KR20050076453A - Anticancer agent containing epigallocatechin gallate and cocoa extract - Google Patents

Abstract

The present invention relates to an anticancer composition comprising EPIGALLOCATECHIN GALLATE (EGCG) and cocoa extract. In particular, the present invention is a cocoa extract EGCG having the effect of restoring the inhibition of intercellular signal transduction through the gap junction channel of EGCG, and enhances the inhibitory effect of matrix metalloproteinas (MMP) It relates to an anticancer composition used in combination with and its use.

Description

ANTICANCER AGENT CONTAINING EPIGALLOCATECHIN GALLATE AND COCOA EXTRACT}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to an anticancer composition comprising EGCG and cocoa extract, and more particularly, has an effect of restoring the inhibition of intercellular signal transduction, which is a side effect of EGCG, and enhancing the inhibitory effect of matrix metalloproteinase. It relates to an anticancer composition using a cocoa extract in combination with EGCG.

[Private Technology]

Recently, various diseases of adult diseases are increasing rapidly due to the improvement of the living environment and dietary changes of modern people, and chronic adult diseases such as cancer, arteriosclerosis, stroke, diabetes, and high blood pressure are greatly increased due to excessive nutrition or disproportionate diet. It is a trend. In particular, cancer has been counted as a major cause of death from the past to the present.

Drug therapy, surgical therapy, and radiation therapy are mainly performed as a method of treating cancer, and various methods have been attempted. However, drug therapy has inevitably caused side effects due to high doses of drugs in order to integrate an appropriate amount of anticancer drugs into the lesion. For example, the use of HolmiuM, which has been shown to be effective against cancer, has been shown to significantly increase anticancer effects when used as a complex with chitosan (Milican, Donghwa Pharm. Co., Ltd.), but has been shown to cause serious side effects. (Watterson. JD et al., J. Urol . 168: 442-445, 2002; Peh, OH et al., Ann. Acad. Med. Singapo re 30: 563-567, 2001).

EGCG, a major polyphenol of green tea, is known to have a very high anticancer effect, but it has been reported to have side effects that inhibit intercellular signaling through a gap binding channel. Inhibition of intercellular signal transduction through gap binding channels is necessary to compensate for these shortcomings because it is associated with the promotion of cancer (Lars-Oliver Klotz et al., Cancer research , 62: 4922-28, 2002). In addition, EGCG is expected to enhance the efficacy of anticancer drugs by developing a substance that can compensate for the side effects of inhibiting intercellular signaling through the interstitial binding channel of EGCG.

In addition, matrix metalloproteinase (hereinafter referred to as "MMP") is an enzyme that is important for cancer migration and metastasis, and is a representative enzyme that dissolves extracellular matrix. To date, it has been confirmed that MMP expression is increased in various types of carcinoma. Gelatinase A (hereinafter referred to as "MMP-2", 72 kD) and Gelatinase B (hereinafter referred to as "MMP-9", 92 kD) are known to be most directly associated with cancer metastasis, In particular, it has been reported to play an important role in metastasis of liver cancer (Kleiner DE et al., Analytical Biochemistry 218; 325-9, 1994; Stetler-Stevenson WG et al., Invasion Metastasis 14; 41-8, 1994; Lin LI et al., Oncology 55 (4); 349-53, 1998; Ming-Chung J et al., Biochemical and Biophysical Research Communication 282; 671-7, 2001; Lisa M et al., Science 295; 2387-92, 2002; Francesca T et al., The FASEB Journal 16, 2002). Accordingly, there is a demand for development of a substance capable of inhibiting MMP activity as an anticancer agent.

Accordingly, an object of the present invention is to provide an anticancer composition in which side effects of inhibiting intercellular signaling through a gap binding channel by EGCG are improved.

In addition, an object of the present invention is to provide an anticancer composition with enhanced MMPs inhibitory effect compared to using EGCG alone.

In order to achieve the above object, the present invention provides an anticancer composition comprising EGCG and cocoa extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention, while studying the anti-cancer drugs that ensure the human safety, while preventing the side effects of the cocoa extract inhibits intercellular signaling through the interstitial binding channel of EGCG not only inhibits the progression of the cancer but at the same time the MMP inhibitory effect of EGCG It was confirmed that can be enhanced, based on this has been completed the present invention.

