KR101722427B1 - Composition for growth inhibition of cancer cell comprising methylated catechin as effective component - Google Patents

Abstract

The present invention relates to a composition for inhibiting the growth of cancer cells containing methylated catechin as an active ingredient, and more particularly, to a composition for inhibiting the growth of cancer cells comprising Methyl-catechin (EGCG-3Me) And can inhibit the proliferation of cancer cells. In particular, since methylated catechin has an effect of inhibiting the proliferation of skin cancer, prostate cancer and breast cancer cells, it can be used as a health functional food or a pharmaceutical composition for inhibiting the growth of cancer cells It can be useful.

Description

[0001] The present invention relates to a composition for inhibiting the growth of cancer cells containing methylated catechin as an active ingredient,

The present invention relates to a composition for inhibiting growth of cancer cells containing methylated catechin as an active ingredient, and more particularly, to a composition for inhibiting the growth of cancer cells containing methylated catechins, which can be used as a health functional food or a pharmaceutical composition, .

Cancer among human diseases is a disease that is difficult to treat despite the development of modern medicine. 80 ~ 90% of the causes are due to environmental factors, and 30 ~ 60% of them are related to diet and nutrition And other factors are known to be caused by smoking, radiation such as UV and X-rays, occupational factors, drinking, endogenous hormones, and the like. Many researchers are conducting various studies to conquer cancer. As a part of this research, efforts are being made to find an ingredient in an organism that blocks or inhibits the growth of cancer in foods or natural medicines.

Examples of the components having typical carcinogenic inhibitory activities contained in foods include phytochemicals such as phenol, indole, aromatic isothiocyanate, methylated flavone, coumarin, plant sterol, selenium salt, proteolytic inhibitor, ascorbic acid, tocopherol, ) Is known. Most of these phytochemicals have a high antioxidant activity and act as a blocking agent to prevent the carcinogen in the initial stage of carcinogenesis or prevent the carcinogen from reaching the target site or act on the final stage to prevent cancer.

On the other hand, catechin belongs to flavan-3-ol in chemical structure, and catechin or pyrogallol substituent at position 2 or epigallocatechin gallate (catechol or pyrogallol) depending on presence or absence of galloyl group at position 3 epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG) and epicatechin (EC). These catechins have been shown to inhibit the lipid peroxidation (Kinura et al., J. Jpn. Soc. Nutr. Food Sci., 37, 223-232 (1984)), antioxidant activity in oils (Okuda, T. et al. Anti-hypertensive, antimicrobial, hyperglycemic, cholesterol-elevating, anti-inflammatory (Fukuyo, M. et al., J. Clin. Pharmacol. Bull., 31, 1625-1631 (1983)). Soc. Nutr. Food Sci., 39, 495-500 (1986)).

Korean Patent No. 0788331 discloses an anticancer composition comprising epigallocatechin gallate and conjugated linoleic acid as an active ingredient, Korean Patent Registration No. 0633357 discloses an anticancer composition comprising epigallocatechin gallate and Lt; RTI ID = 0.0 > cocoa < / RTI > However, the composition for inhibiting growth of cancer cells containing the methylated catechin of the present invention as an active ingredient is not known.

SUMMARY OF THE INVENTION The present invention provides a composition for inhibiting growth of cancer cells containing methylated catechin as an active ingredient. The methylated catechin (EGCG-3Me) of the present invention inhibits protein expression of MMP-1 (Matrix metalloproteinase-1) induced by ultraviolet rays and inhibits the proliferation of skin cancer, prostate cancer and breast cancer cells, .

In order to achieve the above object, the present invention provides a health functional food composition for inhibiting the growth of cancer cells, comprising methylated catechin or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for inhibiting the growth of cancer cells containing methylated catechin or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention relates to a composition for inhibiting the growth of cancer cells containing methylated catechin as an active ingredient, wherein the methylated catechin (EGCG-3Me) of the present invention inhibits the protein expression of MMP-1 (Matrix metalloproteinase-1) And has an effect of inhibiting the proliferation of skin cancer, prostate cancer and breast cancer cells, thereby being useful as a health functional food or a pharmaceutical composition for inhibiting the growth of cancer cells.

