KR100553266B1 - Composition for inhibiting liver cancer containing doxorubicin and quercetin - Google Patents

Abstract

 The present invention relates to an anticancer composition comprising doxorubicin and quercetin. In particular, the effect of not only preventing the side effects of intracellular signal transduction inhibition of doxorubicin but also enhancing the inhibition of activity of the matrix metalloproteinase relates to an anticancer composition comprising the use of quercetin in combination with doxorubicin and its use.

Doxorubicin, quercetin, anticancer, intracellular signaling, matrix metalloproteinase

Description

Composition for inhibiting liver cancer containing doxorubicin and quercetin {COMPOSITION FOR INHIBITING LIVER CANCER CONTAINING DOXORUBICIN AND QUERCETIN}

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, c is a group treated with 100 μM of doxorubicin + 15 μM of quercetin, and d Is a group treated with 100 μM of doxorubicin + 30 μM of quercetin.

Figure 2 is a graph showing the recovery of intercellular signal transduction through the gap binding channel according to the quercetin treatment.

Figure 3 confirms the effect of inhibiting MMP activity when treated with doxorubicin, doxorubicin and quercetin mixtures respectively in the cancer cell line SK-Hep-1.

Figure 4 shows the cell killing effect when treated with doxorubicin, doxorubicin and quercetin mixture, respectively, to the cancer cell line SK-Hep-1.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a composition for inhibiting liver cancer comprising doxorubicin and quercetin, and more particularly, to restore the inhibition of intracellular signal transduction, which is a side effect of doxorubicin, and to enhance the inhibitory effect of matrix metalloproteinase. It relates to a composition for inhibiting liver cancer using a quercetin having a doxorubicin mixed with.

[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).

On the other hand, while inhibition of intercellular signaling through gap junction channels is recognized as an important biochemical indicator of cancer development, agents that induce this process are well known as agents that promote and promote 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).

Quinone compounds that are used to treat solid and many cancers (Nutter LM et al., Biochem Pharmacol. Doxorubicin, one of 41 (9): 1283-92, 1991, has been reported to have side effects that inhibit intercellular signaling through interstitial channels (Kotb A et al., European Journal of Biochemistry, ps01-). 0688, 2003). Therefore, it is expected that by developing a substance that can compensate for the intercellular signal transmission inhibitory effect, it is possible to further enhance the efficacy of the anticancer agent.

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.

In order to solve the problems of the prior art, an object of the present invention is to provide a composition for inhibiting liver cancer with improved intercellular signal transduction inhibitory action through a gap binding channel by doxorubicin.

In addition, an object of the present invention is to provide a composition for inhibiting liver cancer enhanced MMPs inhibitory effect compared to doxorubicin alone.

In order to achieve the above object, the present invention provides a composition for inhibiting liver cancer comprising doxorubicin and quercetin as an active ingredient.

 The inventors of the present invention, while researching anti-cancer drugs that ensure human safety, quercetin prevents the inhibition of intracellular signaling through gap junctions, which is a side effect of doxorubicin, not only inhibits cancer progression but also enhances the MMP inhibitory effect of doxorubicin. It has been confirmed that the present invention was completed based on this.

The present invention relates to a composition for inhibiting liver cancer comprising doxorubicin and quercetin as active ingredients. Doxorubicin and quercetin are each readily available by purchasing a commercially available compound or by preparing or extracting the compound by a known method.

The ratio of doxorubicin and quercetin contained in the composition for inhibiting liver cancer of the present invention is preferably adjusted according to the method of use of the composition, the condition of the doser, the type of disease and the severity of the disease. For example, the ratio of doxorubicin and quercetin 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 the therapeutic and prophylactic purposes. However, if the ratio is out of the range, the anticancer effect may be somewhat insignificant.