The present invention relates to an anticancer composition comprising EGCG and cocoa extract as an active ingredient. Each of EGCG and cocoa extract can be easily purchased by purchasing a commercially available material, or by manufacturing or extracting a known method. For example, the cocoa extract may be prepared by removing cocoa butter from cacao beans and extracting the remaining fraction (cacao) or cacao beans with acetone or alcohol water. The alcoholic water may be an alcohol having 1 to 5 carbon atoms or a diluted alcohol thereof, preferably ethanol, methanol or dilution water thereof. More specifically, the cocoa extract is obtained by removing 4% by weight of 50% acetone, 50% ethanol or 50% methanol aqueous solution by removing cocoa butter from cacao beans and 1 part by weight of the remaining fraction (cacao) or cacao beans. After stirring under reflux for 4-6 hours at a temperature of −70 ° C., the supernatant obtained by centrifugation was collected and the remaining residue was repeatedly extracted, and then the extract was filtered and concentrated under reduced pressure to prepare a cocoa extract.

The ratio of EGCG and cocoa extract contained in the anticancer composition of the present invention is preferably adjusted according to the method of use of the anticancer composition, the condition of the doser, the type of disease and the severity of the disease. For example, the ratio of EGCG and cocoa extract may be in a 10: 1 to 1:10 weight ratio, which is not limited to the above because it is preferably adjusted according to a therapeutic purpose and a preventive purpose. However, if the ratio is out of the range, the anticancer effect may be somewhat insignificant.

In addition to the active ingredient, the anticancer composition of the present invention may further include a pharmaceutically acceptable carrier, diluent or excipient, wherein the content of the active ingredient may be 0.001 to 99% by weight of the total composition, preferably 0.1 to 50 wt%. Carriers, excipients or diluents which may be used include lactose, dextrose, sucrose, sorbitol, mannitol, xyitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, which may be used one or more. In addition, the anticancer composition may further include fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives.

In addition, the anticancer composition comprising the EGCG and cocoa extract of the present invention as an active ingredient, may be used as a drug, food, food additives, beverages, beverage additives and the like. The anticancer composition may be an anticancer agent when used as a medicine, and when used as a food, a food additive, a beverage or a beverage additive, various foods, meats, beverages, chocolate, snacks, confectionary, pizza, ramen, other noodles, gums, ice cream Alcohols, alcoholic beverages, vitamin complexes, alcoholic beverages, and other health supplements, but are not limited thereto. At this time, the anticancer composition may be included in 0.001 to 50% by weight in the final drug, food or beverage, but is not limited thereto.

The dosage form of the anticancer composition may be in a preferred form depending on the method of use, and is preferably formulated by employing methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. . Examples of specific formulations include PLASTERS, GRANULES lotions, LPTIONS, LINIMENTS, LIMONADES, AROMATIC WATERS, POWDERS, Syrups. ), OPHTALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, Emulsions , SUSPENSIONS, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, SSUPPOSITIORIES, INJECTIONS, SPIRITS, CATAPLSMA , Capsules (CAPSULES), creams (CREAMS), troches (TROCHES), tinks (TINCTURES), pasta (PASTES), pills (PILLS), soft or hard gelatin capsules.

The dosage of the anticancer composition according to 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 anticancer composition of the present invention may be administered at least once at 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 is not limited to the above range.

Anticancer composition comprising EGCG and cocoa extract according to the present invention as an active ingredient significantly improves the side effect of inhibiting cell signaling through gap binding of EGCG, and has a superior MMP inhibitory effect compared to using EGCG alone. Therefore, the anticancer composition of the present invention can be used for the purpose of preventing and treating cancer progression and metastasis in the early stage of cancer development, and can also be applied to various cancers such as liver cancer, breast cancer and colon cancer.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention is not limited to the scope of the present invention.

In the following experiments the solids and solid mixtures, the liquids and liquids, and the percentages for the liquids and solids are based on weight / weight, volume / volume and weight / volume, respectively, and all reactions were performed at room temperature unless otherwise noted.

Example 1: Verification of the effect of restoring intercellular signaling mechanism through the gap binding channel by cocoa extract

Cocoa butter was removed from cacao beans, and 5 parts by weight of 50% ethanol was added to 1 part by weight of the remaining fraction (cacao) or cacao beans, and the mixture was refluxed and stirred at a temperature of 50 ° C. for 5 hours. Thereafter, the supernatant was obtained by centrifugation, and the remaining residue was repeatedly extracted, and then the supernatant was collected, filtered and concentrated under reduced pressure, and dried to prepare a cocoa extract.

To determine whether cocoa extracts have the effect of restoring intercellular signaling through gap binding channels inhibited by EGCG, the Scrape Loading / Dye Transfer assay; Upham BL et al., Carcinogenesis, 18:37 -42, 1997).

