KR100633452B1 - Anticancer agent containing doxorubicin and cocoa extract - Google Patents

Abstract

The present invention relates to an anticancer composition comprising doxorubicin (DOXORUBICIN) and cocoa extract. In particular, the anticancer composition of the present invention can compensate for the side effects of doxorubicin by restoring inhibition of intercellular signal transduction through gap binding, which is a side effect of doxorubicin, and is a matrix metalloproteinase that is mainly involved in metastasis of liver cancer cells ( Matrix metalloproteinase (MMP) is effective in inhibiting liver cancer by enhancing the activity inhibitory effect, as well as can be used as a drug, food, beverage and alcohol.

Doxorubicin, cocoa extract, anticancer, gap binding, intercellular signaling, matrix metalloproteinase

Description

ANTICANCER AGENT CONTAINING DOXORUBICIN AND COCOA EXTRACT}

Figure 1a to 1d is a photograph confirming the inhibition of intercellular signal transduction through the gap binding channel, a is a positive control group, b is a negative control, c is a group treated with doxorubicin 100 μM + cocoa extract 10 ㎍ / ml And d is a group treated with 100 μM of doxorubicin + 50 μg / ml of cocoa extract.

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

3a to 3c confirm the inhibition of the activity of MMP when treated with doxorubicin, cocoa extract, and mixtures thereof in cancer cell line SK-Hep-1, respectively.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to an anticancer composition comprising a doxorubicin and a cocoa extract, and more particularly, a cocoa extract having an effect of restoring intercellular signaling inhibition, which is a side effect of doxorubicin, and enhancing the inhibition of activity of the matrix metalloproteinase. It relates to an anticancer composition using a mixture.

[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), but it has been found 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).

On the other hand, while inhibition of intercellular signaling through gap junction channels is recognized as an important biochemical indicator of cancer development, substances that inhibit this process are recognized as substances that have the effect of preventing and inhibiting cancer. (Eghbali B. et al., Proc. Natl. Acad. Sci. 87: 1328-1331, 1990; Ara C et al., Cell Mol Life Sci. 59 (10): 1758-65, 2002; Evert M et al , Carcinogenesis 23 (5): 697-703, 2002; Muramatsu A et al., Carcinogenesis 23 (2): 351-8, 2002).

However, quinone compounds (Nutter LM et al., Used to treat various types of cancer, including solid cancer, breast cancer, ovarian cancer and liver cancer). Biochem Pharmacol. Doxorubicin, one of 41 (9): 1283-92, 1991, has been reported to have side effects that inhibit intercellular signal transduction through gap binding channels (Kotb A et al., European Journal of Biochemistry, ps01-). 0688, 2003). Therefore, it is expected that the anticancer activity can be further enhanced by developing a substance that can complement the intercellular signal transduction inhibitory effect.

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.

It is an object of the present invention to provide an anticancer composition capable of improving the inhibition of intercellular signaling through a gap binding channel which is a side effect of doxorubicin.

Another object of the present invention is to provide an anticancer composition having enhanced MMPs inhibitory effect as compared to doxorubicin alone.

In order to achieve the above object, the present invention provides an anticancer composition comprising doxorubicin 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 drug that ensures the safety of the human body, the cocoa extract prevents the inhibition of intercellular signaling through gap binding, which is a side effect of doxorubicin, not only inhibits the progression of the cancer but at the same time enhances the MMP inhibitory effect of doxorubicin It was confirmed that it can be, based on this, the present invention was completed.

The present invention relates to an anticancer composition comprising doxorubicin and cocoa extract as an active ingredient.

Each of doxorubicin and cocoa extract can be easily purchased by purchasing a commercially available compound or extract, or by preparing or extracting the same by a known method. All. 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. The alcoholic water may be an alcohol having 1 to 5 carbon atoms or a diluted alcohol thereof, preferably ethanol, methanol or a dilution water of 30 to 99% 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 doxorubicin and cocoa extract 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 doxorubicin 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.

The anticancer composition of the present invention may further comprise a pharmaceutically acceptable carrier, diluent or excipient in addition to the active ingredient, wherein the active ingredient is 0.001 to 99% by weight, preferably 0.1 to 50% by weight of the total composition May be included.

As the carrier, excipient or diluent, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, 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 doxorubicin and cocoa extract of the present invention as an active ingredient, may be used as a drug, food, food additives, beverages, or beverage additives. When used as a medicament, it may be an anticancer agent, and when used as a food, food additive, beverage or beverage additive, various foods, meat, beverages, chocolate, snacks, confectionary, pizza, ramen, other noodles, gum, ice cream , Alcoholic beverages, vitamin complexes, alcoholic beverages and other health supplements, but is 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, LOTION, LPTIONS, LINIMENTS, LIMONADES, AROMATIC WATERS, POWDERS, Syrup ( SYRUPS), OPHTALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, Emulsion ), SUSPENSIONS, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, Suppositories (SUPPOSITIORIES), INJECTIONS, SPIRITS, CATASLSMA ), 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. In one embodiment, the anticancer composition of the present invention can 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 is not limited to the above range.