The composition for inhibiting liver cancer of the present invention may further include a pharmaceutically acceptable carrier, diluent or excipient in addition to the active ingredient, wherein the content of the active ingredient is preferably 0.001 to 99% by weight of the total composition. 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 composition for inhibiting liver cancer comprising doxorubicin and quercetin of the present invention as an active ingredient may be used as a drug, food, food additive, beverage, or beverage additive. The composition may be an anticancer agent when used as a medicament, 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 Alcohols, alcoholic beverages, vitamin complexes, alcoholic beverages, and other health supplements, but are not limited thereto. In this case, the composition may be included in 0.001 to 50% by weight in the final drug, food or beverage, but is not limited thereto.

The formulation of the composition for inhibiting liver cancer may be in a preferred form depending on the method of use, and in particular, it may be formulated by adopting methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It is good. 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 composition for inhibiting liver cancer according to the present invention may be determined in consideration of the administration method, the age, sex and weight of the recipient, and the severity of the disease. For example, the 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.

The composition for inhibiting liver cancer comprising doxorubicin and quercetin according to the present invention significantly improves the side effect of inhibiting cell signaling through gap binding caused by doxorubicin, and has an excellent effect of inhibiting MMP compared to doxorubicin alone. Therefore, the 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 includes cancer diseases to which doxorubicin is applied, such as solid cancer, gastric cancer, breast cancer, ovarian cancer and liver cancer. Applicable to various cancers.

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 Intracellular Signal Transduction Mechanism Through the Quercetin-Induced Gap Binding Channel

To determine whether quercetin has 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 penicillin and 100 μg / ml 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 × 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 + 15 μM of quercetin, Or 100 μM of doxorubicin + 30 μM of quercetin. Positive controls were treated with culture, negative controls were treated with 100 μM of doxorubicin. 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 1d 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 a group treated with doxorubicin 100 μM + quercetin 15 μM, d is Doxorubicin 100 μM + quercetin 30 μM.

As shown in FIG. 1, when doxorubicin alone is treated with cells, intercellular signaling is rapidly inhibited through a gap binding channel, but when quercetin is treated together, signal transduction 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.

Figure 2 is a graph showing the recovery of intercellular signal transduction through the gap binding channel according to the quercetin treatment. In FIG. 2, it was confirmed that intercellular signal transduction, which was inhibited in doxorubicin treatment, was restored to about 87 to 103% due to 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 doxorubicin.

Example 2: Determination of MMP Inhibitory Effect of Doxorubicin and Mixture of Quercetin in MMP Inhibition

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 concentration of doxorubicin 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 a 10% SDS-PAGE gel containing 0.1% gelatin. After the end of 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 doxorubicin and quercetin, 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 0.5 μM + Quercetin 30 μM 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 activity of MMP-9 and MMP-2 is inhibited by doxorubicin, its inhibitory effect is further enhanced by the addition of quercetin.

Example 3 Anti-cancer Effect Change Test According to Quercetin Addition to Doxorubicin

To determine the cancer cell proliferation inhibitory efficacy of the doxorubicin and quercetin mixtures, MTT assay (JA Radosevich et al., Virchows Arch. B. Cell Pathol. Incl, Mol. Pathol . 63: 345-350, 1993) was performed.

Human cancer cell line SK-Hep-1 cells (Korea Cell Line Bank) were cultured in a 5% CO ₂ , 37 ° C. incubator (Forma) using DMEM medium supplemented with 10% FBS, 100 IU / mL of penicillin and 100 μg / mL of streptomycin. Scientific Co., Marjetta, OH, USA). The medium compositions used for cell culture were all used by GIBCO BRL (Grand Island, NY, USA).

In order to investigate the growth inhibitory effect of the cancer cell line by the addition of the mixture, the cultured cancer cell SK-Hep-1 was dispensed in a 96 well plate at a concentration of 1 × 10 ⁴ / well and doxorubicin (Sigma) Aldrich) and quercetin (Sigma Aldrich) were added individually or in combination, followed by incubation for 72 hours. Four hours before 72 hours, 20 μl / well of MTT reagent (Sigma Aldrich) was added to each well, and after incubation, all supernatants were removed and only cells were left. The remaining stained cells were lysed with 200 μl of DMSO (dimethyl sulfuroxide) and the absorbance was measured at 570 nm. This experiment was repeated three times.

