KR20180016457A - The composition containing ginsenoside F2 for preventing and treating liver cancer - Google Patents

Abstract

The present invention relates to a composition containing ginsenoside F2 as an active ingredient for preventing or treating liver cancer. More specifically, a composition according to the present invention does not affect normal cells but kills liver cancer cells and specifically inhibits liver cancer incidence. Also, the composition reduces expression of alanine aminotransferase (ALT) or aspartate aminotransferase (AST), thereby being usefully used as a liver cancer therapeutic agent.

Description

[0001] The present invention relates to a composition for prevention or treatment of liver cancer comprising ginsenoside F2,

The present invention relates to a pharmaceutical composition for preventing or treating liver cancer comprising ginsenoside F2 as an active ingredient.

The present invention also relates to a health functional food composition for preventing or ameliorating liver cancer comprising ginsenoside F2 as an active ingredient.

The present invention also relates to a method for the prophylaxis or treatment of liver cancer comprising the step of administering to a subject a composition comprising ginsenoside F2.

According to the data from the National Cancer Information Center of the National Cancer Information Center (2013), the death rate of 24.1 deaths per 100,000 cancer patients per year is 74.8 men and 15.6 women per 100,000 Koreans aged 40-60. Reference).

Currently, the best way to treat liver cancer is surgical resection and liver transplantation. However, in the case of liver transplantation, it is difficult to obtain donors. In the case of surgical resection, the number of patients who can undergo surgery is limited to within 20% of all patients.

Other therapies include systemic chemotherapy, hepatic artery chemoembolization, hepatic arterial chemo injection, and radiation therapy. However, these treatments have deadly side effects that kill not only hepatocarcinoma cells but also normal cells.

Nexavar, a sorafenib ingredient, is the world's first oral treatment drug for hepatocellular carcinoma (HCC). It has been reported that side effects such as pancreatic enlargement are reduced when taken over a long period of time. .

On the other hand, ginsenoside is saponin in ginseng. There are more than 30 kinds of saponin, and it is classified into PPD (protopanaxadiol) and PPT (protopanaxatrio) depending on the structure. ( Curr Vasc Pharmacol . 2009 July; 7 (3): 293-302). Among the ginsenosides, minor ginsenosides present in ginseng in a very small amount are difficult to separate, and thus their research is insufficient.

Thus, the present inventors have made intensive efforts to find a compound having excellent therapeutic effect on liver cancer. As a result, it has been confirmed that ginsenoside F2 can inhibit the formation and proliferation of liver cancer without affecting normal cells. Respectively.

KR 10-1360231 B1 KR 10-1433661 B1

The main object of the present invention is to provide a pharmaceutical composition for preventing or treating liver cancer comprising ginsenoside F2 as an active ingredient.

Another object of the present invention is to provide a health functional food composition for preventing or ameliorating liver cancer comprising ginsenoside F2 as an active ingredient.

It is yet another object of the present invention to provide a method of preventing or treating liver cancer, which comprises administering to a subject a composition comprising ginsenoside F2.

The inventors of the present invention found that treatment of liver cancer-induced mice with ginsenoside F2 inhibited the formation of liver cancer without affecting the survival of normal cells, ALT and AST, suggesting the damage of the liver, and thus the composition containing the ginsenoside F2 showed excellent therapeutic effect of liver cancer for the first time.

In order to achieve the above object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating liver cancer comprising ginsenoside F2 as an active ingredient.

In the present invention, the term "ginsenoside F2" means a kind of saponin which is a main active ingredient of ginseng or red ginseng and means ginsenoside belonging to PPD (protopanaxadiol) series.

The ginsenoside F2 may be purchased commercially, or may be separated from ginseng cultivated or harvested in the nature, or converted from ginsenosides separated from the ginseng. Alternatively, ginsenoside F2 synthesized by a synthetic method can be used. However, any ginsenoside F2 that exhibits the preventive or therapeutic effect of liver cancer of the present invention can be used without limitation.

In one embodiment of the present invention, crude saponin was isolated from ginseng and then ginsenoside F2 was prepared from ginsenosides obtained from crude saponin.

The term "prophylactic" in the present invention may mean all actions that inhibit or delay the onset of liver cancer by administering ginsenoside F2 according to the present invention to a subject.

In the present invention, the term "treatment" may mean all the actions of allowing the composition of the present invention to be administered to suspected individuals of hepatocellular carcinoma, thereby improving or alleviating the symptoms of hepatocellular carcinoma.

