KR101530910B1 - Anti-cancer Composition Using an Extract of Adonis amurensis or an Effective Compound Isolated Therefrom - Google Patents

Abstract

The present invention discloses an anticancer agent composition using a herbal extract of herbal cancers exhibiting anticancer activity in cell experiments and animal experiments or an effective substance thereof.

Description

[0001] The present invention relates to an anticancer composition using an anticancer composition or an effective substance thereof,

The present invention relates to an anticancer composition using an extract of Adonis multiflora or an effective substance thereof.

Cancer generally refers to malignancy in which the cell cycle of a cell that constitutes a human tissue is abnormal and the cell does not normally differentiate and can not control growth.

Cancer occurs through three stages: initiation, promotion, and progression. Cells are mutated by the carcinogens contained in the environment or food, and these cells are constantly stimulated by carcinogens, It is known to continue the proliferation to form cancer tissue.

Research methods of cancer drugs to treat cancer include a method of searching for a direct cytotoxic substance to cancer cells, a method of searching for a substance that regulates immune ability of a living body, a method of searching for a substance inhibiting cancer cell metastasis, And a method for searching for a substance inhibiting angiogenesis.

The currently used anticancer drugs can be roughly classified into biological agents such as enzymes and vaccines, chemical synthetic drugs, and medicines derived from natural materials. Of these, biological agents such as enzymes and vaccines are not in a practical stage, (Gillman, et al., 18, pp1202, 1986), and it is pointed out as a problem in the treatment of cancer due to various toxic side effects (Chung, et al., J. Wonkwang Medical Sci., 3, pp 13-34, 1987).

In order to minimize the side effects of such chemically synthesized anticancer drugs and to increase the therapeutic effect, development of anticancer drugs using plant extracts has been continuously tried.

Many plant extracts with anticancer activity have been reported (Pujol M, Gavilondo J, Ayala M, Rodriguez M, Gonzalez EM, Perez L. 2007 Trends Biotechnol 25 (10): 455-459). For example, Angelica gigas ) extracts were isolated from colon cancer (KanWL, Cho CH, Rudd JA, Lin G. 2008. 120 (1): 36-43), colon cancer (Lu J. Kim SH Jiang C, Lee H Guo J. 2007. Acta Pharmacol Sin (9): 1365-1372), brain cancer (Tasi NM, Chen YL, Lee CC, Lin PC, Cheng YL, Chang WL, Lin SZ, Ham HJ 2006. J Neurochem 99 (4): 1251-1262) Have been reported to have anticancer effects against various kinds of cancer such as Glycyrrhiza glabra ) was detected in prostate cancers (Kanazawa M, Satomi Y, Mizutani Y, Ukimura O, Kawauchi A, Sakai T, Baba M, Okuyama T. Nishino H, Miki T. 2003. Eur Urol 43 (5): 580- 586) or breast cancer (Jo EH, Lim SH, Ra JC, Kim SR, Cho SD, Jung JW, Yang SR, Park JS, Hwang JW, Aruoma OI, Kim TY, Lee YS, Kang Ks 2005. Cancer Lett. (2): 239-247).

The present invention has been completed by confirming the anticancer activity of the herbal extracts and their active substances through cell experiments and / or animal experiments.

It is an object of the present invention to provide an anticancer agent composition.

Other and further objects of the present invention will be described below.

As shown in the following Examples and Experimental Examples, the present inventors prepared an 80% ethanol extract of three kinds of herbal extracts, suspending the 80% ethanol extract in distilled water, and washing it with n-hexane, dichloromethane (CH ₂ Cl ₂ ), ethyl acetate (EtOAc) and butanol (BuOH), and then the anticancer activity of the 80% ethanol extract and each fraction extract was firstly examined through cell experiments. As a result, The cell growth inhibitory activity of the cancer cell line A549, gastric cancer cell line AGS, colon cancer cell line HCT-15, breast cancer cell line MDA-MB-231 and hepatoma cell line SK-Hep1 was confirmed As a result of animal experiments with ethyl acetate fraction extract, it was found that HF (Hollow fib using Hap (Fib) cancer cell line A549, gastric cancer cell line AGS or hepatoma cell line SK- er) assay model, the ethyl acetate fraction showed anticancer activity in all tumor cells used in the experiment in a concentration-dependent manner. In particular, in the nude mouse animal model in which hepatocellular carcinoma cell line SK-Hep-1 or HepG-2 was transplanted It was confirmed that the anticancer activity was generally dependent on the concentration.

