KR20120058395A - Composition Comprising Extract of Aurantii Immatri Pericarpium for Treatment of Prostate Cancer and Functional Food Comprising This - Google Patents

Abstract

PURPOSE: A composition and functional food containing Aurantii Immatri Pericarpium organic solvent extract is provided to effectively prevent and treat prostate cancer. CONSTITUTION: A composition for preventing and treating prostate cancer contains organic solvent extract of Aurantii Immatri Pericarpium as an active ingredient. The organic solvent is ethanol. The ethanol extract is prepared at 50°C for 24 hours. The composition contains pharmaceutically acceptable carrier or diluent. A functional food for preventing prostate cancer contains the extract and sitologically acceptable food additive.

Description

Composition Comprising Extract of Aurantii Immatri Pericarpium for Treatment of Prostate Cancer and Functional Food Comprising This}

The present invention relates to a novel use of the skin extract. Specifically, the present invention relates to a therapeutic composition and functional food containing the extract as an active ingredient showing an excellent prophylactic or therapeutic effect against prostate cancer.

The prostate is an organ that exists only in males that makes up part of the semen and is located below the bladder and adjacent to the rectum. Prostate cancer is a malignant tumor that occurs in the prostate gland, and is one of the most common cancers among men in the West, and it is the fastest growing cancer in men due to recent dietary habits in Korea. do. In developed countries such as North America and Western Europe, it is the most common cancer that accounts for about 20% of male cancers, and the United States has the highest frequency of male cancers occurring annually, and ranks second after lung cancer among cancer deaths. In Korea, the incidence of prostate cancer has increased significantly in recent years due to an increase in life expectancy, an increase in the elderly, westernization of a diet, development of diagnostic technology, and increased awareness of prostate cancer. According to the central cancer registration data, the cancer incidence fraction of prostate cancer was 6th in 2001, 2.7% of male cancers and 3.0% in 2002, and it is the fastest growing in recent years and is expected to increase further. to be. Prostate cancer is known to be caused by hormones, dietary habits, and chemicals in addition to genetic predisposition.

Most prostate cancers are cancers of gland cells in the prostate, which spread well to lymph nodes and bones. About 90% of prostate cancers are proliferated by male hormones that are produced in your body. For this reason, hormonal therapies that inhibit cancer growth and kill some of the cancer cells by inhibiting the action of male hormones are most commonly used in the treatment of prostate cancer (Greenlee, R. T et al., Cancer statistics.CA Cancer J. Clin 50: 7 (2000).

Treatments for prostate cancer include (a) radical prostatectomy (b) radiation (c) chemotherapy (d) hormonal therapy. The most widely used treatment is hormonal therapy, which is a so-called androgen removal method that inhibits male hormone secretion or blocks hormone production by surgery, since a significant portion of prostate cancer cells multiply androgen-dependently. This method has a temporary therapeutic effect in more than 80% of patients, such as cancer suppression or lesion reduction. However, even patients who responded well to transient hormonal therapy progressed to hormone-refractory prostate cancer (HRPC) after a certain period of time, and those who die within one year of the mean diagnosis. In the case of HRPC, conventional anticancer drugs, chemotherapy, or radiation therapy do not show a great effect, so a new treatment method is required (Fong, L. et al., Induction of tissue-specific autoimmune prostatitis with prostatic acid phosphatase immunization: implications for immunotherapy of prostate cancer.J. Immunol. 159: 3113. (1997).

Recently, with the possibility of immunotherapy using cytokines and dendritic cells, immunotherapy using dendritic cell therapy (DC vaccine) has emerged as the next generation treatment for HRPC patients (Fong, L. et al., Dendritic cells in cancer immunotherapy. Annu. Rev. Immunol. 18: 245. (2000); Xue BH. Et al., Induction of human cytotoxic T lymphocytes specific for prostate specific antigen.Prostate. 30: 73.78. (1997)). For the clinical trial of immunotherapy using dendritic cells, the effectiveness and safety of the animal model should be confirmed first. However, there is no animal model for prostate cancer to evaluate the efficacy of dendritic cell vaccine against human prostate cancer.

