WO2014051359A1 - Pharmaceutical composition comprising neferine as active ingredient for preventing or treating hepatoma - Google Patents

Abstract

The present invention relates to a novel use of neferine for treating hepatoma, and more particularly, to a pharmaceutical composition and to a health functional food comprising neferine as an active ingredient for preventing or treating hepatoma. The neferine of the present invention can have an anticancer effect through the activities of stopping a cell cycle, inducing apoptosis and autophagy, and inhibiting angiogenesis and cell migration of hepatocarcinoma cells. In particular, since neferine has cytotoxicity specificity for hepatocarcinoma cells caused by viral infection even among hepatocarcinoma cells, the present invention has an effect optimized for treating hepatocarcinoma cells caused by viral infection. Thus, the neferine of the present invention can be used as a health functional food, as well as a medicine such as a pharmacological composition used as a material which can prevent or treat hepatoma. Therefore, it is expected that the value of the present invention is very high when the present invention is applied to the food industry and the pharmaceutical industry. Furthermore, since the neferine of the present invention does not show cytotoxicity for cells other than certain hepatocarcinoma cells, the composition of the present invention, which comprises neferine as an active ingredient, is beneficial in consideration of the safety of long-term use.

Description

Pharmaceutical composition for preventing or treating liver cancer, including neferin as an active ingredient

The present invention relates to a novel use for the treatment of liver cancer of neferine (neferine), and more particularly to a pharmaceutical composition, health functional food for preventing or treating liver cancer containing neferin as an active ingredient.

The liver is the largest organ in the body, 800-1,200g in adults, where various types of malignant tumors occur. Hepatocellular carcinoma and cholangiocarcinoma account for 95%. The remaining 5% are very rare, such as hepatoblastoma (hepatoblastoma), hepatocellular carcinoma in children, hepatocellular carcinoma, mixed cholangiocarcinoma, undifferentiated cancer, cholangiocystic adenocarcinoma, carcinoid tumor.

Currently, the majority of adult liver cancer (90% or more) is hepatocellular carcinoma (HCC). Hepatocellular carcinoma is the fifth most common tumor in the world, with half a million deaths each year (Okuda 2000). Survival rates of hepatocellular carcinoma patients have not improved over the last 20 years and have almost the same incidence of mortality (Marrero, Fontana et al. 2005). The average age of onset of hepatocellular carcinoma (HCC) is approximately 55 years, most of which were found to be between 40-60 years.

There are several causes of hepatocellular carcinoma on a global scale, but most cases in Korea are suspected to be caused by hepatitis virus. Hepatitis viruses include A, B, C, D, E, and F. Very recently, hepatitis G has been discovered. There are three types of viruses that are a problem in our country, A, B, and C. Of these, two are related to liver cancer: B and C. According to statistics since 1990, when hepatitis B and C viruses became available, 85% of those who received liver cancer were infected with hepatitis B or C virus. It is estimated that hepatitis B and C viruses act on normal hepatocytes, causing mutations to increase the chance of developing cancer. Therefore, in Korea, people infected with hepatitis virus are classified as a high risk group of liver cancer.

Surgery, radiotherapy, and chemotherapy have been used to treat liver cancer for the past several decades. At the same time, research and development and commercialization of liver cancer, hepatitis, and hepatitis vaccines have progressed. It is not. Regarding the treatment of hepatocellular carcinoma, the therapeutic agents known to date are epirubicin, cisplatin, 5-fluorouracil, etoposide, etc., but have low therapeutic efficiency, and long-term use of these chemotherapeutic agents may cause chemotherapy resistance. . Therefore, there is an urgent need to develop a liver cancer treatment agent having high sensitivity without side effects.

Neferine, on the other hand, is one of the major bisbenzylisoquinline alkaloids from the lotus ( Nelumbo nucifera Gaerth.) Embryo. Alkaloids abundant in these green plant embryos have been reported to have a variety of biological activities, including anti-arrhythmia, anti-hypertension, relaxants, anti-diabetic, cholinesterase inhibition, sedatives and anti-multi-drug resistance. In particular, the lotus embryo has been widely used as a traditional herbal medicine for neurological diseases, insomnia, continuous fever and cardiovascular diseases such as hypertension and arrhythmia.

However, there has been no study on the effect of preventing or treating liver cancer of a compound called neferine derived from lotus embryos.

Therefore, while the present inventors are studying the pharmacological effects of nephrine extracted and purified from lotus embryos, nephrine can stop the cell cycle progression of hepatocellular carcinoma caused by viral infection, induce apoptosis, autophagy. The present invention was completed by confirming that it is useful for preventing, improving or treating liver cancer as it has anticancer effects through autophagy induction, angiogenesis inhibition, and cell migration inhibitory activity.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition which is particularly effective in preventing or treating liver cancer by liver cancer cell death due to excellent cytotoxicity against liver cancer cells caused by viral infection.

Still another object of the present invention is to provide a health functional food that is effective in preventing or improving liver cancer by having excellent cytotoxicity to liver cancer cells caused by viral infection.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for preventing or treating liver cancer comprising Neferine (Neferine) as an active ingredient.

In one embodiment of the invention, the neferine (Neferine) may be derived from the lotus embryo.

In one embodiment of the present invention, the liver cancer may be hepatocellular carcinoma caused by viral infection.

In one embodiment of the invention, the pharmaceutical composition is through the cell cycle arrest (cell cycle arrest), induction of apoptosis (apoptosis), autophagy induction, angiogenesis inhibition and cell migration inhibitory activity of cancer cells It can have an anticancer effect.

The present invention also provides a health functional food for the prevention or improvement of liver cancer comprising Neferine (Neferine) as an active ingredient.

In one embodiment of the invention, the neferine (Neferine) may be derived from the lotus embryo.

In one embodiment of the present invention, the liver cancer may be hepatocellular carcinoma caused by viral infection.

In one embodiment of the invention, the food is selected from the group consisting of beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements Can be.

Neferine of the present invention can stop the cell cycle progression of liver cancer cells, and have anticancer effects through induction of apoptosis, induction of autophagy, inhibition of angiogenesis and cell migration. Can be. In particular, neferrin has a therapeutic effect optimized for hepatocellular carcinoma induced by viral infection, as it has cytotoxicity specifically to hepatocellular carcinoma cells caused by viral infection among hepatic cancer cells.