The present inventors have further succeeded in isolating the active substance from the ethyl acetate fraction and identifying the substance as a novel compound as a compound of the following formula (I).

≪ Formula 1 >

In one aspect, the present invention provides compounds of formula (I) or a pharmaceutically acceptable salt thereof. In another aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, Can be identified as an anticancer composition comprising the compound of the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

As used herein, "anti-cancer" is meant to include cancer cell death, cancer cell proliferation inhibition, cancer cell metastasis inhibition, improvement of pathological symptoms of cancer, treatment or inhibition / delay of onset of such pathological symptoms.

In the present specification, the term "active ingredient" alone means an ingredient which exhibits the desired activity or can exhibit activity together with a carrier which is not itself active.

In the present specification, the term "herb extracts" refers to extracts of three or more kinds of herbs, leaves, stems, flowers, roots or mixtures thereof to be extracted, , An extract obtained by extracting with hexane, ethyl acetate, chloroform, dichloromethane, water or a mixed solvent thereof, and an extract obtained by fractionating the extract with one or more of the above-listed solvents. As long as it is extracted through the step of immersing in the extracting solvent, the extracting method may be any method such as freezing, refluxing, heating, ultrasonic irradiation or the like, It should be understood that all of them can be applied. Nevertheless, the above-mentioned "three-stage herbal extracts" is preferably obtained by extracting leaves, stems, flowers, roots or mixtures thereof, which are the object of extraction, with water, ethanol or a mixed solvent thereof, Extract or solid form of the extract, the extract of the solid form of the fraction, the extract of the solid form, or the solid form extract obtained by fractionating the solid form of the extract with water, hexane, water and dichloromethane, water and ethyl acetate or water and butanol, , Followed by sequential fractionation using hexane, dichloromethane, ethyl acetate and butanol. Here, "sequential fractionation" means that the fraction of the water layer is continuously used after fractionation and is fractionated into the solvent of the above-mentioned order.

The compound of formula (I) of the present invention can be used in the form of a pharmaceutically acceptable salt. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid can be used. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Derived from organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, and the like. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate. The acid addition salt in the present invention can be obtained by a conventional method, for example, by dissolving the compound of formula (1) in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, Followed by filtration and drying. Alternatively, the solvent and excess acid may be distilled off under reduced pressure, followed by drying or crystallization in an organic solvent.

In addition, the compound of formula (I) of the present invention can be prepared into a pharmaceutically acceptable metal salt using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

The compounds of formula (I) of the present invention may be used in the form of solvates, hydrates, stereoisomers, and the like, as well as pharmaceutically acceptable salts thereof.

In the anticancer composition of the present invention, it is preferable that the anticancer activity is an anticancer activity against lung cancer or liver cancer, and the effective ingredient is methylene chloride fraction extract, ethyl acetate fraction extract or Butanol extract. Particularly, the active ingredient is preferably a methylene chloride fraction extract or an ethyl acetate fraction extract. Most preferably, the anticancer activity is an anticancer activity against liver cancer and the active ingredient is an ethyl acetate fraction extract.

The anticancer composition of the present invention may contain any amount (effective amount) of the active ingredient as long as it can exhibit an anticancer activity depending on the purpose of formulation, purpose of formulation, etc., and a typical effective amount is 0.001 To 15% by weight. The term "effective amount" as used herein refers to an amount of an effective amount of a compound of the present invention which is effective in the mammal, preferably a human, to which the compound is administered, for the prevention of cancer cell death, cancer cell proliferation inhibition, cancer cell metastasis inhibition, Of the active ingredient. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art. The subjects to which the anticancer composition of the present invention can be applied (prescription) are preferably mammals and humans, especially humans.

The anticancer composition of the present invention can be used as a pharmaceutical composition in a specific embodiment.

The pharmaceutical composition of the present invention may be in the form of oral formulations (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations (sterile injectable aqueous or non- Ointments, ointments, solutions, creams, ointments, gels, lotions, patches, and the like).