WB-F344 cells (University of Michigan, USA) were prepared using DMEM medium supplemented with 10% FBS, 100 IU / mL of penicillin and 100 μg / mL of streptomycin, and a 5% CO ₂ , 37 ° C. incubator (Forma Scientific Co. , Marjetta, OH, USA). The medium compositions used for cell culture were all used by GIBCO BRL (Grand Island, NY, USA).

Cultured cells were dispensed at 1 x 10 ⁴ per ml in a 2 ml plastic culture plate and incubated for about 48 hours. When the cells were 90% full after culturing, 20 μg / ml + cocoa extract of EGCG was added to the cells cultured in each culture plate. 10 μg / ml, EGCG 20 μg / ml + cocoa extract 20 μg / ml, EGCG 20 μg / ml + cocoa extract 40 μg / ml. Positive control group was treated with culture solution, negative control group was treated with EGCG 20 ㎍ / ㎖. After 1 hour staining with lucifer yellow, the degree of intercellular signaling through the gap binding channel was observed by confocal microscopy (BioRad, Hercules, CA, USA).

Figures 1a to 1e is a picture confirming the inhibition of intercellular signaling through the gap binding channel, a is a positive control group, b is a negative control group, c is treated with EGCG 20 ㎍ / ㎖ + cocoa extract 10 ㎍ / ㎖ D is a group treated with 20 μg / ml EGCG + 20 μg / ml cocoa extract, and e is a group treated with 20 μg / ml EGCG 20 μg / ml + cocoa extract. In FIG. 1, when EGCG alone was treated to cells, intercellular signaling was rapidly inhibited through a gap binding channel, but when cocoa extracts were treated together, the effect of inhibiting signaling was observed.

In addition, in order to more clearly confirm the recovery of intercellular signaling inhibition through the interstitial binding channel induced by EGCG, the number of cells in which intercellular signaling was performed was measured and the results are shown in FIG. 2. In Figure 2 it can be seen that the intercellular signaling that was suppressed during EGCG treatment, recovered to about 50 to 85% level due to the cocoa extract treatment.

Therefore, the addition of cocoa extract has the effect of preventing intercellular signaling inhibition through the gap binding channel, which is a side effect of EGCG.

Example 2 MMP Inhibition Effect of EGCG and MMP Inhibition Effect of the Mixture Added with Cocoa Extract

SK-Hep-1 cells, which are human liver cancer cells, were cultured for 24 hours, washed with PBS, incubated for 2 hours with serum-free media, and mixed with EGCG and cocoa extract for 48 hours. Were further incubated. Culture supernatants were collected, centrifuged (vision, DN-5500), quantified (Beckman, DU650), and the same amount of total protein was electrophoresed on a 10% SDS-PAGE gel containing 0.1% gelatin. After the electrophoresis, the gel was washed three times with 2.5% Triton X-100. The gel was added to 40 mM Tris, 200 mM NaCl and 10 mM CaCl ₂ mixed solution, treated at 37 ° C. for 18 hours, and then stained with 0.1% Coomassie brilliant blue. At this time, the gel is dyed blue as a whole, the gelatin decomposed portion appears as a white band. Gel staining pictures and the results are shown in Figures 3a to 3c.

Figure 3a shows the MMP inhibitory effect of the EGCG and cocoa extract mixture, lane 1 is an untreated control, lane 2 is EGCG 1 ㎍ / ㎖ treated group, lane 3 is EGCG 1 ㎍ / ㎖ + cocoa extract 10 ㎍ / The treated group was 4 ml, and the lane 4 was EGCG 1 µg / ml + cocoa extract 20 µg / ml, and the lane 5 was EGCG 1 µg / ml + cocoa extract 40 µg / ml. In Figure 3, the first band shows the activity of MMP-9, the second band shows the activity of MMP-2.

3B is a graph comparing MMP-9 activity for each experimental group among the results of FIG. 3A, and FIG. 3C is a graph comparing MMP-2 activity for each experimental group.

As shown in Figure 3a to 3c, the effect of inhibiting the activity of MMP-9 and MMP-2 by EGCG was further enhanced by the addition of cocoa extract.

Example 3: Anticancer Agent Preparation

3-1. Powder

1 g of lactose was mixed with 2 g of the mixture of EGCG / cocoa extract (10: 1 weight ratio) and filled into an airtight cloth to prepare a powder.

3-2. refine

Tablets were prepared by mixing 100 mg of EGCG / cocoa extract (10: 1 weight ratio) mixture, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate, followed by compression according to a conventional tablet preparation method.

3-3. Capsule

A capsule was prepared by mixing 100 mg of EGCG / cocoa extract (10: 1 weight ratio), 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate, and then filling the gelatine capsule.

