KR101284279B1 - The extract of the bark of Acer barbinerve Max with antimetastasis effect and composition thereof - Google Patents

Abstract

The present invention relates to a composition containing cheongsi shut bark extract, and this represents the activity of inhibiting cancer metastasis, cheongsi shut tree (Acer barbinerve Whereas as Max) in the xylem of the extract is insignificant cancer metastasis inhibitory activity, of the present invention cheongsi shut tree (Acer barbinerve Max ) bark extract showed significant cancer metastasis inhibitory activity below the cell death concentration.

Description

Extract of the bark of bark tree bark showing cancer metastasis inhibitory activity and composition containing the same {The extract of the bark of Acer barbinerve Max with antimetastasis effect and composition

The present invention relates to a blue bark bark extract exhibiting cancer metastasis inhibitory activity and a composition containing the same.

Recently, as the economic level of our country has risen, as the living environment has been improved and the diet has been enriched, western diets have been widely spread. As a result, chronic adult diseases such as cancer, arteriosclerosis, stroke, diabetes, and high blood pressure, which are presumed to be caused by excessive nutrition or an unbalanced diet, are increasing.

To date, various methods have been tried to treat cancer, and cancer treatment can be roughly classified into drug therapy, surgical therapy, radiation therapy, and the like. Among them, the drug therapy oral or intravenous administration of the anticancer drug during drug treatment has a problem that only a small amount accumulates in the lesion since the drug spreads throughout the bloodstream and reaches the lesion. Therefore, in order to accumulate an appropriate amount in the lesion, a large amount of drug must be administered, which may cause serious side effects.

On the other hand, in the case of radiation therapy, there is a problem that safety is not guaranteed. For example, a substance such as Holmium, which is effective for liver cancer, has been produced as chitosan complex (Milican, Donghwa Pharm. Co., Ltd.), and its effects have been proved, but the side effects are also serious and still have problems. (Watterson. J. D. et al., J. Urol. 168: 442-445, 2002; Peh, O. H. et al., Ann. Acad. Med. Singapore 30: 563-567, 2001).

In recent years, research has been focused on finding substances or foods that effectively inhibit cancer in the long-term progression of cancer as a cancer prevention agent, rather than directly controlling and eliminating cancer. (Kang Jin-Seok et al., Chemical Cancer Prevention, Korean Medicine, 2000; Surh, Y. J., Mutat. Res. 428: 305-327, 1999; Sporn, M. B., Lancet. 347: 1377-1381, 1996).

Meanwhile, metastasis refers to a condition in which cancer proliferates after attaching to another organ or tissue, leaving the first organ or tissue. Metastasis includes contact metastasis, hematogenous metastasis via blood vessels and lymphatic metastasis via lymphatic vessels. If the cancer leaves the site of origin and progresses to metastasis, it is usually considered to be at serious stage, so preventing metastasis is very important in the treatment of cancer.

Meanwhile, Cheongdak Tree ( Acer barbinerve Max ) is a deciduous broad-leafed arborescent with maple leaves, about 10 m high. The leaves are opposite, broad egg-shaped or round, divided into 3 ~ 5 branches, and have a lot of fine hair on the back side. Stems and branches grow in medicinal and deep mountains, and are distributed in Korea, Japan, and Manchuria. Bark is green or greyish brown with yellow branches, but sometimes reddish, with hairs.

Many studies have been conducted on natural products or extracts having anticancer activity, and studies on the cancer metastasis inhibiting function of the green bark tree extract are currently insignificant.

The present invention is a blue-green tree ( Acer barbinerve Max ) is to separate the extract having a cancer metastasis inhibiting function to develop and provide as an anticancer drug composition.

The present invention is the first form of green pine ( Acer barbinerve After extracting the skin of the bark of Max ) by hot water, the extract is provided with a cancer metastasis inhibiting activity obtained by separating the extract. According to experiments of the present invention to shut cheongsi tree (Acer barbinerve Max ) It was confirmed that the hot water extract of the skin of skin has a significant effect of inhibiting cancer metastasis. Whereas the bark has a significant effect on inhibiting cancer metastasis, the wood parts did not show a significant effect on inhibiting cancer metastasis.