The term "pharmaceutically acceptable" as used herein means that the application (subject) does not have an adaptive or higher toxicity (sufficiently low toxicity) without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (e.g. corn starch, potato starch, etc.), cellulose, derivatives thereof (e.g. sodium carboxymethylcellulose, ethylcellulose, etc.) malt, gelatin, talc, (E.g., peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyol (e.g., propylene glycol, glycerin and the like), alginic acid, emulsifiers (e.g., TWEENS), humectants Such as sodium lauryl sulfate, a coloring agent, a flavoring agent, a tableting agent, a stabilizer, an antioxidant, a preservative, water, a saline solution, and a phosphate buffer solution. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention.

The excipient may be selected according to the formulation of the pharmaceutical composition of the present invention. For example, when the pharmaceutical composition of the present invention is prepared by an aqueous suspension, suitable excipients include sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose , Sodium alginate, polyvinylpyrrolidone, and the like. Suitable excipients when prepared from injection solutions include Ringer's solution, isotonic sodium chloride, and the like.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in some cases, can be administered topically.

The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention Should not be understood to be.

In another specific embodiment, the present invention can be identified as a food composition.

The food composition of the present invention may contain sweetening agents, flavoring agents, physiologically active ingredients, minerals and the like in addition to the active ingredients thereof.

Sweetening agents may be used in an amount that sweetens the food in a suitable manner, and may be natural or synthetic. Preferably, natural sweeteners are used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavors may be used to enhance taste or flavor, both natural and synthetic. Preferably, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. Examples of natural flavoring agents include those obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or those obtained from green tea leaves, Asiatica, Daegu, Cinnamon, Chrysanthemum leaves and Jasmine. Also, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like can be used. The natural flavoring agent may be a liquid concentrate or a solid form of extract. Synthetic flavors may be used depending on the case, and synthetic flavors such as esters, alcohols, aldehydes, terpenes and the like may be used.

Examples of the physiologically active substance include catechins such as catechin, epicatechin, gallocatechin and epigallocatechin, and vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine and riboflavin.

As the mineral, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium and zinc can be used.

In addition, the food composition of the present invention may contain preservatives, emulsifiers, acidifiers, thickeners and the like as needed in addition to the above sweeteners.

Such preservatives, emulsifiers and the like are preferably added in a very small amount as long as they can attain an application to which they are added. The term " trace amount " means, when expressed numerically, in the range of 0.0005% by weight to about 0.5% by weight based on the total weight of the food composition.

Examples of the preservative which can be used include calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate and EDTA (ethylenediaminetetraacetic acid).

Examples of the emulsifier which can be used include acacia gum, carboxymethyl cellulose, xanthan gum, pectin and the like.

Examples of the acidulant that can be used include acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. Such an acidulant may be added so that the food composition has a proper acidity for the purpose of inhibiting the growth of microorganisms other than the purpose of enhancing the taste.

Agents that may be used include suspending agents, sedimentation agents, gel formers, bulking agents and the like.

In addition, various herbal medicines can be added to the food composition of the present invention in order to improve flavor and palatability and to add other functionalities (for example, osteoporosis prevention). Examples of herbal medicines that can be added include mulberry extract, Examples of such extracts include extracts, soil extracts, water extracts, medicinal extracts, garlic extracts, corn oil extracts, Gugija extracts, licorice extracts, Angelica keiskei extracts, Puerariae Radix extracts, Gangjin extracts, Ganoderma extracts,

As described above, according to the present invention, it is possible to provide an anticancer composition comprising an extract of the herbal extracts or an effective substance thereof as an active ingredient. The anticancer composition of the present invention has indications for lung cancer, stomach cancer, colon cancer, breast cancer, liver cancer and the like, and can be commercialized as a pharmaceutical composition or a food composition.