In one embodiment of the present invention, the size, number, and weight of liver cancer tissue after treatment with ginsenoside F2 in liver cancer-induced mice were significantly reduced compared to the carrier treatment group , And the number was reduced more than twice (Figs. 4A to 4C). In addition, there was no difference in body weight in both treatment groups, but it was confirmed that the weight of liver tissue was reduced in the group treated with ginsenoside F2 compared to the group treated with carrier (Figs. 4D to 4F).

Therefore, it has been found that the pharmaceutical composition can be effectively used as a preventive or therapeutic agent for liver cancer.

In the present invention, the pharmaceutical composition may exhibit apoptotic effect specifically on liver cancer cells, and the hepatocarcinoma cells may be Hep3B, a human liver cancer cell line, or Hepa1-6, a mouse liver cancer cell line. However, It is possible to show killing effect on all cells.

In one embodiment of the present invention, the treatment of ginsenoside F2 with human liver cancer cell line (Hep3B) revealed that the survival rate of hepatocellular carcinoma cell line was decreased in a concentration-dependent manner of ginsenoside F2 (Figs. 1A, ). In addition, the treatment of ginsenoside F2 with mouse liver cancer cell line (Hepa1-6) revealed that the survival rate of hepatocellular carcinoma cell line was decreased depending on the concentration of ginsenoside F2 (FIGS. 1B and 3). However, treatment of normal hepatocytes with ginsenoside F2 showed no effect on normal hepatocyte survival (Fig. 1C).

Therefore, the pharmaceutical composition containing the above-mentioned ginsenoside F2 as an active ingredient shows a cell killing effect specifically on liver cancer cells, but has no effect on normal cells and can be used as a preventive or therapeutic agent for liver cancer without side effects .

In the present invention, the pharmaceutical composition may reduce the expression of an alanine amino transferase (ALT) or an aspartate aminotransferase (AST).

The term "alanine aminotransferase (ALT)" in the present invention refers to an enzyme in hepatocyte that forms glutamic acid by transferring the amino group of alanine to 2-oxoglutaric acid as a serum glutamyl pyruvic acid amino group transferase Serum Glutamic Pyruvate Transaminase (SGPT) or alanine amino transferase (ALAT).

Since the alanine amino group transfer enzyme is leaked to the serum when the hepatocyte is damaged, it can be used as an index for judging whether or not the hepatocyte is damaged. Specifically, the present invention can be used as an index for determining whether hepatocytes are damaged by liver cancer.

The term "aspartate aminotransferase (AST)" in the present invention refers to an enzyme in hepatocytes in which glutamic acid is formed by transferring an amino group of aspartic acid to 2-oxoglutaric acid. As an enzyme, serum glutamic acid oxaloacetate trans It is used in conjunction with Aminase (Serum Glutamic Oxaloacetic Transaminase (SGOT) or aspartate aminotransferase (ASAT).

Since the aspartic acid amino group transfer enzyme is leaked to the serum when the hepatocyte is damaged, it can be used as an index for judging whether or not the hepatocyte is damaged together with the alanine amino group transferase. Specifically, in the present invention, And the like.

In one embodiment of the present invention, the liver cancer-induced mouse is treated with ginsenoside F2, and the alanine aminotransferase (ALT) in the serum of the blood collected from the abdominal aorta of the mouse, the aspartic acid amino transferase Aspartate aminotransferase (AST) expression levels were measured. As a result, the expression level of the protein was decreased in the ginsenoside F2-treated group compared to the control group (FIGS. 5A and 5B).

Therefore, it was found that the pharmaceutical composition containing ginsenoside F2 as an active ingredient exhibited an effect of preserving liver function.

In the present invention, the pharmaceutical composition may increase the number of immune cells.

The term "immune cell" in the present invention collectively refers to a cell that produces an antibody. The immune cell may be any one or more selected from the group including CD8 T cells, spontaneous T cells, and macrophages, It is not.

In one embodiment of the present invention, after treatment of ginsenoside F2 with liver cancer-induced mice, immune cells were isolated from the liver of mice, and cells were analyzed using markers specific to various cells. As a result, The ginsenoside F2 treatment group had increased CD8 T cells, spontaneous T cells, and macrophages compared to the carrier treatment group (Fig. 6B).

