KR20130081984A - Pharmaceutical composition for anticancer comprising extract of lysimachia foenum-graecum as effective component - Google Patents

Abstract

PURPOSE: A pharmaceutical composition for prevention or treatment of cancer containing Lysimachia foenum-graecum extract is provided to have the low possibility to cause a side effect as the active ingredient is derived from a natural substance, and to be able to be usefully used for prevention, treatment and alleviation of cancer as the composition has no cytotoxicity. CONSTITUTION: A pharmaceutical composition for prevention or treatment of cancer contains Lysimachia foenum-graecum extract as an active ingredient. The Lysimachia foenum-graecum extract is ethanol extract of Lysimachia foenum-graecum. The ethanol extract of Lysimachia foenum-graecum is ethanol extract of pH 3-pH 11. Said cancer is breast cancer, colon cancer, skin cancer or liver cancer. Food for prevention or alleviation of cancer contains Lysimachia foenum-graecum extract as an active ingredient. [Reference numerals] (AA) MCF-7, Lysimachia davurica pH 3, 6 h

Description

[0001] The present invention relates to a pharmaceutical composition for anticancer comprising an extract of Yeongran fragrance as an active ingredient, an extract of Lysimachia foenum-graecum as an effective component,

The present invention relates to Lysimachia foenum - graecum ) as an active ingredient, and a food for preventing or improving cancer.

Cancer occurs when mutations occur in genes due to environmental factors such as food, chemical contaminants, and the like. These mutations lead to uncontrolled cell proliferation, the formation of cell masses called tumors, and some of them penetrate into other tissues to cause metastasis, which makes malignant tumors, , Which is a group of diseases that can destroy the life of an individual.

According to the latest statistics from the World Health Organization, more than 10 million cancer cases occur annually worldwide, and about 1,300,000 new cases occur in the United States and about 550,000 people die from cancer. Even in Korea, the number of cancer patients is steadily increasing every year, and it accounts for the highest percentage of deaths caused by diseases. In addition, the incidence and patterns of various cancers are gradually changing to advanced countries.

There are many internal factors such as genetic factors, immunological factors, and external factors such as viruses, environment, and chemicals. It is known that the recent increase in cancer incidence is more likely to be attributed to external factors such as the environment or eating habits than to internal factors such as genetic factors, and the incidence and mortality rate of cancer will continue to increase unless people's diet changes Seems to be. In particular, the treatment of cancer is difficult to be cured unless it is detected early, and the importance of prevention and early treatment of cancer is becoming more important because of financial and mental loss due to disease progression. Therefore, it is considered necessary to develop a substance that can prevent cancer from natural substances, not chemical substances that cause drug resistance when treated for a long period of time.

On the other hand, the Youngryung scent of the present invention belongs to the primrose and has an ovate-like leaf which is offset from the circumferential stem, and the length of the stem is 40 to 60 cm. Leaf length is about 4 ~ 6cm, the tip is pointed and has long petiole, peduncle is relatively thin and soft. It has strong fragrance, it has sweet taste and it is known to be effective in relieving the pain by promoting the circulation of the period, and relieving chills, toothache, diarrhea and inflammation.

Korean Patent No. 0863524 discloses a composition for prevention and treatment of metabolic diseases containing 'Youngran Scent Extract as an active ingredient', Korean Patent Publication No. 2010-0076869 discloses a composition containing 'Youngran Scent Extract as an active ingredient Compositions for the prevention and treatment of diabetes ". However, as described in the present invention, there is no disclosure of a pharmaceutical composition for anticancer chemotherapy which contains the extract of Longran fragrance as an active ingredient.

The present invention is derived by the request as described above, the present inventors youngreung direction (Lysimachia foenum - graecum) human breast cancer cells by the ethanol extract (MCF-7), colon cancer cells (HT-29), skin cancer cells (B16F10) And hepatocellular carcinoma cells (Hep3B), and the expression level of mTOR (Mammalian target of rapamycin), COX-2 (Cyclooxygenase-2) associated with cancer cell tumor proliferation and VEGF (Vascular endothelial growth factor) of the present invention is reduced, thereby preventing, treating, and improving the cancer of the Korean native rind extract derived from a natural product having no side effects.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises Youngran Scent Extract as an active ingredient.

In addition, the present invention provides a food for preventing or improving cancer, which contains Youngran Scent Extract as an active ingredient.

According to the present invention, a pharmaceutical composition for preventing or treating cancer, which comprises Youngran Scent Extract as an active ingredient, is a substance derived from a natural product and has a low possibility of occurrence of side effects and is free from cytotoxicity. Therefore, Can be usefully used.