Meanwhile, Aurantii Immatri Pericarpium is dried fruit peel of unripe fruit of citrus or unripe fruit of tangerine. It has antitussive, expectorant effect, strengthens digestive tract, It is known to have action and anti-allergic agents. Also, Citrus unshiu Marcovich or Citrus reticulata Blanco is a young or immature rind of the fragrance and tangerine. It is a drug that has been traditionally used for the treatment of diseases of the digestive system and hepatobiliary system due to poor pain, epigastric pain, etc. [Kang, Byung-su et al., Herbology (2000)].

In addition, according to Hye-Am Hwang Do-yeon, the medicinal herb used Igigeopungsan (利 氣 祛風 散), which consists of 15 kinds of herbal medicines including Cheongpi, for the treatment of stroke and Guanwa. In oriental medicine, it has been used as an aromatic placebo or cough sputum, and constituents of blue skin include flavonoids such as hesperidin, apigenin, and naringin, and essential oils such as limonene and lemon acid. In general, the main components of skin peel are known as nobiletin, 5-hydroxyauranetin. Until now, the pharmacological action of cheongpi has been promoted digestion, anti-allergic, antibacterial action in oriental medicine has been used as a drug to help improve stomach function and liver pain, planting, breast pain and liver function.

Patents using this plant include extracts and components as cholesterol lowering agents (Korea Patent Registration No. 213895, Korea Patent Registration No. 254906, Korea Patent Registration No. 291145, and Korea Patent Registration No. 291144) ), And patents using components as a composition for inhibiting platelet aggregation (Korea Patent Registration No. 284130, Korea Patent Registration No. 298124, Korea Patent Registration No. 284129, Korea Patent Registration No. 276979), and the like. , Literature (Huang YT. Et al., Life Sciences, 57 (22), pp2011-2020, 1995), described on the effect of improving the blood pressure of cheongpi. In addition, there was a study of blood glucose levels due to dietary fiber supplements containing fiber of tangerine [Frape DL, Jones AM., Br J Nutr. 73 (5): 733-51 (1995)], fruit juices including tangerines were studied [Bolt on RP, Heaton KW, Burroughs LF. Am J Clin Nutr. 34 (2): 211-7 (1981).] And other pharmacological studies on health.

However, nothing has been disclosed or disclosed anywhere in the literature in relation to prostate cancer-related diseases of the dermal skin.

Accordingly, the present inventors completed the present invention by confirming that the extract of the skin can effectively kill prostate cancer cells during the study of herbal medicine on the skin.

It is an object of the present invention to provide a composition for treating prostate cancer and a functional food containing the extract as an active ingredient.

In order to achieve the above object, the present invention provides a composition for the prevention and treatment of prostate cancer containing the organic solvent extract of Aurantii Immatri Pericarpium as an active ingredient.

In addition, the present invention provides a functional food for preventing prostate cancer containing an organic solvent extract of Aurantii Immatri Pericarpium containing a food supplement acceptable additive as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for preventing and treating prostate cancer, which contains an organic solvent extract of Aurantii Immatri Pericarpium as an active ingredient. The composition of the present invention comprises a skin extract as an active ingredient, and may further include a pharmaceutically acceptable carrier or diluent.

In the composition for preventing and treating prostate cancer of the present invention, the organic solvent is preferably ethanol, wherein the ethanol extract is more preferably extracted at 50 ° C. for 24 hours or dried and concentrated at 45 ° C. under reduced pressure, and also Most preferably, the ethanol is 95%.

The skin extract of the present invention is possible at any part of the skin, but is preferably extracted from the skin. The extraction solution may be obtained by extraction with water or an organic solvent, and examples of the organic solvent may include lower alcohols, acetone, chloroform, methylene chloride, ether, ethyl acetate, and hexane. Lower alcohols include methanol, ethanol, propanol and butanol, with ethanol being most preferred.

Specifically, 1 to 5 times, preferably 3 times, 95% ethanol is added to the dried skin or powder, and 10 to 100 hours, preferably 15 to 15, at a temperature of 20 to 100 ° C, preferably 40 to 60 ° C. Extraction for 40 hours, more preferably 24 hours, and then filtered to prepare the ethanol extract of the skin. Preferably, the filtrate obtained by filtering the extract may be concentrated under reduced pressure. In the above extraction methods, the extraction process may be repeated two or more times as necessary, and the extract obtained after filtration may be lyophilized or dried under reduced pressure to obtain a powder form.

The "pharmaceutically acceptable carrier" is a pharmaceutically acceptable substance such as a liquid or solid filler, diluent, excipient or solvent which serves to transport the active ingredient from one organ or part of the body to another organ or part of the body. , Composition or vehicle.