Therefore, it has been found that the pharmaceutical composition comprising the above-mentioned ginsenoside F2 as an active ingredient can inhibit the formation of liver cancer by increasing immune cells near the liver cancer tissue.

The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" may mean a carrier, excipient or diluent which does not disturb the biological activity and properties of the compound to be injected, without irritating the organism. Specifically, occuring carrier. The type of the carrier that can be used in the present invention is not particularly limited, and any carrier conventionally used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more.

The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. 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.

In particular, solid preparations 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, lactose , Gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the non-aqueous solution and suspension include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

In addition, the pharmaceutical compositions of the present invention may comprise a pharmaceutically effective amount of ginsenoside F2. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and is generally in the range of 0.001 to 1000 mg / kg, To 200 mg / kg, more specifically, 0.1 to 100 mg / kg, may be administered once to several times per day. For purposes of the present invention, however, the specific therapeutically effective amount for a particular patient will depend upon the nature and extent of the reaction to be achieved, the particular composition, including whether or not other agents are used, the age, weight, Sex and diet of the patient, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used or concurrently used with the specific composition, and similar factors well known in the medical arts.

The pharmaceutical composition of the present invention can be used as a therapeutic agent for hepatocellular carcinoma, an adjuvant additive for improving the efficacy of the anti-cancer agent, and an auxiliary therapeutic agent (for example, anticancer adjuvant) for improving the therapeutic efficacy.

Another aspect of the present invention provides a food composition for preventing or improving liver cancer comprising ginsenoside F2 as an active ingredient.

The term "ginsenoside F2" and "prevention" in the present invention are the same as described above.

As used herein, the term "improvement" may mean any action that at least reduces the degree of symptom associated with the condition being treated.

The term "food" in the present invention refers to any product such as dairy products including meat, sausage, bread, chocolate, candy, staple, confectionery, pizza, ramen and other noodles, gums, ice cream, various soups, drinks, tea, Beverages, vitamin complexes, health functional foods, and health foods, all of which include foods of ordinary meaning.

The term "functional food" as used herein is the same term as "food for special health use" (FoSHU). In addition to nutritional supplementation, functional foods are processed so as to efficiently show the biological control function, It means food. Here, the term "function (surname)" means that the structure and function of the human body have a beneficial effect for health use such as controlling nutrients or physiological action. The food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients which are conventionally added in the art. In addition, the formulations of the food can also be produced without restrictions as long as they are formulations recognized as food. The composition for food of the present invention can be manufactured in various forms, and unlike general pharmaceuticals, it has the advantage that there is no side effect that may occur when a drug is used for a long period of time, and is excellent in portability, Can be ingested as an adjuvant to enhance the immune enhancing effect.

The health food refers to a food having an active health promotion effect or a health promotion effect compared to a general food, and a health supplement food refers to a health supplement food. In some cases, the terms health functional foods, health foods, and health supplements are used.

Specifically, the health functional food is a food prepared by adding ginsenoside F2 to a food material such as beverage, tea, spice, gum or confection, or encapsulated, powdered or suspended, But it has the advantage that there is no side effect that can occur when the food is used as a raw material and long-term use of the drug.

Since the food composition of the present invention can be routinely ingested, a high liver cancer improving effect can be expected, which is very useful.

The composition may further include a physiologically acceptable carrier. The carrier is not particularly limited and any carrier conventionally used in the art may be used.

In addition, the composition may contain additional ingredients which are commonly used in food compositions and which can improve odor, taste, vision and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid and the like. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu) It may also include amino acids such as lysine, tryptophan, cysteine, valine, and the like.

In addition, the composition can be used in combination with a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate), a disinfectant (such as bleaching powder and highly bleached white powder, sodium hypochlorite), an antioxidant (butylhydroxy anisole (BHA) (Sodium hypophosphate), bleach (sodium sulfite), seasoning (sodium MSG glutamate, etc.), sweeteners (hemicellulose, cyclamate, saccharin, A food additive such as a flavor (vanillin, lactones), a swelling agent (alum, D-tartrate, potassium hydrogen), an emulsifier, a thickening agent (glue), a covering agent, a gum base agent, a foam inhibitor, and food additives. The additives may be selected and used in appropriate amounts depending on the type of food.

The ginsenoside F2 can be added intact or used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the food composition of the present invention may be added in an amount of not more than 50 parts by weight, specifically not more than 20 parts by weight, based on the food or beverage, when the food or drink is prepared. However, in case of long-term ingestion for health and hygiene purposes, the active ingredient may be contained in an amount not exceeding the above range and there is no problem in terms of safety.

