KR20060121623A - Composition containing jaceosidin for inhibiting breast cancer - Google Patents

Abstract

A composition for inhibiting breast cancer comprising jaceosidin is provided to treat breast cancer without toxicity to breast cells by inhibiting activity, infiltration and mobility of matrix metalloproteinase(MMP). The composition for inhibiting breast cancer comprises jaceosidin(5,7,4'-tetramethoxy 6,3'-dimethoxyflavone) represented by the formula(1) as an effective ingredient, wherein the effective concentration of jaceosidin is 10-50 mu M. The composition is the pharmaceutical or food composition.

Description

Composition containing Jaceosidin for inhibiting breast cancer

Figure 1 shows the results of measuring the cytotoxicity of jaciosidin against H-ras transformed human breast epithelial cell line H-ras MCF10A cells.

Figures 2a and 2b is the result of measuring the efficacy of jaciosidin inhibit the activity of matrix metalloproteinase (MMP) in H-ras MCF10A cells.

3A and 3B show the results of measuring the efficacy of jaciosidin inhibiting invasiveness of H-ras MCF10A cells.

4A and 4B show the results of measuring the efficacy of jaciosidin inhibiting the mobility of H-ras MCF10A cells.

The present invention relates to a breast cancer inhibitor containing jaciosidin as an active ingredient that can effectively inhibit the progression and metastasis of breast cancer by inhibiting the activity, invasiveness, and mobility of MMP without toxicity to breast cells.

Recently, as the economic level of our country has risen, as the living environment has been improved and the diet has been enriched, western diets have been widely spread. As a result, chronic adult diseases such as cancer, arteriosclerosis, stroke, diabetes, and high blood pressure, which are presumed to be caused by excessive nutrition or an unbalanced diet, are increasing.

Many methods have been tried to treat cancer until now, and cancer treatment can be roughly classified into drug therapy, surgical therapy, radiation therapy, and the like. When oral or intravenous administration of anticancer drugs during drug treatment, the drug spreads throughout the bloodstream and reaches the lesion, so only trace amounts accumulate in the lesion.

Therefore, in order to accumulate an appropriate amount in the lesion, a large amount of drugs must be administered, and the side effects thereof are emerging as serious problems. In addition, there is a problem that a generally effective cancer inhibitor has a greater side effect such as pain or fever.

In particular, radiation therapy is more problematic for its safety. For example, a substance such as Holmium, which is effective for liver cancer, has been produced with a complex with chitosan (Milican), and its effects have been proved, but the side effects are serious and still have problems (Watterson. JD et al., J. Urol . 168: 442-445, 2002; Peh, OH et al., Ann. Acad. Med. Singapore 30: 563-567, 2001).

Recently, in the search for cancer inhibitors, substances and foods having an inhibitory effect on the long-term progression of cancer as a realistic and practical cancer preventive agent rather than a strategy for inhibiting it at the initiation stage of the irreversible and short-term cancer occurring in recent years. Research has been focused on finding chemistry (Kang Jin-suk et al., Chemical Cancer Prevention, Korea Medicine, 2000; Surh, YJ, Mutat. Res. 428: 305-327, 1999; Sporn, MB, Lancet. 347: 1377-1381, 1996).

Matrix metalloproteinases (MMPs) are important enzymes involved in the migration and metastasis of cancer and are representative enzymes that dissolve extracellular matrix. To date, MMP expression is increased in several types of carcinoma, and gelatinase A (MMP-2, 72 kD) and gelatinase B (MMP-9, 92 kD) are the most directly related to cancer metastasis. Known.

In particular, MMP-2 has been reported to play an important role in the metastasis of breast cancer, and substances that inhibit MMP have recently been spotlighted as novel cancer inhibitors (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).

On the other hand, studies on breast cancer inhibiting effect of jaciosidine (5,7,4'-tetramethoxy 6,3'-dimethoxyflavone) have not been reported yet.

Therefore, the present inventors searched for breast cancer inhibitors derived from natural foods or plants that ensure human safety, and jaciosidin (5,7,4'-tetramethoxy 6,3'-dimethoxyflavone) isolated from wormwood was used to metabolize breast cancer. The present invention was completed by discovering that MMPs play an important role in inhibiting the progression and metastasis of breast cancer by inhibiting the activity, invasiveness, and mobility.

Accordingly, it is an object of the present invention to provide a breast cancer inhibitor comprising jaciosidin as an active ingredient.