The composition for treating prostate cancer of the present invention may be prepared as a medicament by adding one or more pharmaceutically acceptable carriers together with the active ingredient. The carrier may include, but is not limited to, saline, buffered saline, water, glycerol and ethanol, and any suitable agent known in the art (Remingtons's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA) may be used. .

Formulations for the formulation of the extract of the present invention can be administered orally during clinical administration and can be used in the form of general pharmaceutical preparations, when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, It is prepared using diluents or excipients such as surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and liquid preparations for oral use include suspensions, solvents, emulsions, and syrups. In addition to liquids and paraffins, various excipients may be included, such as wetting sweeteners, fragrances, preservatives and the like.

In addition, the herbal medicine that may be added to the composition of the present invention may be any pharmaceutically acceptable herbal medicine, for example, Angelica tenuissimae Radix, Gastrodiae Rhizoma, Bapleuri Radix, Angelica ( Angelicae gigantis Radix, Persicae Semen, Cinnamomi Ramulus, Rhubarb (Rhei Rhizoma), Licorice (Glycyrrhizae Radix), Cnidii Rhizoma, Aurantii nobilis Pericarpium, Taxa (Alismatis Rhizoma) Coptidis Rhizoma, Scutellariae Radix, Hoelen, Peeoniae Radix, Atractylodis Rhizoma alba, Phellodendri Cortex, Gardeniae Fructus, Pinelliae Tuber, Ramulu Set (Uncaria) Uncus, Ponciri Fructus, Ginseng (Gingseng), Liriopis Tuber, Polygalae Radix, Acori graminei Rhizoma, Atractylodis Rhizoma alba, Chrysanthemi Flos, Windproof (Ledebouri) ), Ginger (Aurantii Immatri Pericarpium crudus), forget-me-not (Natrii sulfas), Zizyphi Fructus, Salviae Radix, Mautan Radicis Cortex, Rehmanniae Radix, Minthae Herba, Dioscoreae Rhizoma, Polyporus, Polygoni multiflori Radix, Guja Allii tuberosi Semen, Cassiae Semen, Lycii Fructus, Araliae cordatae Radix, Eucommiae Cortex, Hyedotis Herba, Saururus Herba, Artemisiaecapillaris Herba (Anemarrhenae Rhizoma), Safflower (Carthami Flos), Astragali Radix, Lycopodium, Ginkgonis Folium, Polygonati Rhizoma, Nelumbinis Semen, Keel (Fossilia ossis Mastodiyc, Bones) radicis Cortex, Achyranthis Radix, Rehmanniae Radix preparata, Perillae Semen, Thujae Semen, Malt (Hordei Fructus germinatus), Cuscutae Semen, Morindae Radix, Sea Song Pini koraiensis Radix) can be used alone or in combination. .

The composition of the present invention may be administered in various parenteral formulations during actual clinical administration, and solid preparations include tablets, pills, powders, granules, capsules, and the like. In addition to water, liquid, and paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Specifically, preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like. In addition, calcium or vitamin D ₃ may be added to enhance the efficacy of the treatment. Such compositions may be presented in unit-dose (single) or multi-dose (several) containers, such as sealed ampoules and vials, and immediately before use, sterile liquid carriers such as injectable water. Can be stored under freeze-drying conditions requiring only the addition of. Immediate injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

The formulations of the present invention can be applied differently depending on the age, sex, condition of the subject, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the drug used in combination. The invention also includes formulations of dosage units. The formulations are present in individual dosage forms, such as tablets, coated tablets, capsules, pills, suppositories, and ampoules, wherein the amount of active compound in the drug corresponds to the fraction or multiple of the individual dosage. Dosage units may contain, for example, one, two, three or four times the individual dosage, or 1/2, 1/3 or 1/4 times. The individual dosages preferably contain an amount in which the active compound is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dosage.

As used herein, the term "extract" refers to an active ingredient isolated from natural products. The extract may be obtained by an extraction process using water, an organic solvent, or a mixed solvent thereof, and includes an extract, a dry powder thereof, or any form formulated using the same.

In a specific embodiment of the present invention, the ethanol extracts of the dermis kill 54% and 66% of PC-3 or DU 145 prostate cancer cells at 100 μg / ml, respectively. The above results demonstrate that the skin extract of the present invention has excellent killing activity of PC-3 or DU 145 prostate cancer cells and further has prostate cancer treatment and prophylactic activity.