Fig. 1 is the 1 H NMR spectrum of the active substance separated into three fractions.
Fig. 2 is a 13C NMR spectrum of the active substance separated into three fractions.
FIG. 3 is a summary of 1 H NMR and 13 C NMR spectral data of the active substances separated into three fractions.
Fig. 4 is a DEPT NMR spectrum of the active substance separated into three or more species.
Figure 5 is the HH COZY spectrum of the active substance isolated in three hens.
Fig. 6 is the HSQC spectrum of the active substance separated into three or more species.
Fig. 7 shows the HMBC spectrum of the active substance separated into three or more species.
Fig. 8 shows the results of experiments on the inhibition of cancer cell proliferation by the MTT method of 80% ethanol extract of cancodes.
FIG. 9 is a graph showing changes in body weight of experimental animals during an experimental period of anticancer activity against SK-Hep-1 transplanted into a nude mouse.
FIG. 10 is a graph showing changes in tumor volume during an experiment for anticancer activity against SK-Hep-1 transplanted into a nude mouse.
FIG. 11 is a graph showing changes in tumor weight during an experiment for anticancer activity against SK-Hep-1 transplanted into a nude mouse.
12 is a graph showing changes in body weight of experimental animals during an experimental period of anticancer activity against HepG-2 transplanted into a nude mouse.
FIG. 13 is a graph showing the change in tumor volume during an experiment for antitumor activity against HepG-2 transplanted into a nude mouse.
14 is a graph showing changes in tumor weight during an experimental period of anticancer activity against HepG-2 transplanted into a nude mouse.
Fig. 15 shows the results of measurement of the proliferation inhibitory activity of SK-Hep-1, which is an active substance isolated from three different herbs, by the MTT method.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > Three claws  Extract and Fraction  Preparation and isolation of its active substances

< Example  1> Three claws  Extract and Fraction  Produce

One kilogram of three meats was transferred to 80% ethanol and immersed for 48 hours. After repeated extraction three times, the resulting extract was concentrated under reduced pressure to remove the solvent and lyophilized to obtain a powdery extract.

The obtained ethanol extract was suspended and dissolved in 10 parts by weight of distilled water and then sequentially fractionated with n-hexane, dichloromethane (CH ₂ Cl ₂ ), ethyl acetate (EtOAc) and butanol (BuOH) , A dichloromethane layer, an ethyl acetate layer and a butanol layer, and a residue layer.

< Example  2> Isolation and Identification of Active Substances

8 g of the ethyl acetate fraction was mixed with Celite 545 and concentrated under reduced pressure to prepare a mixture, which was subjected to open column chromatography. Five small fractions were obtained using n-hexane, methylene chloride (DE), diethyl ether (EA), ethyl acetate (EA) and methanol (MeOH) as mobile phases. Among them, DE (800 mg) fractions were subjected to silica gel column chromatography. Chloroform / MeOH (9/2) was used as the mobile phase and flowed at a flow rate of 2.5 ml per minute to obtain a total of 18 small fractions. Of these, fraction 14 (DE-14) was briefly purified to obtain a single compound (compound 1).

To analyze the structure of the separated material, 10 mg of a single compound was dissolved in methanol-d to measure the NMR spectrum. Three methyl groups were identified in the 1H NMR spectrum (FIG. 1 and FIG. 3), and all of the three methyl groups were expected to be bonded to the quaternary carbon in the singlets. Based on 4 ppm signals, one or more benzene rings Respectively. It was also expected that more than one sugar would be combined due to the complex signals around 3.7-3.3ppm. A total of 21 carbons were predicted to exist in the 13 C NMR (FIGS. 2 and 3) and DEPT NMR (FIG. 4) spectra, and anomeric carbon was confirmed at 105.8 ppm.

It was confirmed that the proton (H-11, δ 6.40) of the benzene ring and the protons (H-15, δ2.2) of the methyl group were coupled in the H-H COZY spectrum (FIG. 5). Thus, the H-15 methyl group is bonded to the benzene ring and the anomeric hydrogen (H-1 ', delta 105) and the benzene ring carbon (C- 9, &amp;bull; 146). The methyl group linked to the benzene ring was coupled with C-9 (δ146), C-10 (δ129), and C-11 (δ116). C-9 and C-7 are ortho, C-12 and meta, C-7, respectively, because benzene ring hydrogen (H-11, And -8 were in the para position. Methyl group hydrogen (H-14, delta 1.7) is coupled with benzene ring carbon (C-7, delta 125), quaternary carbon (C-6, delta 74.4) and C- (H-13, δ1.2) was coupled with quaternary carbon (C-2, δ74.5) and C-1 (δ29.6) Respectively. The H-1 (δ2.3) and the H-4 (δ1.16) were C-3 (δ75.5) and C-7 (δ125) .02) were coupled with C-3 (δ75.5), and C-1 to C-8 were expected to form macrocycles. In addition, although C-2 and C-6 are quaternary carbons in the form of single bonds, chemical shifts have shifted to the author's field. Therefore, it was confirmed by NMR spectrum analysis that the separated compound was composed of one β-glucose, benzene ring, and octahedron ring, and the expected structure was found to be a novel substance not reported in the literature. > Multioside, a novel substance.

