WO2021215891A1 - Hepatic regeneration promoting composition comprising 6-o-trans-feruloyl catalpol as active ingredient - Google Patents

Abstract

The present invention relates to hepatic regeneration-promoting use of a composition comprising 6-O-trans-feruloyl catalpol or pharmaceutically acceptable salts thereof and, more particularly, to: a pharmaceutical composition, for promoting hepatic regeneration or inhibiting liver damage, which comprises 6-O-trans-feruloyl catalpol or pharmaceutically acceptable salts thereof; and a functional health food composition for promoting hepatic regeneration or inhibiting liver damage. The 6-O-trans-feruloyl catalpol of the present invention has efficacy for promoting hepatic regeneration and thus can be used for developing a composition for treating or enhancing hepatic regeneration after a partial hepatectomy.

Description

Composition for promoting liver regeneration comprising 6-O-trans-feruloyl catalpol as an active ingredient

The present invention relates to a use for promoting liver regeneration comprising 6-O-trans-feruloyl catalpol or a pharmaceutically acceptable salt thereof. Specifically, the present invention provides 6-O-trans-feruloyl catalpol or a pharmaceutically acceptable salt thereof. Or a pharmaceutical composition for promoting liver regeneration or inhibiting liver damage comprising a pharmaceutically acceptable salt thereof; And it relates to a health functional food composition for promoting liver regeneration.

The liver is a living organ in which enzymatic reactions and energy metabolism occur continuously, and plays a pivotal role in the metabolism of substances and substances in the body. In addition, the liver is located between the digestive system and the systemic circulatory system in the human body, thereby performing a function of defending the whole body from external substances introduced from the outside. Therefore, once the ex vivo substance that enters the body passes through the liver, the liver has a higher risk of being exposed to many toxic substances in addition to nutrients than other organs, so the probability of damage is higher than that of other organs.

The liver is an organ with excellent regenerative capacity, and in case of slight damage, it is sufficiently restored to normal. However, if it is continuously damaged, a part of the liver tissue is completely destroyed, and the damaged part cannot be restored to normal and the connective tissue accumulates excessively. As a result, scars are formed in the liver tissue, and liver function cannot be restored to normal. In addition, when liver cancer or liver tumor occurs and the liver function does not function normally, a treatment method in which a part of the liver is resected through a surgical method is being used.

Partial hepatectomy (PHx) is widely used as a general treatment method for treating diseases such as liver cancer and liver tumors. promote Such liver regeneration must occur essential for restoration to a normal liver condition. However, many patients have difficulties in recovery after liver resection due to the decreased ability of the liver to proliferate. Therefore, the need for the development of therapeutic agents to promote liver regeneration after liver resection is increasing, and through research and experiments, the efficacy of natural substances such as silymarin, ursolic acid, quercetin and resveratol to protect liver cells and prevent liver disease has been demonstrated. It is being revealed (Korean Patent 10-0693613, Korean Patent 10-1840950).

Under this background, the present inventors continued their efforts to develop a novel therapeutic agent for promoting liver regeneration, and as a result, 6-O-trans-feruloyl catalpoly iridoid compound has an excellent effect of promoting liver regeneration after liver resection. By confirming that there is, the present invention was completed.

The present inventors first identified the liver regeneration promoting effect of 6-O-trans-feruloyl catalpol, and 6-O-trans-feruloyl catalpol or a pharmaceutically acceptable salt thereof for promoting liver regeneration or a pharmaceutical composition for inhibiting liver damage; And a health functional food composition for promoting liver regeneration has been developed to complete the present application.

One object of the present invention is to provide a pharmaceutical composition for promoting liver regeneration or inhibiting liver damage comprising 6-O-trans-feruloyl catalpol or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a health functional food composition for promoting liver regeneration or inhibiting liver damage, comprising 6-O-trans-feruloyl catalpol or a pharmaceutically acceptable salt thereof.

Another object of the present invention is liver resection comprising administering to a subject in need thereof a pharmaceutical composition comprising 6-O-trans-feruloyl catalpol or a pharmaceutically acceptable salt thereof A method for promoting liver regeneration or inhibiting liver damage is provided.

Another object of the present invention is 6-O-trans-feruloyl catalpol or a nutraceutical composition comprising a pharmaceutically acceptable salt thereof to an individual in need thereof, the liver A method for promoting regeneration or inhibiting liver damage is provided.

Another object of the present invention is to prepare a pharmaceutical composition for promoting liver regeneration or inhibiting liver damage after liver resection, including 6-O-trans-feruloyl catalpol or a pharmaceutically acceptable salt thereof It provides the use of the composition.