Anticancer composition comprising doxorubicin and cocoa extract according to the present invention as an active ingredient significantly improves the side effect of inhibiting cell signaling through gap binding caused by doxorubicin, and MMP inhibitory effect is superior to doxorubicin alone. Therefore, the anticancer composition of the present invention can be used for the prevention and treatment of cancer progression and metastasis in the early stage of cancer development, and also cancer diseases to which doxorubicin is applied, such as solid cancer, stomach cancer, breast cancer, ovarian cancer and liver cancer. Applicable to various cancers, including.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and 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 restoring intercellular signaling mechanism through the gap binding channel by cocoa extract

1-1. Preparation of Cocoa Extracts

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

1-2. Evaluation of Intercellular Signal Transduction through Coupling Channel by Cocoa Extracts

To determine whether the cocoa extract had the effect of restoring intercellular signaling through interstitial channels inhibited by doxorubicin, the SL / DT (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.When the cells were 90% full after incubation, 100 μM of doxorubicin + 10 ㎍ of cocoa extract was added to the cultured cells in each culture plate. / ml, or doxorubicin 100 μM + cocoa extract 50 μg / ml, respectively. In addition, the positive control group was treated only with the culture medium, the negative control group was treated with only 100 μM of doxorubicin, respectively. 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 is a photograph confirming whether the intercellular signaling is inhibited through the gap binding channel, a is a positive control group, b is a negative control group, c is a group treated with doxorubicin 100 μM + cocoa extract 10 ㎍ / ml , d is a group treated with 100 μM of doxorubicin + 50 μg / ml of cocoa extract. As shown in FIG. 1, when doxorubicin is treated with cells alone, intercellular signaling is rapidly inhibited through a gap binding channel. However, when cocoa extracts are treated together, signaling inhibition is inhibited.

 In addition, in order to more clearly confirm the recovery of intercellular signaling inhibition through the gap binding channel induced by doxorubicin, 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 that was suppressed during doxorubicin treatment recovered to about 70 to 80% level due to the cocoa extract treatment.

Therefore, it can be seen that the cocoa extract acts to prevent intercellular signal transduction through interstitial binding, which is a side effect of doxorubicin.

Example 2 Measurement of MMP Inhibitory Effect of Doxorubicin and MMP Inhibitory Effect of a Mixture Added with Cocoa Extracts

After culturing SK-Hep-1 cells, which are human liver cancer cells, for 24 hours, washed with PBS, incubated for 2 hours with serum-free media, and then treated with concentrations of doxorubicin and cocoa extract. Incubate further for hours. 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 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 whole background is dyed blue, and the gelatin decomposed portion appears as a white band.

Figure 3a shows the MMP inhibitory effect of the mixture of doxorubicin and cocoa extract, lane 1 is the untreated control, lane 2 is doxorubicin 0.1 μM treated group, lane 3 is doxorubicin 0.5 μM treated group, lane 4 is doxorubicin 0.1 μM + 10 μg / ml treated group with cocoa extract, lane 5 shows 0.1 μM of doxorubicin + 20 μg / ml treated group with cocoa extract, lane 6 shows 0.1 μM of doxorubicin + 40 μg / ml of cocoa extract, and the first band is MMP -9 activity, the second band is 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.

In Figures 3a to 3c, doxorubicin shows the inhibitory effect of MMP-9 and MMP-2 activity, but this inhibitory effect can be confirmed that significantly enhanced when combined with cocoa extract.

Example 3: Anticancer Drug Preparation

3-1. Powder

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

3-2. refine

Doxorubicin / Cocoa Extract Mixture 100 mg Corn starch, 100 mg lactose, and 2 mg magnesium stearate were mixed and tableted according to a conventional tablet preparation method.

3-3. Capsule

100 mg of doxorubicin / cocoa extract mixture, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed and then filled into gelatin capsules to prepare capsules.

3-4. Injection

Injectable drugs were prepared by adding a suitable amount of distilled water for injection to 100 mg of doxorubicin / cocoa extract 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.

After that, 30% by weight brown rice, 15% by weight of barley, 20% by weight of barley, 9% by weight of glutinous rice, 7% by weight of perilla, 8% by weight of black soybeans, 7% by weight of black sesame, 3% by weight of doxorubicin / cocoa extract mixture, 0.5% of ganoderma lucidum Wire was prepared by mixing% and 0.5% by weight of sulfur.

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 doxorubicin / cocoa extract mixture.

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 doxorubicin / cocoa extract mixture.

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 spraying salt, 7.27 wt% of spray oil, and 1 wt% of doxorubicin / cocoa extract mixture.

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 doxorubicin / 1 wt% of the cocoa extract mixture was blended 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% by weight doxorubicin / cocoa extract mixture Sausage was prepared.

4-7. Health Supplement

55% by weight of spirulina, 10% by weight of guar gum enzyme, 0.01% by weight of vitamin B ₁ 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 doxorubicin / cocoa extract mixture 10% by weight to prepare a dietary dietary supplement by a conventional method.

4-8. mainstream

0.5% mixture of doxorubicin / cocoa extract is mixed with soju, beer, liquor or fruit liquor to make an emulsion, followed by centrifugation at 7,000 rpm for 15 minutes in vacuum or at 9,000 rpm with a high speed mixer to mix vitamin K ₃ / quercetin This containing liquor was prepared.

As described above, the cocoa extract mixture according to the present invention effectively prevents intercellular signal transduction through gap binding, which is a side effect of doxorubicin, and enhances the effect of inhibiting MMP activity. Therefore, the anticancer composition using cocoa extract in combination with doxorubicin minimizes the side effects of doxorubicin and shows an improved anticancer effect.

Claims (5)

Anticancer composition comprising doxorubicin and cocoa extract as an active ingredient. The anticancer composition according to claim 1, wherein the doxorubicin and cocoa extract are included in a weight ratio of 10: 1 to 1:10. The anticancer composition of claim 1, wherein the anticancer composition is in the form of a medicament, a food, a food additive, a beverage, or a beverage additive. The anticancer composition of claim 1, wherein the anticancer composition is effective for solid cancer, gastric cancer, breast cancer, ovarian cancer, or liver cancer. According to claim 1, wherein the content of the active ingredient in the anticancer composition is an anticancer composition of 0.1 to 50% by weight.

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