ / Mg ²⁺ HBSS) using a lower level 10 and ㎖ Mixed well. 6 ml of 0.3% BSA (HBSS without Ca ²⁺ / Mg ²⁺ ) was carefully flowed over the cell solution so as not to be mixed with the lower layer very slowly. And centrifuged at 1400 g for 20 minutes. 6 ml of HSC was extracted from the puffy portion between the upper layer of Nycodenz and the BSA layer and centrifuged at 1200 g for 3 minutes using 5 ml of DMEM medium containing 1% antibiotic and 10% FBS. After inhaling the supernatant, HSC was loosely dissolved in 200 μl of DMEM. 2 wells of DMEM and 100 占 퐇 of primary HSC were mixed with 6 well plates, and a total of 2100 ml were cultured in two wells for 7 days in an incubator in which 5% CO ₂ was supplied. On the 7th day, the HSC form was observed after treatment with Zawang extract 0.5 mg / ml for 24 hours.

As a result, as shown in FIG. 2, HSC collagen and fibronectin were observed before the treatment of the human milk extract, but collagen decreased and the cells were decomposed after the treatment with the human milk extract. These results suggest that the extracts of Zaanjang showed cell inhibition and abilities in HSC-T6 and primary HSC.

4-1. Cell cycle analysis

Conduct HSC-T6 cells (15 x 10 ⁵ cells / well) were cultured in DMEM medium supplemented for example as described in 2-1. The sample material was evaluated in an atmosphere of 5% CO ₂ and 95% humidity at 37 ° C for 24 hours with a concentration of 0.1% and 0.5 mg / ml of human milk ethanol extract, respectively. After 24 hours, the cells were washed twice with PBS and suspended in 1 ml of a cold solution of Propidium iodide (PI) (50 μg / ml PI and 100 μg / ml RNase A). The cells were then blocked with light, kept at room temperature for 30 minutes, and analyzed with a flow cytometer. The silymarin treated group was used as a positive control group.

As a result, as shown in FIGS. 3A and 3B, ATE 100 group treated with 0.1 mg / mL of human milk extract alone did not differ from NT group treated with nothing, but 0.5 mg / In the case of the ATE 500 group treated with ㎖ of Javanese extract, 2.58% of cells were found in the sub-G1 group as compared with the NT group (Fig. 3 (B)). Both G0 and S were found to show a slight difference, and Annexin V and PI double staining were performed to confirm the ability of the extracts to induce apoptosis.

4-2. Cell death analysis

HSC-T6 cells were treated with either silymarin (0.05 mg / ml) or human-derived extract (0.1 and 0.5 mg / ml) for 24 hours and saponin was removed with annexin V-FITC and PI (FICS annexin V cell death detection kit I, BD Biosciences, USA). The detection procedure was performed according to the manufacturer's instructions. Data analysis was performed with the CellQuest software (Beckton Dickinson), which allows evaluation of only specific populations. Individualization by gate was in accordance with size (FSC), graininess (SSC), and fluorescence (FL) parameters. Early apoptotic cells (annexin V ⁺ and PI ^-) and late apoptotic cells (annexin V ⁺ and PI ^-) included in the measuring cell death.

As a result, as shown in FIG. 4, 0.5 mg / ml of the extract of Zygomycin showed about twice the cytotoxic activity as that of the NT group. It was confirmed that the extract of Zykwan reduced HSC-T6 cells and induced cell death through cell cycle intervention.

5-1. Experimental animal  And experimental design

Thirty male Sprague Dawly (SD) male rats (6 weeks old, 190-210 g) were purchased from Orient Bio (Gyeonggi Province, Korea) and used. The experimental animals were housed in a conventional cage under controlled conditions of a light and dark cycle of 23 ± 3 ° C, 50 ± 20% relative humidity and 12 hours / 12 hours. After one week of adaptation, rats were randomly divided into 5 groups of 6 rats each in the following order: normal, TAA (treated with TAA only), ATE 500 (treated with TAA and 500 mg / (Treated with TAA and 100 mg / kg of humanized extract) and positive control (treated with TAA and 50 mg / kg of silymarin).