As an example of the food composition of the present invention, it can be used as a health beverage composition. In this case, various flavors or natural carbohydrates can be added as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose; Polysaccharides such as dextrin, cyclodextrin; Xylitol, sorbitol, erythritol, and the like. Sweeteners include natural sweeteners such as tau Martin and stevia extract; Synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acid, protective colloid thickener, pH adjuster, stabilizer, Alcohols or carbonating agents, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks, or vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The food composition of the present invention may be contained at various weight percentages as long as it can exhibit the effect of preventing or ameliorating liver cancer, but specifically includes 0.00001 to 100% by weight or 0.01 to 80% by weight of ginsenoside F2 relative to the total weight of the food composition .

Another aspect of the present invention provides a method of preventing or treating liver cancer comprising administering to a subject a composition comprising ginsenoside F2.

In the present invention, the term "ginsenoside F2" is the same as described above.

The term "administering" in the present invention means introducing the composition of the present invention into a subject by any suitable method, and the administration route of the composition can be administered through any conventional route so long as it can reach the target tissue. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal administration.

The term "individual" in the present invention means all animals such as rats, mice, livestock and the like, including humans, who are in need or need of prevention or treatment of liver cancer. Specifically, it may be a mammal including a human.

The composition may be administered in a pharmaceutically effective amount.

The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level will vary depending on the species and severity, age, sex, The time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. For example, the pharmaceutical composition comprising ginsenoside F2 may be administered at a daily dose of 0.0001 to 1000 mg / kg, particularly 0.001 to 100 mg / kg.

The above composition may be administered daily or intermittently, and the number of times of administration per day may be administered once or two or three times. In addition, the composition can be used alone or in combination with other drug therapy for the prevention or treatment of liver cancer. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The composition according to the present invention can kill liver cancer cells and specifically inhibit the formation of liver cancer without affecting normal cells and can also inhibit the production of alanine aminotransferase (ALT) or aspartate aminotransferase , AST), and thus can be usefully used as a therapeutic agent for liver cancer.

FIG. 1A is a graph showing the cytotoxic effect of ginsenoside F2 on human liver cancer cell line (Hep3B). FIG.
1B is a graph showing the cytotoxic effect of ginsenoside F2 on mouse liver cancer cell line (Hepa1-6).
1C is a graph showing that ginsenoside F2 does not affect survival of mouse normal hepatocytes.
2A is a photograph showing the cytotoxic effect of ginsenoside F2 on human liver cancer cell line (Hep3B) through a phase contrast microscope and a fluorescence microscope.
FIG. 2B is a graph showing the amount of human hepatoma cell line induced by apoptosis by ginsenoside F2 through fluorescence.
FIG. 3 is a graph showing that the fragmented DNA increases in the mouse liver cancer cell line (Hepa1-6) upon treatment with ginsenoside F2.
4A is a photograph showing the size of cancer formed when ginsenoside F2 or vehicle was administered to a mouse in which liver cancer was induced.
4B is a graph showing the size of cancer formed upon administration of ginsenoside F2 or carrier to liver cancer-induced mice.
FIG. 4C is a graph showing the number of cancer cells formed upon administration of ginsenoside F2 or carrier to liver cancer-induced mice. FIG.
FIG. 4D is a graph showing the weight of a mouse when ginsenoside F2 or carrier is administered to liver cancer-induced mice.
FIG. 4E is a graph showing the weight of tumor when administering ginsenoside F2 or carrier to liver cancer-induced mice. FIG.
FIG. 4f is a graph showing the weight of the tumor relative to the total weight upon administration of ginsenoside F2 or carrier to liver cancer-induced mice.
FIG. 5A is a graph showing expression levels of alanine amino transferase (ALT) upon administration of ginsenoside F2 or carrier to liver cancer-induced mice.
FIG. 5B is a graph showing the expression levels of aspartate aminotransferase (AST) upon administration of ginsenoside F2 or carrier to liver cancer-induced mice.
FIG. 6A is a photograph showing inhibition of the formation of liver cancer and increase of infiltration of immune cells upon administration of ginsenoside F2 or carrier to liver cancer-induced mice.
FIG. 6b shows the results of immunohistochemical staining for CD4 T cells, CD8 T cells, natural killer cells, spontaneous killer T cells, Tregs, neutrophils (Neu), neutrophils And the number of macrophages (Mac). The unit of 1 x 10 ³ / g means the number of cells present per liver tissue weight (g). % Refers to the ratio of the number of cells in the number of immune cells isolated from liver tissue.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Preparation of ginsenoside F2

The production method of ginsenoside F2 is as follows.

