WO2014077437A1 - Composition containing pharbitidis semen extract for renal cancer treatment and functional food - Google Patents

Abstract

The present invention relates to a novel use of a Pharbitidis Semen extract, in which a composition for treatment and functional food contains, as an active ingredient, a Pharbitidis Semen extract exhibiting excellent preventative and treatment effects on renal cancer. The Pharbitidis Semen extract according to the present invention suppresses growth and induces apoptosis of renal cancer cells, and therefore can be used effectively for renal cancer treatment and prevention.

Description

Composition for the treatment of kidney cancer containing black shaft extract and health functional food

The present invention relates to a novel use of black shaft extract. Specifically, the present invention relates to a therapeutic composition and a functional food containing a black shaft extract as an active ingredient, which shows an excellent prophylactic or therapeutic effect against kidney cancer.

Among the major diseases of modern people, researches on the treatment and diagnosis of cancer are being conducted mainly on lung cancer, liver cancer, and stomach cancer, which have high incidence.

The kidney is an important urinary system that has the role of excreting waste products in vitro by filtering blood to produce urine. At the same time, it is also an important endocrine organ that produces hormones such as angiotensin, which regulates blood pressure, and erythropoietin, a red blood cell hematopoietic factor. Renal tumors include kidney cell cancer in adults, Wilms' tumor in children, and sarcoma as a rare tumor, but the most common malignancy below Renal cell cancer, a tumor, is called kidney cancer. The incidence of renal cancer is about 2.5 per 100,000 population, and male to female ratio is 2 to 3: 1. Among urological malignancies, there are many tumors following prostate cancer and bladder cancer.

Genetic factors are also known as risk factors for kidney cancer, but generally include smoking and excessive fat intake. It is also known that the incidence of such tumors is high in patients undergoing long-term dialysis. In kidney cancer, when the tumor has a maximum diameter of 5 cm or less, some subjective symptoms rarely occur and are often found by CT scan or the like at the time of examination. Larger tumors may include hematuria, abdominal tumors, and pain. In addition, systemic symptoms may cause fever, weight loss, anemia and the like, and rarely, endocrine factors may cause erythrocytosis, hypertension, hypercalcemia, and the like. On the other hand, there may be cases in which vein veins of the abdominal body surface or venous varicose veins occur due to the progression of kidney cancer into the inferior vena cava. About 20% of kidney cancers are found from lung or bone metastases. In kidney cancer, tumors tend to spread in veins, and metastasis to other organs is likely to occur.

Ultrasound, CT, angiography, etc. are examined for kidney cancer, but specific biochemical markers are not known. Therefore, examination using a device is necessary. In addition, although several types of kidney cancer can be classified pathologically, the cause of the development of clear cell type kidney cancer, which accounts for about 90%, is known to be a defect in the von-Hippel-Lindau (VHL) gene, a cancer suppressor gene. . Deletion of the VHL gene results in transcriptional activation of the hypoxic inducible gene group through association of HIF-α / VHF, leading to enhanced expression of genes such as VEGF and TGFβ.

The main treatment for kidney cancer is surgical therapy. Regardless of the timing of the disease, extraction of the kidneys or partial extraction of the kidneys is the most common where possible, and even if there is metastasis, surgical removal of the kidneys may be considered. Arterial embolization of the renal artery may be used as a method other than the surgical therapy. This method may be performed prior to surgery in cases where it is impossible to extract or when a large tumor is removed. Recent advances in burn diagnostics have led to early detection of renal cancer, even at very small sizes, resulting in more than 90% healing in early cancer. However, since the therapeutic results of large tumors larger than 5 cm or tumors with metastases are poor (the 5-year survival rate of all renal cell carcinomas is about 50 to 60%), the importance of early detection is recognized.