1 is a graph showing the survival rate of cells after treatment with neprene for each concentration of Hep3B, Sk-Hep1 and THLE-3 cancer cell lines for 24 hours.

Figure 2 shows the change in nuclear condensation of cells through DAPI staining after treatment with Hep3B cells neferin (20, 25μM) at 6 hour intervals.

Figure 3 after treatment with Hep3B cells neferin (15, 20, 25μM) for 24 hours is analyzed the cell cycle using a flow cytometer.

Figure 4 shows the expression level of proteins related to the cell cycle after treatment with Heper3B cells for 24 hours at different concentrations (15, 20, 25μM) by Western blot.

FIG. 5 shows that p21 ^{Waf1 / Cip1} protein expression levels detected by immunofluorescence analysis after treatment of Hep3B cells with 15 μM concentration of nephrin for 12 hours using a fluorescence microscope (× 1000) (p21 ^{Waf1 / Cip1).} : green, nucleic: blue, cytoskeletal actin: red).

FIG. 6A shows that hep3B cells were treated with nephrine at different concentrations (15, 20, 25 μM) for 24 hours, and then mitochondrial membrane-related proteins (Bim, BID, Bax, Bak, Puma) were measured by Western blot.

Figure 6B shows the amount of apoptotic protein (cleaved caspase-8, -3, -6, -7 and PARP) involved in apoptosis after treatment with Hep3B cells for 24 hours at different concentrations (15, 20, 25 μM) Was measured via Western blot.

FIG. 7A shows the cleaved caspase-3 protein expression level detected by immunofluorescence analysis after treatment with Hep3B cells for 15 hours at 15 μM concentration of neferin (cleaved caspase-3). : green, nucleic: blue, cytoskeletal actin: red).

FIG. 7B shows the cleaved caspase-8 protein expression level detected by immunofluorescence assay after treatment with Hep3B cells for 15 hours at 15 μM concentration of neferin (cleaved caspase-8). : green, nucleic: blue, cytoskeletal actin: red).

8 is a Western blot expression of vesicle stress-related chaperon proteins (Bip, calnexin, PDI, calpain2 and caspase-12) over time after treatment with 20 μM concentration of neprine in Hep3B cells.

FIG. 9 is a caspase-12 protein expression level detected by immunofluorescence analysis after treatment with Hep3B cells for 15 hours at 15 μM concentration of nephrin using a fluorescent microscope (× 1000) (caspase-12: green , nucleic: blue, cytoskeletal actin: red).

FIG. 10 shows the degree of autophagosomes formation in the cytoplasm with fluorescence microscopy (× 1000) after treatment with 20 μM of nephrine in Hep3B cells transformed with GFP-LC3B plasmid. .

FIG. 11 shows the expression of LC3B protein after Western blot treatment of Hep3B cells with nephrine at different concentrations (15, 20, 25 μM) for 12 hours.

FIG. 12A is a measurement of the degree of tube formation using an inverted microscope (× 40) after 18 hours of treatment with concentrations (10, 20, 30 μM) of HUVECs cells, and FIG. 12B shows the total tube based on the results. This is a graph of lengths converted into percentages using Wimasis Image Aalysis software.

FIG. 13A is a measure of cell migration over time after treatment with Hep3B cells at 15 μM using an inverted microscope (× 40), and FIG. 13B is based on the results of cell migration using Wimasis Image Aalysis software. This graph is then converted to cell-covered area percent.

The present invention is characterized by providing a pharmaceutical composition for preventing or treating liver cancer comprising Neferine (Neferine) represented by the following formula (1) as an active ingredient.

<Formula 1>

The 'neferine' of the present invention is one of the main bisbenzylisoquinline alkaloids from the lotus (Nelumbo nucifera Gaerth.) Embryo. Nefferin is 4-[[(1R) -6,7-Dimethoxy-2-methyl-3,4-dihydro-1H-isoquinolin-1-yl] methyl] -2-[[(1R) -6-methoxy- Also called 1-[(4-methoxyphenyl) methyl] -2-methyl-3,4-dihydro-1H-isoquinolin-7-yl] oxy] phenol, the molecular formula of this substance is C ₃₈ H ₄₄ N ₂ O ₆ and has a molecular weight Is 624.77.

Alkaloids contained in green plant embryos have been reported to have a variety of biological activities including anti-arrhythmia, anti-hypertension, relaxants, anti-diabetic, cholinesterase inhibition, sedatives and anti-multi-drug resistance. In particular, the lotus embryo has been widely used as a traditional herbal medicine for neurological diseases, insomnia, continuous fever and cardiovascular diseases such as hypertension and arrhythmia.

However, there has been no study on the effect of preventing or treating liver cancer of the compound called neferine.

The present inventors can stop the cell cycle progression of hepatocellular carcinoma caused by nephrine-induced viral infection, and anti-cancer effect through induction of apoptosis, induction of autophagy, inhibition of angiogenesis and cell migration. As a substance useful for the prevention, improvement or treatment of liver cancer, as well as pharmaceuticals such as pharmacological composition, as well as health functional food, it was first identified the first.

In Experimental Example 1 of the present invention, in order to confirm the cytotoxicity of the nephrine against cancer cell lines, the cell viability was measured using Hep3B, Sk-Hep1 and THLE-3 cell lines, and only the Hep3B cell line was treated with the neferrin treatment concentration. Independently, cell viability has been shown to be markedly reduced. These results confirm that neferrin has a specific cytotoxic sensitivity to Hep3B cells (see Fig. 1).

In addition, in Experimental Example 2 of the present invention, in order to examine whether apoptosis of hep3B, a hepatocellular carcinoma cell line of neferrin, was performed, a DAPI assay was performed. 6 hours after the treatment, it was confirmed that nuclear membrane changes such as condensation and blebbing of cells appeared in the experimental group. In particular, apoptosome formation, which is typical of apoptotic cells, was observed in the experimental group 24 hours after neferrin treatment (see FIG. 2).