On the other hand, the present invention in the second form Cheongdak wood ( Acer barbinerve Max ) bark skin was extracted by hot water extraction, the extracts were separated, fractionated with ethyl acetate, and the extracts having the cancer metastasis inhibiting activity obtained by separating the fractions were provided. According to the experiment of the present invention, among the various fractions, ethyl acetate fraction showed the best cancer metastasis inhibitory activity.

On the other hand, the present invention provides an anticancer composition comprising the extract of the first or second form of the present invention as an active ingredient in a third form. In the present invention, "included as an active ingredient" means that the extract of the first or second form of the present invention is added to the composition to the extent that it can exhibit an anticancer effect. That is, when the composition is a pharmaceutical composition, it can be formulated in various forms by adding a variety of ingredients as a sub-component for drug delivery and stabilization, etc., if the form of the composition is a food or cosmetic composition, various formulations It means that the formulation base and excipient can be added to prepare the.

The term 'extract' in the present invention is used in the sense including both the liquid form containing the extraction solvent and the form of the remaining solid powder volatilize the extraction solvent.

On the other hand, the present invention provides a food composition for preventing cancer or cancer metastasis, comprising the first or second form of the present invention extract or dried powder thereof as an active ingredient, the first or second embodiment of the present invention The bimodal extract is preferably added in the composition at a concentration of 1-20 μg / mL relative to dry weight.

Food composition for cancer or cancer metastasis prevention of the present invention is preferably meat, cereals, caffeine beverage, general beverage, chocolate, bread, snacks, confectionary, pizza, jelly, noodles, gum, ice cream, alcoholic beverage, alcohol, vitamin complex And other health supplements may be any one selected from, but is not necessarily limited thereto.

On the other hand, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the first or second form of the present invention extract or dried powder thereof as an active ingredient. The content of the extract of the first or second form of the present invention or the dry powder thereof included in the pharmaceutical composition for preventing or treating cancer of the present invention may be used in the method of preventing and treating the agent, the condition of the user, the type of disease, and the severity of the disease. It is preferable to adjust according to, preferably added in the composition at a concentration of 1 to 20 μg / mL relative to the dry weight.

Meanwhile, the pharmaceutical composition for preventing or treating cancer of the present invention may further include a pharmaceutically acceptable carrier, diluent or excipient in addition to the active ingredient. Examples of usable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. These may be used singly or in combination. When the preventive and therapeutic agent is a pharmaceutical agent, a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent or an antiseptic agent may be additionally included.

On the other hand, the formulation of the pharmaceutical composition for preventing or treating cancer of the present invention may be formulated in a desired form according to the method of use, and in particular, to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It is desirable to formulate by employing methods known in the art. Examples of specific formulations include PLASTERS, GRANULES, LPTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, Syrups SYRUPS, OPHTALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS, ), Suspensions, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, SUPPOSITIORIES, INJECTIONS, SPIRITS, CATAPLSMA, ), Capsules, CREAMS, TROCHES, TINCTURES, PASTES, PILLS, soft or hard gelatin capsules.

On the other hand, the dosage of the pharmaceutical composition for preventing or treating cancer of the present invention may be determined in consideration of the method of administration, the age, sex and weight of the recipient and the severity of the disease. For example, the cancer prevention or treatment agent of the present invention may be administered one or more times 0.1 to 100 mg / kg (body weight) per day based on the active ingredient. However, the above dosage is only one example to illustrate, and may vary depending on the doctor's prescription depending on the condition of the patient.

As confirmed by the present invention, the green tree ( Acer barbinerve Whereas as Max) in the xylem of the extract is insignificant cancer metastasis inhibitory activity, cheongsi shut tree (Acer barbinerve Max ) bark extract showed significant cancer metastasis inhibitory activity below the cell death concentration.

1 is an MTT assay result for examining cell proliferation ability (killing ability).
Figure 2 is a result showing the effect of the bark of the tree bark extract on the mobility of prostate cancer cells DU145 cells.
Figure 3 is a result showing the effect of the ethyl acetate (Etoac) fraction of bark of the tree bark on the mobility of DU145 cells, prostate cancer cells.
4 is a result showing the effect of ethyl acetate fraction of the bark of the tree bark on the activity of MMP-9 and MMP-2.
5 is a result showing the effect of ethyl acetate fraction of the bark of the tree bark on the activity of uPA.
Figure 6 is a Western blot showing the effect of ethyl acetate fraction of the bark of the tree bark on the activity of MMP-9 and uPA.