HR-FABMS analysis was performed to determine the molecular weight of the separated material. The molecular weight of the novel substance was 426.46 and analyzed by HR-FABMS analysis as 449.1791 (Calcd for C ₂₁ H ₃₀ O ₉ Na: 449.1788) in which sodium was bonded, confirming that the expected molecular structure was correct.

< Experimental Example > Three claws  The extract and Fraction  Anticancer activity experiment

< Experimental Example  1> MTT  Experiments to inhibit the proliferation of cancer cells by the method

<1> Experimental method

The 80% ethanolic extract of the herbal cancers in the examples of 5 kinds of cancer cells (lung cancer: A549, gastric cancer: AGS, colorectal cancer: HCT-15, breast cancer: MDA-MB-231, liver cancer: SK- Hep1) The inhibitory effect was assessed using the MTT method. For this, the number of cells was adjusted at a concentration of 3-5 × 10 ⁴ / mL for each cancer cell, and the cells were placed in each well of a 96-well plate for 24 hours. After 24 hours, the samples were treated for 3 days, MTT reagent was added, and the cells were further cultured for 4 hours. Plate was centrifuged at 1,000 rpm for 10 minutes, carefully removed the medium, and then the absorbance was measured at 540 nm after addition of DMSO.

<2> Experimental results

The experimental results are shown in Fig. 8, Table 1 and Table 2. Fig.

As shown in FIG. 8, A549 and SK-Hep1 inhibited cancer cell proliferation by using A549, AGS, HCT-15, MDA-MB- Hep1 showed more than 80% inhibition. As shown in Table 1 and Table 2, when A549 and SK-Hep1 were treated with each fraction of the herbal extracts of various herbal extracts, the cell proliferation inhibitory effect was shown dose-dependently on A549 and SK-Hep1.

Based on these experimental results, animal experiments were conducted using the most effective EtOAc fraction (AAM-EA).

< Experimental Example  2> AAM - EA of Hollow fiber  ( HF ) assay  Experiments on anticancer activity in model

<1> Experimental method

&Lt; 1-1 > Administration method and frequency of administration

The test substance was administered intrathecally for 7 days once a day after 1 HF transplantation using a disposable syringe with a sonde for oral administration.

<1-2> Group composition and administration dose

<1-2-1> Group composition

The group composition is shown in [Table 3] below.

&Lt; 1-2-2 >

The dose of the test substance was set at 25, 50 and 100 mg / kg, and the negative control group was administered the injectable water as an excipient. The volume of administration of the negative control group and the test substance administration group was set to 10 mL / kg, and the amount of the individual administration solution was calculated based on the weight measured recently once a week.

<1-3> Observation and weight measurement

<1-3-1>. General symptoms observation

The general symptoms were observed immediately after the administration once a day throughout the administration period. Observations of general symptoms were recorded according to the type of death, symptom type and degree, and the days of onset.

<1-3-2> Weight measurement

All animals were measured once a week for the time of entry, at the time of administration, at the time of administration, and during the administration period.

<1-4> HF assay

<1-4-1> Cell culture

The human-derived cancer cell lines used in the HF assay after the cytotoxicity test on the five cell lines using the test substance (supplementary data) were selected as follows; A549 (lung cancer), AGS (stomach cancer), SK-Hep1 (liver cancer).

A549 and AGS were added to RPMI 1640 medium, SK-Hep1 was added to DMEM medium with sodium bicarbonate, L-glutamine, penicillin / streptomycin, and FBS was added to a final concentration of 10% and incubated in humidified CO ₂ incubator. Cells were grown in HF (Cellmax Implant Membrane, Spectrum) at a concentration of 1 × 10 ⁶ cells / ml for 24 hr in humidified CO ₂ incubator.

<1-4-2> Implantation

Nude mice were anesthetized with zoletil and rompun, and the abdominal wall was slightly incised. After 3 days of HF transplantation for 24 hr, the test material was started.