鈴)이라는 이름은 씨앗의 형태를 의미하며 분증(盆甑)과 구이(狗耳)라는 이름은 꽃잎의 모양에 따라 붙여졌다. 깨어서 보면 약간 냄새가 있고 자극성이며 맛은 쓰고 성질은 차며 독이 있다. [苦寒有毒] 견우자는 사하(瀉下)작용과 이뇨작용이 강하고 기를 잘 내려 몸이 부을 때 , 만성신우신염, 간경화 등으로 복수가 찰 때 사용한다. 줄기가 왼쪽으로 감아 올라 가면서 3m안팎까지 뻗어 나가고 줄기에는 밑을 향한 털이 있다. 7~8월에 꽃이 홍자색, 흰색, 붉은색 등 여러 가지 색으로 피는데 잎겨드랑이에 1~3개의 꽃이 달린다. 꽃부리는 지름이 10~23 ㎜이며 깔때기 모양이고, 꽃봉오리는 붓끝같은 모양으로 오른쪽으로 말리는 주름이 있다. 또한 5개의 수술과 1개의 암술이 달린다. 열매는 속의 3실에 각각 2개의 씨가 들어 있고 9~10월에 까맣게 익는다. 생김새는 구슬을 세로로 4~6등분한 모양을 하고 있으며 바깥면은 흑색~회적갈색 또는 회백색이며 매끈하거나 약간 오무라져 있다. 확대경으로 보면 씨앗 껍질의 바깥 면에는 짧은 털이 밀생하고 융기선의 아래쪽에는 제점이 오므라져 있다. 횡절 면은 부채 모양이고 엷은 황갈색 ~ 엷은 회갈색을 나타내며 질은 치밀하다. 씨앗 껍질은 얇고 그 바깥층은 어두운 회색, 안쪽 층은 엷은 회색이다. 다른 이름으로 흑축(黑丑), 구이초(狗耳草), 백견우(白牽牛), 백축(白丑), 분증초(盆甑草), 초금령(草

鈴), 흑견우(黑牽牛) 등이 있으며 자양, 보신의 효과가 있다.On the other hand, the black shaft (black-haired, Pharbitidis Semen ) refers to the seeds of the morning glory ( Pharbitis nil choisy) (buckthorn and convolvulaceae), which are found in Chinese herbal medicine and folklore. Secret, alluvial abdominal pain, swelling, abdominal bloating, constipation was used. Morning glory is native to tropical Asia such as India and is known to have about 256 varieties. Black seed is also known as black axis (黑 丑), white seed is white axis (白 丑). prohibition(

The name 鈴) refers to the form of seeds, and the names of sorghum and roast are named after the shape of the petals. Wake up a little smell, irritating, taste bitter, cold and poisonous. [苦寒 有毒] The dogs have a lowering (瀉 下) and diuretic effect is strong and well down the body when swollen, chronic pyelonephritis, liver cirrhosis, etc. is used to fill the revenge. The stem winds up to the left and extends in and out of 3m, and the stem has downward hairs. Flowers bloom in July-August in various colors such as reddish purple, white, and red, with one to three flowers hanging on the axilla. The corolla is 10 ~ 23 ㎜ in diameter and has a funnel shape, and the bud has a wrinkle like a brush tip. It also runs five surgeries and one pistil. The fruit contains two seeds in each of the three chambers of the genus and ripens in September-October. The appearance is divided into 4 ~ 6 equal lengths of the beads. The outer surface is black to gray brown or greyish white and smooth or slightly crusted. In the magnifier, the outer surface of the seed husk is dense with short hairs, and the bottom of the ridge is closed. Transverse plane fan-shaped, pale yellowish brown to pale grey-brown, and the quality is dense. Seed shell is thin, the outer layer is dark gray, and the inner layer is light gray. Other names are Black Axis, Guicho, White Dog, White Axis, Bunpumcho, Chogeumryeong

鈴), black dog (黑 牽 牛), etc., has the effect of nutrition and Bosin.