Another object of the present invention is to prepare a health functional food composition for promoting liver regeneration or inhibiting liver damage, 6-O-trans-feruloyl catalpol or a composition comprising a pharmaceutically acceptable salt thereof provides the use of

Since the 6-O-trans-feruloyl catalpol of the present invention has the effect of promoting liver regeneration, it can be used to develop a composition for improving or treating liver regeneration after liver resection.

1A is a graph showing the liver/body weight ratio in an experimental mouse model.

1B is a photograph comparing the size of each group.

2 is a graph showing the levels of ALT and AST in serum for each group.

3A is a photograph confirming cyclinD, cyclinE, and c-myc by group through electrophoresis.

3B is a graph confirming cyclinD, cyclinE, and c-myc by group through protein quantification.

3C is a graph confirming cyclinD, cyclinE, and c-myc by group through mRNA expression.

4A is a photograph confirming pYSTAT3, STAT3, Haploglobin, and Fibrinogen by group through electrophoresis.

4B is a graph confirming pYSTAT3, Haploglobin, and Fibrinogen by group through protein quantification.

4C is a graph confirming pYSTAT3, Haploglobin, and Fibrinogen by group through mRNA expression.

5A is a photograph confirming p-IKKIα, p-IκBα, IκBα, p-NFκB p65, and NFκB p65 by group through electrophoresis.

5B is a graph confirming p-IKKIα, p-IκBα, and p-NFκB p65 through protein quantification.

6A is a graph confirming c-Jun and c-Fos through mRNA expression.

6B is a graph identifying HGF, EGFR, TGF-α, NF-κB, IL-6, and C/EBP-β through mRNA expression.

7A is a photograph confirming p-Akt, Akt, p-ERK, ERK, p-JNK, JNK, p-p38, and p38 by group through electrophoresis.

7B is a graph confirming p-Akt, P-ERK, p-JNK, and p-p38 through protein quantification.

8A is a photograph confirming p-IκBα and IκBα through electrophoresis after treatment with HGF and 6-O-trans-feruloyl catalpol.

8B is a graph confirming P-IκBα through protein quantification after treatment with HGF and 6-O-trans-feruloyl catalpol.

8C is a photograph confirming p-Akt, Akt, p-ERK, ERK, p-JNK, JNK, p-p38, and p38 through electrophoresis after treatment with HGF and 6-O-trans-feruloyl catalpol .

8D is a photograph confirming p-Erk, p-JNK, p-p38, and p-Akt through protein quantification after treatment with HGF and 6-O-trans-feruloyl catalpol.

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

Meanwhile, each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein are within the scope of the present invention. In addition, it cannot be said that the scope of the present invention is limited by the specific descriptions described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be encompassed by the present invention.

One embodiment of the present invention for achieving the above object provides a pharmaceutical composition for promoting liver regeneration or inhibiting liver damage, comprising 6-O-trans-feruloyl catalpol or a pharmaceutically acceptable salt thereof .

Since 6-O-trans-feruloyl catalpol according to the present invention exhibits excellent liver regeneration promotion and liver damage inhibition efficacy, it may be included in the composition for the purpose of promoting liver regeneration or inhibiting liver damage after liver resection, A high effect can be expected for promoting liver regeneration or inhibiting liver damage.

As used herein, the term “liver regeneration” refers to a phenomenon in which a part of the liver is replaced when the part is lost.

As used herein, the term “promoting” means helping something happen quickly.

As used herein, the term "liver damage" refers to a disease or injury to the liver.

As used herein, the term “inhibition” refers to a phenomenon in which biological activity or activity is reduced.

Specifically, the regeneration of the liver may be made after liver resection, but is not limited thereto, and the regeneration may include an increase in cell cycle regulatory protein expression, an increase in STAT3 or NF-κB protein activity, an increase in gene expression in the early regeneration phase, and regeneration. It may be due to increased expression of related growth factors or transcription factors, increased Akt, ERK, JNK, and p38 phosphorylation, and more specifically, the cell cycle regulatory protein may be c-myc, cyclinD, or cyclinE, and the initial The regeneration stage gene may be c-Jun or c-Fos, and the regeneration-related growth factor or transcription factor may be HGF, EGFR, TGF-α, IL-g, NF-κB, or C/EBF-β, However, the present invention is not limited thereto.

In addition, the composition of the present invention may further include HGF, but is not limited thereto.