In addition, in Experimental Example 3 of the present invention, as a result of examining the cell cycle arrest of Hep3B, a hepatocellular carcinoma cell line of neferrin, sub-G1 was proportional to the neferrin treatment concentration (15, 20, 25 μM). Cell population increased (see FIG. 3); It was observed that the expression of proteins associated with the cell cycle is regulated as evidence of the suspension of cell cycle progression (see FIG. 4); In the cytoplasm of neprene-treated Hep3B cells, it was confirmed that the expression level of p21 ^{Waf1 / Cip1} protein (regulator capable of inhibiting the cell cycle) of the nucleus was significantly increased (see FIG. 5).

In general, the cell cycle is performed in a certain order by a mechanism defined in the cell. However, if the abnormality occurs in this order, the maintenance of the cell cycle becomes difficult, and when the abnormality of the cell cycle is induced, a regulator that acts to restore it acts, such as cyclin (cyclin), cyclin- Cyclin-dependent kinase (hereinafter abbreviated 'CDK') and cyclin-dependent kinase inhibitor (hereinafter abbreviated 'CDKI').

In the cell cycle, CDK4, 6, 8, etc. are activated by the type of cells in the early stage of G1 phase, CDK2 in the late stage of G1 and early stage of S1, and the progression from G2 to M CDK1 is known to play an important role. Binding to cyclins is essential for the activity of CDK. CDK4, 6 and 8 are activated by binding to cyclin D, and CDK2 binds to cyclins A and E. CDK1 binds to cyclins B and A, in addition to cyclins G, F and the like. At each period of the cell cycle, the specific cyclin-CDK complex is activated and the proteins phosphorylated specifically for CDK are responsible for the progression of the cell cycle.

In addition, CDK acts as an essential factor for cyclin activity. Activated CDK-cyclin is divided into cyclin regulatory unit and CDK active unit, and cyclin CDK regulation can be seen in two ways. The combination of cyclin and CDK induces structural changes in the protein, making the arrangement of the ATP phosphate group easy to transfer to the substrate protein. In addition, the position of the T loop that prevents the protein substrate from accessing the CDK is changed, thereby facilitating access to the substrate. The activity of CDK is activated only at certain times of the cell cycle because of cyclin synthesis that occurs specifically in the cell cycle. In addition, cyclin D is most likely synthesized in the middle of G1, and is mainly induced by mitogens such as cell growth factors. Cyclin D has three types of subtypes (D1, 2, and 3), and the degree of expression varies depending on the type of cell. For example, inhibiting the synthesis of cyclin D stops the cell cycle G1, and overexpressing cyclin D shortens the G1 group and starts the cell cycle without mitogen.

The ^{p15 INK4B, p16 INK4A, p18 INK4C} , p19 INK4D, p27 Kip1 and p21 ^{WAF1 / Cip1} and cell cycle regulation such as characters are known as core control to inhibit cyclin D1 / CDK4,6 or Cyclin E / CDK2 complexes. Therefore, such cell cycle regulators are called negative cell cycle regulators, and when they are expressed, cell cycle progression can be inhibited.

In Experimental Example 4 of the present invention, as a result of examining the induction of apoptosis of Hep3B, a hepatocellular carcinoma cell line of nephrine, Bcl-2 family proteins (Bim, Bid, known to regulate the mitochondrial-mediated apoptosis pathway) It was confirmed that the expression of Bax, Bak and Puma was increased, and changes of apoptotic proteins (cleaved caspase-8, -3, -6, -7 and PARP) accompanying apoptosis due to vesicle stress were observed (FIG. 6). In addition, cleaved caspase-8 and -3 were found to be prominently expressed in neprine-treated Hep3B cells compared to the untreated group, which was not treated with neferin, and apoptosomes induced by neferin were caspase in the cytoplasm. It could be deduced that activating -3 and caspase-8 causes apoptosis (see FIG. 7).

In addition, in Experimental Example 5 of the present invention, as a result of confirming that nephrine can induce autophagy as an anticancer effect on liver cancer cells, one of the morphological characteristics of autophagy over time in the experimental group treated with neferrin Vacuole formation occurred in the phosphorus cytoplasm. Autophagy also forms vesicles enclosed in a double membrane called autophagosomes, which show strong fluorescence spots (GFP-LC3B "dots") over time in the nephrine treated group. It was confirmed that the increase in the cytoplasm (see Figure 10).

In addition, in Experimental Example 6 of the present invention, as a result of testing the neovascularization inhibitory effect of neferrin in HUVECs cells, it was confirmed that the tube length (px) of HUVECs is reduced depending on the concentration of neferrin treatment.

In addition, in Experimental Example 7 of the present invention, it was confirmed that the cell migration is suppressed depending on the neferoline treatment concentration as a result of confirming that nephrine can induce cell migration inhibition as an anticancer effect on liver cancer cells (FIG. 13).

Through these results, the present inventors have found that nepherin has cytotoxicity to liver cancer cells induced by virus infection among hepatic cancer cells, cell cycle arrest, induction of apoptosis and autophagy of liver cancer cells. It has been experimentally demonstrated to have anticancer effects through induction, angiogenesis inhibition and cell migration inhibitory activity.

Therefore, the composition of the present invention comprising nephrine as an active ingredient can effectively prevent or treat liver cancer, and in particular, as it has cytotoxicity specifically to liver cancer cells induced by viral infection, liver cancer caused by viral infection It has a therapeutic effect optimized for cells.

The neferrin according to the present invention can be used in the form of salts, preferably pharmaceutically acceptable salts. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and an organic acid and an inorganic acid may be used as the free acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

The neferrin according to the present invention may be commercially available, or may be one that is separated from nature or manufactured by chemical synthesis known in the art.

When separating the neferrin of the present invention from natural products, there is no limitation to the separation method, any known method can be used. For example, in the following examples of the present invention, the N. nucifera embryo was extracted under reflux with methanol, and concentrated by vacuum drying to obtain a lotus embryo methanol extract. The extract thus obtained was suspended in distilled water, followed by dichloromethane and ethyl acetate. And sequentially fractionated with n-butanol, and the dichloromethane fraction was subjected to silica gel column chromatography again to purify the final neferrin.

The composition of the present invention may be prepared using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to such an active ingredient as a pharmaceutical composition comprising the neferin as an active ingredient, the adjuvant, an disintegrant, Sweetening agents, binders, coatings, expanding agents, lubricants, lubricants, or flavoring agents may be used.

