KR20060121998A - Composition containing eupatilin for inhibiting breast cancer - Google Patents

Abstract

A composition comprising eupatilin is provided to inhibit activity, saturation, and mobility of matrix metalloproteinase(MMP), thereby being usefully used for inhibiting progress and metastasis of breast cancer. The composition for inhibiting progress or metastasis of breast cancer comprises eupatilin represented by the formula(1) and is used for preparing medicines, processed foods, functional foods, drinks and liquors having the breast cancer progress and metastasis inhibition function.

Description

Composition containing eupatilin for inhibiting breast cancer

Figure 1 shows the results of measuring the cytotoxicity of eupatini to H-ras transformed human breast epithelial cell line H-ras MCF10A.

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

Figures 3a and 3b is the result of measuring the efficacy of eupatilin to inhibit the invasiveness of H-ras MCF10A cells.

Figures 4a and 4b is the result of measuring the efficacy of eupatilin to inhibit the mobility of H-ras MCF10A cells.

The present invention relates to a breast cancer inhibitor comprising eupatini as an active ingredient that can effectively inhibit the progression and metastasis of breast cancer by inhibiting the activity of MMP and inhibiting invasiveness and mobility 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 demonstrated to be effective by making a complex with chitosan (Milican), but its 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).

On the other hand, matrix metalloproteinase (MMP) is an enzyme that is important in the movement and metastasis of cancer, and is a representative enzyme to dissolve the 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, research on breast cancer inhibitory effect on eupatilin (eupatilin) has not been reported yet.

Therefore, the present inventors, while searching for breast cancer inhibitors derived from natural foods or plants that ensure human safety, by inhibiting the activity, invasiveness, and mobility of MMPs that eupatilin isolated from wormwood plays an important role in the metastasis of breast cancer The present invention has been completed by discovering the efficacy of inhibiting the progression and metastasis of breast cancer.

Accordingly, it is an object of the present invention to provide a medicament for inhibiting breast cancer progression or metastasis using as an active ingredient eupatillin.

In addition, another object of the present invention is to provide a food having an effect of inhibiting the progression or metastasis of breast cancer using the eupatini 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 the eupatin 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 eupatillin as an active ingredient in order to achieve the above another object.

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

In the present invention, the cytotoxicity of eupatillin was first measured, and cytotoxicity was 6.2%, 10.83%, and 14,53% at treatment concentrations of 10 μM, 20 μM, and 50 μM, respectively. Therefore, the eupatini of the present invention shows little cytotoxicity at a concentration of 50 μM or less.

On the other hand, the present invention looked at the effect of inhibiting MMP activity of eupatillin, shown in Figure 2a (lane 1 is a non-treated control; lane 2 is a 10 μM treated group of eupatillin; lane 3 is a 50 μM treated group of eupatin) As described above, it was confirmed that MMP-2 activity was inhibited (the band was clouded in the eufatlin treatment) when eupatillin was treated.

In addition, in the present invention, in order to quantitatively examine the MMP inhibitory activity of eupatillin, the inhibition of activity was measured using an NIH image analysis program (NIH homepage, USA), as shown in FIG. 2B. It was confirmed that exhibits inhibitory activity against MMP-2. In particular, it was confirmed that the concentration of 50 μM inhibits the activity of MMP-2 by 80% or more.

On the other hand, the present invention also confirmed the effect of inhibiting the invasiveness of eupatillin, as shown in Figure 3a and 3b, it was confirmed that the invasiveness of breast cancer cells at a concentration of 50μM 75% or more.

In addition, the present invention also confirmed the effect of inhibiting the cell mobility of eupatilin, as shown in Figures 4a and 4b when treated with human breast cancer epithelial cells (H-ras MCF10A cell line) 10, 20, 50 μM, as time passes Cell mobility was inhibited compared to the untreated group.

From the above results, it was confirmed that the eupatillin 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, the eupatillin of the present invention may 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 formulation 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 eupatillin, which can inhibit the progression and metastasis of breast cancer by inhibiting the activity, invasiveness, and mobility of MMPs.

Examples of the foods to which the upartylline of the present invention can be added to exhibit the above effects include, for example, various foods, meats, beverages, chocolates, snacks, confectionery, pizzas, ramen noodles, other noodles, gums, ice creams, and alcoholic beverages. , Vitamin complexes, alcoholic beverages and other health supplements, but are not necessarily limited thereto.

Eupatilloin of the present invention can be added to the raw material during food manufacturing or properly mixed with the cooked food to prepare the above-mentioned health-promoting food or drink, in this case the content of the eufatlin in the finally produced food or drink 0.01 to 50% by weight.