As used herein, the term "prevention" means any action that inhibits or delays the development of prostate cancer by administration of the composition.

As used herein, the term "treatment" means any action that improves or advantageously changes the symptoms of prostate cancer by administration of the composition.

Cheongpi extract in the present invention can be extracted by using water, an organic solvent, or a mixed solvent thereof. Preferably it is extracted using an organic solvent, in particular ethanol. The extracted solution can be used directly or can be concentrated and / or dried. When extracted with an organic solvent, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof may be used and extracted by room temperature or warming under conditions where the active ingredient of the herbal medicine is not destroyed or minimized. Depending on the organic solvent to be extracted, the degree of extraction and loss of the active ingredient of the drug may vary, so select an appropriate organic solvent. The extraction method is not particularly limited, and examples thereof include cold needle extraction, ultrasonic extraction, reflux cooling extraction, and the like. Filtration is a process of removing the suspended solid particles from the extract, it may be used to filter the particles using cotton, nylon or the like, or may be used, such as ultrafiltration, cryofiltration, centrifugal separation, but is not limited thereto.

Concentration of the extract may be used, such as concentrated under reduced pressure, reverse osmosis concentration. The drying step after concentration includes freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying, infrared drying, and the like. If desired, a process of grinding the final dried extract may be added.

In addition, the extract can perform an additional fractionation process. Preferably, the extract is suspended in distilled water to obtain a nonpolar solvent soluble layer by extraction and separation with a nonpolar organic solvent such as hexane, ether, dichloromethane, chloroform, ethyl acetate, or a mixed solvent thereof. It can be used by concentrating and / or drying it.

In the present invention, the term "pharmaceutically acceptable salts" means salts derived from pharmacologically or physiologically acceptable inorganic acids, organic acids and bases. Examples of suitable acid include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, Formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, ammonium and the like.

The pharmaceutical composition for preventing and treating prostate cancer diseases of the present invention comprises 0.1 to 50% by weight of the extract or compound based on the total weight of the composition. In addition, the composition does not increase the efficacy, but may include additional ingredients that are commonly used in the pharmaceutical composition to improve the smell, taste, time and the like. In addition, the composition adds inorganic and organic additives such as vitamins B1, B2, B6, C, E, niacin, carnitine, betaine, folate pantothenic acid, biotin, zinc, iron, calcium, chromium, magnesium, and mixtures thereof. It can be included as. In addition, the composition may include a substance having a therapeutic activity against prostate cancer, used alone or previously used.

As used herein, the term "patient" refers to humans and horses, sheep, pigs, goats, and camels that have a disease caused by prostate cancer and its direct and indirect causes, and whose symptoms may be improved by administering the composition of the present invention. Means animals such as nutrition, dogs. By administering to the patient a composition comprising the extract of the present invention, the above-mentioned prostate cancer can be effectively prevented and treated. The composition of the present invention can be administered in parallel with existing prostate cancer therapies.

As used herein, the term "administration" means introducing a predetermined substance into a patient by any suitable method, and the route of administration of the composition of the present invention is oral or parenteral via any general route as long as the target tissue can be reached. May be administered. In addition, the composition may be administered by any device in which the active agent may migrate to the target cell.

The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to a patient's sexually transmitted disease, age, severity, and drug activity. , Drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. It may be single or multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. The method of administering the composition comprising the extract or compound prepared according to the preparation method of the present invention is preferably oral administration or intravenous administration. In general, when the effective dose is oral administration, it is usually 1 to 1 time per adult. 500 mg / kg is preferred, and in the case of intravenous administration, 1 to 100 mg / kg is preferred, and may be administered 2-3 times a day. Dosage levels for a particular patient may vary depending on sex, age, skin condition, diet, time of administration, method of administration, drug mixture, patient's condition and the extent of neurological disease.

In addition, the present invention is a sheltered ( Arrantii) comprising a food acceptable food supplement additive Immatri It provides a functional food for the prevention of prostate cancer containing the organic solvent extract of Pericarpium ) as an active ingredient.

The functional food of the present invention preferably contains 0.1 to 5% by weight, more preferably 1% by weight of the organic solvent extract of the skin peel. Functional food of the present invention is not particularly limited in the formulation, for example, dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, Various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, etc., and includes all of the health food in the usual sense.