Specifically, ginseng leaves and roots including Korean ginseng, Korean ginseng, and Korean ginseng extract were extracted twice with 20 times volume of 80% alcohol and dried to obtain crude saponin. The crude saponin was dissolved again in water, adsorbed on HP-20 resin, and washed with 100% water to remove sugar. After the sugar was removed, the protopanaxatriol series ginsenosides Re and Rg1 were preferentially removed by primary rinsing with 40% alcohol. Thereafter, it was washed with 80% alcohol and dried to obtain ginsenosides Rb1, Rb2, Rc, and Rd, which were eluted protopanaxadiol series. Using the protopanaxadiol ginsenoside mixture as a substrate, more than 70% of ginsenoside F2 was obtained according to the method described in Korean Patent Publication No. 2013-0134930. After the ginsenoside F2 was adsorbed using an ODS resin, the ginsenoside F2 fraction having a purity of 95% or more was obtained by continuously flowing the alcohol having the proper concentration to obtain ginsenoside F2 in a concentration sphere distribution .

Example 2: Cancer cell killing effect of ginsenoside F2

Example 2-1: Comparison of killing effect on human liver cancer cell line, mouse liver cancer cell line, and normal hepatocyte

The ginsenoside F2 prepared in Example 1 was treated with human liver cancer cell line Hep3B cells, mouse liver cancer cell line Hepa1-6 cells and mouse normal hepatocytes at various concentrations (10 to 60 μM), and after 24 hours, Survival rates of the cells were measured according to the experimental method provided by the manufacturer using EZ-CYTOX (Daeil Lab Service, Korea) used for measuring cell viability.

As a result of the measurement, it was confirmed that the survival rate of human liver cancer cell line (Hep3B) and mouse liver cancer cell line (Hepa1-6) was decreased depending on the concentration of ginsenoside F2 (Figs. 1a to 1b) And almost no change was observed (FIG. 1C).

Therefore, it was confirmed that ginsenoside F2 exhibited cytotoxic effect on hepatocarcinoma cell line and showed cell killing effect, but did not show cytotoxicity on normal cell, and showed cell killing effect specifically on hepatocarcinoma cell.

Example 2-2: Killing effect on human liver cancer cell line

The killing effect of ginsenoside F2 against human liver cancer cell line (Hep3B) was confirmed using a phase contrast microscope and a fluorescence microscope.

Specifically, the ginsenoside F2 prepared in Example 1 was treated with human liver cancer cell line (Hep3B) at various concentrations (10 to 60 μM), and after 24 hours, the cells were observed under a phase contrast microscope , DAPI (4 ', 6-diamidino-2-phenylindole, Vector laboratory, USA) and TUNEL (terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling, Roche life science, USA). The staining was carried out according to the method provided by the manufacturer, and the degree of staining was observed with a fluorescence microscope.

As a result of observing with a phase contrast microscope, it was confirmed that the cell death of human liver cancer cell line (Hep3B) was induced by ginsenoside F2 (Fig. 2a). Furthermore, the fluorescence microscopic observation revealed that there was a difference in the number of cells stained with DAPI However, it was confirmed that the number of cells stained with TUNEL increased depending on the concentration of ginsenoside F2 (FIGS. 2A and 2B).

Therefore, it was once again confirmed that ginsenoside F2 exhibited cytotoxicity against hepatocarcinoma cell line and showed apoptosis.

Example 2-3: killing effect on mouse liver cancer cell line

The degree of DNA degradation was confirmed, and the killing effect of ginsenoside F2 against the mouse liver cancer cell line (Hepa1-6) was confirmed.

Specifically, the ginsenoside F2 prepared in Example 1 was treated with mouse liver cancer cell line (Hepa1-6) at various concentrations (10 to 60 μM), and after 24 hours, the cells were collected and subjected to genomic DNA extraction kit (iNtRON, USA), genomic DNA was extracted according to the experimental method provided by the manufacturer, and then gel electrophoresis was performed.

As a result, it was confirmed that the genomic DNA fragmentated by ginsenoside F2 appeared, and in particular, the fragmented genomic DNA was increased as the concentration of ginsenoside F2 increased (FIG. 3).