Example 1 Cytotoxicity Test of Eupatilin

In order to measure the cytotoxicity of eupatini, 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 inhibition effect of the cancer cell line by the addition of upatillin, the cultured breast cancer epithelial cell H-ras MCF10A was dispensed in a 96 well plate at a concentration of 1 × 10 ⁴ / well, Tilin was added at various concentrations and then incubated for 72 hours. Four hours before 72 hours, 20 μl / well of MTT reagent (Sigma Aldrich) was added to each well and incubated. 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 then absorbance was measured at 570 nm. This experiment was repeated three times.

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

Treatment concentration (μM) Cytotoxicity (%) 0 0 10 6.2 20 10.83 50 14.53

As shown in FIG. 1 and Table 1, the eufatlin of the present invention showed little cytotoxicity at a concentration of 50 μM or less.

Example 2 Inhibitory Effect of Eupatillin 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 eufatlin, and further cultured for 48 hours. It was. 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 eupatin, lane 1 is the untreated control; Lane 2 is the eupatin 10 μM treated group; Lane 3 is the eupatin 50 μM treated group; The band represents the activity of MMP-2.

In addition, the MMP inhibitory activity of eupatillin was measured using an NIH image analysis program (NIH homepage, USA), and is shown in FIG. 2B.

As shown in Figures 2a and 2b, it was confirmed that eupatillin exhibits inhibitory activity against MMP-2 at a concentration of 10 μM or more. In particular, it was confirmed that the inhibition of MMP-2 activity by 80% or more at 50 μM of eupatini.

Example 3 Invasion Inhibitory Effect of Eupatillin

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 ( 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 eupatini inhibits the invasiveness of breast cancer cells by more than 75% at a concentration of 50 μM.

Example 4 Inhibition of Cell Mobility by Eupatillin

After culturing H-ras MCF10A cells in a 6-well plate, when the cells are filled up completely, the medium is removed and mitomycin C (sigma) is dissolved in serum-free media and pretreated for 30 minutes. Inhibits proliferation After removing the medium, draw an injury line on the bottom of the well using a yellow tip, wash it with PBS, and add a medium mixed with eupatillin. 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, when eutectilin was treated with human breast cancer epithelial cells (H-ras MCF10A cell line) at 10, 20, and 50 μM, the inhibitory effect on cell mobility was observed as time passes. .

Therefore, it was confirmed that the eupatin 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, the eupatillin of the present invention can be used in the form of preparations such as powders, tablets, capsules, injections, solutions, etc. according to the method commonly used pharmaceutically or in combination with excipients used alone or pharmaceutically.

Formulation examples are illustrated below.

Production Example 1 Powder

2 g of eufatlin

1 g lactose

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

Preparation Example 2 Tablet

Eupatillin 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

Eupatillin 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

Eupatillin 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 in a known manner, and then ground to a powder having a particle size of 60 mesh.

Granules were prepared by combining the grains, seeds and eupatylin prepared above in the following proportions.

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,

Eufatillin 3 parts by weight, ganoderma 0.5 parts by weight, sulfuric acid 0.5 parts by weight

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, and 2 parts by weight of water and 0.1 part by weight of eupatillin.

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 eufatlin.

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 To prepare a biscuit in a conventional manner, 0.29 parts by weight of spraying salt, 7.27 parts by weight of spray oil, and 1 part by weight of eufatlin.

<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 riboflavin sodium hydrochloride, 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 and eupatini By blending 1 part by weight to prepare a health drink in a conventional manner.

<Manufacture example 10> Sausage

Pork 65.18 parts by weight, broiler 25 parts by weight, starch 3.5 parts by weight, soy protein 1.7 parts by weight, salt 1.62 parts by weight, glucose 0.5 parts by weight, glycerin 1.5 parts by weight and eutectilin 1 parts by weight to prepare a sausage It was.

Production Example 11 Health Supplement

Spirulina 55 parts by weight, guar gum enzyme digest 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 by combining 10 parts by weight of eu Patillin to prepare a tablet-type health supplement in a conventional manner.

<Manufacture example 12> Alcohol

0.15 to 0.7 wt% (w / v) of eupatillin is mixed with soju, beer, liquor or fruit liquor to make an emulsion, followed by separating at 7,000 rpm with a centrifuge for 15 minutes in vacuum or mixing at 9,000 rpm with a high speed mixer. Liquor containing tilin was prepared.

As described above, the eufatlin according to the present invention has an effect of effectively inhibiting the progression and metastasis of breast cancer by inhibiting the activity, invasiveness, and mobility of MMPs.

Claims (2)

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

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

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