A component called parbitin contained in the hypothalamic-nuts is broken down by the bile sera in the intestine to produce Ipulolic acid. It gives intense stimulation to the intestinal tract, increasing peristalsis in the intestine, causing redness in the intestinal mucosa and increasing secretion. Therefore, when you do not see the stool is firmly effective. This is especially effective when the body's essences do not see dry stools or patients who usually have stools. It is also good for the body as well as the body. Effective for seawater and asthma and used for stubborn constipation and parasite removal. Intestinal interlocking action as a pharmacological action has been reported to have a strong lower action.

The black axis contains about 11% of fatty oil and 2% of pharbitin, a resinous glycoside, and is known to have hypothalamic, diuretic and insecticidal effects (KP Part 2 651). It is also effective in improving sea water, asthma and constipation, and is used as a cosmetic composition for massage to remove impurities from the skin. It also works when you don't see diuretics-urine. Especially used for Gochang disease. Can be used when revenge is cold and swelling in the body. Expectant effect on sputum, cough, asthma, etc. It is used as a remedy for insecticidal action-roundworms, tapeworms, etc., and it is used to alleviate various symptoms caused by parasites or abdominal pain.

However, nothing is disclosed or disclosed in this document in connection with kidney cancer-related diseases of the axial axis.

Accordingly, the present inventors completed the present invention by confirming that the black shaft extract can effectively kill kidney cancer cells during the study of herbal medicine for black shaft.

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

In order to achieve the above object, the present invention provides a composition for preventing and treating kidney cancer comprising an active ingredient extracted from the black shaft with an organic solvent. It is preferable that the said organic solvent is ethanol.

In another aspect, the present invention provides a functional food for preventing kidney cancer containing black ethanol extract containing a food supplement acceptable food additive as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for the prevention and treatment of kidney cancer containing black ethanol extract as an active ingredient. The composition of the present invention comprises a black shaft extract as an active ingredient, and may further include a pharmaceutically acceptable carrier or diluent.

In the composition for preventing and treating kidney cancer of the present invention, the ethanol extract is preferably extracted at 50 ° C. for 24 hours, wherein the ethanol extract is more preferably dried and concentrated at 45 ° C. under reduced pressure, and the ethanol Is most preferably 95%.

In addition, in the composition for preventing and treating kidney cancer of the present invention, the kidney cancer is preferably renal cell cancer.

Although the black shaft extract of this invention is possible in any site of black shaft, it is preferable to extract from a root preferably. 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 black shaft dry matter 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 produce an ethanol extract of black shaft. 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 kidney 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 pharmaceutical formulation of the black shaft extract of the present invention can be administered orally during clinical administration and can be used in the form of general pharmaceutical formulations, 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 Radix ), Ginger (Zingiberis Rhizoma crudus), forget-me-not (Natrii sulfas), control (Ziz) yphi Fructus, Salviae Radix, Mautan Radicis Cortex, Rehmanniae Radix, Mint Herba, Dioscoreae Rhizoma, Polyporus, Polygoni multiflori Radix, Gusi (Allii tube) Semen, Cassiae Semen, Lycii Fructus, Araliae cordatae Radix, Eucommiae Cortex, Hedyotis Herba, Saururus Herba, Artemisiaecapillaris Herba, Anemarrhen Rhizoma, Carthami Flos, Astragali Radix, Lycopodium, Ginkgonis Folium, Polygonati Rhizoma, Nelumbinis Semen, Fossilia ossis Mastodi, Cortexi radics ), Achyranthis Radix, Rehmanniae Radix preparata, Perillae Semen, Thujae Semen, Malt (Hordei Fructus germinatus), Cuscutae Semen, Mordaedae Radix, Sea Song (Pini koraiensis) Radix) and the like 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. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used. 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, ethanol extract of black shaft killed 97.3% of ACHN kidney cancer cells at 100 μg / ml, and IC ₅₀ (half maximal inhibitory concentration) was 4.4 μg / ml. The above results demonstrate that the black shaft extract of the present invention has excellent killing activity of ACHN kidney cancer cells and further has therapeutic and prophylactic activity for kidney cancer.