In the present invention, "functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functional" refers to the structure of the human body And ingestion for the purpose of obtaining nutrients for function or for obtaining useful effects in health uses such as physiological actions.

In the present invention, when the dermal extract of the present invention was injected into PC-3 or DU 145 prostate cancer cells, it was confirmed that PC-3 or DU 145 prostate cancer cells were killed (see FIGS . 1 and 2 ). In addition, as a result of flow cytometry and Western blotting, extracts of blue blood extracts prostate cancer cells of human prostate cancer cell lines PC-3 and DU 145 cells to stop cell division in a specific cell cycle, thereby apoptosis (apoptosis). ) Process (see FIGS. 3, 4 and 8 ). In addition, the extract extracts affect the Myc, AP-1 cell signal and NF-κB cell signal of prostate cancer cells (see FIGS. 5, 6 and 7 ). Therefore, it can be seen that the extract of the skin extract has a prostate cancer treatment effect. Thus, in the present invention, the present invention was completed by preparing a functional food for preventing prostate cancer, which contains the extract as an active ingredient (see Preparation Example 2 ).

As discussed above, the extract of the present invention inhibits the growth of prostate cancer cells and induces apoptosis. Therefore, the composition for treating prostate cancer according to the present invention will be very effective for the treatment of patients with prostate cancer.

1 is a result of Alamar Blue analysis to determine the effect of the introduction of the dermal extract obtained in Example 1 on the growth of prostate cancer cells in PC-3 cells, a human prostate cancer cell line, wherein the X-axis is the concentration of the dermal extract, The Y axis shows the survival rate of surviving human prostate cancer PC-3 cells.
Figure 2 is the result of Alamar Blue analysis to determine the effect of the introduction of the dermal extract obtained in Example 1 on the growth of prostate cancer cells in DU 145 cells, a human prostate cancer cell line, wherein the X-axis is the concentration of the extract of the skin, Y The axis shows the survival rate of surviving human prostate cancer DU 145 cells.
Figure 3 is a flow cytometry result to determine the effect of the introduction of the dermal extract obtained in Example 1 in PC-3 cells, a human prostate cancer cell line to stop the cell cycle and apoptosis of prostate cancer cells.
Figure 4 is a flow cytometry result to determine the effect of the introduction of dermal extract obtained in Example 1 in human prostate cancer cell line DU 145 to stop the prostate cancer cells cell cycle and apoptosis.
5 is a result of analyzing luciferase activity by the Myc reporter assay method to determine the effect of the introduction of the extract of the skin in PC-3 human prostate cancer cell line Myc signal of the prostate cancer cells.
Figure 6 shows the results of analyzing luciferase activity in the AP-1 reporter assay to determine the effect of the introduction of dermal extract in PC-3 human prostate cancer cell line AP-1 cell signal of prostate cancer cells.
Figure 7 is the result of analyzing luciferase activity in the NF-κB reporter assay to determine the effect of the introduction of the extract of the skin extract in PC-3 human prostate cancer cell line NF-κB cell signal of prostate cancer cells.
8 is a result of Western blotting analysis of the quantitative change of Bax induced the apoptosis of prostate cancer cells by the introduction of dermal extract in PC-3, a human prostate cancer cell line.

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

Example 1 Preparation of Extract

3 kg of cheongpi (Chinese) purchased from Seoul medicinal herb was dried and ground for 5 days at the shade and room temperature. The ground skin was soaked in 30 L of 95% ethanol and extracted at 50 ° C. for 24 hours. This was filtered through filter paper, dried and concentrated under reduced pressure of 45 ° C. to obtain 285 g of the total extract, and stored at −20 ° C.

Example 2 Preparation and Treatment of Human Prostate Cancer Cell Line

The human prostate cancer cell lines PC-3 and DU 145 used in the present invention were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA) and used for experiments. Specifically, PC-3 and DU 145 cell lines were treated with DMEM (Dulbeco's Modified Eagle's Medium) medium containing 10% fetal bovine serum (FBS) (Welgene) at 37 ° C. and 5% CO ₂ . It was kept as it was, and it was passaged for 2-3 days.