<1-4-3> Measurement of HF Extraction and Cancer Cell Proliferation Inhibition

After the end of the test, the mouse was sacrificed by cervical disassembly, and the transplanted fiber was removed to remove the foreign substances on the surface. Then, the surface was immobilized in a humidified CO ₂ incubator for 30 minutes in a medium set at 37 ° C. and MTT (final conc. / Ml) and then cultured for 4 hours.

After the incubation, the cells were washed with 2.5% protamine sulfate, and the fibers were immersed in fresh 2.5% protamine sulfate, followed by O / N at 4 ° C. After adding fresh 2.5% protamine sulfate, the reaction was allowed to proceed for 4 hours. The fiber was cut longitudinally and then O / N dried. After confirming that the fiber was completely dried, DMSO was added and the formazan salt was dissolved by shaking at room temperature for 4 hours, and the absorbance was measured using an ELISA reader.

<1-5> Statistical processing of data

Comparisons between the excipient control group and the test substance-administered group were generally performed using the Student-t test. Expression of edema rate and inhibition rate is generally expressed as a percentage. The statistical method was the SPSS 12.1K program, which is a widely used statistical package.

<2> Experimental results

The results are shown in Table 4.

As shown in [Table 4], HF assay using A549, AGS and SK-Hep1 showed significant inhibitory effect on cell proliferation in the pre-treatment group. In particular, SK-Hep1 inhibited cancer cell proliferation to the highest level .

< Experimental Example  3> AAM - EA Of nude mice SK - Hep -1 Anticancer Activity Test

<1> Experimental method

&Lt; 1-1 > Administration method and frequency of administration

The administration of the test substance was forcibly administered in a disposable syringe equipped with a sonde for oral administration once a day, for 4 weeks, in a total of 28 times.

The positive control was administered to the abdomen 8 times a week for 4 weeks using a disposable syringe (26G, 1 mL, Doowon meditec. Corp., Korea).

<1-2> Group composition and administration dose

<1-2-1> Group composition

The group composition is shown in [Table 5].

&Lt; 1-2-2 >

The dose of the test substance was set at 50, 200 and 500 mg / kg, and the dose of the positive control substance (Cis-Diamine platinum (II) dichloride) was set at 2 mg / kg. Negative control group received injectable water, an excipient. The dose of the negative control, the test substance administration group and the positive control group was set to 10 mL / kg, and the amount of the individual administration liquid was calculated based on the body weight measured close to the administration day.

<1-3> Observation and weight measurement

<1-3-1> General symptom observation

During the observation period, general symptoms such as appearance, behavior, and excretion were observed once a day, and dead animals were identified.

<1-3-2> Weight measurement

Body weight was measured twice daily (Tue, Fri) from the day of initiation of administration, before administration.

<1-4> Transplantation of tumor

The tumor mass formed in "Attachment. Succeeding generations on transplanted tumor in nude mice" (Study No .: B10999) was made into a piece of about 3 × 3 × 3 mm ³ . The animals were anesthetized with Isoflurane, and the animals were incised about 4 mm in the anterolateral region of the left fascia. The tumor pieces were placed at the end of the trocar, and the trocar was passed through the left fusiform incision to the back region . Next, the trocar was rapidly rotated 360 degrees and the skin surface was observed to confirm the location of the tumor. Then, the left incisor of the animal was placed in a povidone iodine solution (Lot No .: 1005, Dongin-dang Pharm Co., Ltd., Korea).

&Lt; 1-5 > Evaluation of tumor growth inhibition

<1-5-1> Measurement of tumor volume

The maximum length (L) and the perpendicular width (W) of the tumor were measured using a caliper twice a week during the observation period and the tumor volume tumor volume, TV) were calculated.

TV (mm ³ ) = L (mm) x W ² (mm ² ) x 1/2

The volume of the tumor before administration of each individual was set to the value measured when the group was separated.

<1-5-2> Tumor Extraction and Weighing

At the end of the observation period, the animals were anesthetized with isoflurane (ASJ9AE, Choongwae Pharma Corp., Korea) and the tumor was weighed and weighed.

<1-6> Statistical processing of data

Body weights, tumor volume, and tumor weights obtained in the experiment were assayed using SAS (Version 9.2, SAS Institute Inc., U.S.A.).