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

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

In the present invention, the black shaft extract may be extracted and used 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 kidney 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 kidney cancer used alone or used in the past.

As used herein, the term "patient" refers to humans, horses, sheep, pigs, goats, and camels having a disease caused by kidney 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 black shaft extract of the present invention, it is possible to effectively prevent and treat the above-mentioned kidney cancer. The composition of the present invention can be administered in parallel with existing kidney cancer treatments.

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. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, 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 gender, age, shrinkage, diet, time of administration, method of administration, drug mixture, the condition of the patient, and the extent of neurological disease.

In another aspect, the present invention provides a functional food for preventing kidney cancer containing black ethanol extract containing a food supplement acceptable food additive as an active ingredient.

Functional food of the present invention preferably contains 0.1 to 5% by weight, more preferably 1% by weight of the ethanol extract relative to the total weight. 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 black shaft extract of the present invention was injected into ACHN kidney cancer cells, it was confirmed that ACHN kidney cancer cells were killed (see FIG. 1 ). Therefore, it can be seen that the black shaft extract has a renal cancer treatment effect. Thus, in the present invention, the present invention was completed by preparing a functional food for preventing kidney cancer containing black shaft extract as an active ingredient (see Preparation Example 2 ).

As described above, the black shaft extract of the present invention inhibits the growth of kidney cancer cells and induces apoptosis. Therefore, the composition for treating renal cancer according to the present invention will be very effective in the treatment of renal cancer patients.

1 is a result of Alamar Blue analysis to determine the effect of the introduction of the black axis on the growth of kidney cancer cells in ACHN cells, human kidney cancer cells, wherein the X axis is the concentration of the black axis extract, the Y axis is surviving human kidney cancer cells Survival rate.

2 is an EdU assay result to determine whether the introduction of the black axis obtained in Example 1 in the human kidney cancer cell line ACHN cells to stop the cell cycle and inhibit proliferation of kidney cancer cells.

Figure 3 compares the activity of c-Myc cell signal in a reporter assay to determine the effect of the introduction of the black axis obtained in Example 1 on the c-Myc cell signal of kidney cancer cells in ACHN cells, a human kidney cancer cell line The result of the analysis.

Figure 4 compares the activity of AP-1 cell signal in a reporter assay to determine the effect of the introduction of the black axis obtained in Example 1 on the AP-1 cell signal of kidney cancer cells in ACHN cells, a human kidney cancer cell line The result of the analysis.

Figure 5 compares the activity of NF-κB cell signal in a reporter assay to determine the effect of the introduction of the black axis obtained in Example 1 on the NF-κB cell signal of kidney cancer cells in ACHN cells, a human kidney cancer cell line The result of the analysis.

Figure 6 shows the effect of the introduction of the black axis obtained in Example 1 on the major cell signal that promotes the proliferation of kidney cancer cells in ACHN cells, a human kidney cancer cell line p-ERK, Ras, β- This is the result of quantitative changes such as actin protein.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited to the following examples and may be changed to other embodiments equivalent to substitutions and equivalents without departing from the technical spirit of the present invention. Will be apparent to those of ordinary skill in the art.

Example 1 Preparation of Black Axis Extract

3 kg of black shaft (Chinese) purchased from Seoul medicinal herb was dried and ground for 5 days at the shade and room temperature. The ground black shaft was immersed in 30 L of 95% ethanol and extracted at 50 ° C. for 24 hours. This was filtered through a filter paper, dried and concentrated under reduced pressure of 45 ° C. to obtain 294 g of the total extract, and stored at −20 ° C.

Example 2 Effect of Black Axis Extract on the Growth of Kidney Cancer Cells

In order to determine the effect of the black shaft extract extracted in Example 1 on the growth of kidney cancer cells, ACHN cells, which are human kidney cancer cells, were treated with ethanol extract of black shaft 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.