Hereinafter, the content of the present invention will be described in detail in the following Examples and Experimental Examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

Experimental Example  One: MTT Assay  Cell through Proliferative ability ( Death ) Research

In this experimental example, DU145 cells, which are human prostate cancer cells, were treated with a fraction of bark of the bark of tree bark at a concentration of 20 μg / mL to investigate cell death ability (proliferative capacity) by MTT assay.

DU145 cells were cultured in a CO ₂ incubator at 37 ° C. using a medium containing 10% FBS, 1% penicillin streptomycin in DMEM / F12 medium. When the cells were about 70-80% full of the culture dish, the cells were washed with PBS (phosphate-buffered saline, pH 7.4), and then subcultured with 0.25% trypsin 2.56 mmol / L EDTA. Exchanged every day.

The DU145 cells incubated as described above were diluted in DMEM / F12 medium containing 10% FBS and dispensed into 24 well-plates at a density of 50,000 cells / well. After 24 hours of incubation, the cells were further incubated for 24 hours with a serum-deprivation medium (SDM) containing 1% FBS. Thereafter, the cells were exchanged with SDM containing 20 μg / mL concentrations of bark seedling bark fraction, and after 4 hours, the number of living cells was measured by the MTT assay method.

As a result of confirming the proliferative capacity of the cells, it was confirmed that they did not affect the proliferation of the cells. (FIG. 1).

Experimental Example  2: Investigate Cell Mobility

Blue Tak Tree ( Acer barbinerve Max ) bark extract did not affect the proliferation of cells at a concentration of 20 μg / mL, this experiment was to investigate the mobility of DU145 cells within this range.

The migration of cancer cell lines was examined in a Transwell cell culture chamber. First, the bottom of the transwell membrane was coated with 15 μL of 0.1% type A gelatin. 0.1% BSA was added to the lower part of the transwell cell culture chamber, and cells and celadon bark fractions were added to the upper part of the transwell, followed by incubation at 37 ° C for 4 hours. Cells on the upper part of the filter that did not migrate were removed with a cotton swab, and the cells migrated through a polycarbonate filter were stained by hematoxylin-eosin staining. The number of migrated cells was quantified using a microscope (× 100).

As a result of the experiment, it was confirmed that the migration of the cells was reduced by the extract of the bark tree bark.

However, since the effect was the best in the ethyl acetate (Etoac) fraction (Fig. 2), the ethyl acetate fraction was treated with 0 ~ 20 μg / mL to further observe the mobility of the cells. Further experiments showed that cell migration was reduced in a concentration-dependent manner. (FIG. 3).

Experimental Example  3: MMP -9, 2 active probes

It was confirmed in Example 2 that the movement of DU145 cells, which are human prostate cancer cells, was reduced by the bark extract of Cheongdak tree, in this example, matrix metalloproteinase (MMP)- The effect on the activity of 9 was to be measured using gelatin zymography. As the sample, the ethyl acetate fraction having the best cell migration effect was used among the bark extracts.

Cells were cultured by adding the bark ethyl acetate fraction to the concentration of 0, 5, 10, 20 μg / mL. After 24 hours, the medium was collected and concentrated 100 times by centrifugal ultrafiltration using 'Amicon Ultra-15'.

Concentrated medium was electrophoresed on 0.2% gelatin-containing SDS-PAGE, washed twice with renaturing buffer (2.5% Triton X-100) for 30 minutes, and then 50 mmol / L Tris-HCl (pH). 7.5), 0.15 mmol / L NaCl, 10 mmol / L CaCl ₂ , and a developing buffer containing 0.02% Brij 35 at 37 ° C. for 48 hours. After the reaction, the gel was stained with 0.25% Coomassie Brilliant Blue solution for 2 hours, and then decolorized to observe the degree of degradation of gelatin.

As a result, it was found that the activity of MMP-9 and MMP-2 decreased when the bark ethyl acetate fraction was treated, and the by-products of MMP-2 activity also decreased. (Fig. 4).

Experimental Example  4: uPA  Active investigation

In this experiment, we attempted to determine whether the activity of uro-kinase type plasminogen activator (uPA), which is known to play an important role in cell migration, was reduced by treatment with bark ethyl acetate fraction. It was.