As used herein, the term "6-O-trans-Feruloyl catalpol" of the present invention refers to a compound having the structure of formula (I).

The 6-O-trans-feruloyl catalpol may be a fraction from the extract of Gaedong tree, but is not limited thereto. The liver regeneration promoting efficacy of the 6-O-trans-feruloyl catalpol and Gaeonia extract was not known, and was first identified by the present inventors.

The composition containing 6-O-trans-feruloyl catalpol may include a range of derivatives expected by some skilled in the art from 6-O-trans-feruloyl catalpol, and as long as the same effect in the present invention is obtained included without limitation. 6-O-trans-feruloyl catalpol can be obtained from an extract comprising it. Moreover, it can be synthesize|combined and used by a well-known method.

As used herein, the term "partial hepatectomy (PHx)" is widely used as a general treatment method for treating diseases such as liver cancer and liver tumors.

In the present invention, the pharmaceutical composition for promoting liver regeneration comprising the 6-O-trans-feruloyl catalpol is characterized in that it promotes liver regeneration. Specifically, in one embodiment of the present invention, the efficacy of promoting regeneration was confirmed by measuring the liver/body weight after liver resection of the 6-O-trans-feruloyl catalpol-treated group of mice (FIG. 1). , 6-O-trans-feruloyl catalpol treatment group confirmed the decrease in the expression of the liver damage markers ALT and AST (Fig. 2), and confirmed the increase in the level of the cell cycle-related proteins c-myc and cyclinD,E. (FIG. 3), confirming the change in the activity of several factors for liver regeneration, including NF-κB and STAT3 in the early stage after liver resection (FIGS. 4 to 6). The phosphorylation of growth factors was confirmed (FIG. 7), and by treating with HGF, the increase in cell proliferation and regeneration-related factors through synergistic efficacy was confirmed (Table 1, Table 2, and FIG. 8), 6-O-trans- It was confirmed that feruloyl catalpol aided liver regeneration by inducing liver cell proliferation after liver resection in mice.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt in a form that can be used pharmaceutically among salts, which are substances in which cations and anions are bonded by electrostatic attraction. salts, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), an aluminum salt or the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine salts with, etc.; salts with inorganic acids may be salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like; salts with organic acids may be salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; Salts with basic amino acids may be salts with arginine, lysine, ornithine and the like; The salt with an acidic amino acid may be a salt with aspartic acid, glutamic acid, or the like.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent, which may include a non-naturally occurring carrier.

More specifically, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate , calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid (Poly Lactic Acid), poly-L-lactic acid, mineral oil, and the like.

The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral dosage forms, external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. The carrier may include various amorphous carriers, microspheres, nanofibers, and the like.

In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract and its fractions, for example, starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The content of 6-O-trans-feruloyl catalpol included in the pharmaceutical composition of the present invention is not particularly limited.

Another embodiment of the present invention for achieving the above object provides a health functional food composition for promoting liver regeneration or inhibiting liver damage comprising 6-O-trans-feruloyl catalpol or a pharmaceutically acceptable salt thereof do.

In this case, the descriptions of "6-O-trans-feruloyl catalpol", "liver regeneration", "promotion", "liver damage" and "inhibition" are the same as those described above.

Since 6-O-trans-feruloyl catalpol according to the present invention exhibits excellent liver regeneration promoting efficacy, it may be included in a food composition for the purpose of promoting liver regeneration after liver resection, and the food composition is routinely ingested Because it is possible to do it, a high effect can be expected for the promotion of liver regeneration.

As used herein, the term "health functional food" means a food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 of the Health Functional Food Act, and 'functionality' refers to the structure of the human body. and regulating nutrients for function or obtaining useful effects for health applications such as physiological action. On the other hand, health food refers to food that has an active health maintenance or promotion effect compared to general food, and health supplement refers to food for the purpose of health supplementation. can be mixed.

The 6-O-trans-feruloyl catalpol of the present invention may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.

The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. Specifically, the food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used. In addition, the food composition contains food additives such as preservatives, fungicides, antioxidants, colorants, coloring agents, bleaching agents, seasonings, sweeteners, flavoring agents, expanding agents, strengthening agents, emulsifiers, thickeners, filming agents, gum base agents, foam inhibitors, solvents, and improving agents. may include The additive may be selected according to the type of food and used in an appropriate amount.

In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur during long-term administration of the drug using food as a raw material, and is excellent in portability, so the present invention Foods of can be consumed as a supplement to promote liver regeneration.