Example 3 Effect of Cheongpi Extract on Growth of Prostate Cancer Cells

In order to examine the effect of the extract extracted in Example 1 on the growth of prostate cancer cells, PC3 and DU 145 cells, which are human prostate cancer cells, were treated with ethanol extract of the skin for 48 hours and subjected to Alamar Blue analysis. . The Alamar Blue assay is a modified form of the MTT assay, in which a specific enzyme degrades a living cell and then measures the fluorescence intensity of the product as the compound breaks down to determine the relative number of living cells after treatment. I am an experimental method. It will be described in more detail below.

96 well plates were seeded with 4 × 10 ³ PC-3 and DU 145 cells per well and incubated for 24 hours. Blue skin of Example 1 dissolved in DMSO (dimethyl sulfoxide) When the ethanol extract was treated at concentrations of 0 to 100 μg / ml (specifically, concentrations of 0, 3.125, 6.25, 12.5, 25, 50, and 100 μg / ml, respectively) for 48 hours, the degree of inhibition of cell growth was confirmed. (Table 1 and Table 2). After treatment with each concentration of extract, 20 μl of Alamar Blue reagent was added to 0.2 ml of cell culture filled in each well in a 96-well plate, and the plates were incubated for 2 hours in an incubator. The plate was slowly shaken to evenly react the cells of each well, and the intensity of fluorescence was measured by a Fluorescence Microplate Reader (Molecular Devices Corp.) at 590 nm while irradiating irradiated light at a wavelength of 544 nm. Survival rates of -3 and DU 145 human prostate cancer cells are also shown in FIGS. 1 and 2.

Bark Concentration of Example 1 Experiment 1 Experiment 2 Experiment 3 PC-3 cell viability (average) Standard Deviation 0 μg / ml 100% 100% 100% 100% 0% 3.125 μg / ml 101% 105% 101% 102% 2% 6.25 μg / ml 107% 98% 98% 101% 5% 12.5 μg / ml 103% 99% 97% 100% 3% 25 μg / ml 95% 91% 90% 92% 3% 50 μg / ml 75% 72% 68% 72% 3% 100 μg / ml 47% 50% 41% 46% 4%

Bark Concentration of Example 1 Experiment 1 Experiment 2 Experiment 3 DU 145 cell viability (average) Standard Deviation 0 μg / ml 100% 100% 100% 100% 0% 3.125 μg / ml 104% 106% 89% 99% 9% 6.25 μg / ml 107% 108% 91% 102% 9% 12.5 μg / ml 107% 102% 92% 101% 7% 25 μg / ml 91% 91% 82% 88% 5% 50 μg / ml 65% 65% 62% 64% 2% 100 μg / ml 34% 36% 33% 34% 2%

As a result, as shown in Table 1 and Table 2, the growth of prostate cancer cells decreased as the treatment concentration of cheongpi increased, indicating that cheongpi has a prostate cancer treatment effect. That is, in Table 1, prostate cancer cells were killed at 8% at 25 μg / ml, 28% at 50 μg / ml, and 54% at 100 μg / ml. In Table 2, prostate cancer cells were killed at 12% at 25 μg / ml, 36% at 50 μg / ml, and 66% at 100 μg / ml. Therefore, it is judged that the therapeutic effect of skin extract extracted in Example 1 is more excellent. Table 1 and Table 2 describe the relative cell numbers of prostate cancer cells after 48 hours according to the concentration of each skin treatment, based on the number of survival rates of prostate cancer cells of the control group not treated with dermal skin. As such, the skin extract of the present invention has excellent PC-3 and DU 145 prostate cancer cell killing activity and further demonstrates prostate cancer treatment and prophylactic activity.

Example 4 Effect of Cheongpi Extract on Cell Division Cycle of Prostate Cancer Cells

Flow Cytometry was performed to investigate the effect of extracts on cell division cycle of cancer cells. Flow cytometry is a method to quickly identify the characteristics of a cell as it passes through the detection zone. When staining the cell nucleus with a fluorescent material, the flow cytometry can be determined. .