Figure 1 shows the results of UVB-induced expression of MMP-1 (Matrix metalloproteinase-1) in fibroblasts (HS68).
Fig. 2 shows the results of inhibition of protein expression of MMP-1 induced by UVB upon treatment of methylated catechin (EGCG-3Me) in fibroblast (HS68). EGCG is epigallocatechin gallate; EGCG-3Me is a methylated catechin.
FIG. 3 is a graph showing inhibitory activity of skin cancer, prostate cancer and breast cancer cell proliferation according to the methylated catechin (EGCG-3Me) treatment of the present invention. Control is a negative control without any treatment; EGCG-3Me is a methylated catechin.
4 is an optical microscope photograph showing the inhibitory activity of skin cancer, prostate cancer and breast cancer cell proliferation according to the methylated catechin (EGCG-3Me) treatment of the present invention. NT negative controls that did not process anything; EGCG-3Me is a methylated catechin.

In order to accomplish the object of the present invention, the present invention provides a health functional food composition for inhibiting the growth of cancer cells containing methylated catechin or a pharmaceutically acceptable salt thereof as an active ingredient.

The methylated catechins of the present invention may be EGCG-3Me (epigallocatechin-3-0- (3-0-methyl) gallate) or EGCG-4Me (epigallocatechin-3-0- And preferably EGCG-3Me (epigallocatechin-3-0- (3-O-methyl) gallate).

The cancer of the present invention may be skin cancer, prostate cancer or breast cancer, preferably skin cancer, but is not limited thereto.

The methylated catechins of the present invention are preferably separated from leaves of green tea, but are not limited thereto.

The concentration of the methylated catechin for the composition of the present invention may be 40-100 mu M, but is not limited thereto.

The health functional food composition according to the present invention inhibits the growth of cancer cells by inhibiting the expression of UV-induced MMP-1 and pro-MMP-1 protein.

The health functional food composition according to the present invention may further comprise at least one selected from the group consisting of gallic acid (GA), gallocatechin (GC), epigallocatechin (EGC), catechin (C) But are not limited to, one or more of epigallocatechin gallate (EGCG), epicatechin (EC), gallocatechin gallate (GCG) and epicatechin gallate (ECG) Do not.

The health functional food composition according to the present invention may be prepared in any form of powder, granule, ring, tablet, capsule, candy, syrup and beverage, but is not limited thereto.

The health functional food composition is not particularly limited as long as it can be ingested to inhibit the growth of cancer cells.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is, or may be used together with other food or food ingredients, and suitably used according to a conventional method. The active ingredient may be suitably used depending on its use purpose. Generally, the health functional food composition of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight based on the raw material, when the food or beverage is produced. However, in the case of long-term intake for health purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount in the above range.

There is no particular limitation on the kind of the health functional food. Examples of the foods to which the health functional food composition can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, soups, Drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

In addition, the health functional food composition of the present invention can be produced as a food, particularly a functional food. The functional food of the present invention includes components that are ordinarily added in food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, in the case of a drink, a natural carbohydrate or a flavoring agent may be included as an additional ingredient in addition to the active ingredient. The natural carbohydrate may be selected from the group consisting of monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, For example, xylitol, sorbitol, erythritol, etc.). The flavoring agent may be a natural flavoring agent (e.g., tau Martin, stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In addition to the above health functional food composition, it is also possible to use various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in beverages, and the like. Although the ratio of the above-mentioned ingredients is not critical, it is generally selected in the range of 0.01 to 0.1 part by weight based on 100 parts by weight of the health functional food composition of the present invention.

The present invention also provides a pharmaceutical composition for inhibiting the growth of cancer cells containing methylated catechin or a pharmaceutically acceptable salt thereof as an active ingredient.

The salt is not particularly limited as long as it is pharmaceutically acceptable so long as it is pharmaceutically acceptable and includes, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, , Benzenesulfonic acid, toluenesulfonic acid, and naphthalenesulfonic acid. In addition to acid addition salts, additional base salts such as sodium hydroxide, potassium hydroxide, triethylamine, tertiary-butylamine may also be used.