6-O-trans-feruloyl catalpol of the present invention may be included in various weight % in a food composition if it can exhibit efficacy for promoting liver regeneration or inhibiting liver damage. Specifically, it may be included in an amount of 0.00001 to 100% by weight or 0.01 to 80% by weight relative to the total weight of the food composition, but is not limited thereto. When ingested for a long period of time for health and hygiene purposes, it may contain an amount below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

Another object of the present invention is liver resection comprising administering to a subject in need thereof a pharmaceutical composition comprising 6-O-trans-feruloyl catalpol or a pharmaceutically acceptable salt thereof A method for promoting liver regeneration or inhibiting liver damage is provided.

The description of "6-O-trans-feruloyl catalpol", "pharmaceutically acceptable salts", "liver resection", "liver regeneration", "promotion", "liver damage" and "inhibition" above As described above.

In the present invention, the subject is an individual suspected of having liver damage after liver resection, and may be a mammal including a mouse, livestock, etc. including humans that has or may develop the disease, but the 6-O-trans- Subjects that can be treated with a pharmaceutical composition comprising feruloyl catalpol or a pharmaceutically acceptable salt thereof are included without limitation. In addition, humans may be excluded from the subject of the present invention, but is not limited thereto.

The method of the present invention may include administering a pharmaceutical composition comprising 6-O-trans-feruloyl catalpol or a pharmaceutically acceptable salt thereof in a pharmaceutically effective amount. A suitable total daily amount may be determined by the treating physician within the scope of sound medical judgment, and may be administered once or divided into several doses. However, for the purposes of the present invention, a specific therapeutically effective amount for a particular patient depends on the type and extent of the response to be achieved, the specific composition, including whether other agents are used, if necessary, the specific composition, the patient's age, weight, general health, It is preferable to apply differently depending on various factors including sex and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together or concurrently with a specific composition, and similar factors well known in the pharmaceutical field.

Another object of the present invention is 6-O-trans-feruloyl catalpol or a nutraceutical composition comprising a pharmaceutically acceptable salt thereof to an individual in need thereof, the liver A method for promoting regeneration or inhibiting liver damage is provided.

The description of "6-O-trans-feruloyl catalpol", "health functional food", "individual", "liver regeneration", "promotion", "liver damage" and "inhibition" is described above. like a bar

The method of the present invention may include administering a nutraceutical effective amount of a health functional food composition comprising 6-O-trans-feruloyl catalpol or a pharmaceutically acceptable salt thereof. A suitable total daily amount may be determined by the treating physician within the scope of correct food and medical judgment, and may be administered once or divided into several doses. However, for the purposes of the present invention, a specific therapeutically effective amount for a particular patient depends on the type and extent of the response to be achieved, the specific composition, including whether other agents are used, if necessary, the specific composition, the patient's age, weight, general health, It is preferable to apply differently depending on various factors including sex and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together or concurrently with a specific composition, and similar factors well known in the pharmaceutical field.

Another object of the present invention is to prepare a pharmaceutical composition for promoting liver regeneration or inhibiting liver damage after liver resection, including 6-O-trans-feruloyl catalpol or a pharmaceutically acceptable salt thereof It provides the use of the composition.

The description of "6-O-trans-feruloyl catalpol", "pharmaceutically acceptable salts", "liver resection", "liver regeneration", "promotion", "liver damage" and "inhibition" above As described above.

Another object of the present invention is to prepare a health functional food composition for promoting liver regeneration or inhibiting liver damage, 6-O-trans-feruloyl catalpol or a composition comprising a pharmaceutically acceptable salt thereof provides the use of

The descriptions of "6-O-trans-feruloyl catalpol", "health functional food", "liver regeneration", "promotion", "liver damage" and "inhibition" are the same as those described above.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Experimental Example 1: Use of experimental animals

A 10-week-old male mouse C57BL/6J was used for the experiment, and all procedures were performed in compliance with the guidelines approved by the Ewha Womans University Animal Care Committee. Mice were placed in a temperature-controlled cage with a 12 h light/12 h dark cycle and 6-O-trans-Feruloyl catalpol or 10 mg/mL of silymarin was administered 7 preoperatively. Daily oral administration was observed from day one until sacrifice.

In addition, a sham-surgery group that did not undergo liver resection but was treated with the compound, a vehicle-treated group that underwent liver resection and treated with PBS instead of the compound, and a group administered with silymarin as a positive control group were prepared and compared.