As shown in FIG. 5, TAA (200 mg / kg) was intraperitoneally injected three times a week for 13 weeks in the remaining groups except for the normal group (physiological saline, intraperitoneal injection) to induce hepatic fibrosis. Gastric juice (100 ㎎ / ㎏ or 500 ㎎ / ㎏), silymarin (50 ㎎ / ㎏), or distilled water was administered by gavage six times a week starting from week 7 and body weight was measured twice a week.

After the last drug administration, the rats were fasted for 18 hours and then blood was collected from heart puncture under CO ₂ anesthesia. The liver was separated and the absolute and relative weights were measured.

A portion of liver tissue stored at -80 ° C was separated for hydroxyproline, GSH, protein expression determinations. Fixed liver tissue in Bouin solution was processed for histomorphological study. A small portion of liver tissue fixed in RNAlater solution was stored at -80 ° C for gene expression studies. Animal experiments conducted in the present invention were carried out in accordance with the guidelines of the Experimental Animal Ethics Committee of Konkuk University (IACUC No. KU15148).

5-2. Liver fibrosis  Analysis of liver morphology changes in animal models

On the last day of the experiment, the rats were opened for each group and the liver was extracted, and the size and changes of the liver were visually confirmed. As shown in Fig. 6, TAA group (B) showed no liver and lesion in comparison with normal group (A), liver of group (C and D) And the lesion was alleviated.

5-3. Serum biochemical analysis

On the last day of the experiment, blood was collected from cardiac puncture under CO ₂ anesthesia and the serum was separated by centrifugation (3,000 xg, 15 min). Serum levels of AST (aspartate transaminase) and ALT (alanine transaminase) were measured using a GOT-GTP assay kit (Asan Pharmaceutical, Korea) after blood coagulation.

As a result, as shown in FIG. 7, the levels of serum AST and ALT were significantly increased in the TAA group, whereas the levels of serum AST and ALT in the AE100 group and the ATE 500 group Respectively. In particular, the ATE 500 group (0.5 ㎎ / ㎏) recovered serum AST and ALT levels similar to the silymarin treated group, which is a positive control group.

6-1. Glutathione Assay  ( GSH  assay)

Total GSH was measured according to the Ellman method. Briefly, after cutting liver tissue to 100 mg, the liver tissues were homogenized at 10% (w / w) in PBS and centrifuged at 10000 x g for 15 minutes to transfer the supernatant to a new tube. A double 50 [mu] l aliquot of the sample (or GSH standard) was mixed with a previously prepared 80 [mu] l DTNB and NADPH mixture (10 ul 4 mM DTNB and 70 ul 0.3 mM NADPH) prepared in 96 well plates. Finally, 20 [mu] l (0.06 U) GSH reductase solution was added to each well and absorbance was measured at 405 nm after 5 minutes.

As a result, as shown in FIG. 8, the total GSH content of the TAA group was significantly increased compared with that of the normal group, but the ATE 100 group and the ATE 500 group recovered the total GSH level to the normal group level. In particular, the ATE 500 group recovered GSH level to a higher level than the positive control group. It was confirmed that the extract of Zyklon protects the liver more effectively than the positive control.

6-2. Hydroxyproline Assay  ( Hydroxyproline  Assay)

Hydroxyproline measurement was performed as follows using the slightly modified method in the conventional method. In summary, liver tissue (156 mg) stored at -70 ° C was homogenized in 1 ml of 6N HCl and stored at 100 ° C for one day. The acid hydrolyzate was filtered through a filter paper (Toyo Roshi Kaisha, Tokyo, Japan), and 50 μl samples or hydroxyproline standards dissolved in 6N HSL were naturally dried. The dried sample was dissolved in methanol (50 μl) and then 1.2 ml of 50% isopropanol and 200 μl of chloramine-T solution were added to each and stored at room temperature for 10 minutes. Ehrlish solution (1.3 ml) was added and the sample was stored at 50 ° C for 90 minutes. The optical density of the reaction product was read at 558 nm using a spectrophotometer (Tecan, USA). Standard curves were constructed using serial dilutions of 0.5 mg / ml hydroxyproproline solution.