In addition, the pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the production of pharmaceutical compositions.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Various compounds or mixtures including cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

A suitable dose of the pharmaceutical composition of the present invention may be variously prescribed by factors such as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient .

The pharmaceutical composition of the present invention may be administered orally or parenterally. In the case of parenteral administration, the composition may be administered topically to the skin, intravenously, subcutaneously, intramuscularly, intraperitoneally, or transdermally.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Example  One. Of methylated catechin (EGCG3-Me) MMP -One( Matrix metalloproteinase -1) Inhibitory Effect on Protein Expression

In general, exposure to ultraviolet light destroys vitamins C and E and causes lipid peroxidation, inducing oxidative stress, damaging nucleic acids, causing skin aging and skin diseases, and increasing the activity of MMPs (matrix metalloproteinases). This increased activity of MMPs is associated with a variety of pathological conditions, including neoplastic disease. Activation of MMPs degrades the extracellular matrix (ECM), which plays a major role in the proliferation of cancer cells. Therefore, the growth of cancer cells can be inhibited by inhibiting the increase of protein expression of MMP-1 induced by ultraviolet rays.

Therefore, the HS68 fibroblasts were treated with UVB (0, 50, 75, 100 mJ / cm ² ) and after 72 hours, the protein expression level of MMP-1 was confirmed by western blot analysis. As a result, even compared with the group without irradiation of UVB as described in 1, in the group a review of 50mJ / cm ² was the MMP-1 protein expression by about 16-fold, group review of 75mJ / cm ² is 24 times, 100 mJ / cm ² , the expression of MMP-1 protein was increased 36-fold. These results indicate that when exposed to ultraviolet light, the expression of MMP-1 is increased and the ECM (Extracellular matrix) protein can be degraded. In addition, since the pro-MMPs are structurally modified and activated after cleavage of the amino terminal region, a 43 kDa blot is a form of active form of MMPs protein, and a 55 kDa blot is a form of inactivated form (proform). In Example 1, it was confirmed that the expression of inactivated proform and activated form of MMP-1 was increased upon UVB irradiation.

When the methylated catechin (EGCG-3Me) of the present invention was treated, 250 μM of EGCG-3Me was pretreated and UVB was irradiated to observe the degree of inhibition of MMP-1 protein expression by UVB irradiation. 72 hours later, -1 was analyzed. As a result, in the case of treatment with EGCG-3Me as shown in FIG. 2, the expression of MMP-1 decreased to 1/56 as compared with the case without treatment, and the expression of MMP-1 in the case of EGCG treatment was reduced to 1/28 , Indicating that EGCG-3Me exhibits higher proteolytic (metalloproteinase) inhibitory activity than EGCG. In addition, proteolytic activity, which is a characteristic of MMPs, was examined by gelatinolytic assay. As a result, the expression of EGCG-3Me was not treated with EGCG-3Me but treated with EGCG-3Me In one group, the clear zone formed a very low band, and proteolytic activity was inhibited by pretreatment of EGCG-3Me. In addition, the expression of MMP-1 (form, 43 kDa) as well as inactivated MMP-1 (proform, 55 kDa) was inhibited by treatment with EGCG-3Me. Inhibitory activity of inactivated MMP-1 EGCG-3Me showed better tendency than EGCG.

Therefore, the methylated catechin (EGCG-3Me) of the present invention inhibits the increase of the protein expression of MMP-1 due to photoaging, thereby inhibiting the degradation of ECM (extracellular matrix) and inhibiting the growth of cancer cells.