All surgeries were performed by one person and standardized. After anesthesia, the central and left lateral lobes of the liver were removed and collected (middle 2/3), blood was collected and centrifuged at 3000 g for 5 minutes to separate serum, - Stored at 80°C.

Experimental Example 2: Quantification of mRNA expression

Total RNA was isolated from tissue using TRIzol solution (Invitrogen), and analyzed by RT-PCR and real-time PCR using ABI PRISM 7700 system (PE Biosystems), and data was quantified through GAPHD mRNA.

Experimental Example 3: Immunoblot

Equal amounts of protein from individual mice were collected for each group. More specifically, the mouse tissue was homogenized through a homogenizer together with a protein lysis buffer, and the debris was removed, followed by centrifugation at 15,000 rpm, 4° C. for 15 minutes. The supernatant was dissolved in polyacrylamide SDS gel and electrophoresed with a polyvinylidene difluoride membrane. The membrane was treated with 5% BSA, and then incubated with a primary antibody. Thereafter, after enrichment with a chemiluminescent reagent (Ab Frontier), immune complexes were detected using a horseradish peroxidase-conjugated secondary antibody (Bio-Rad). A large amount of the target protein was quantified by immunoblot densitometry analysis.

Experimental Example 4: Cell culture

Hep3B human hepatocellular carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA), and in DMEM containing 10% fetal bovine serum (HyClone) and 1% penicillin-streptomycin, 5% CO2, cells at 37°C. was cultured. In addition, the morphology of the cells was examined under a microscope and subcultured at 80% confluence. Hep3B cells (1x106/mL) were serum-free 16 h prior to HGF (10ng/mL) stimulation.

Experimental Example 5: Cell proliferation assay

For cell proliferation studies, cell number counting was measured using a hemocytometer in monolayer culture in 12-well culture dishes. Hep3B cells were inoculated as much as 10 μM at a concentration of 0.5x105/mL in a culture medium containing 0.5 μM to 10 μM of the compound and only DMSO as a control, and trypsinized every 24 hours to count the number of live cells three times. The experiment was conducted for 4 days, and the dose-dependent effect of the compound of the present invention on proliferation was confirmed through three independent experiments.

Experimental Example 6: Material Preparation

6-O-trans-feruloyl catalpol was isolated from the bark of the bald eagle, and the purity was evaluated by analytical HPLC (purity >99%). Silymarin was also purchased from Sigma (St. Louis, MO, USA).

Example 1: Measurement of liver/body weight ratio and ALT/AST activity in serum

In order to confirm the effect of 6-O-trans-feruloyl catalpole on liver regeneration, through the method of Experimental Example 1, 6-O-trans-feruloyl catalpole was administered daily from 7 days before surgery to sacrifice. The liver/weight ratio of the experimental groups administered orally was measured.

As a result, as shown in FIG. 1 , the liver weight compared to the body weight of the group administered with 6-O-trans-feruloyl catalpol was approximately doubled after 48 hours and about 1.3 after 72 hours compared to the vehicle-treated group. doubled. This was similar to that of silymarin (FIG. 1A), and the group administered with 6-O-trans-feruloyl catalpol did not induce liver overgrowth even after 14 days of liver resection (FIG. 1B).

Through this, it was confirmed that 6-O-trans-feruloyl catalpol had excellent regeneration promoting efficacy in the early stage of liver regeneration, especially from post-surgery to 72 hours, and had excellent efficacy in the recovery of the initial stage. , it was confirmed that it did not cause overgrowth of the liver.

Example 2: ALT/AST in the serum of rats

Alanine aminotransferase (ALT; alanine aminotransferase) and aspartate aminotransferase (AST) are known as markers indicative of liver damage, and the values of ALT and AST in rat serum during liver regeneration were analyzed through its kit. did.

As a result, as shown in FIG. 2 , it was confirmed that the liver damage marker level was reduced by about 1/2 in the group administered with 6-O-trans-feruloyl catalpol than in the vehicle-treated group 12 hours after liver resection. and it was confirmed that the inhibition was continued even after 12 hours (FIG. 2). In addition, it was confirmed that the efficacy was similar to the result of administration of silymarin.

Through this, it was thought that 6-O-trans-feruloyl catalpol inhibited liver damage by about 1/2.

Example 3: c-myc, cyclinD, and cyclinE expression

Example 3-1: Protein Quantification

To determine whether 6-O-trans-feruloyl catalpol promotes cell cycle return to normal during liver regeneration, the protein of hepatic cell cycle protein was quantified and analyzed.