As a result, as shown in FIG. 9, the level of hydroxyproline in the TAA group was significantly increased as compared with that in the control group. All of the chalice extract-treated groups had a concentration-dependent decrease in hydroxyproline levels compared to the TAA group. In particular, the ATE 500 group reduced hydroxyproline levels to the same level as the silymarin treated group.

Hydroxyproline is a substance that occurs during the post-transcriptional process and is related to the stability of the collagen, meaning that collagen is stabilized and increased when the amount of hydroxyproline is increased (meaning collagen level in the liver). Thus, the results of this example confirm that collagen is reduced in the liver by the human milk extract.

Liver tissue  Histopathology

7-1. Hematoxylin and eosin (H & E) staining

For histopathological examination, fixed Bouin solution liver tissue was fixed to paraffin and cut into 5 ㎛ thick flakes. After drying, liver slices were stained with hematoxylin and eosin (H & E).

Histological changes of liver tissue were observed by TAA-induced hepatotoxicity. Blue represents nucleus and red represents cytoplasm. As a result, as shown in Fig. 10, white lesions which were not found in the normal group (A) were observed in the TAA group (B), and the cell structure was also changed. However, in the ATE 500 group (C) and ATE 100 group (D), it was visually confirmed that the degree of lesion was alleviated.

7-2. Mason Trichrome  process of dyeing

Based on the above results, mason trichrome staining was performed to confirm more accurate histological changes. For semi-quantitative analysis of collagen expression, the area stained blue with mason trichrome stained slices was measured with an image analyzer (Image J, NIH). Blue represents fibronectin, red represents muscle and cytoplasm, and black blue represents nucleus.

As a result, collagen accumulation (blue portion) widely distributed in TAA group was observed as shown in Fig. 11, but collagen accumulation of liver flakes was remarkably decreased in ATE 500 group (Fig. 11 (A)), In addition, it was decreased in the group treated with the human milk extract, especially in the ATE 500 group (FIG. 11 (B)).

TAA - induction liver Liver fibrosis  Related gene analysis

In order to confirm the anti - fibrotic effect of the Zaanwan extract, real - time PCR was used to analyze the genes associated with liver fibrosis. Total RNA was extracted from liver tissue samples and HSC-T6 cells using TRIzol reagent (QIAGEN, Calif.). CDNA was synthesized from total RNA (2)) of 20 ㎕ reactant using a high performance cDNA reverse kit (Applied Biosystems, USA). The primers used in this Example are shown in Table 1 below.

order α-SMA Forward: AACACGGCATCATCACCAACT (SEQ ID NO: 1)
Reverse: TTTCTCCCGGTTGGCCTTA (SEQ ID NO: 2) ColT1A1 Forward: CCCAGCGGTGGTTATGACTT (SEQ ID NO: 3)
Reverse: GCTGCGGATGTTCTCAATCTG (SEQ ID NO: 4)  TGF-β1 Forward: GGAGACGGAATACAGGGCTTT (SEQ ID NO: 5)
Reverse: AGCAGGAAGGGTCGGTTCAT (SEQ ID NO: 6) β-actin Forward: TAAGGCCAACCGTGAAAAGAT (SEQ ID NO: 7)
Reverse: ACCAGAGGCATACAGGGACAA (SEQ ID NO: 8)

Each gene was compared and measured based on β-actin. As a result, as shown in Fig. 12 (A), expression of TGF-beta was elevated in the TAA group, but remarkably decreased in the group treated with the human milk extract, and as shown in Figs. 12 (B) and (C) The expression of SMA gene was significantly decreased in the dose - Thus, it was confirmed that the extract of JiaoYang showed anti-fiber activity in the TAA-induced liver.