The pharmaceutical composition may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredient for administration.

Formulation forms of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include but are not limited to natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component, as appropriate in the art.

In one embodiment of the present invention, the neferrin of the present invention may be included in the composition in a concentration of 1 to 300μM, and the neferrin compound of the present invention may be included in 0.1 to 95% by weight relative to the total weight of the composition.

The present invention also provides the use of a composition comprising neferine (neferine) as an active ingredient for the manufacture of a medicament for preventing or treating liver cancer. The composition of the present invention comprising the neferrin as an active ingredient can be used for the manufacture of a medicament for the prevention or treatment of liver cancer.

The present invention also provides a method for preventing or treating liver cancer comprising administering neferine to a mammal.

As used herein, the term "mammal" refers to a mammal that is the subject of treatment, observation or experimentation, preferably human.

As used herein, the term “therapeutically effective amount” means an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, doctor or other clinician, which Amounts that induce alleviation of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dosages and frequency of administrations for the active ingredients of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by one skilled in the art and includes the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, general health of the patient. , Sex and diet, time of administration, route of administration and rate of composition, duration of treatment, and drugs used concurrently. In the treatment method of the present invention, in the case of an adult, the neferine of the present invention is preferably administered at a dose of 0.01 mg / kg to 250 mg / kg once or several times a day.

In the treatment method of the present invention, the composition comprising the neferine of the present invention as an active ingredient is an oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal route. Can be administered in a conventional manner.

The present invention also provides a health functional food for preventing or improving liver cancer comprising neferine as an active ingredient.

The health functional food of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like for the purpose of preventing and improving liver cancer.

In the present invention, "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to the Health Functional Food Act No. 6767, and nutrients for the structure and function of the human body. It is meant to be consumed for the purpose of regulating or obtaining a useful effect for health use such as physiological action.

The health functional food of the present invention may include a conventional food additive, and the suitability as a food additive, unless otherwise specified, in accordance with the General Regulations of the Food Additives and General Test Methods approved by the Food and Drug Administration, etc. Judging by the standards and standards.

Examples of the items listed in the "Food Additive Reduction" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark blue pigment, licorice extract, crystalline cellulose, high color pigment and guar gum; And mixed preparations such as sodium L-glutamate, algae additives, preservatives and tar dyes.

For example, the health functional food in the form of tablets is a mixture of neferine, an active ingredient of the present invention, with an excipient, a binder, a disintegrant, and other additives, followed by granulation in a conventional manner, and then a lubricant and the like. Compression molding, or the mixture may be directly compression molded. In addition, the health functional food in the form of tablets may contain a mating agent or the like as necessary.

Hard capsules among the health functional foods in the form of capsules may be prepared by filling a mixture of additives such as excipients with neferine, which is the active ingredient of the present invention, in a conventional hard capsule, and the soft capsules are neferine ) Can be prepared by filling a capsule base such as gelatin with a mixture of additives such as excipients. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.

The cyclic health functional food can be prepared by molding a mixture of neferine, an active ingredient of the present invention, an excipient, a binder, a disintegrant, and the like by using a conventionally known method. It may be encapsulated with a skin coating, or the surface may be coated with a material such as starch, talc.

The health functional food in the form of granules can be prepared by granulation of a mixture of neferine, an excipient, an excipient, a binder, a disintegrant, and the like, which is an active ingredient of the present invention. A mating agent and the like.

The health functional food may be beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

<Example>

reagent

DMSO, BSA and propidium iodide were purchased from Sigma Aldrich (St. Louis, MO, USA); EZ-Cytox Cell Viability Assay Solution WST-1 was purchased from Daeil Lab Service (Jong-No, Seoul, Korea); DAPI was purchased from Roche (Pleasanton, CA, USA); Lysis buffer [50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1 mM DTT, 0.5% NP-40, 1% Triton X-100, 1% deoxycholate, 0.1% SDS] and proteinase inhibitors (PMSF, EDTA, Aprotinin, Leupeptin, and Prostatin A) were purchased from Intron biotechnology (Gyeonggi, Korea); The Protein Quantification Kit (CBB solution) was purchased from Dojindo Molecular Technologies (Rockville, MD, USA); Nitrocellulose membranes were purchased from PALL Life Sciences (Ann Arbor, MI, USA); Enhanced chemiluminescent (ECL) detection solutions were purchased from Pierce (Rockford, IL, USA); antibodies against caspase-12 and cyclin D1 were purchased from Abcam (Cambridge, MA, USA); And c-Myc, Cyclin D3, CDK4, E2F-1, p21Waf1 / Cip1, phospho-cdc2 (Tyr15), GAPDH, Bim, Bid, Bax, Bak, Puma, β-actin, cleaved caspase-8, caspase-3, cleaved caspase-3, cleaved caspase-6, cleaved caspase-7, cleaved PARP, Bip, calnexin, PDI, Calpain 2, LC3B, SirT6, DFF45 / DFF35, Lamin A / C, HRP conjugated anti-rabbit with anti-mouse, Antibodies to anti-mouse IgG (H + L), F (ab ') 2 fragment (Alexa Fluor 555 Conjugate) and anti-rabbit IgG (H + L), F (ab') 2 fragment (Alexa Fluor 488 Conjugate) Were purchased from Cell Signaling Biotechnology (Beverly, MA, USA).

statistics

All experimental results were expressed as mean ± standard deviation and one-way ANOVA according to Tukey's test using statistical software Sigma plot v.12.3 (Systat Software, Inc., San Jose, CA, USA) for statistical analysis of the results. Was performed. P = <0.001 was determined to be statistically significant.

< Example  1> Neferrin ( neferine Tablets

2.0 kg of lotus ( N. nucifera ) embryos were extracted under reflux for 3 hours in 3.0 L methanol and concentrated through vacuum drying at 40 ° C. to obtain 543.5 g of lotus embryo methanol extract. The extract obtained through the above process was suspended in distilled water and sequentially partitioned into dichloromethane, ethyl acetate and n-butanol to obtain 13.8 g of dichloromethane fraction, 2.2 g of ethyl acetate fraction and 58.3 g of butanol fraction, respectively. Water obtained 469.0 g. The dichloromethane fraction (13.8 g) in the fraction was again subjected to chromatography on a silica gel column (120 mm id), whereby finally, 920 mg of neferine was obtained. At this time, a mixed solvent of benzene-ethyl acetate-diethylamine (7: 2: 1, isocratic) was used as the elution solvent.