More specifically, the activation of proto-oncogenes such as c-myc, which is known to be active during the regeneration process and utilized as an initial marker of liver regeneration, and cyclinD and cyclinE, which are known to be necessary for liver cell growth, are liver cell cycle markers. Because it is known, its activity was measured through electrophoresis through a conventional method, and protein quantification through the method of Experimental Example 3.

As a result, as shown in FIGS. 3A to 3B, it was confirmed that cyclinD was increased after liver resection in the group administered with 6-O-trans-feruloyl catalpol compared to the vehicle-treated group, which was treated with silymarin. It was confirmed that the increase was superior to that of the group.

In addition, it was confirmed that cyclinE and c-myc increased, and similar efficacy was confirmed in the group treated with silymarin (FIG. 3B).

Through this, when 6-O-trans-feruloyl catalpol was administered, it was confirmed that the liver regeneration cycle marker increased in the initial stage, and it was possible to infer that the liver regeneration occurred actively, which was more enhanced than silymarin. It was confirmed that there is an effect of promoting liver regeneration.

Example 3-2: mRNA expression

In order to confirm whether 6-O-trans-feruloyl catalpol promotes the return of the cell cycle to a normal cycle during liver regeneration, mRNA of liver cell cycle protein was analyzed by the method of Experimental Example 2.

As a result, as shown in FIG. 3C , it was confirmed that cyclinD and cyclinE increased after liver resection in the group administered with 6-O-trans-feruloyl catalpol compared to the vehicle-treated group, and silymarin-treated group A similar effect was also confirmed.

Through this, when 6-O-trans-feruloyl catalpol was administered, it was confirmed that the liver regeneration cycle marker increased in the early stage, and it was possible to infer that liver regeneration occurred actively, which is similar to silymarin. It was confirmed that there is a regeneration promoting effect.

Example 4: STAT3 activity

Example 4-1: Protein Quantification

Cytokines and growth factors activate STAT3 (signal transducer and activator of transcription 3), and the activated STAT3 is known to be dimerized and migrated to the nucleus to bind to a target gene and induce transcription. The STAT3 is known to play an important role in liver regeneration. Therefore, in order to confirm the liver regeneration efficacy of 6-O-trans-feruloyl catalpol, electrophoresis and experiments were performed on the expression of STAT3, tyrosine phosphorylated pYSTAT3, Haptoglobin (HapG), and fibrinogen, which are increased in proportion to the target of pYSTAT3. It was confirmed through the method of Example 3.

As a result, as shown in FIGS. 4A to 4B, the pYSTAT3/STAT3, HapG/GAPDH and Fibrinogen/GAPDH ratios after liver resection in the group administered with 6-O-trans-feruloyl catalpol compared to the vehicle-treated group. It was confirmed that this increased, and it was confirmed that it increased better than the group treated with silymarin (FIG. 4B).

Example 4-2: mRNA expression

In order to confirm the liver regeneration efficacy of 6-O-trans-feruloyl catalpol, the mRNA of Haptoglobin (HapG) and Fibrinogen, which is proportionally increased as the target of pYSTAT3 identified above, was analyzed through the method of Experimental Example 2. .

As a result, as can be seen in Figure 4C, the mRNA expression of HapG and Fibrinogen gradually increased in the vehicle-treated group, the group administered with 6-O-trans-feruloyl catalpol, and the group treated with silymarin. It was confirmed that the mRNA increase rate was higher in the group administered with 6-O-trans-feruloyl catalpol and silymarin compared to the vehicle-treated group ( FIG. 4C ).

Through this, it was confirmed that when 6-O-trans-feruloyl catalpol was administered, the mRNA expression of HapG and Fibrinogen, liver regeneration markers, was significantly higher than that of the vehicle-treated group. It was confirmed that tyrosine phosphorylation was significantly increased, and it was confirmed that the efficacy was superior to that of silymarin, thereby confirming the excellent liver regeneration promoting efficacy of the composition of the present invention.

Example 5: NF-κB activity

The activity of NF-κB was confirmed to occur within a few hours after liver resection, which is thought to play an important role in liver regeneration, and is known to prevent apoptosis and promote the survival of liver cells. In addition, p-IKKα, p-IκBα, and p-NFκB p65 are known as the above-mentioned NF-NFκB markers. Therefore, in order to confirm the liver regeneration efficacy of 6-O-trans-feruloyl catalpol, the expression of p-IKKα, p-IκBα, and p-NFκB p65, which are the NF-κB markers, was analyzed by electrophoresis and Experimental Example 3 It was confirmed through the method of

As a result, as shown in FIG. 5 , it was confirmed that the p-IKKα/GAPDH ratio increased in the group administered with 6-O-trans-feruloyl catalpol compared to the vehicle-treated group after liver resection, and silymarin It was also increased in the treated group, but it was confirmed that the increase in the group administered the composition of the present invention was higher.