3-4. Injection

Injectable drugs were prepared by adding a suitable amount of distilled water for injection to 100 mg of the EGCG / cocoa extract (10: 1 weight ratio) mixture, adjusting the pH to about 7.5, and then filling and sterilizing a 2 ml ampoule.

Example 4: Functional Food Preparation

4-1. Wire

Brown rice, barley, glutinous rice, and yulmu were alphatized by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh using a grinder. 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, 30% by weight brown rice, 15% by weight barley, 20% by weight barley, 9% by weight glutinous rice, 7% by weight perilla, 8% by weight black soybean, 7% by weight black sesame, EGCG / cocoa extract (10: 1 weight ratio) 3 A wire was prepared by mixing the wt%, ganoderma lucidum 0.5 wt%, and sulfur wt% 0.5 wt%.

4-2. Chewing gum

Chewing gum was prepared in a conventional manner by combining 20% by weight of gum base, 76.9% by weight of sugar, 1% by weight of perfume, 2% by weight of water, and 0.1% by weight of EGCG / cocoa extract (10: 1 weight ratio).

4-3. candy

Candy was prepared by the conventional method by combining 60% by weight of sugar, 39.8% by weight of starch syrup, 0.1% by weight of perfume, and 0.1% by weight of EGCG / cocoa extract (10: 1 weight ratio).

4-4. 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% , Biscuits were prepared in a conventional manner by combining 0.29 wt% of spray salt, 7.27 wt% of spray oil, and 1 wt% of EGCG / cocoa extract (10: 1 weight ratio).

4-5. Health drink

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 sodium hydrochloride, 0.0001% by weight of pyridoxine hydrochloride, 0.001% by weight of inositol, 0.002% by weight of orthoic acid, 98.7362% by weight of water and EGCG / 1 wt% of cocoa extract (10: 1 weight ratio) was formulated to prepare a health beverage in a conventional manner.

4-6. sausage

65.18% pork, 25% poultry, 3.5% starch, 1.7% soy protein, 1.62% salt, 0.5% glucose, 1.5% glycerin and 1% EGCG / cocoa extract (10: 1 weight ratio) Sausage was prepared by combining the conventional method.

4-7. Health Supplement

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 And EGCG / cocoa extract (10: 1 weight ratio) by combining 10% by weight to prepare a tablet-type health supplement in a conventional manner.

4-8. mainstream

0.5% EGCG / cocoa extract (10: 1 weight ratio) is mixed with soju, beer, liquor or fruit liquor to make an emulsion, and then centrifuged at 7,000 rpm for 15 minutes in vacuum or mixed at 9,000 rpm with a high speed mixer. A liquor containing the cocoa extract mixture was prepared.

As described above, the cocoa extract according to the present invention is treated in combination with EGCG, effectively prevents intercellular signaling inhibition through gap binding channel, which is a side effect of EGCG, and enhances the effect of inhibiting MMP activity. Therefore, the anticancer composition to which cocoa extract is added to EGCG is an ideal anticancer agent that minimizes side effects as well as excellent anticancer effect.

1A to 1E are photographs confirming whether intracellular signaling is inhibited through a gap binding channel, a is a positive control group, b is a negative control group, and c is a EGCG 20 ㎍ / ml + cocoa extract 10 ㎍ / ml One group, d was treated with 20 μg / ml of epigallocatechin gallate (EGCG) + 20 μg / ml of cocoa extract, e was treated with 20 μg / ml of EGCG + 40 μg / ml of cocoa extract It's a military.

2 is a graph quantifying the results of FIG. 1.

Figures 3a to 3c confirms the effect of inhibiting MMP activity when the EGCG, EGCG and cocoa extract mixture respectively treated cancer cell line SK-Hep-1.

Claims (6)

Epigallocatechin gallate (EPIGALLOCATECHIN GALLATE; EGCG) and anti-cancer composition comprising a cocoa extract as an active ingredient. The anticancer composition of claim 1, wherein the composition comprises EGCG and cocoa extract in a 10: 1 to 1:10 weight ratio. According to claim 1, wherein the composition is an anticancer composition comprising 0.1 to 50% by weight of the active ingredient. The anticancer composition according to claim 1, wherein the cocoa extract is extracted from cacao beans with a solvent selected from the group consisting of acetone, ethanol, methanol and diluting water thereof. The anticancer composition of claim 1 wherein the composition is a medicament, a food, a food additive, a beverage, or a beverage additive. The anticancer composition according to claim 1, wherein the anticancer composition is applied to stomach cancer, breast cancer, colon cancer or liver cancer.