In order to confirm that the material finally obtained through the above process was neferrin, spectra including ¹ H- and ¹³ C-NMR were analyzed, and the analytical data was compared with the previously published data. Was verified.

¹ H-NMR (400 MHz, CDCl 3): δ 6.88 (2H, d, J = 8.4 Hz, H-11 / H-15), 6.81 (1H, d, J = 8.1 Hz, H-14 ′), 6.66 (3H, d, J = 8.2 Hz, H-15 ′ / H-12 / H-14), 6.62 (1H, s, H-5), 6.51 (1H, m, H-11 ′), 6.49 (1H , s, H-5 ′), 6.35 (1H, s, H-8), 5.98 (1H, s, H-8 ′), 3.79 (3H, s, 6-OCH3), 3.76 (3H, s, 7 ′ -OCH3), 3.70 (3H, s, 13-OCH3), 3.60 (2H, m, H-1 / H-1 ′), 3.52 (3H, s, 6′-OCH3), 3.15-2.95 (4H, m, H-3 / H-3 ′ / H-9 / H-9 ′), 3.04 (1H, dd, J = 12.0, 5.0 Hz, H-9), 2.98 (1H, dd, J = 12.0, 5.3 Hz, H-9 ′), 2.832.53 (8H, m, H-3 / H-3 ′ / H-4 / H-4 ′ / H-9 / H-9 ′), 2.47 (3H, H- 2), 2.43 (3H, s, H-2 '); ¹³ C-NMR (100 MHz, CDCl 3): δ 157.7 (C-13), 148.8 (C-6), 147.1 (C-7 ′), 146.2 (C-6 ′), 145.5 (C-13 ′), 144.7 (C-12 '), 142.7 (C-7), 131.6 (C-10'), 131.3 (C-10), 130.8 (C-8a), 130.3 (C-4a), 130.3 (C-11) , 130.3 (C-15), 129.0 (C-4a ′), 125.5 (C-8a ′), 125.1 (C-11 ′), 119.8 (C-8), 119.1 (C-15 ′), 115.6 (C -14 '), 113.3 (C-12), 113.3 (C-14), 112.2 (C-5), 111.0 (C-5'), 110.9 (C-8 '), 64.6 (C-1'), 64.3 (C-1), 55.7 (7'-OCH3), 55.6 (6'-OCH3), 55.3 (6-OCH3), 55.0 (13-OCH3), 47.0 (C-3), 46.5 (C-3 ' ), 42.5 (C-2), 42.3 (C-2 '), 40.5 (C-9), 39.7 (C-9'), 26.0 (C-4), 25.0 (C-4 ').

< Example  2> Cell line and culture

HCC Hep3B, Sk-Hep1, THLE-3 (cells immortalized by infecting normal hepatocytes with SV40 large T antigen) and HUVEC (human venous venous endothelial cells) cells were obtained from the American Tissue Culture Collection (Manassas, VA, USA) .

Hep3B and Sk-Hep1 cells were cultured using minimum essential medium (MEM / EBSS) (HyClone, Logan, UT, USA) containing Earle's Balanced Salts, and THLE-3 cells were gentamicin / Empotericin and epinephrine were removed and cultured using Bronchial Epithelia Cell Growth Medium (Clonetics BEGM bullet kit; CC3170, Lonza, Walkersville, MD, USA) medium containing 500 ml basal medium, 5 ng / ml EGF, 70 ng. Culture was added by adding / ml phosphoethanolamine. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured using endothelial basal medium-2 (EBM-2) supplemented with EGM-2 kit (Lonza). All cell lines in this experiment were 37 ° C., 5% CO in media containing 10% heat inactivated FBS (HyClone), 100 U / ml penicillin, and 100 μg / ml streptomycin (PAA Laboratories GmbH, PA, Austria). _It was incubated in the conditions of ₂ . THLE-3 cells were coated with 0.01 mg / ml BSA, 0.01 mg / ml fibronectin and 0.03 mg / ml bovine collagen type Ι (BD biosciences, San Jose, Calif., USA) for 2 hours at 37 ° C and then incubated.

< Experimental Example  1>

In this experiment, the cell viability assay was performed using Hep3B, Sk-Hep1, and THLE-3 cell lines cultured in Example 2 to confirm the cytotoxicity of the cancer cell line of nephrin obtained through Example 1. .

First, Hep3B, Sk-Hep1 and THLE-3 cells were each resuspended in 100 μl medium at 1 × 10 ⁴ cells density and dispensed into three 96-well plates and incubated for 24 hours. Then, neferrin at various concentrations (5, 10, 15, 20, 25, 30 μM) was treated with the cells, incubated for 24 hours, exchanged with fresh medium, and then added with 10 μl of WST-1 solution for 5 hours. Reacted for a while. Reaction absorbance was measured at 460 nm using an ELISA reader (Molecular Devices, Sunnyvale, Calif., USA). The results were expressed as mean ± standard deviation through three independent experiments, and the inhibition graph was derived using the mean values obtained from each concentration associated with the control values.

As a result, as shown in FIG. 1, only Hep3B cell lines among Hep3B, Sk-Hep1 and THLE-3 cell lines showed a significant decrease in cell viability depending on the neferrin treatment concentration. In particular, there was a marked cell suppression in the experimental group treated with neferrin concentration of 10 μM or more. On the other hand, Sk-Hep1 and THLE-3 cells did not show cytotoxicity according to the neferrin treatment, and in the 30 μM concentration of the nephrine treated group showing less than 10% cell viability in the Hep3B cell line, the Sk-Hep1 and THLE-3 cells. The survival rate of the cells was measured to reach about 90%.

These results confirm that neferrin has a specific cytotoxic sensitivity to Hep3B cells. In other words, neferin did not show cytotoxicity to both Hep3B and Sk-Hep1 liver cancer cell lines, and showed specific cytotoxicity to Hep3B cell line, suggesting that hepatic cancer cells may have anticancer activity in different types.