<2-1> Preparation and Treatment of Human Kidney Cancer Cell Line

ACHN cells, a kidney cancer cell line used in the present invention, were distributed from Korean Cell Line Bank (KCLB) and used for experiments. Specifically, ACHN kidney cancer cells (Minimum essential medium) containing 10% FBS (fetal bovine serum, fetal bovine serum) (Welgene) and 25 mM HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) Subcultured in medium.

<2-2> Cell growth inhibition measurement of ACHN kidney cancer cells

The black shaft extract extracted in Example 1 was confirmed to inhibit the growth of ACHN cells, which are kidney cancer cells. Specifically, 6.0 x 10 per well in a 96 well plate³ The black axis dissolved in DMSO (dimethyl sulfoxide) after seeding and incubating for 24 hours in ACHN kidney cancer cells 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 FIG. 1). 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 in each well, and the intensity of fluorescence was measured at 590 nm with a Fluorescence Microplate Reader (Molecular Devices Corp.) while irradiating irradiation light at a wavelength of 544 nm. The survival rate of is shown in FIG.

Table 1 Black shaft concentration Kidney Cancer Cell Survival (Average) Standard Deviation 0 μg / ml 1.000 0.000 3.125 μg / ml 0.837 0.024 6.25 μg / ml 0.157 0.009 12.5 μg / ml 0.049 0.003 25 μg / ml 0.013 0.001 50 μg / ml 0.016 0.001 100 μg / ml 0.027 0.001

As a result, as shown in Table 1 and Figure 1, the higher the treatment concentration of the black axis, the growth of kidney cancer cells was reduced, from which it can be seen that the black axis has a renal cancer treatment effect. Ie 16.3% at 3.125 μg / ml, 84.3% at 6.25 μg / ml, 95.1% at 12.5 μg / ml, 98.7% at 25 μg / ml, 98.4% at 50 μg / ml, 97.3% at 100 μg / ml Kidney cancer cells were killed. In addition, the IC ₅₀ (half maximal inhibitory concentration) was determined to be 4.4 μg / ml. Table 1 describes the relative cell numbers of kidney cancer cells after 48 hours according to each of the treatment rates, based on the number of survival rates of the kidney cancer cells of the control group not treated with the black axis. As such, the black shaft extract of the present invention has excellent ACHN cell killing activity, and further demonstrates the therapeutic and prophylactic activity of kidney cancer.

Example 3 Acute Toxicity Test by Black Axis Extract

Although the black axis used in the present invention was widely used as a medicinal herb, 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 suspended orally administered at a dose of 5 g / kg in the black shaft 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. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities.

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 of the black shaft extracts did not show toxic changes 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.

Example 4 Cell Proliferation Test (EDU Assay)

EdU assay is the same test method as BrdU assay and is an improved assay. Halogenated nucleotides such as the pyrimidine analog bromodeoxyuridine (BrdU) are useful for staining DNA in living cells or tissues. In phase S, which is doubled in the nucleus of the cell cycle and doubled in volume, BrdU is used instead of thymidine in the replicating DNA, and the amount of synthesized DNA can be confirmed by staining with BrdU antibody.

In the test method, black shafts were treated by concentration in renal cancer cells in culture, and after 6 hours, EdU was added to the medium and the cells were cultured. After 18 hours, the medium containing EdU was removed and the cells were fixed with 3.7,% paraformaldehyde and washed twice with PBS. After permeation of cells with 0.1% Triton X-100, Alexa Fluor 488 azide was reacted with EdU. Stained with Hoechst 33342 to identify the nuclei of the cells. As shown in Fig. 2, the nucleus of the whole cell is the first one, of which the nucleus of the newly generated cell after the time of treatment of black axis is the second one. The third is the sum of these.

As shown in FIG. 2, when the black axis was treated at each concentration, it can be seen that the number of newly generated cells was smaller than that of the untreated control group. This shows that the black axis has the effect of inhibiting the proliferation of kidney cancer cells.