Therefore, it was once again confirmed that ginsenoside F2 exhibited cytotoxicity against hepatocarcinoma cell line and showed apoptosis.

Example 3: Inhibitory effect of ginsenoside F2 on liver cancer formation

Example 3-1: Preparation of mice inducing liver cancer formation

Diethylnitrosamine (DEN), a hepatocyte inducer, was injected intraperitoneally at 14 mg / kg of male mice at 20 mg / kg to induce the formation of liver cancer. Carriers (olive oil) or ginsenoside F2 (50 mg / kg) were treated three times a week at 20 weeks of age and sacrificed 20 weeks later to confirm the inhibition of liver cancer formation.

Example 3-2: Inhibitory effect of ginsenoside F2 on liver cancer formation

The mice sacrificed in the above Example 3-1 were opened and the liver was extracted. The size, number and weight of the cancer tissues in the carrier treatment group and the ginsenoside F2 treatment group were compared.

As a result of comparison, it was confirmed that the group treated with ginsenoside F2 significantly decreased the size of the cancerous tissue in the liver as compared to the group treated with the carrier, and the number was reduced more than twice (Figs. 4A to 4C). In addition, there was no difference in body weight in both treatment groups, but it was confirmed that the weight of liver tissue was reduced in the group treated with ginsenoside F2 compared to the group treated with carrier (Figs. 4D to 4F).

Therefore, it was confirmed that ginsenoside F2 inhibits the formation and proliferation of liver cancer.

Example 3-3: Preservation of liver function of ginsenoside F2

Blood was collected from the abdominal aorta of the mouse sacrificed in Example 3-1, and centrifuged at 3,300 rpm for 10 minutes using a centrifuge to separate only the serum. Subsequently, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglyceride were purchased from manufacturers using IDEXX Laboratories (USA) (VetTEST, IDEXX, USA) according to the experimental method provided.

As a result of the measurement, it was confirmed that ALT and AST values suggesting injury of hepatocytes were reduced in the ginsenoside F2 treatment group as compared with the carrier treatment group (FIGS. 5A and 5B).

Thus, it was confirmed that ginsenoside F2 inhibits the formation of hepatocarcinoma and preserves liver function.

Example 3-4: Immune cell increase effect of ginsenoside F2

The mice sacrificed in Example 3-1 were lyophilized and the liver was harvested. H & E (hematoxin and eosin) staining was performed to observe histological changes in liver.

As a result, it was confirmed that the group treated with ginsenoside F2 inhibited the formation of liver cancer and increased the infiltration of immune cells in the vicinity of liver cancer tissue (Fig. 6A).

In order to specifically measure the activity of the immune cells, immune cells were isolated from the extracted liver, and the markers shown in Table 1 below, which exhibited characteristics in various cells, were stained using a flow cytometer (LSRII, BD bioscience) Cells were analyzed.

Cell type Use marker CD4 T cells CD3e, CD4 CD8 T cells CD3e, CD8 Natural kill cells, natural killer T cells CD3e, NK1.1 Regulatory T cells (Treg) CD4, CD25, Foxp3 Neutrophil (Neu) CD11b, Gr.1 Macrophages (Mac) CD11b, F4 / 80

As a result, it was confirmed that the group treated with ginsenoside F2 showed an increase in CD8 T cells, spontaneous T cells, and macrophages among the immune cells as compared with the group treated with the carrier (Fig. 6B).

Therefore, it was confirmed that ginsenoside F2 has an effect of increasing CD8 T cells, natural killer T cells, and macrophages, which inhibit the proliferation of liver cancer, in the vicinity of liver cancer tissue.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (7)

A pharmaceutical composition for preventing or treating liver cancer comprising ginsenoside F2 as an active ingredient.
2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition shows a cytotoxic effect specifically on liver cancer cells.
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition reduces the expression of an alanine amino transferase (ALT) or an aspartate aminotransferase (AST).
The pharmaceutical composition of claim 1, wherein the pharmaceutical composition increases the number of immune cells.
5. The pharmaceutical composition according to claim 4, wherein the immune cell is at least one selected from the group consisting of CD8 T cells, spontaneous T cells, and macrophages.
A health functional food composition for preventing or ameliorating liver cancer comprising ginsenoside F2 as an active ingredient.
A method for preventing or treating liver cancer, comprising administering a composition comprising ginsenoside F2 to a subject other than a human.

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