In addition, the ratio of p-IκBα/IκBα and p-NFκB p65/NFκB p65 was increased, which was confirmed to be superior to that of the silymarin-treated group ( FIG. 5B ).

Through this, it was confirmed that when 6-O-trans-feruloyl catalpol was administered, the activity of NF-κB, which indicates liver activity, appeared immediately after liver resection, and had superior efficacy than the vehicle-treated and silymarin-treated groups. , it was confirmed that there is an effect of promoting liver regeneration through increased liver activity.

Example 6: Expression of genes and factors in the early stages of liver regeneration

Example 6-1: c-Jun and c-Fos

The c-Jun and c-Fos genes are components of activator protein-1 (AP-1) that play an important role in the early stage of liver regeneration, and can be used as markers of the liver regeneration stage, and their mRNA expression was confirmed.

As a result, as shown in FIG. 6A, it was confirmed that c-Jun mRNA increased after liver resection in the group administered with 6-O-trans-feruloyl catalpol compared to the vehicle-treated group, and silymarin-treated group. Similar efficacy was also confirmed in the group.

In addition, the mRNA of C-Fos was also increased, and it was confirmed that similar efficacy appeared in the group treated with silymarin.

Through this, it was confirmed that 6-O-trans-feruloyl catalpole significantly increased the expression of c-Jun and c-Fos, which are genes that play an important role in the early stage of liver regeneration, to have the effect of inducing liver regeneration.

Example 6-2: growth factors and transcription factors

By confirming the mRNA expression of a number of growth factors and transcription factors involved in liver regeneration, liver regeneration was confirmed.

More specifically, the expression of hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR), TGF-α, IL-g, NF-κB, and C/EBF-β (CCAAT enhancer-β) was confirmed.

As a result, as shown in FIG. 6B, it was found that HGF mRNA, EGFR mRNA and C/EBP-β increased after liver resection in the group administered with 6-O-trans-feruloyl catalpol compared to the vehicle-treated group. It was confirmed, and it was confirmed that it was significantly increased compared to the group treated with silymarin.

In addition, NF-κB, IL-6 and TGF-α were increased, and similar efficacy was confirmed in the group treated with silymarin.

Through this, the expression of HGF, EGFR, TGF-α, IL-g, NF-κB, and C/EBF-β, which are growth factors and transcription factors that 6-O-trans-feruloyl catalpole promotes liver regeneration In particular, it was confirmed that the expression of HGF, EGFR, and C/EBF-β was significantly increased compared to the silymarin-treated group, confirming the excellent liver regeneration inducing efficacy of the composition of the present invention.

Example 7: Akt, ERK, JNK, and p38 phosphorylation

Akt serine/threonine kinase is an important cell survival mediator in response to growth factor stimulation, and activation of the phosphatidylinositol 3-kinase (PI3 K)/Akt pathway is known to inhibit apoptosis. In addition, since ERK, JNK, and p38 are important signal transduction mediators in liver regeneration, phosphorylated Akt, ERK, JNK, and p38 in the 6-O-trans-feruloyl catalpol-treated group after liver resection were transduced. It was analyzed by phoresis and protein quantitation.

As a result, as shown in Figure 7, after liver resection in the group administered with 6-O-trans-feruloyl catalpol compared to the vehicle-treated group, p-Akt/Akt, p-Erk/Erk, p- The ratio of JNK/JNK and p-p38/38 was increased, respectively, and similar efficacy was confirmed in the group treated with silymarin ( FIGS. 7A and 7B ).

Through this, it was confirmed that 6-O-trans-feruloyl catalpol promoted the phosphorylation of Akt, ERK, JNK, and p38, which are survival mediators and regenerative signal transduction mediators, and had a liver regeneration induction effect similar to that of silymarin.

Example 8: Synergistic efficacy by treatment with HGF

In Example 6, it was confirmed that the expression of the HGF growth factor was significantly increased, and the synergistic effect by the additional treatment of the HGF growth factor was confirmed.

