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WO2014051359A1 - Composition pharmaceutique comprenant de la néférine comme ingrédient actif de prévention contre un hépatome ou de traitement de ce dernier - Google Patents

Composition pharmaceutique comprenant de la néférine comme ingrédient actif de prévention contre un hépatome ou de traitement de ce dernier Download PDF

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WO2014051359A1
WO2014051359A1 PCT/KR2013/008639 KR2013008639W WO2014051359A1 WO 2014051359 A1 WO2014051359 A1 WO 2014051359A1 KR 2013008639 W KR2013008639 W KR 2013008639W WO 2014051359 A1 WO2014051359 A1 WO 2014051359A1
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cells
neferine
liver cancer
neferrin
cell
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Korean (ko)
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윤진수
김군도
최재수
최영현
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Pukyong National University
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Pukyong National University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/62—Nymphaeaceae (Water-lily family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • 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.
  • HCC hepatocellular carcinoma
  • 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.
  • Hepatitis viruses include A, B, C, D, E, and F.
  • hepatitis G has been discovered.
  • viruses that are a problem in our country, A, B, and C. Of these, two are related to liver cancer: B and C.
  • 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.
  • liver cancer treatment agent having high sensitivity without side effects.
  • Neferine 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.
  • 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.
  • 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.
  • 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.
  • the present invention provides a pharmaceutical composition for preventing or treating liver cancer comprising Neferine (Neferine) as an active ingredient.
  • Neferine Neferine
  • the neferine may be derived from the lotus embryo.
  • the liver cancer may be hepatocellular carcinoma caused by viral infection.
  • 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.
  • Neferine Neferine
  • the neferine may be derived from the lotus embryo.
  • the liver cancer may be hepatocellular carcinoma caused by viral infection.
  • 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.
  • 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, 25 ⁇ M) at 6 hour intervals.
  • 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, 25 ⁇ M) by Western blot.
  • FIG. 5 shows that p21 Waf1 / Cip1 protein expression levels detected by immunofluorescence analysis after treatment of Hep3B cells with 15 ⁇ M 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 ⁇ M) 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 ⁇ M) 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 ⁇ M 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 ⁇ M concentration of neferin (cleaved caspase-8). : green, nucleic: blue, cytoskeletal actin: red).
  • vesicle stress-related chaperon proteins (Bip, calnexin, PDI, calpain2 and caspase-12) over time after treatment with 20 ⁇ M 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 ⁇ M 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 ⁇ M of nephrine in Hep3B cells transformed with GFP-LC3B plasmid. .
  • 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 ⁇ M) 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.
  • 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.
  • Neferine represented by the following formula (1) as an active ingredient.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the cell cycle is performed in a certain order by a mechanism defined in the cell.
  • 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').
  • 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.
  • the specific cyclin-CDK complex is activated and the proteins phosphorylated specifically for CDK are responsible for the progression of the cell cycle.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.
  • 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.
  • the separation method 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.
  • 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.
  • 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.
  • the active ingredient may be combined with an oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • 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.
  • the neferrin of the present invention may be included in the composition in a concentration of 1 to 300 ⁇ M, 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.
  • 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.
  • mammal refers to a mammal that is the subject of treatment, observation or experimentation, preferably human.
  • 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.
  • 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.
  • 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.
  • 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.
  • Food Additive Reduction examples 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.
  • 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.
  • 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 )
  • 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 are examples of neferine, an excipient, an excipient, a binder, a disintegrant, 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.
