KR20050076452A - Anticancer agent containing epigallocatechin gallate and quercetin - Google Patents

Abstract

 The present invention relates to EPIGALLOCATECHIN GALLATE (EGCG) and quercetin anticancer agents. In particular, the present invention relates to quercetin, which has the effect of restoring intercellular signal transduction inhibition through interstitial binding channels, a side effect of EGCG (epigallocatechin gallate), and enhancing the inhibitory effect of matrix metalloproteinase and EGCG. It provides an anticancer composition comprising using in combination.

Description

ANTICANCER AGENT CONTAINING EPIGALLOCATECHIN GALLATE AND QUERCETIN}, including epigallocatechin gallette and quercetin

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to an anticancer composition comprising EGCG and quercetin, and more particularly, quercetin having the effect of restoring the inhibition of intracellular signal transduction, which is a side effect of EGCG, and enhancing the activity inhibitory effect of matrix metalloproteinase. It relates to an anticancer composition used 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 signaling through gap junction channels is necessary to compensate for these drawbacks because they are associated with cancer promotion (Lars-Oliver Klotz et al., Cancer research , 62: 4922-28, 2002). EGCG is expected to further enhance the efficacy of anticancer drugs by developing a substance that can compensate for the side effects of inhibiting intercellular signaling through the gap binding channel.

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 having improved side effects of inhibiting intercellular signaling through a gap binding channel by EGCG.

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 quercetin as an active ingredient.

 Hereinafter, the present invention will be described in detail.

 The inventors of the present invention, while studying the anticancer drugs that ensure the human safety, quercetin prevents the inhibition of intercellular signaling through the gap binding channel of EGCG not only inhibits the progression of cancer, but also can enhance the MMP inhibitory effect of EGCG It was confirmed that the present invention was completed based on this.

The present invention relates to an anticancer composition comprising EGCG and quercetin as an active ingredient. Each of EGCG and quercetin can be easily purchased by purchasing a commercially available compound or preparing or extracting the compound by a known method.

The ratio of EGCG and quercetin contained in the anticancer composition of the present invention is preferably adjusted according to the method of using 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 quercetin may be in a 20: 1 to 1:20 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 quercetin of the present invention as an active ingredient, may be used as a drug, food, food additives, beverages, or beverage additives. 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.

The anticancer composition comprising EGCG and quercetin as an active ingredient according to the present invention significantly improves the side effect of inhibiting intercellular signaling through the gap binding channel of EGCG, and has an excellent MMP inhibitory effect compared to 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, but the protection scope of the present invention is not limited to the following examples.

In the following examples solids and solid mixtures, liquids and liquids, and percentages for 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 Intracellular Signal Transduction Mechanism Through the Quercetin-Induced Gap Binding Channel

To determine whether quercetin has 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).

The cultured cells were dispensed at 1 x 10 ⁴ per ml in a 2 ml plastic culture plate and incubated for about 48 hours. After culturing, the cells cultured in each culture plate were EGCG 20 ㎍ / ml + quercetin 1 Μg / ml, EGCG 20 μg / ml + quercetin 5 μg / ml, EGCG 20 μg / ml + quercetin 10 μg / ml. The positive control group was treated with the culture solution, the 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).

1A to 1D 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 group treated with 20 μg / ml + quercetin 1 μg / ml of EGCG. D is a group treated with 20 μg / ml EGCG + 5 μg / ml of quercetin. That is, when EGCG alone in the cell treatment in Figure 1, intercellular signaling through the gap binding channel is sharply inhibited, but when quercetin is 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 was confirmed that the intercellular signaling, which was inhibited by EGCG treatment, recovered to about 65-98% level upon quercetin treatment. Therefore, the addition of quercetin has the effect of preventing the suppression of intercellular signaling through the gap junctions, a side effect of EGCG.

Example 2 Determination of MMP Inhibitory Effect of EGCG and MMP Inhibitory Effect of a Quercetin-Added Mixture

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 further cultured for 48 hours by treatment with a mixture of EGCG and quercetin. It was. Culture supernatants were collected, centrifuged (vision, DN-5500), quantified (Beckman, DU650), and the same amount of total protein was electrophoresed on 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 mixture of EGCG and quercetin, lane 1 is the untreated control, lane 2 is the EGCG 1 ㎍ / ㎖ treated group, lane 3 is EGCG 1 ㎍ / ㎖ + quercetin 1 ㎍ / ㎖ treatment Lane 4 is the EGCG 1 μg / ml + quercetin 5 μg / ml treatment group, and lane 5 is the EGCG 1 μg / ml + quercetin 10 μg / ml treatment group. In FIG. 3A, the first band shows the activity of MMP-9 and 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 Figures 3a to 3c, the effect of inhibiting the activity of MMP-9 and MMP-2 by EGCG is further enhanced by the addition of quercetin.

Example 3: Anticancer Agent Preparation

3-1. Powder

1 g of lactose was mixed with 2 g of a mixture of EGCG / quercetin (20: 1 weight ratio) and filled into an airtight cloth to prepare a powder.

3-2. refine

A tablet was prepared by mixing 100 mg of EGCG / quercetin (20: 1 weight ratio), 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 / quercetin (20: 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 / quercetin (20: 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 soybeans, 7% by weight black sesame, EGCG / quercetin mixture (20: 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 / Quercetin mixture (20: 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 / quercetin mixture (20: 1 weight ratio).

4-4. Biscuits

Force 1st class 25.59 wt%, gravity 1st class 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 an EEGG / Quercetin mixture (20: 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 sodium riboflavinate, 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 the quercetin mixture (20: 1 weight ratio) was combined to prepare a health beverage in a conventional manner.

4-6. sausage

Pork 65.18%, broiler 25%, starch 3.5%, soy protein 1.7%, salt 1.62%, glucose 0.5%, glycerin 1.5% and EGCG / quercetin mixture (20: 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 / quercetin mixture (20: 1 weight ratio) by combining 10% by weight to prepare a tablet health supplement in a conventional manner.

4-8. mainstream

0.5% EGCG / Quercetin mixture (20: 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 a vacuum or at 9,000 rpm with a high speed mixer. Liquor containing the EGCG / Quercetin mixture was prepared.

As described above, the quercetin according to the present invention has an effect of effectively preventing intercellular signal transduction through the gap binding, which is a side effect of EGCG, and enhancing the effect of inhibiting MMP activity when used in combination with EGCG. Therefore, the anticancer composition having quercetin added to EGCG is an ideal anticancer agent that minimizes side effects as well as excellent anticancer effect.

Figure 1a to 1d is a photograph confirming the inhibition of intercellular signaling through the gap binding channel, a is a positive control group, b is a negative control, c is treated with EGCG 20 ㎍ / ㎖ + quercetin 1 ㎍ / ㎖ D is a group treated with 20 μg / ml of EGCG + 5 μg / ml of quercetin.

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

3a to 3c confirm the effect of inhibiting MMP activity when the EGCG, EGCG and quercetin mixtures respectively treated in the cancer cell line SK-Hep-1.

Claims (5)

Epigallocatechin gallate (EPIGALLOCATECHIN GALLATE; EGCG) and an anticancer composition comprising quercetin as an active ingredient. The anticancer composition of claim 1, wherein the EGCG and quercetin are included in a weight ratio of 20: 1 to 1:20. The anticancer composition of claim 1, wherein the anticancer composition comprises 0.1 to 50% by weight of the active ingredient. 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 of claim 1, wherein the anticancer composition is applied to gastric cancer, breast cancer, colon cancer, or liver cancer.