Fig. 1 shows the cytotoxicity of fibroblast of Youngryung incense ethanol extract. The cytotoxicity of the extracts of Youngryung fragrance was measured by MTT method using ethanol. In the graph, "None" represents the untreated control, and the horizontal axis represents the dose (ug / ml) of the Youngran Scent extract and the vertical axis represents the survival rate of the fibroblast.
FIG. 2 shows the results of measuring the anti-cancer effect of the extract of Youngryung incense on breast cancer cell (MCF-7) death. The MCF-7 cell line was used to measure the anticancer effect of the extract of Youngryung fungus according to the ethanol pH by MTT assay. In the graph, the horizontal axis represents the concentration of the Youngran Scent extract and the vertical axis represents the relative cell survival rate.
FIG. 3 shows the results of measuring the anticancer effect on the colon cancer cell (HT-29) of the extract of Youngryung incense. The HT-29 cell line was used to measure the anticancer effect of the extract of Yeongran extract according to the ethanol pH by MTT assay. In the graph, the horizontal axis represents the concentration of the Youngran Scent extract and the vertical axis represents the relative cell survival rate.
Fig. 4 shows the results of measuring the anti-cancer effect of the extract of Youngryung incense on the skin cancer cell (B16F10). The antitumor effect of the extract of Youngryung fungus according to the ethanol pH was measured by MTT assay using B16F10 cell line. In the graph, the horizontal axis represents the concentration of the Youngran Scent extract and the vertical axis represents the relative cell survival rate.
FIG. 5 shows the results of measuring the anti-cancer effect of hepatocyte cancer cells (Hep3B) on the ethanol extract of Youngryung incense. The hepatitis B virus (Hep3B) cell line was used to measure the anticancer effect of the Korean herb extract on the MTT assay. In the graph, the horizontal axis represents the concentration of the Youngran Scent extract and the vertical axis represents the relative cell survival rate.
FIG. 6 shows the results of measuring the protein inhibitory effect of cancer cell tumor proliferation of Korean herb extract against ethanol. Western blot analysis of the protein expression of p-mTOR, COX-2 and p-AMPKα1 (AMP-activated protein kinase α1) was performed to confirm the anticancer effect of the ethanol extract of Youngranung flavor using MCF-7 cell line Respectively. The numerical value on the right vertical axis represents the protein size (kDa), the left vertical axis represents the name of the protein concerned, and the horizontal axis represents the dose (쨉 g / ml) of the Korean native extract.

According to an aspect of the invention, the invention is youngreung direction (Lysimachia The present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises an extract of Foenum- Graecum as an active ingredient.

In the pharmaceutical composition according to one embodiment of the present invention, the Youngran Scent Extract may be Youngran Scent ethanol extract, but is not limited thereto.

In the pharmaceutical composition according to an embodiment of the present invention, the Korean herb extract may be an extract of ethanol having a pH of 3 to 11, but is not limited thereto.

In the pharmaceutical composition according to an embodiment of the present invention, the cancer may be breast cancer, colorectal cancer, skin cancer, liver cancer, gastric cancer, cervical cancer, lymphoma, hepatoblastoma or lung cancer and preferably breast cancer, colorectal cancer, skin cancer, But is not limited thereto.

The pharmaceutical composition for preventing or treating cancer according to the present invention may include 0.02 to 80% by weight, preferably 0.02 to 50% by weight, of the extract of Youngran Scent with respect to the total weight of the pharmaceutical composition, but is not limited thereto.

The pharmaceutical composition comprising the extract of Yeungran Flavor of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions.

The pharmaceutical dosage forms of the extract of Yeongran Flavor of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmacologically active compounds as well as in suitable aggregates.

The pharmaceutical composition comprising the extract of Yeongran Flavor according to the present invention can be administered orally or parenterally in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, oral preparation, suppository, Can be used. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Various compounds or mixtures including silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The preferred dose of the Korean herb extract of the present invention varies depending on the condition and body weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg per day, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose does not in any way limit the scope of the invention.

The extract can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In addition, the present invention provides a food for preventing or improving cancer, which contains Youngran Scent Extract as an active ingredient.

In the food according to one embodiment of the present invention, the Youngran Flavor Extract may be, but is not limited to, Youngran Flavor Ethanol Extract.

In the food according to an embodiment of the present invention, the Korean herb extract may be an ethanol extract having a pH of 3 to 11, but is not limited thereto.

In a food according to an embodiment of the present invention, the cancer may be breast cancer, colon cancer, skin cancer, liver cancer, stomach cancer, cervical cancer, lymphoma, hepatoblastoma or lung cancer, preferably breast cancer, colon cancer, skin cancer or liver cancer But is not limited thereto.

The food is not particularly limited as long as it can be ingested to increase anticancer activity.

When the Youngran Scent Extract of the present invention is used as a food additive, the Youngran Scent Extract can be directly added or used together with other food or food ingredients, and can be suitably used according to a conventional method. The mixed amount of the active ingredient can be suitably determined according to the purpose of use (prevention, health or therapeutic treatment). Generally, the extract of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of foods to which the Youngran Scent Extract can be added include dairy products such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice cream, Drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the extract of the present invention.