In addition, another object of the present invention is to provide a food having an inhibitory effect of breast cancer comprising jaciosidin as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for inhibiting the progression or metastasis of breast cancer, characterized in that it contains jaciosidin represented by the formula (1) as an active ingredient.

In addition, the present invention provides a food composition for inhibiting the progression or metastasis of breast cancer, characterized in that it comprises jaciosidin as an active ingredient in order to achieve the above another object.

Hereinafter, the configuration of the present invention will be described in more detail.

In the present invention, the cytotoxicity of jaciosidine was first measured, and cytotoxicity was 5.33%, 9.41%, and -5.94% at treatment concentrations of 10 μM, 20 μM, and 50 μM, respectively. Therefore, the jaciosidin of the present invention can be said to show little cytotoxicity at a concentration of 50 μM or less.

On the other hand, it was confirmed that the effect of inhibiting the MMP activity of jaciosidin, which is shown in Figure 2a (lane 1 is a non-treated control; lane 2 is a 10 μM treated group of jaciosidin; lane 3 is a 50 μM treated group) As shown, the effect of suppressing the MMP (by blurring of the band) of jaciosidine was confirmed. In addition, in order to quantitatively confirm the MMP inhibitory activity of zaciosidine, the result was measured using an NIH image analysis program (NIH homepage, USA), and the results are shown in FIG. 2B. As a result, it was confirmed that jaciosidin exhibits inhibitory activity against MMP-2 at a concentration of 10 μM or more. In particular, it was confirmed that at 50 μM inhibits the activity of MMP-2 by more than 90%.

On the other hand, it was confirmed that the inhibitory effect of jaciosidine, as shown in Figures 3a and 3b, it was confirmed that inhibits the invasiveness of breast cancer cells at a concentration of 50μM.

In addition, it was confirmed that the effect of inhibiting the cell mobility of jaciosidin, as shown in Figure 4a and 4b when treated with 10, 20, 50 μM of jaciosidin in human breast cancer epithelial cells (H-ras MCF10A cell line), time is As time progressed, it showed an inhibitory effect on the mobility of cells compared to the untreated group.

Accordingly, the jaciosidine of the present invention may be usefully used to inhibit the progression and metastasis of breast cancer by inhibiting the activity, invasiveness, and mobility of MMPs.

On the other hand, the jaciosidine of the present invention can be mixed with a suitable pharmaceutically acceptable carrier or excipient according to a conventional method or diluted with a diluent to prepare a pharmaceutical composition having the above function. Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, 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.

The pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. The pharmaceutical compositions of the invention may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

The formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like.

In addition, the present invention provides a functional food or beverage composition containing an effective amount of jaciosidin, which can inhibit the progression and metastasis of breast cancer by inhibiting the activity of MMP. Examples of the food to which the jaciosidin of the present invention can be added to exhibit the above effects include, for example, various foods, meat, beverages, chocolate, snacks, confectionery, pizza, ramen, other noodles, gums, ice creams, and alcohols. Beverages, vitamin complexes, alcoholic beverages and other health supplements, but are not necessarily limited thereto.

The jaciosidin of the present invention can be added to the raw material during food preparation or properly mixed with the cooked food to prepare the above-mentioned health-promoting food or drink, in which case the final preparation of The content is in the range of 0.01 to 50% by weight.

Example 1 Cytotoxicity Experiment of Jaciosidine (5,7,4'-tetramethoxy-6,3'-dimethoxyflavone)

To measure the cytotoxicity of jaciosidin, MTT assay (JA Radosevich et al., Virchows Arch. B. Cell Pathol. Incl, Mol. Pathol . 63: 345-350, 1993) was performed.

The human breast cancer cell line, H-ras MCF10A cells, 5% horse serum, penicillin (penicillin) 100 IU / ㎖ and streptomycin (streptomycin) 100 ㎍ / ㎖ using a DMEM / F12 medium supplemented with addition of 5% CO _2, 37 ℃ The cells were cultured in an incubator (Forma 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 jaciosidin, the cultured breast cancer cells H-ras MCF10A were dispensed in a 96 well plate at a concentration of 1 × 10 ⁴ / well, and then Seedin was added at various concentrations and then incubated for 72 hours. Four hours before 72 hours, each well was incubated with 20 μl / well of MTT reagent (Sigma Aldrich). After incubation, all of the supernatant was removed, leaving only the remaining cells. The remaining stained cells were lysed with 200 μl of DMSO (Dimethyl sulfoxide) and the absorbance was measured at 570 nm. This experiment was repeated three times.