Example 8-1: Cell proliferation in vitro

The synergistic efficacy of HGF and 6-O-trans-feruloyl catalpol was evaluated through human liver cells Hep3B (Table 2). Cell viability was evaluated through Hep3B cells treated with 10 ng/mL HGF in complete medium, and 10 μM 6-O-trans-feruloyl catalpol, DMSO was used as a negative control, and silymarin was used as a positive control. did.

As a result, as shown in Table 1, counting the number of cells every 24 hours, and treating the Hep3B cells with 6-O-trans-feruloyl catalpol during the culture period of 4 days, the cell proliferation rate compared to the control group From day 1 to day 4, it was confirmed that there was an increase of 19%, 76%, 39%, and 37%, respectively. In addition, it was confirmed that silymarin also increased the number of cells (12%, 15%, 7%, and 21%, respectively), but it was confirmed that the increase rate was lower than when 6-O-trans-feruloyl catalpol was treated.

In addition, as shown in Table 2, as a result of culturing for 4 days by treating Hep3B cells with silymarin and 6-O-trans-feruloyl catalpol at different concentrations, 6-O-trans-feruloyl catalpole was 0.5, When treated with 1, 5, and 10 μM, growth in Hep3B cells was increased by 10.9%, 12.1%, 29.7%, and 41.7%, respectively, compared to the control group, and silymarin treated with 0.5, 1, 5, and 10 μM increased by 4.1%, respectively. , it was confirmed that 7.0%, 6.9%, and 15.5% increase.

On the other hand, it was confirmed that there was no difference in cell adhesion or morphology during cell culture by the two compounds.

Through this, when HGF and 6-O-trans-feruloyl catalpol were treated together, the proliferation rate of cells was further increased continuously/concentration-dependently without affecting cell adhesion or morphology, and its efficacy compared to silymarin By confirming this remarkably excellent, it was confirmed that the composition of the present invention has superior liver regeneration efficacy than conventionally known silymarin.

Example 8-2: Protein phosphorylation in vitro

The synergistic efficacy of HGF and 6-O-trans-feruloyl catalpol was analyzed by electrophoresis and protein quantification for phosphorylation of IκBα, Akt, ERK, JNK, and p38.

More specifically, 10ng/mL of HGF growth factor was treated and the expression rates of these factors were compared over time in the vehicle-treated group and the 6-O-trans-feruloyl catalpol group.

As a result, as shown in FIG. 8, p-IκBα/IκBα, p-ERK/ERK, p-JNK in the group administered with 6-O-trans-feruloyl catalpol + HGF rather than the vehicle-treated + HGF group. The ratios of /JNK, p-Akt/Akt and p-p38/p38 each gradually increased significantly, indicating that the phosphorylation of the 6-O-trans-feruloyl catalpol + HGF group was significantly higher than that of the vehicle-treated + HGF group. It was confirmed that it occurs more actively (FIG. 8).

Through this, when HGF and 6-O-trans-feruloyl catalpol were treated together, phosphorylation of Akt, ERK, JNK, and p38 was further increased, confirming the synergistic effect of promoting liver cell proliferation.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Claims (10)

1. A pharmaceutical composition for promoting liver regeneration or inhibiting liver damage, comprising a compound represented by Formula I or a pharmaceutically acceptable salt thereof:

   [Formula I]

   
2. The pharmaceutical composition for promoting liver regeneration according to claim 1, wherein the regeneration is made after liver resection.
3. The pharmaceutical composition for promoting liver regeneration according to claim 1, wherein the regeneration is promoted by increased expression of cell cycle regulatory proteins.
4. The pharmaceutical composition for promoting liver regeneration according to claim 1, wherein the regeneration is promoted by an increase in STAT3 or NF-κB protein activity.
5. The pharmaceutical composition for promoting liver regeneration according to claim 1, wherein the regeneration is promoted by an increase in gene expression in the early regeneration stage.
6. The pharmaceutical composition for promoting liver regeneration according to claim 1, wherein the regeneration is promoted by an increase in expression of a regeneration-related growth factor or transcription factor.
7. The pharmaceutical composition for promoting liver regeneration according to claim 1, wherein the regeneration is made by increasing phosphorylation of Akt, ERK, JNK, or p38.
8. The pharmaceutical composition for promoting liver regeneration according to claim 1, wherein the composition further comprises HGF.
9. The pharmaceutical composition for promoting liver regeneration according to claim 1, wherein the compound is fractionated from an extract of Gaedongtong tree.
10. A health functional food composition for promoting liver regeneration or inhibiting liver damage, comprising a compound represented by Formula I or a pharmaceutically acceptable salt thereof:

    [Formula I]

    

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