In order to investigate the uPA activity, the concentrated medium prepared in Experimental Example 3 was electrophoresed in SDS-PAGE containing 0.2% fibrinogen and 10 NIH units thrombin, followed by a renaturing buffer (50 mmol / L Tris-HCl). (pH 7.4), 2.5% Triton X-100), washed twice for 30 minutes, then washed three times for 10 minutes with tertiary distilled water, followed by develpping buffer containing 30 mM Tris-HCL (pH 7.4), 0.02% NaN3. (developing buffer) was reacted for 48 hours at 37 ℃. After the reaction, the gel was stained with 0.25% Coomassie Brilliant Blue solution for 30 minutes, and then decolorized to observe the degree of fibrin degradation.

As a result, it was observed that both high molecular weight uPA and low molecular weight uPA decreased by treatment of the bark ethylacetate fraction. (Fig. 5)

Experimental Example  5: MMP -9 and uPA of Western Blat

In the present experimental example, Western blot was used to determine the decrease in secretion of MMP-9 and uPA by treatment of the bark ethyl acetate fraction.

The concentrated medium (100-fold concentrated, 50 μg protein) prepared in Experimental Example 3 was separated by 4-20% concentration gradient SDS-PAGE and transferred to PVDF (polyvinylodene fluoride) membrane. The resulting membrane was blocked in 5% non-fat milk-TBST (20 mmol / L Tris-HCl, pH 7.6, 150 mmol / L NaCl, 0.1% Tween 20) for 1 hour and then 3 times with TBST for 10 minutes. Rinsed. After MMp-9 and uPA antibodies were added to the membrane and incubated at 4 ° C. for 24 hours, they were rinsed three times for 10 minutes with TBST. The membrane was then incubated for 1 hour with the addition of HRP-binding anti-rabbit, mouse or goat IgG, then rinsed three times with TBST for 10 minutes. Signals of the proteins bound to the antibody were visualized for bands using Immobilon Western Chemiluminescent HRP substrates.

As a result of the experiment, it was confirmed that the protein secretion of both MMP-9 and uPA was reduced by treatment of the bark ethyl acetate fraction. (Fig. 6)

From the above experiments, the treatment of the bark ethyl acetate fraction of blue bark tree decreased cell migration in DU145 cells, which are human prostate cancer cells, and decreased the activity and secretion of MMP-9 and uPA, which play an important role in cell migration. I could confirm it. These results indicate that the bark ethyl acetate fraction of blue bark tree is an effective substance for inhibiting cancer (prostate cancer) metastasis.

Example  1: Preparation of pharmaceutical composition for preventing or treating cancer

In Example 1, a pharmaceutical composition for preventing or treating cancer was prepared as follows.

(1) Manufacture of powders

Blue Tak Tree ( Acer barbinerve Max ) bark skin was extracted with hot water, and then 1 g of lactose was mixed with 2 g of dry powder of the fraction obtained by fractionation with ethyl acetate, and filled with an airtight cloth to prepare a powder.

(2) Preparation of tablets

Blue Tak Tree ( Acer barbinerve Max ) bark skin was extracted with hot water, and then 100 mg of dry powder, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed with ethyl acetate. The tablets were prepared by tableting according.

(3) Manufacture of capsules

Blue Tak Tree ( Acer barbinerve Max ) bark skin was extracted with hot water, and then 100 mg of dried powder, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed with ethyl acetate, and then filled into gelatin capsules. Capsules were prepared.

(4) Injection preparation

Blue Tak Tree ( Acer barbinerve Max ) bark skin was extracted with hot water, and then 100 ml of dry powder of the fraction obtained by fractionation with ethyl acetate was added for dissolution of distilled water for injection. The pH was adjusted to about 7.5, followed by a 2 ml ampoule. Injections were made by filling and sterilizing.

Example  2: Preparation of food composition for cancer prevention

In Example 2, a food composition for preventing cancer was prepared as follows.

(1) Manufacturing of wire

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh. Black beans, black sesame seeds and perilla were each steamed and dried in a known manner, then roasted and ground to prepare a powder having a particle size of 60 mesh. Then, the brown rice 30 weight%, coix 15 weight%, barley 20 weight%, and 9% by weight of glutinous rice, perilla 7 weight%, 8% Black soybean, black sesame 7 weight%, cheongsi shut tree (Acer barbinerve Max ) bark skin was extracted with hot water, and then, 3% by weight of the dry powder, 0.5% by weight of Ganoderma lucidum and 0.5% by weight of sulfuric acid were obtained by fractionation with ethyl acetate.