1 and Table 1 show the results of measuring the cytotoxicity of jaciosidin.

Treatment concentration (μM) Cytotoxicity (%) 0 0 10 5.33 20 9.41 50 -5.94

As shown in FIG. 1 and Table 1 below, the jaciosidin of the present invention showed little cytotoxicity at concentrations of 50 μM or less.

Example 2 Inhibitory Effect of Jaciosidine on MMP Activity

After culturing H-ras MCF10A cells, which are human breast cancer cells, for 24 hours, washed with PBS, incubated for 2 hours with serum-free media, treated with concentrations of jiosidin, and further treated for 48 hours. Incubated. 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 2a shows the effect of inhibiting the MMP of jaciosidin, lane 1 is the untreated control; Lane 2 was treated with jaciosidine 10 μM; Lane 3 was treated with 50 μM treatment of jaciosidine; The band represents the activity of MMP-2.

In addition, the MMP inhibitory activity of jaciosidin is measured in the NIH image analysis program (NIH homepage, USA) is shown in Figure 2b.

As shown in Figure 2a and 2b, it was confirmed that jaciosidin exhibits inhibitory activity against MMP-2 at a concentration of 10 μM or more. In particular, it was confirmed that at least 50% inhibition of the activity of MMP-2 at 50 μM of jiosidin.

Example 3 Inhibitory Effect of Jaciosidin

A 24-well transwell (Corning Costar, Cambridge, Mass.) With a polycarbonate filter is used to coat Type 1 collagen on the bottom of the filter and Matrigel (Collaborative Research, Lexington, KY) on the top. The lower wells were filled with serum-free media containing 0.1% BSA, the H-ras MCF10A cells (human breast cancer epithelial cells) were incubated for 17 hours in the upper wells, fixed with methanol and hematoxylin , Sigma Aldrich) for 10 minutes and then Eosin (Eosin, Sigma Aldrich) for 4 minutes. Remove the filter with a razor blade and designate zone 13 through a 200X microscope to count the number of infiltrated cells on the underside of the filter.

As shown in Figures 3a and 3b, it was confirmed that jaciosidine inhibits invasiveness of breast cancer cells at a concentration of 50 μM.

Example 4 Inhibitory Effect of Jaciosidine on Cell Mobility

After culturing the H-ras MCF10A cells in 6-well plates, when the cells were filled up completely, the medium was removed and mitomycin C (Sigma Aldrich) was dissolved in serum-free media and pretreated for 30 minutes. Inhibits proliferation of cells. After removing the medium, draw an injury line on the bottom of the well using a yellow tip, wash it with PBS, and add the medium mixed with jaciosidin. Place the cells in the incubator while culturing the cell's mobility around the line drawn at intervals and take pictures with a 200X microscope.

As shown in FIGS. 4a and 4b, as the time elapsed when 10, 20, and 50 μM of the treatment of human breast cancer epithelial cells (H-ras MCF10A cell line) 10, 20, 50 μM over time showed an inhibitory effect on the cell mobility compared to the untreated group.

Therefore, it was confirmed that the jaciosidine of the present invention effectively inhibits the progression and metastasis of breast cancer by inhibiting the activity, invasiveness, and mobility of MMP at concentrations that do not exhibit cytotoxicity.

On the other hand, jaciosidine of the present invention can be used in the form of preparations such as powders, tablets, capsules, injections, solutions, etc. according to the methods commonly used pharmaceutically or in combination with excipients used alone or pharmaceutically. .

The formulation examples are given below.

Production Example 1 Powder

2 g of jaciosidine

1 g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

Preparation Example 2 Tablet

Jaciosidine 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components was prepared by tableting according to the conventional tablet production method.

Preparation Example 3 Capsule

Jaciosidine 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above ingredients was filled in gelatin capsules according to the conventional capsule preparation method to prepare a capsule.

Production Example 4 Injection

Jaciosidine 100 mg

Suitable amount of distilled water for injection

pH adjuster

Injectables were prepared by dissolving the active ingredient in distilled water for injection and adjusting the pH to about 7.5 according to a conventional injection method, and then filling the whole with a 2 ml ampoule with sterilized water for injection and sterilizing.