4 is a graph showing cytotoxicity results when doxorubicin and quercetin were respectively or mixed. In FIG. 4, the inhibition rate of cancer cell growth was 93.88% in the experimental group treated with doxorubicin alone at 5 μM, and the inhibition rate of cancer cell growth was 92.68% in the experimental group mixed with quercetin 30 μM. In the experimental group treated with only 30 μM of quercetin, it was confirmed that there was little effect of inhibiting cancer cell proliferation. That is, quercetin alone does not have a cancer cell proliferation inhibitory effect, and did not have a significant level of effect on doxorubicin's cancer cell proliferation inhibitory effect.

Example 4: Anticancer Agent Preparation

4-1. Powder

1 g of lactose was mixed with 2 g of a mixture of doxorubicin / quercetin (5: 2 weight ratio) and filled into an airtight cloth to prepare a powder.

4-2. refine

Tablets were prepared by mixing 100 mg of doxorubicin / quercetin (5: 2 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.

4-3. Capsule

A capsule was prepared by mixing 100 mg of doxorubicin / quercetin (5: 2 weight ratio), 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate, and then filling the gelatine capsule.

4-4. Injection

Injectable drugs were prepared by adding a suitable amount of distilled water for injection to 100 mg of a doxorubicin / quercetin (5: 2 weight ratio) mixture, adjusting the pH to about 7.5, and then filling and sterilizing a 2 ml ampoule.

Example 5: Functional Food Preparation

5-1. 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, doxorubicin / quercetin mixture (5: 2 weight ratio) 3 A wire was prepared by mixing the wt%, ganoderma lucidum 0.5 wt% and the turmeric sulfur 0.5 wt%.

5-2. 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 / quercetin mixture (5: 2 weight ratio).

5-3. candy

Candy was prepared by the conventional method by combining 60 wt% of sugar, 39.8 wt% of starch syrup, 0.1 wt% of fragrance, and 0.1 wt% of doxorubicin / quercetin mixture (5: 2 weight ratio).

5-4. Biscuits

Force 1st grade 25.59 wt%, gravity 1st grade 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%, 0.01 wt% calcium phosphate , Biscuits were prepared in a conventional manner by combining 0.29% by weight of spray salt, 7.27% by weight of spray oil, and 1% by weight of doxorubicin / quercetin mixture (5: 2 weight ratio).

5-5. 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 doxorubicin / 1 wt% of the quercetin mixture (5: 2 weight ratio) was combined to prepare a health beverage in a conventional manner.

5-6. sausage

65.18 weight% of pork, 25 weight% of broil, 3.5 weight% of starch, 1.7 weight% of soy protein, 1.62 weight% of salt, 0.5 weight% of glucose, 1.5 weight% of glycerin and 1 weight% of doxorubicin / quercetin mixture (5: 2 weight ratio) Sausage was prepared by combining the conventional method.

5-7. 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 doxorubicin / quercetin mixture (5: 2 weight ratio) by combining 10% by weight to prepare a tablet-type dietary supplement by a conventional method.

5-8. mainstream

0.5% of the mixture of doxorubicin / quercetin (5: 2 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 state or mixed at 9,000 rpm with a high speed mixer. Liquor containing a doxorubicin / quercetin mixture was prepared.

As described above, the quercetin according to the present invention may be used in combination with an anticancer drug, doxorubicin, to effectively prevent intercellular signal transduction through gap binding, which is a side effect of doxorubicin, and to enhance the effect of inhibiting MMP activity. Thus, anticancer compositions in which quercetin is added to doxorubicin are ideal anticancer agents with not only excellent anticancer effects but also minimized side effects.

Claims (5)

A composition for inhibiting liver cancer comprising doxorubicin and quercetin as active ingredients. The anticancer composition according to claim 1, wherein the doxorubicin and quercetin are included in a weight ratio of 10: 1 to 1:10. The anticancer composition of claim 1, wherein the anticancer composition is a medicament, a food, a food additive, a beverage, or a beverage additive. delete The anticancer composition according to claim 1, wherein the content of the active ingredient in the anticancer composition is 0.1 to 50% by weight.

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