Through the above experiment, the nephrine of the present invention confirmed specific cytotoxicity to Hep3B cell line even in hepatocellular carcinoma cell line. In the following experiment, experiments were conducted on various mechanisms related to anticancer activity in Hep3B cell line.

< Experimental Example  2> Neferrin  Induction of Apoptosis on Liver Cancer Cells

In this experiment, we conducted a DAPI assay to examine the ability of nephrine to induce apoptosis on Hep3B, a liver cancer cell line.

DAPI is an abbreviation of 4, 6-diamidino-2-phenylindole dihydrochloride hydrate and is a probe that can be permeated to cells and used to stain living cell nuclei in a fixed state. It forms a bond with DNA and a minor groove, and prefers to bind with AT rich DNA, and becomes stable through hydrogen bonding. DAPI bound to DNA is about 20 times brighter than non-DNA bound and emits light by ultraviolet light under a fluorescence microscope. When combined with DNA, it exhibits an absorption maximum at 358 nm and an emission maximum at 461 nm. In this experiment, hepatic cancer cells were stained with DAPI for detection of nuclear condensation and apoptosome formation following neferrin treatment and analyzed under fluorescence microscope (× 1000).

For this experiment, Hep3B cells were first incubated in a cover glass bottom culture dish for 24 hours, treated with neferin (20, 25 μM) at 6 hour intervals, and washed once with PBS buffer, followed by DAPI solution (1 μg / ml). Staining was added. Incubated in the dark at 37 ° C. for 20 minutes, and the cells were washed once again with methanol. Nuclear changes were observed using an ECLIPSE 50i fluorescence microscope (Nikon, Tokyo, Japan).

As a result, as shown in FIG. 2, nuclear membrane changes such as shrinkage and blebbing of cells were observed in the experimental group 6 hours after the neferrin treatment compared to the non-nephrrine-treated group. It was confirmed that it appeared. In particular, a typical characteristic of apoptotic cells, nuclear condensation (apoptotic formation) was observed in the experimental group 24 hours after neferrin treatment.

< Experimental Example  3> Neferrin  Cell cycle arrest for liver cancer cells cell cycle arrest ) Judo

In this experiment, to investigate the cell cycle arrest induction of hep3B, hepatic cancer cell line, cell cycle analysis; Protein expression analysis associated with cell cycle via Western blot; And immunofluorescence analysis.

<3-1> Cell Cycle Analysis

Hep3B cells were treated with nephrine at different concentrations (15, 20, 25 μM) for 24 hours, and then treated with trypsin, and then fixed overnight using 70% ethanol at 4 ° C. The cells were then resuspended in PBS buffer containing 0.2 mg / ml RNase A and then further incubated at 37 ° C. for 1 hour. The cells were stained with 40 μg / ml propidium iodide (PI) for 30 minutes in the dark. Dispersion of subgenomic DNA content was analyzed using flow cytometer (BD biosciences).

As a result, as shown in Figure 3, it was confirmed that the sub-G1 cell population increases in proportion to the neferrin treatment concentration (15, 20, 25μM). In particular, the sub-G1 cell population showed 1.35% in the untreated group without neferrin, while 4.78% in the experimental group treated with 15 μM concentration of neferrin, and 16.01% in the experimental group treated with 20 μM concentration of neferin. Increased. In addition, it was confirmed that the G1 / S phase was increased approximately 9% in the experimental group treated with 20 μM concentration of neferrin compared to the untreated group.

Through these results, it was confirmed that when hep3B cells were treated with neferrin, cell growth of hepatocellular carcinoma was inhibited through the suspension of sub-G1 and G1 phases.

<3-2> Analysis of Protein Expression Related to Cell Cycle

Western blots were performed for expression analysis of cell cycle-related proteins (c-Myc, cyclin D1, cyclin D3, CDK4, E2F-1, P21 ^{Waf / Cip1} , p-cdc2).

For this experiment, first, Hep3B cells were treated with neferin by concentration (15, 20, 25 μM) for 24 hours, washed twice with cold PBS, and then lysed with cold lysis buffer. Then, the reaction was carried out on ice for 30 minutes, followed by centrifugation at 14,000 rpm for 20 minutes to remove insoluble matters. Protein content of the cell lysate was measured using a Protein Quantification Kit (CBB solution). Western blots were performed using 30 μg of protein isolated via electrophoresis on a 12% polyacrylamide gel, transferred to nitrocellulose membrane and subjected to immune reaction with indicator antibody. Chemiluminescence was detected using chemiluminescent (ECL) reagent.

As a result, as shown in Figure 4, the dephosphorylation of cdc2 in the experimental group treated with neferrin for each concentration; down regulation of c-Myc, cyclin D1, D3, CDK4, E2F-1; And p21 ^{Waf1 / Cip1} protein expression was increased. These results are in agreement with Experimental Example <3-1> as evidence of cell cycle arrest.

<3-3> Immunofluorescence

Cultured Hep3B cells were reacted for 12 hours by treatment with 15 μM concentration of neferrin on a cover-glass bottom dish. Cells were pretreated with 1 μg / ml DAPI for 20 minutes at 37 ° C., then fixed with 4% formaldehyde (JUNSEI Chemical Co., Japan) at 25 ° C. for 15 minutes, then 5% mice and 0.3% Triton X-100 It was blocked for 1 hour with a blocking solution containing normal rabbit serum (Santa Cruz Biotechnology Inc.) with (Sigma-Aldrich, St. Louis, MO, USA). Fixed and blocked cells were reacted with the primary antibody (p21Waf1 / Cip1) and then washed three times with PBS buffer. After washing, the cells were treated with 0.1 μg / ml anti-mouse IgG (H + L), F (ab ') 2 fragment (Alexa Fluor 555 Conjugate) and anti-rabbit IgG (H + L), F (ab') 2. Fragment (Alexa Fluor 488 Conjugate) was treated for 1 hour. Stained cells were transferred to slides with Prolong Gold Antifade Reagent (Invitrogen, Grand Island, NY, USA) and measured using an ECLIPSE 50i fluorescence microscope equipped with a charged-coupled device (CCD) camera. Images were captured and processed with High-Content Analysis Software (Cambridge Healthtech Ins., Needham, MA, USA).