  • 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
  • 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.
  • a mixed solvent of benzene-ethyl acetate-diethylamine (7: 2: 1, isocratic) was used as the elution solvent.
  • 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.
  • MEM / EBSS minimum essential medium
  • 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
  • HAVECs Human Umbilical Vein Endothelial Cells
  • EBM-2 endothelial basal medium-2
  • EGM-2 kit Lignogen kit
  • 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 ⁇ g / 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 ⁇ (BD biosciences, San Jose, Calif., USA) for 2 hours at 37 ° C and then incubated.
  • Example 2 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. .
  • Hep3B, Sk-Hep1 and THLE-3 cells were each resuspended in 100 ⁇ l 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 ⁇ M) was treated with the cells, incubated for 24 hours, exchanged with fresh medium, and then added with 10 ⁇ l 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.
  • neferrin has a specific cytotoxic sensitivity to Hep3B cells.
  • 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.
  • the nephrine of the present invention confirmed specific cytotoxicity to Hep3B cell line even in hepatocellular carcinoma cell line.
  • experiments were conducted on various mechanisms related to anticancer activity in Hep3B 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).
  • Hep3B cells were first incubated in a cover glass bottom culture dish for 24 hours, treated with neferin (20, 25 ⁇ M) at 6 hour intervals, and washed once with PBS buffer, followed by DAPI solution (1 ⁇ g / 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).
  • 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.
  • a typical characteristic of apoptotic cells nuclear condensation (apoptotic formation) was observed in the experimental group 24 hours after neferrin treatment.
  • Hep3B cells were treated with nephrine at different concentrations (15, 20, 25 ⁇ M) 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 ⁇ g / ml propidium iodide (PI) for 30 minutes in the dark. Dispersion of subgenomic DNA content was analyzed using flow cytometer (BD biosciences).
  • PI propidium iodide
  • the sub-G1 cell population increases in proportion to the neferrin treatment concentration (15, 20, 25 ⁇ M).
  • the sub-G1 cell population showed 1.35% in the untreated group without neferrin, while 4.78% in the experimental group treated with 15 ⁇ M concentration of neferrin, and 16.01% in the experimental group treated with 20 ⁇ M concentration of neferin. Increased.
  • the G1 / S phase was increased approximately 9% in the experimental group treated with 20 ⁇ M concentration of neferrin compared to the untreated group.
  • Hep3B cells were treated with neferin by concentration (15, 20, 25 ⁇ M) 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 ⁇ g 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.
  • ECL chemiluminescent
  • 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.
  • Bcl-2 family proteins Bim, Bid, Bax, Bak and Puma
  • 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.
  • apoptotic proteins cleaved caspase-8, -3, -6, -7 and PARP
  • 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.
  • 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.
  • 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- ⁇ 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.
  • GFP-LC3B green fluorescent protein-light chain 3B plasmids were prepared, and the present plasmid was transformed by introducing Hep3B cells.
  • 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).
  • GFP-LC3B plasmid 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 ⁇ M 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.
  • 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 ⁇ m 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.
  • the cell migration was suppressed depending on the neferrin treatment concentration.
  • the cell coverage area was reduced by about 20% in the experimental group treated with neferrin at a concentration of 15 ⁇ M after 72 hours of treatment, compared to the increase in cell migration in the untreated group without nephrin treatment. 13B).
  • HCC hepatocellular carcinoma
  • CDKs Cyclin-dependent kinases
  • ER endoplasmic reticulum
  • HBV hepatitis B virus
  • 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
  • PARP poly (ADP-ribose) polymerase
  • 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.

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CN116712449A (zh) * 2023-08-01 2023-09-08 北大荒集团齐齐哈尔医院(北大荒集团齐齐哈尔妇幼保健院) 一种治疗肾性高血压的药物组合物

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KR102348878B1 (ko) * 2014-08-13 2022-01-06 주식회사 엘지생활건강 네페린 또는 이의 약학적으로 허용가능한 염을 포함하는 피부 미백, 탄력, 주름개선, 또는 보습용 화장료 또는 약학 조성물
WO2020225611A1 (fr) * 2019-05-07 2020-11-12 Sardeshmukh Sadanand Prabhakar Composition herbo-lipidique et son procédé de préparation

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WO2017142283A1 (fr) * 2016-02-15 2017-08-24 서울대학교산학협력단 Composition permettant de traiter ou de prévenir le cancer du foie
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CN109069513B (zh) * 2016-02-15 2021-12-28 硕氏医科有限公司 用于治疗或预防肝癌的组合物
CN116712449A (zh) * 2023-08-01 2023-09-08 北大荒集团齐齐哈尔医院(北大荒集团齐齐哈尔妇幼保健院) 一种治疗肾性高血压的药物组合物

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