(2) Production of chewing gum

Gum base 20%, 76.9% sugar by weight and 1% by weight of spices, 2% water and cheongsi Doc trees (Acer barbinerve Max ) bark skin was extracted with hot water, and then 0.1% by weight of the dry powder of the fraction obtained by fractionation with ethyl acetate was prepared to prepare chewing gum in a conventional manner.

(3) Candy manufacturing

60 wt% of sugar, 39.8 wt% of starch syrup, 0.1 wt% of fragrance, and 0.1 wt% of dry powder of the fraction obtained by fractionation with ethyl acetate after hydrothermal extraction of the bark of Acer barbinerve Max The candy was prepared by the conventional method by combining.

(4) Manufacturing of biscuits

Force 1st class 25.59 wt%, 1st class gravity 22.22 wt%, white sugar 4.80 wt%, salt 0.73 wt%, glucose 0.78 wt%, palm shortening 11.78 wt%, ammonium 1.54 wt%, sodium bicarbonate 0.17 wt%, sodium bisulfite 0.16 wt %, Rice flour 1.45 wt%, Vitamin B₁0.0001 wt%, Vitamin B20.0001 wt%, Milk flavor 0.04 wt%, Water 20.6998 wt%, Whole milk powder 1.16 wt%, Substitute milk powder 0.29 wt%, Calcium phosphate 0.03 wt% , 0.29 wt% salt spray and spray oil 7.27% by weight and cheongsi shut tree (Acer barbinerve Max ) bark skin was extracted with hot water, and then 1% by weight of dry powder of the fraction obtained by fractionation with ethyl acetate was prepared to prepare a biscuit in a conventional manner.

(5) health drink manufacturing

0.26% by weight of honey, 0.0002% by weight of thioctoamide, 0.0004% by weight of nicotinic acid, 0.0001% by weight of riboflavin hydrochloride, 0.0001% by weight of pyridoxine hydrochloride, 0.001% by weight of inositol, 0.002% by weight of ortic acid, 98.7362% by weight of water and cyansid Hot water was extracted from the bark of the trees ( Acer barbinerve Max ), and then 1% by weight of the dry powder of the fraction obtained by fractionation with ethyl acetate was prepared to prepare a health beverage in a conventional manner.

(6) Production of sausages

Pork 65.18%, poultry 25%, starch 3.5%, soy protein 1.7%, salt 1.62%, glucose 0.5% and glycerin 1.5% and bark of Acer barbinerve Max ) Was extracted with hot water, and then 1% by weight of the dry powder of the fraction obtained by fractionation with ethyl acetate, sausage was prepared in a conventional manner.

(7) Health supplement manufacturing

55% by weight of spirulina, 10% by weight of guar gum enzyme digestion, 0.01% by weight of vitamin B₁ hydrochloride, 0.01% by weight of vitamin B6 hydrochloride, 0.23% by weight of DL-methionine, 0.7% by weight of magnesium stearate, 22.2% by weight of lactose and 1.85% by weight of corn starch Hot water was extracted from the bark of Acer barbinerve Max , and 10% by weight of the dry powder of the fraction obtained by fractionation with ethyl acetate to prepare a tablet health supplement in a conventional manner. .

(8) Liquor production

Green Tree (Acer barbinerve Max0.5% by weight of the dry powder of the fraction obtained by fractionation with ethyl acetate was mixed with soju, beer, liquor or fruit wine to make an emulsion, and then vacuum at 7,000 rpm. The liquor was prepared by centrifugation for 15 minutes or mixing at 9,000 rpm with a high speed mixer.

Claims (3)

An anticancer agent comprising an extract having cancer metastasis inhibiting activity obtained by separating hot water extracts from the bark of Acer barbinerve Max and separating the extracts as an active ingredient.
An anticancer agent comprising an extract having cancer metastasis inhibiting activity obtained by separating hot water extract of bark of Acer barbinerve Max and separating the fractions fractionated with ethyl acetate as an active ingredient.
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