As a result, as shown in Figure 5, compared to the non-neferrin-treated group, hep3B cells treated with neferin was found to significantly increase the expression level of p21 ^{Waf1 / Cip1} protein around the nucleus in the cytoplasm. .

< Experimental Example  4> Neferrin  Apoptosis on Liver Cancer Cells apoptosis ) Judo

In this experiment, we performed Western blot to measure the degree of Bcl-2 family protein expression and caspase activation related to apoptosis in order to investigate whether apoptosis of Hep3B, a hepatic cancer cell line of nephrin, was induced. Immunofluorescence was further performed.

<4-1> Weston Blot  analysis

Western blot was performed to analyze the degree of Bcl-2 family protein (Bim, BID, Bax, Bak, Puma) expression and caspase activation associated with apoptosis regulation.

The experimental procedure was performed in the same manner as in Experimental Example <3-2>.

FIG. 6A shows Western blot results of Bcl-2 family proteins (Bim, Bid, Bax, Bak and Puma) known to modulate mitochondrial-mediated apoptosis pathways, whereby Bak and Puma protein expression was treated with nephrin. It was shown that the concentration was significantly increased, and Bim, Bid and Bax were found to be slightly increased in expression. In addition, Figure 6b shows the Western blot results of apoptotic proteins (cleaved caspase-8, -3, -6, -7 and PARP) associated with apoptosis due to endoplasmic reticulum stress, through which caspase-3 protein expression is neferin It was found that the expression of cleaved caspase-6 and cleaved PARP protein was markedly increased while it was shown to decrease depending on the treatment concentration.

<4-2> Immunofluorescence

In order to further confirm the results shown in Experimental Example <4-1>, the expression levels of several apoptosis-related proteins were observed through immunofluorescence in this experiment.

The experimental procedure was performed in the same manner as in Experimental Example <3-3> except that cleaved caspase-3 and cleaved caspase-8 were used as primary antibodies.

As a result, as shown in Figure 7, cleaved caspase-8, and -3 was found to be markedly expressed in Hep3B cells treated with neferrin compared to the untreated group not treated with neferrin. These results suggest that nephrine-induced apoptosomes induce apoptosis by activating caspase-3 and caspase-8 in the cytoplasm.

< Experimental Example  5> Neferrin  Vesicular Stress and Autophagy on Liver Cancer Cells autophagy ) Judo

Autophagy has mechanisms by which eukaryotic cells, old organelles, damaged organelles, and cytoplasm are phagocytized, digested, and reused, which are involved in the turnover of basic cellular components or changes in the external environment, ie nutrients. It may also be induced by depletion, pathogen infiltration and oxygen starvation. Recently published studies have reported that cancer cells may have anti-cancer effects by inducing autophagy. Autophagy has been reported to be activated by tumor therapies in many tumor cell lines, but the association between cancer formation and inhibition has not been elucidated.

In this experiment, to determine whether nephrine can induce autophagy as an anticancer effect on hepatocellular carcinoma cells, we investigated whether nephrine causes vesicle stress on Hep3B, a hepatocellular carcinoma cell line, and whether autophagy is present.

<5-1> In neferrin  Induction of Vesicular Stress in Liver Cancer Cells

First, in order to confirm the induction of endoplasmic reticulum stress by neferin, Hep3B cells were treated with neferin and analyzed for Western blot expression of endoplasmic reticulum chaperone protein. Western blot analysis was performed in the same manner as in Experimental Example <3-2>.

As a result, as shown in Figure 8, the neferin-treated experimental group was found to significantly up-regulate Bip, calnexin, PDI, calpain2 and caspase-12 protein expression levels depending on time. These results mean that the expression of chaperon protein is increased by intracellular mechanisms for endoplasmic reticulum stress relief because nephrin of the present invention causes endoplasmic reticulum stress in liver cancer cell line.

In addition, in order to demonstrate the induction of the endoplasmic reticulum stress of the neferin, after treatment with Hep3B cells for 15 hours at 15 μM concentration of neferin, the expression level of the protein caspase-12 located on the endoplasmic reticulum membrane was analyzed by immunofluorescence. As a result, the expansion of the endoplasmic reticulum was observed as shown in FIG. These results indicate that the cytoplasmic vacuoles induced by nephrin may expand the endoplasmic reticulum to cysternae. The experimental procedure was performed in the same manner as in Experimental Example <3-3> except that caspase-12 was used as the primary antibody.

<5-2> In neferrin  Caused by liver cancer cells Self-feeding ( autophagy ) Judo

LC3 is an important component in the development of autophagy and is used as a specific marker for autophagy by participating in the formation of autophagosome double membranes. LC3 exists in two forms in cells. LC3-Ι is pre-existing in the cytoplasm and is transformed into LC3-II when stressed inside or outside the cell, which is involved in the formation of autophagosome bilayers. Therefore, when much LC3-II is expressed, autolysates are also in a correlation.

In this experiment, we examined the expression distribution of plasmid GFP-LC3 over time after treatment with 20 μM of nephrine in Hep3B cells to determine whether LC3 is related to autophagy induced by nephrine. The degree of autophagosome formation in the cytoplasm was confirmed.

First, GFP-LC3B (green fluorescent protein-light chain 3B) plasmids were prepared, and the present plasmid was transformed by introducing Hep3B cells. Specifically, PCR was used to amplify LC3B full-length sequences from human liver cDNA libraries (Clontech, Mountain View, CA, USA). Primer sequences used for PCR amplification are shown in Table 1 below. Purified PCR products were introduced into the pGEM-T Easy vector (Promega, Madison, Wis., USA) and then cleaved back with HindIII and Bam Hl and fused with N-terminal GFP in the pEGFP-N2 vector (Clontech). The structure thus prepared was confirmed through DNA sequencing and named GFP-LC3B plasmid. Meanwhile, Hep3B cells were cultured in sterile 6-well plates for 24 hours, and then transformed into GFP-LC3B plasmid using Fugene 6 reagent (Roche Diagnostics GmbH, Mannheim, Germany) mixture in FBS-free medium. Transformed cells were incubated in regular complete medium for one day, then treated with 20 μM neferrin at regular intervals (6, 12, 18, 24), washed with cold PBS buffer and fixed in cold methanol for 5 minutes at room temperature. I was. The cells were washed again with PBS buffer twice and placed on coverslips with Gold Antifade Reagent. The cells were observed using an ECLIPSE 50i fluorescence microscope.