In addition, health foods and alcoholic beverages were prepared in the following manner.

<Manufacture example 5> Wire type

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to make a powder having a particle size of 60 mesh. Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The grains, seeds and jaciosidine prepared above were combined at the following ratio to make granules.

Cereals: 30 parts by weight brown rice, 15 parts by weight of barley, 20 parts by weight of barley, 9 parts by weight of glutinous rice,

Seeds: 7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds,

3 parts by weight of jaciosidine, 0.5 part by weight of ganoderma lucidum, 0.5 part by weight of sulfur

Preparation Example 6 Chewing Gum

Chewing gum was prepared in a conventional manner by combining 20 parts by weight of a gum base, 76.9 parts by weight of sugar, 1 part by weight of perfume, 2 parts by weight of water, and 0.1 parts by weight of jaciosidin.

Production Example 7 Candy

Candy was prepared in a conventional manner by combining 60 parts by weight of sugar, 39.8 parts by weight of starch syrup, 0.1 part by weight of perfume, and 0.1 part by weight of jaciosidin.

Production Example 8 Biscuits

Force Class 1 25.59 parts by weight, Gravity Class 1 22.22 parts by weight, white sugar 4.80 parts by weight, salt 0.73 parts by weight, glucose 0.78 parts by weight, palm shortening 11.78 parts by weight, Ammonium 1.54 parts by weight, 0.17 parts by weight sodium bisulfite 0.16 parts by weight Part, 1.45 parts by weight of rice flour, vitamin B₁0.0001 parts by weight, vitamin B20.0001 parts by weight, milk flavor 0.04 parts by weight, water 20.6998 parts by weight, whole milk powder 1.16 parts by weight, substitute powdered milk 0.29 parts by weight, calcium phosphate 0.03 parts by weight , Biscuits were prepared in a conventional manner by combining 0.29 parts by weight of spray salt, 7.27 parts by weight of spray oil, and 1 part by weight of jiosidin.

<Manufacture example 9> Healthy drink

0.26 parts by weight of honey, 0.0002 parts by weight of chioctosanamide, 0.0004 parts by weight of nicotinic acid amide, 0.0001 parts by weight of sodium riboflavinate, 0.0001 parts by weight of pyridoxine hydrochloride, 0.001 parts by weight of inositol, 0.002 parts by weight of orthoic acid and 98.7362 parts by weight of water 1 part by weight of Dean was combined to prepare a health beverage in a conventional manner.

<Manufacture example 10> Sausage

Pork 65.18 parts, chicken 25 parts by weight, starch 3.5 parts, soy protein 1.7 parts, salt 1.62 parts, glucose 0.5 parts by weight, glycerin 1.5 parts by weight and 1 part by weight of jiosidin Prepared.

Production Example 11 Health Supplement

Spirulina 55 parts by weight, guar gum degradant 10 parts by weight, vitamin B ₁ hydrochloride 0.01 parts by weight, vitamin B6 hydrochloride 0.01 parts by weight, DL-methionine 0.23 parts by weight, magnesium stearate 0.7 parts by weight, lactose 22.2 parts by weight and corn starch 1.85 parts by weight And 10 parts by weight of jaciosidine were formulated to prepare a dietary supplement.

<Manufacture example 12> Alcohol

Mix jaxyosidine 0.15 ~ 0.7% (w / v) with soju, beer, liquor or fruit liquor and make it into an emulsion state.After separating for 15 minutes under vacuum, centrifuge at 7,000 rpm or mix at 9,000 rpm with a high speed mixer. Liquor containing cidin was prepared.

<Manufacture example 12> Alcohol

Mix jaxyosidine 0.15 ~ 0.7% (w / v) with soju, beer, liquor or fruit liquor and make it into an emulsion state.After separating for 15 minutes under vacuum, centrifuge at 7,000 rpm or mix at 9,000 rpm with a high speed mixer. Liquor containing cidin was prepared.

As described above, jaciosidine according to the present invention can effectively inhibit the progression and metastasis of breast cancer by inhibiting the activity, invasiveness and mobility of MMP.

Claims (2)

A pharmaceutical composition for inhibiting the progression or metastasis of breast cancer, characterized in that it contains jaciosidin represented by Formula 1 as an active ingredient.

Food composition for inhibiting the progression or metastasis of breast cancer, characterized in that it contains jaciosidin as an active ingredient.

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