Bartlett test was performed to determine the equipotentiality (significance level: 0.05). In the case of equal distribution, one-way analysis of variance (ANOVA) was performed (significance level: 0.05). When significance was observed, multiple tests of Dunnett's t-test were conducted to confirm significance of each test group for negative control (Significance level: 0.05, 0.01), and Kruskal-wallis test (significance level: 0.05) was used when the equal distribution was rejected. When the significance was observed, test.

<2> Experimental results

The results are shown in FIG. 9 to FIG.

There was no statistically significant difference in body weight between the test group administered with 50 mg / kg of test substance and the negative control group. 200 and 500 mg / kg of the test substance-administered group was statistically significantly increased on the 28th day after administration compared with the negative control group. The positive control group with a dose of 2 mg / kg showed no statistically significant difference compared to the negative control group, but showed a tendency to decrease.

The dose of 50, 200, and 500 mg / kg of the test substance in the tumor volume and the positive control of the dose of 2 mg / kg were statistically significantly inhibited from 7 to 28 days after administration compared with the negative control.

Tumor weights were significantly smaller in the 50, 200, and 500 mg / kg doses of the test substance-administered and 2 mg / kg doses of the positive control compared to the negative control.

Histopathological examination showed that the necrosis level of the tumors was 50%, 200%, and 500 mg / kg, respectively, compared to the negative control group. However, And the overall evaluation could not be carried out. Apoptosis also disappeared in most of the tumors and necrosis occurred in the excised tumors and could not be compared with the negative control.

In conclusion, in the anti-cancer test for SK-HEP-1, a human tumor cell line transplanted into nude mice, AAM-EA, a test substance, showed a remarkable effect of inhibiting tumor growth at 50, 200 and 500 mg / .

< Experimental Example  4> AAM - EA Of nude mice HepG -2 on the anticancer activity test

<1> Experimental method

&Lt; 1-1 > Administration method and frequency of administration

The administration of the test substance was forcibly administered in a disposable syringe equipped with a sonde for oral administration once a day, for 4 weeks, in a total of 28 times.

The positive control was administered to the abdomen 8 times a week for 4 weeks using a disposable syringe (26G, 1 mL, Doowon meditec. Corp., Korea).

<1-2> Group composition and administration dose

The group composition is shown in [Table 6].

&Lt; 1-2-2 >

The dose of the test substance was set at 20, 80 and 200 mg / kg, and the dose of the positive control substance was set at 2 mg / kg. Negative control group received injectable water, an excipient. The dose of the negative control, the test substance administration group and the positive control group was set to 10 mL / kg, and the amount of the individual administration liquid was calculated based on the body weight measured close to the administration day.

<1-3> Observation and weight measurement

<1-3-1> General symptom observation

During the observation period, general symptoms such as appearance, behavior, and excretion were observed once a day, and dead animals were identified.

<1-3-2> Weight measurement

Body weight was measured twice daily (Tue, Fri) from the day of initiation of administration, before administration.

<1-4> Transplantation of tumor

The tumor mass formed in "Attachment. Succeeding generations on transplanted tumor in nude mice" (Study No .: B10999) was made into a piece of about 3 × 3 × 3 mm ³ . The animals were anesthetized with Isoflurane, and the animals were incised about 4 mm in the anterolateral region of the left fascia. The tumor pieces were placed at the end of the trocar, and the trocar was passed through the left fusiform incision to the back region . Next, the trocar was rapidly rotated 360 degrees and the skin surface was observed to confirm the location of the tumor. Then, the left incisor of the animal was placed in a povidone iodine solution (Lot No .: 1005, Dongin-dang Pharm Co., Ltd., Korea).

&Lt; 1-5 > Evaluation of tumor growth inhibition

<1-5-1> Measurement of tumor volume

The maximum length (L) and the perpendicular width (W) of the tumor were measured using a caliper twice a week during the observation period and the tumor volume tumor volume, TV) were calculated.

TV (mm ³ ) = L (mm) x W ² (mm ² ) x 1/2

The volume of the tumor before administration of each individual was set to the value measured when the group was separated.

<1-5-2> Tumor Extraction and Weighing

At the end of the observation period, the animals were anesthetized with isoflurane (ASJ9AE, Choongwae Pharma Corp., Korea) and the tumor was weighed and weighed.