Table 1 Primer sequence used in PCR

gene	Primer sequence
LC3B	sense	5'-CCGGAATTCCATGCCCTCAGACCGGCCTTT-3 '
	antisense	5'-CGCGGATCCTCAGAAGCCGAAGGTTTCCTG-3 '

As a result, as shown in FIG. 10, in the experimental group treated with neferrin, formation of vacuole in the cytoplasm, which is one of morphological characteristics of autophagy, occurred over time. These bodies were found to grow larger and larger in time. Autophagy also forms vesicles enclosed by a double membrane called autophagosomes, which show strong fluorescence spots (GFP-LC3B "dots") over time in the nephrine treated group. It was confirmed that the increase in the cytoplasm. Although some green fluorescence in the cytoplasm of FBS (-) cells is seen, it is not in the form of autophagosomes.

In addition, as a result of analyzing the increase in the expression of LC3B according to the treatment of the neferrin through Western blot, as shown in FIG. It was confirmed that the concentration increased depending on the neferrin treatment.

< Experimental Example  6> Neferrin HUVECs  Inhibitory effect on neovascularization in cells in in vitro )

In this experiment, a tube formation assay was performed to evaluate the effect of nephrine inhibition of angiogenesis.

Lab-Tek chamber slides (Thermo Fisher Scientific, NY, USA) were coated with 200 μl Matrigel per well (BD Biosciences) and reacted for 30 minutes at 37 ° C. to solidify. Human Umbilical Vein Endothelial Cells (HUVECs) were dispensed at a density of 5 × 10 ⁴ per well on Matrigel and cultured at 5% CO ₂ , at 37 ° C. for 18 hours at varying nepherin concentrations. Incubated at Tube formation was observed at × 40 magnification using phase contrast inverted microscopy (Olympus CKX41; Olympus Optical Co. Ltd, Tokyo, Japan). Tube formation image analysis was performed using the Wimasis Image Analysis software (Wimasis GmbH, Munich, Germany).

As a result, as shown in Figure 12, it was confirmed that the tube length (px) is reduced depending on the neferrin concentration. In detail, it was observed that the suppression of the formation of cellular vascular structures occurred in the neferrin-treated experimental group (see FIG. 12A), and the degree of tube formation was determined using the Wimasis Image Aalysis software. As a result of the conversion to percent, 222 was shown in the experimental group not treated with nephrin, while 216 in the experimental group treated with 10 μM concentration of neferrin, 202 in the experimental group treated with 20 μM concentration of neferrin, and 30 μM concentration of neferin In the experimental group treated with it was confirmed that the reduction to 146 (see Fig. 12B).

Experimental Example 7

Neferrin  Cell migration inhibitory effect on liver cancer cells

In this experiment, to determine whether nephrine can induce cell migration as an anticancer effect on hepatocellular carcinoma cells, wound-healing assay was performed after treating neferin to Hep3B, a hepatic cancer cell line. ) Was performed.

Hep3B (3.5 × 10 ⁴ cells per well) cells were dispensed into an IBIDI culture insert (Ibidi GmbH, M, Germany) chamber consisting of two reservoirs separated by a 50 μm septum, followed by 5% CO ₂ at 24 ° C. Incubate for hours. After incubation, the contents were removed and the cells harvested and re-cultured in culture medium. Cell migration was measured at 12 hour intervals after neferrin treatment using an inverted microscope. Images were calculated as percentage of cell-covered area using Wimasis Image Analysis software.

As a result, as shown in FIG. 13, it was confirmed that cell migration was suppressed depending on the neferrin treatment concentration. In particular, the cell coverage area was reduced by about 20% in the experimental group treated with neferrin at a concentration of 15 μM after 72 hours of treatment, compared to the increase in cell migration in the untreated group without nephrin treatment. 13B).

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Explanation of the sign

HCC: hepatocellular carcinoma

CDKs: Cyclin-dependent kinases

ER: endoplasmic reticulum

HBV: hepatitis B virus

DMSO: Dimethyl sulfoxide

BSA: bovine serum albumin

HUVECs: human umbilical vein endothelial cells

WST-1: 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt

DAPI: 4, 6-diamidino-2-phenylindole dihydrochloride hydrate

GFP: Green fluorescent protein

PBS: phosphate-buffered saline

DFF: DNA fragmentation factor

GAPDH: glyceraldehyde-3-phosphate dehydrogenase

PARP: poly (ADP-ribose) polymerase

PDI: protein disulfide isomerase

LC3B: light chain 3 B

SirT6: sirtuin-6

PCD: programmed cell death

MOMP: mitochondrial outer membrane permeabilization

Neferin of the present invention is a substance capable of preventing or treating liver cancer, as well as pharmaceuticals such as pharmacological compositions, and can be used as a health functional food, etc. It is expected that its value will be very high when applied to food industry and pharmaceutical industry . In addition, since the neferine of the present invention does not show cytotoxicity except certain liver cancer cells, the composition of the present invention comprising the same as an active ingredient has a safe advantage even for long-term use.

Claims (6)

1. A pharmaceutical composition for preventing or treating liver cancer comprising Neferine represented by Formula 1 as an active ingredient;

   <Formula 1>

   
2. The method of claim 1,

   The neferine (Neferine) is a pharmaceutical composition for preventing or treating liver cancer, characterized in that derived from the lotus embryo.
3. The method of claim 1,

   The liver cancer is hepatocellular carcinoma (hepatocellular carcinoma) caused by viral infection, characterized in that for preventing or treating liver cancer.
4. The method according to any one of claims 1 to 3,

   The pharmaceutical composition is characterized in that it has anticancer effect through cell cycle arrest, induction of apoptosis, induction of autophagy, inhibition of angiogenesis and cell migration of cancer cells. Prophylactic or therapeutic pharmaceutical compositions.
5. Health functional food for the prevention or improvement of liver cancer comprising Neferine (Neferine) represented by the formula (1) as an active ingredient;

   <Formula 1>

   
6. The method of claim 5,

   The food is a health functional food, characterized in that selected from the group consisting of beverages, meat, chocolate, food, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements.

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