Statistical analysis

All data values were expressed as mean ± SEM. Statistically significant differences between groups were analyzed using one-way ANOVA followed by Student's t-test. Values of p <0.05 or p <0.01 are considered to be statistically significant.

Manufacturing example  1. Injection

The human milk extract prepared in Example 1-1: 300 mg

Sodium metabisulfite: 3.0 mg

Methylparaben: 0.8 mg

Propyl paraben: 0.1 mg

Sterile sterilized distilled water for injection:

The above ingredients were mixed and made into a final volume of 2 ml by a conventional method, filled in an ampoule and sterilized to prepare an injection.

Manufacturing example  2. Refining

The human milk extract prepared in Example 1-1: 500 mg

Potato starch: 100 mg

Lactose: 100 mg

Colloidal silicic acid: 16 mg

Magnesium stearate:

The ingredients were mixed and tableted according to a conventional tablet preparation method to prepare tablets.

Manufacturing example  3. Capsule

The human milk extract prepared in Example 1-1: 300 mg

Lactose: 50 mg

Starch: 50 mg

Talc: 2 mg

Magnesium stearate:

The above components were mixed according to a conventional capsule manufacturing method and filled in gelatin capsules to prepare capsules.

Manufacturing example  4. Pill

The human milk extract prepared in Example 1-1: 350 mg

Corn starch: 100 mg

Sterilized distilled water: suitable amount

The above ingredients were mixed and pelletized to spheres of appropriate size according to conventional pellet manufacturing methods to produce pellets.

Manufacturing example  5. Health functional foods

1) Health drinks

The honeycomb extract prepared in Example 1-1 was mixed in an appropriate amount to drink materials such as oligosaccharide (2%), liquid fructose (0.5%), sugar (2%), salt (0.5% , And sterilized to prepare a beverage.

2) Functional food

The functionalized food containing the chrysanthemum extract was prepared by mixing the chrysanthemum extract prepared in Example 1-1 with various vitamins and mineral-containing functional foods in an appropriate amount.

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation <120> Composition for prevention, improving or treating hepatic          fibrosis or liver cirrhosis comprising Aster tataricus Linne fil          extract <130> 1061578 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> alpha-SMA forward primer <400> 1 aacacggcat catcaccaac t 21 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> alpha-SMA reverse primer <400> 2 tttctcccgg ttggcctta 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ColT1A1 forward primer <400> 3 cccagcggtg gttatgactt 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ColT1A1 reverse primer <400> 4 gctgcggatg ttctcaatct g 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TGF-beta1 forward primer <400> 5 ggagacggaa tacagggctt t 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TGF-beta1 reverse primer <400> 6 agcaggaagg gtcggttcat 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta-actin forward primer <400> 7 taaggccaac cgtgaaaaga t 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Beta-actin reverse primer <400> 8 accagaggca tacagggaca a 21

Claims (8)

A pharmaceutical composition for the prevention or treatment of liver fibrosis or cirrhosis, which comprises a human milk extract as an active ingredient. [Claim 2] The pharmaceutical composition according to claim 1, wherein the human milk extract is extracted with one or more solvents selected from the group consisting of C ₁ to C ₄ alcohols and water. 3. The pharmaceutical composition according to claim 2, wherein the extraction solvent is 70% ethanol. 2. The pharmaceutical composition according to claim 1, wherein the extract of the human body inhibits activation and proliferation of hepatic stellate cells. A health functional food composition for prevention or improvement of liver fibrosis or liver cirrhosis, which comprises a human milk extract as an active ingredient. The health functional food composition according to claim 5, wherein the human milk extract is extracted with at least one solvent selected from the group consisting of C ₁ to C ₄ alcohols and water. 6. The pharmaceutical composition according to claim 5, wherein the extraction solvent is 70% ethanol. 6. The health functional food composition according to claim 5, wherein the extract of the human body inhibits activation and proliferation of hepatic stellate cells.

Images