In addition to the above, the extract of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the extract of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, embodiments of the present invention will be described in detail. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example  One. Youngrung incense  Preparation of extract

Lysimachia 800 mg of ethanol 3, 800 ml of pH 8 ethanol and 800 ml of ethanol of pH 11 were added to 100 g of powdered foenum - graecum powder, respectively, and they were completely immersed in each extraction solvent and extracted with reflux at room temperature. Each of the Youngran scent extracts prepared by the above extraction method was concentrated under reduced pressure using a vacuum concentrator, and the obtained extract was stored at -80 ° C. The Youngran Scent Extract was dissolved in DMSO before use in the experiment and stored frozen.

Example  2. Cytotoxicity experiment

Fibroblast was purchased from ATCC (America Type Culture Collection, Gaithersburg, Md.) To confirm the cytotoxicity of the extract of Yeongrae perfume prepared in Example 1. DMEM medium (Dulbecco's modified Eagle's medium), FBS (fetal bovine serum), and antibiotics (Gibco / BRL, USA) were used for the culture of the cells. The fibroblasts were cultured in a DMEM medium supplemented with 10% FBS, 1% antibiotic (100 mg / L streptomycin, 100 U / ml penicillin) and cultured in a humidified CO ₂ incubator (5% CO ₂ ) Lt; / RTI > After the cells were cultured to about 80% of the dish, the monolayer of cells was washed with PBS, and then treated with 1 ml of trypsin-EDTA and subcultured every 2 days. The cultured cells were divided into 12 well plates for cell culture at a density of 1 × 10 ⁴ cells / ml and cultured for 24 hours. No treatment was applied to the control. After 24 hours, OD values were measured using the MTT assay.

The MTT assay is an intracellular mitochondria that reduces the yellow water-soluble substrate of MTT tetrazolium to water-soluble MTT formazan crystals with a blue purple color by dehydrogenase action. DMSO, which was confirmed to have no significant effect on cell survival, was used as a solvent for dissolving the Yeongran extract of Example 1. MTT solution was treated with 30 ㎕ of each extract at each concentration, and 1 hour later, the cells were further cultured in a CO ₂ incubator at 37 캜 for 1 hour. Thereafter, after removing the medium, 150 μl of DMSO was dissolved to dissolve formazan crystals, and 100 μl of each was transferred to a 96 well plate to measure cell viability by measuring absorbance at a wavelength of 595 nm using a microplate reader (BIO-RAD).

The results of treating the fibroblasts with Yeongran scent extract for 24 hours are shown in Fig. As shown in Fig. 1, even in the case of treatment for 24 hours, no significant effect was observed on the cell proliferation in both of the control without treatment of the sample and the treatment with the various concentrations of the extract of Yeongran Flavor prepared in Example 1 . Therefore, it was confirmed from the above results that the extract of Youngryung fragrant was not cytotoxic up to 50 / / ㎖.

Example  3. Youngrung incense  Antitumor effect of extracts on cancer cells

(MCF-7), colorectal cancer cells (HT-29), skin cancer cells (B16F10) and hepatocellular carcinoma cells (Hep3B) were cultured in ATCC Respectively. RPMI 1640 (WelGENE lnc., Seul, Korea), FBS (fetal bovine serum), antibiotics (Gibco / BRL, USA) were used for the culture of the cells. MCF-7 cells, HT-29 cells, B16F10 cells and Hep3B cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% antibiotic (100 mg / L streptomycin and 100 U / ml penicillin) Lt; 0 > C in a humidified CO ₂ incubator (5% CO ₂ ). The cells were cultured to about 80% of the culture dish, and the cell monolayer was washed with PBS, and 1 ml of trypsin-EDTA was treated and subcultured every 2 days. The cultured cells were divided into 12 well plates for cell culture at a density of 1 x 10 ⁴ cells / ml and cultured for 24 hours. No treatment was applied to the control. Ethanol pH 8, ethanol pH 11). After 24 hours, OD values were measured using the MTT assay.