<1-6> Statistical processing of data

Body weights, tumor volume, and tumor weights obtained in the experiment were assayed using SAS (Version 9.2, SAS Institute Inc., U.S.A.).

Bartlett test was performed to determine the equipotentiality (significance level: 0.05). In the case of equal distribution, one-way analysis of variance (ANOVA) was performed (significance level: 0.05). When significance was observed, multiple tests of Dunnett's t-test were conducted to confirm significance of each test group for negative control (Significance level: 0.05, 0.01), and Kruskal-wallis test (significance level: 0.05) was used when the equal distribution was rejected. When the significance was observed, test.

<2> Experimental results

Experimental results are shown in Figs. 12 to 14. Fig.

As a result of the body weight measurement, there was no significant difference in the test substance doses of 20 and 80 mg / kg doses compared with the negative control group. In the test substance-administered group of 200 mg / kg dose, 14 to 21 days after administration were significantly increased compared with the negative control group. In the positive control group with a dose of 2 mg / kg, the decrease was significantly reduced at 18-28 days after administration compared to the negative control group.

The volume of the tumor was measured on the 11th and 18th to 25th days after the administration of 20 mg / kg of the test substance, in the test substance-administered group of 80 and 200 mg / kg and in the positive control group of the dose of 2 mg / kg To &lt; 28 &gt; days compared to the negative control group.

Tumor weights showed no significant difference compared to negative control in the 20 mg / kg dose of test substance, but negative in the test substance dose group of 80 and 200 mg / kg dose and in the positive control dose of 2 mg / kg dose Compared with the control group.

Histopathological examination revealed necrosis, degree of apoptosis, and apoptosis in tumor treated group of 20, 80 and 200 mg / kg dose and positive control group of 2 mg / kg dose. Were similar to the negative control group.

In conclusion, AAM-EA, a test substance, is effective in inhibiting tumor growth at doses of 80 and 200 mg / kg in hepatocellular carcinoma cell line HepG2 transplanted into nude mice.

< Experimental Example  4> In three culinary herbs  Of the separated active substance MTT  By method SK - Hep -1 cell proliferation inhibitory activity experiment

The proliferation inhibitory activity of SK-Hep-1 cells was evaluated by the same method as in <Experiment 1>.

The results of the experiment are shown in Fig. 15, which shows that the active substances isolated from the three organs inhibit the proliferation of SK-Hep-1 cells in a concentration-dependent manner.

Claims (9)

Wherein the anticancer agent comprises three herbal extracts as an effective ingredient and exhibits anticancer activity against gastric cancer, colon cancer or liver cancer.
The method according to claim 1,
Wherein the three herbal extracts are obtained by immersing three or more herbs in water, ethanol or a mixed solvent of water and ethanol.
The method according to claim 1,
The herb extract of the three herbal extracts is prepared by suspending the herbal extracts in water, ethanol or a mixed solvent of water and ethanol, suspending the extract in water, and sequentially fractionating the fractions with hexane, dichloromethane, ethyl acetate and butanol. A fraction extract, an ethyl acetate layer fraction extract or a fractional extract of a butanol layer.
The method according to claim 1,
The herb extract of the three herbal extracts is prepared by suspending the herbal extracts in water, ethanol or a mixed solvent of water and ethanol, suspending the extract in water and sequentially fractionating the fractions with hexane, dichloromethane, ethyl acetate and butanol. The ethyl acetate layer fraction extract &Lt; / RTI &gt;
The method according to claim 1,
The herb extract of the three herbal extracts is prepared by suspending the herbal extracts in water, ethanol or a mixed solvent of water and ethanol, suspending the extract in water, and sequentially fractionating the extract with hexane, dichloromethane, ethyl acetate and butanol to obtain a dichloromethane layer fraction extract &Lt; / RTI &gt;
The method according to claim 1,
Wherein the anticancer composition has anticancer activity against liver cancer.
7. The method according to any one of claims 1 to 6,
Wherein the composition is a pharmaceutical composition.
An anticancer composition having anticancer activity against hepatocellular carcinoma comprising the compound of the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
&Lt; Formula 1 >

A compound of the following formula (I) or a pharmaceutically acceptable salt thereof:
&Lt; Formula 1 >

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