Specifically, PC-3 and DU 145 cells were seeded in 6-well plates at about 2.4 × 10 ⁵ cells per well and incubated for 24 hours. Cells were administered 100 μg / ml of dermal extract and left for 24 hours. The culture medium in each well was removed from the 6-well plate and washed twice with PBS (phosphate-buffered saline). The attached cells were suspended by treatment with trypsin-EDTA solution, transferred to microtubes and centrifuged to recover the cells. The recovered cells were washed with PBS, centrifuged and suspended in cold ethanol. Cells suspended in ethanol were stored at 4 ° C to fix the cells, centrifuged and washed with PBS. The washed cells were suspended in 500 μl of PI solution (50 μg / ml of Propidium Iodide, 10 μg / ml of RNase A) and stored at 37 ° C. for 30 minutes to stain nuclei, and 2 ml of PBS was added thereto. Cells suspended in solution were analyzed by flow cytometry (FACS; BD biosciences), and cell cycles and apoptosis of PC-3 and DU 145 human prostate cancer cells were shown in FIGS. 3 and 4, respectively.

As a result, as shown in Figures 3 and 4, the cancer cells treated with the extract were confirmed that cell division was stopped in a specific cell cycle compared to the control group, and the apoptosis process (apoptosis), which is apoptosis process, was in progress. From this, it can be seen that the skin extracts stop the division of cancer cells and lead to apoptosis.

Example 5 Effect of Cheongpi Extract on Cell Signaling of Prostate Cancer Cells

In order to examine the effect of the extract on the cell signal leading to the growth of cancer cells, cell signal inhibition activity in the actual cells was confirmed using a reporter assay (reporter assay).

Specifically, the reporter assay is a human luciferase cell PC-3 luciferase vector (luciferase vector) designed to measure the transcriptional activities of Myc, AP-1, and NF-κB, which have a major effect on the growth of cancer cells. After transfection in 6 hours, it was proceeded by treating the dermal extract at a concentration of 0 to 100 μg / ml and measuring the activity of luciferase after 24 hours. As a result, the degree of inhibition of cellular signals of Myc, AP-1, and NF-κB expressed in human prostate cancer cells is shown in Table 3 below.

Myc signal AP-1 signal NF-kB signal density Average Standard Deviation Average Standard Deviation Average Standard Deviation 0 μg / ml 100% 0% 100% 0% 100% 0% 100 μg / ml 59% 4% 50% 9% 77% 16%

In addition, as shown in Figures 5, 6 and 7, it was confirmed that the carcinoma treated cancer cells lowered the transcriptional activity of Myc, AP-1 and NF-κB leading the growth of cancer cells compared to the control group. Cheongpi extract was found to inhibit the growth of cancer cells.

Example 6 Effect of Cheongpi Extract on Apoptosis of Prostate Cancer Cells

In order to examine the effect of the extract on the apoptosis of cancer cells, the quantitative change of Bax inducing apoptosis process was confirmed by Western blotting method after the extract was treated on human prostate cancer cells.

Specifically, PC-3 cells were cultured to grow 60 to 70% in a 60 mm dish, and then the extract was treated with 0-100 μg / ml concentration. After 24 hours, the culture medium was removed from the cells in culture, washed with PBS, and treated with NP-40 solution to obtain cell lysate. The cell lysate was centrifuged to recover the supernatant in which the cellular protein was dissolved, and the sample was prepared for electrophoresis after measuring the concentration. The prepared samples were electrophoresed and Western blotting was performed to analyze the amount of Bax protein with Bax antibody. The results are shown in FIG. 8.

As shown in Figure 8, the extract was confirmed that the amount of Bax leading to apoptosis of cancer cells increased, through which it can be seen that the cheongpi effectively leads to cancer cells to apoptosis, which has an anticancer effect.

Example 7 Acute Toxicity Test by Extracts

Cheongpi used in the present invention was widely used as a medicinal herb, but it was judged that there would be no problem in stability.

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were administered orally at a dose of 5 g / kg in suspension of the extract of Example 1 of the present invention in 0.5% methylcellulose solution. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and hematological examinations were performed.

As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, all extracts showed no change in toxicity up to 5 g / kg in rats and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of 5 g / kg or more.

Preparation Example 1 Preparation of Prostate Cancer Therapeutic Agents Containing Cheongpi Extract as Active Ingredient

The present inventors have confirmed that the prostate cancer treatment effect of the extract of the blue skin through the above embodiment was prepared to prepare a prostate cancer treatment containing the extract as an active ingredient as follows. In addition, the preparation of the following therapeutic agents can be used for the application of not only therapeutic agents but also functional foods.