MTT assay was used to treat MCF-7 breast cancer cells, HT-29 colorectal cancer cells, B16F10 skin cancer cells and Hep3B hepatoma cells for 24 hours. As shown in the results, it was confirmed that the cell growth was inhibited when the concentration of ethanol extract was increased by the concentration of ethanol for 24 hours. Therefore, it can be seen from the above results that Youngran extract is effective in inhibiting the proliferation of breast cancer cells. In particular, MCF-7 cells showed an inhibitory effect on cancer cell growth evenly in a concentration-dependent manner in all of ethanol extracts of pH 3, 8, and 11 (FIG. 2). In the case of HT-29 cells, the effect of the two extracts (pH 3, 8) up to 100 μg / ml was not significant, but when treated with 150 μg / ml of high concentration, the cell proliferation inhibitory effect was shown (FIG. In addition, the effect of two extracts (pH 3, 8) on B16F10 cells was not significant up to 100 ㎍ / ㎖, but when treated with 150 ㎍ / ㎖, cell proliferation inhibitory effect was confirmed. Especially, Cell proliferation was inhibited (Fig. 4). In Hep3B cells, we examined the effect of ethanol pH 8 extract on cancer cells over time. As a result, it was confirmed that the proliferation of cancer cells was inhibited as the concentration increased. It was confirmed that the treatment was more effective after 48 hours than 24 hours, and as a result, cell proliferation was inhibited depending on the concentration and time. Therefore, it can be seen from the above results that the extract of Yeongran extract according to the pH of ethanol is toxic to cancer cells, and the pH of the extract is changed from acidic to basic to pH 3 and pH 11 ethanol extracts from Yeongran extract showed better inhibition of cancer cell proliferation.

Example  4. Youngrung incense  Measurement of Protein Inhibitory Effect of Extract on Cancer Cell Tumor Proliferation

In order to confirm the inhibitory effect of cancer cell metastasis, that is, cancer cell migration inhibition effect, infiltration inhibition effect and adhesion inhibition effect confirmed in the above-mentioned Examples, western blot analysis was performed on the expression amount of protein related to migration, invasion and adhesion of cancer cells . More specifically, MCF-7 cells were seeded on a 6-well plate for cell culture at a density of 1 × 10 ⁵ cells / ml for 24 hours, treated with Yeongran extract and cultured in a CO ₂ incubator for 6 hours. After the medium was removed to remove the proteins, the cells were washed with PBS, and then RIPA lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 1 mM PMSF) Treated at 150 占 퐇 and dissolved at 4 占 폚 for 5 minutes. Cells were then collected using a scraper, and the supernatant was taken by centrifugation at 4 ° C. for 20 minutes at 14000 rpm. In order to quantify the protein, the ratio of 1: 100 to the Bradford reagent was mixed in a 96 well plate, and the OD value was measured at 595 nm.

The Western blot for confirming the amount of protein expression was performed in the following manner. The sample was loaded on an 8% acrylamide gel, the protein was separated by electrophoresis (SDS-PAGE), and the protein was transferred to the nitrocellulose membrane. The membrane was blocked with 2% BSA (bovine serum albumin). The primary antibody was reacted overnight at 4 ° C and washed six times with TBST for 15 min. Subsequently, the secondary antibody was reacted and washed six times for 15 minutes with TBST, and then photographed using WEST-ZOL to measure the experimental results.

The expression level of mTOR, COX-2, and the migration of cancer cells associated with cancer cell tumor proliferation, that is, the expression level of VEGF related to angiogenesis, decreased in dependence on the concentration of Youngran scent extract (FIG. 6). In addition, the expression level of AMPK, a protein known to induce natural cell death in a variety of cancer cells, was confirmed to be increased in a dose dependent manner. In particular, in the case of mTOR, it was confirmed that the expression remarkably decreased when treated with 50 μg / ml. In addition, β-actin, a housekeeping gene that is not related to the progression of cancer, has similar bands in all experimental groups, and the addition of Youngryung's fragrance extract from the band is not related to cell survival, It has been confirmed that it does not affect proteins that are not associated with cancer development.

From the above results, it can be seen that the extract of Yeongran Fungus according to the pH of ethanol inhibits the expression of protein VEGF related to cancer metastasis and proteins mTOR and COX-2, which are involved in the proliferation of tumor, and significantly inhibits the migration, invasion and adhesion of cancer cells In addition, the expression of AMPK protein, which induces the anticancer effect, was increased, and it was confirmed that it exhibited excellent anticancer effect.

Claims (8)

Lysimachia foenum - graecum ) A pharmaceutical composition for the prevention or treatment of cancer, containing the extract as an active ingredient. The pharmaceutical composition according to claim 1, wherein the Yeongneung-hyang extract is Yeongneung-hyang ethanol extract. The pharmaceutical composition according to claim 2, wherein the Youngneung ethanol extract is an ethanol extract having a pH of 3 to pH 11. The pharmaceutical composition of claim 1, wherein the cancer is breast cancer, colon cancer, skin cancer, or liver cancer. Food for preventing or improving cancer containing Youngneung extract as an active ingredient. The food according to claim 5, wherein the Yeongneung-hyang extract is Yeongneung-hyang ethanol extract. The food product of claim 6, wherein the yeongneung ethanol extract is an ethanol extract pH 3 ~ pH 11. The food product of claim 5, wherein the cancer is breast cancer, colon cancer, skin cancer, or liver cancer.

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