Example  2. Of methylated catechin (EGCG3-Me)  Inhibitory effect on cancer cell proliferation

(LNCaP), a breast cancer cell line (MCF-7), and a skin cancer cell line (A431) were incubated with methylated catechin (EGCG-3Me) to inhibit the growth and proliferation of cancer cells EGCG-3Me) was treated at different concentrations (20, 40 and 80 μM) and cell viability was measured. Therefore, the cancer cell lines were cultured in a 96-well plate, and treated with EGCG-3Me at different concentrations (20, 40 and 80 μM) for 36 hours. After the incubation, 20 μl of MTS [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H- tetrazolium, inner salt] reagent phenazine methosulfate] solution was added to the wells and further cultured for 1 hour (5% CO ₂ , 37 ° C). Cell proliferation in each medium was analyzed using a non-radioactive cell proliferation assay kit (Promega, Madison, Wis.) And assayed using an enzyme-linked immunosorbent assay (ELISA) plate reader And the absorbance was measured at 490 nm.

As a result, it was confirmed that when the methylated catechin (EGCG-3Me) was treated as shown in Fig. 3, cancer cell proliferation was inhibited in a concentration-dependent manner of EGCG-3Me in prostate cancer, breast cancer and skin cancer cell lines.

In the prostate cancer cell line (LNCaP), the cell proliferation inhibitory effect of cancer cell line was not substantially observed when 20 μM of EGCG-3Me was treated, but the cell survival rate was 74.4% at 40 μM and 64.6% at 80 μM. In other words, in prostate cancer cell line (LNCaP) treated with 80 μM of EGCG-3Me, 35.4% of cancer cells were inhibited. In addition, it was confirmed that cancer cell proliferation was inhibited to a level similar to that in the prostate cell line (LNCaP) in the breast cancer cell line (MCF7).

In particular, the cell survival rate of 72.8%, which was about 27.2%, was found to be 66.1% at 40 μM and 52.6% at 80 μM even when 20 μM was used in the skin cancer cell line (A431) And it was confirmed that cancer cell proliferation inhibitory effect was higher than that of the state or breast cancer cell line. Further, as shown in FIG. 4, the number of cells in each well plate after 36 hours from the treatment of each cancer cell line with EGCG-3Me was examined by an optical microscope. As a result, compared with the control without EGCG-3Me treatment The number of cancer cells (viable cells) of EGCG-3Me-treated well plate was significantly decreased, and cancer cell proliferation inhibition rate was the highest in A431, a skin cancer cell line.

Therefore, based on the results of the present study, methylated catechin (EGCG-3Me) is excellent in inhibiting the proliferation and tumor formation of various cancer cells including skin cancer, and may be effectively used in compositions for inhibiting the growth of cancer cells .

Claims (10)

(EN) A health functional food composition for inhibiting the growth of skin cancer, prostate cancer or breast cancer cells containing EGCG-3Me (epigallocatechin-3-O- (3-O-methyl) gallate) or a pharmaceutically acceptable salt thereof as an active ingredient. delete The health functional food composition according to claim 1, wherein the EGCG-3Me is extracted from green tea leaves. The health functional food composition according to claim 1, wherein the EGCG-3Me concentration is 40 to 100 μM. The health functional food composition according to claim 1, wherein the composition inhibits protein expression of MMP-1 (Martix metalloproteinase-1) and pro-MMP-1 induced by ultraviolet light. The method of claim 1, further comprising, in addition to EGCG-3Me, gallic acid (GA), gallocatechin (GC), epigallocatechin (EGC), catechin (C), epigallocatechin Characterized in that it further comprises at least one selected from epigallocatechin gallate (EGCG), epicatechin (EC), gallocatechin gallate (GCG) and epicatechin gallate (ECG) . The health functional food composition according to claim 1, wherein the composition is prepared from one of powder, granule, ring, tablet, capsule, candy, syrup and beverage. A pharmaceutical composition for inhibiting the growth of skin cancer, prostate cancer or breast cancer cells containing EGCG-3Me (epigallocatechin-3-O- (3-O-methyl) gallate) or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition according to claim 8, further comprising a pharmaceutically acceptable carrier, excipient or diluent in addition to the EGCG-3Me. [Claim 9] The pharmaceutical composition according to claim 8, wherein the composition is prepared from any one of a capsule, a powder, a granule, a tablet, a suspension, an emulsion, a syrup and an aerosol.

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