<1-1> Soft Gelatin Capsules Containing Green Skin Extract

Peel Extract 20%

Vitamin C 4.5%

Vitamin D ₃ 0.001%

Manganese Sulfate 0.1%

10% wax

Palm oil 25%

Safflower Seed Oil 30.399%

 <1-2> Preparation of Intravenous Formulation Containing Peel Extract

Peel Extract 0.2%

Mannitol 0.3%

Saline 9.5%

<1-3> tablet containing the skin extract

Green skin extract 35%

Vitamin C 10%

Vitamin D ₃ 0.001%

Manganese Sulfate 0.1%

Crystalline cellulose 25.0%

Lactose 17.999%

Magnesium Stearate 2%

Preparation Example 2 Preparation of Functional Foods Containing Cheongpi Extract as Active Ingredient

The present inventors have confirmed that the extract of the blue skin through the above-mentioned prostate cancer treatment activity was excellent to prepare a functional food containing it as an active ingredient as follows.

<2-1> Preparation of Drink

522 mg of honey

Chioctosanamide 5 mg

Nicotinamide 10 mg

Riboflavin Sodium Hydrochloride 3 mg

Pyridoxine hydrochloride 2 mg

Inositol 30 mg

Orthoic acid 50 mg

Green skin extract 0.48 ~ 1.28 mg

200 ml of water

A beverage was prepared using the above-mentioned composition and content by a conventional method.

<2-2> production of chewing gum

Gum base 20%

Sugar 76.36-76.76%

Green skin extract 0.24 ~ 0.64%

1% fruit flavor

Water 2%

Chewing gum was prepared using the above-mentioned composition and content by a conventional method.

<2-3> Preparation of Candy

Sugar 50-60%

Starch syrup 39.26 ~ 49.66%

Green skin extract 0.24 ~ 0.64%

Orange flavor 0.1%

The composition and the content of the candy were prepared using a conventional method.

<2-4> Preparation of Biscuits

Force Class 1 88 kg

Gravity First Class 76.4 ㎏

16.5 kg per white

2.5 kg of salt

2.7 kg of glucose

Palm shortening 40.5 kg

5.3 kg of ammo

Medium kg 0.6 kg

0.55 kg sodium bisulfite

Rice flour 5.0 kg

Vitamin B1 0.003 kg

0.003 kg of vitamin B2

Milk Flavor 0.16 ㎏

71.1 kg of water

Whole milk powder 4 kg

Substitute powder 1 kg

0.1 kg of calcium phosphate

Spray salt 1 kg

25 kg of spray oil

Peel Extract 0.2 ~ 0.5 kg

Biscuits were prepared using conventional methods with the above composition and content.

<2-5> Preparation of Ice Cream

Milkfat 10.0%

Non-fat solids 10.8%

Sugar 12.0%

Starch syrup 3.0%

Emulsifying stabilizer (span) 0.5%

Spices (Strawberries) 0.15%

Water 63.31 ~ 62.91%

Green skin extract 0.24 ~ 0.64%

Ice cream was prepared using conventional methods using the above composition and content.

<2-6> manufacturing of chocolate

Sugar 34.36-34.76%

Cocoa Butter 34%

Cocoa Mass 15%

Cocoa Powder 15%

Lecithin 0.5%

0.5% vanilla

Green skin extract 0.24 ~ 0.64%

With the above composition and content, chocolate was prepared using conventional methods.

On the other hand, the specific scope of the present invention is defined by the claims rather than the embodiments described above, all changes and modifications derived from the meaning and scope and equivalent concepts of the claims to the scope of the invention It should be interpreted as including.

Claims (7)

A composition for preventing and treating prostate cancer, comprising an organic solvent extract of Aurantii Immatri Pericarpium as an active ingredient.
The composition for preventing and treating prostate cancer according to claim 1, wherein the organic solvent is ethanol.
According to claim 2, wherein the ethanol extract is a composition for preventing and treating prostate cancer, characterized in that extracted for 24 hours at 50 ℃.
The composition for preventing and treating prostate cancer according to claim 2 or 3, wherein the ethanol extract is dried and concentrated at 45 ° C under reduced pressure.
4. The composition for preventing or treating prostate cancer according to claim 2 or 3, wherein the ethanol is 95%.
According to claim 1, wherein the composition is a composition for preventing and treating prostate cancer, characterized in that it comprises a pharmaceutically acceptable carrier or diluent.
A functional food for preventing prostate cancer, which contains an organic solvent extract of Aurantii Immatri Pericarpium containing a food supplement which is acceptable as a food ingredient.

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