Example 5 Cell Signaling Test (Reporter Assay)

A reporter gene assay was performed to determine the effect of black axis on the major cellular signals that control cancer cell growth and proliferation. Reporter gene assay is a test technique designed to confirm the change of the cell signal by introducing a reporter gene that can measure the change of a specific cell signal in the cell.

In the test method, a reporter gene designed to measure a cell signal to be confirmed was transfected into cancer cells, stabilized, and black shafts were treated at each concentration. After 24 hours, the expression level of the reporter gene was compared to 100% in the group without black axis and 0% in the group without transduction.

As a result of confirming the activity of the cell signal, it was confirmed that all major signals for promoting the proliferation of renal cancer cells decreased when treated with the extract of the black axis of 100 ug / ml |

Example 6 Quantitative Change Identification of Signal Proteins (Westernblotting, immunoblotting)

Western blot testing was performed to determine the effect of black axis on signaling proteins involved in the major cellular signals that control cancer cell growth and proliferation. Western blot is a test method that separates the whole protein by molecular weight, and then compares the amount of protein to be detected using the antibody of the protein to be identified.

There is no change in the amount of Ras, but the amount of p-Erk is rapidly decreasing, and c-Myc and p-c-Myc are also decreasing, indicating that the extract of black axis strongly inhibits the activity of Ras and Erk.

<Preparation Example 1> Preparation of a renal cancer therapeutic agent containing the black shaft extract as an active ingredient

The present inventors have confirmed that the renal cancer treatment effect of the black shaft extract is excellent through the above embodiment to prepare a kidney cancer therapeutic agent containing the black shaft 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 black foods.

<1-1> Soft Gelatin Capsules Containing Black Axis Extracts

Black shaft extract 20%, vitamin C 4.5%, vitamin D ₃ 0.001%, manganese sulfate 0.1%, beeswax 10%, palm oil 25%, safflower seed oil 30.399%

 <1-2> Preparation of Intravenous Formulation Containing Black Axis Extract

Black Axis Extract 0.2%, Mannitol 0.3%, Saline 9.5%

<1-3> tablet containing black shaft extract

Black shaft 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 Black Axis Extract as Active Ingredient

The present inventors have confirmed that the black shaft extract is excellent in the renal cancer treatment activity through the above embodiment to prepare a functional food containing it as an active ingredient as follows.

<2-1> Preparation of Drink

The above composition of 522 mg of honey, 5 mg of thioctoamide, 10 mg of nicotinic acid, 10 mg of riboflavin hydrochloride, 2 mg of pyridoxine hydrochloride, 2 mg of inositol, 50 mg of orthoic acid, 0.48-1.28 mg of black shaft extract, and 200 ml of water, and The beverage was prepared using conventional methods by content.

<2-2> production of chewing gum

Chewing gum was prepared using a conventional method using the composition and content of 20% of gum base, 76.36 to 76.76% of sugar, 0.24 to 0.64% of black shaft extract, 1% of fruit flavor, and 2% of water.

<2-3> Preparation of Candy

Candy was prepared using a conventional method with the above composition and content of 50 to 60% of sugar, 39.26 to 49.66% of starch syrup, 0.24 to 0.64% of black shaft extract, and 0.1% of orange flavor.

<2-4> Preparation of Biscuits

Force primary 88 kg, gravity primary 76.4 kg, white sugar 16.5 kg, salt 2.5 kg, glucose 2.7 kg, palm shortening 40.5 kg, ammo 5.3 kg, medium sodium 0.6 kg, sodium bisulfite 0.55 kg, rice flour 5.0 kg, vitamin B1 0.003 kg The composition of vitamin B2 0.003 kg, milk flavor 0.16 kg, water 71.1 kg, whole milk powder 4 kg, substitute milk powder 1 kg, calcium phosphate 0.1 kg, spray salt 1 kg, spray oil 25 kg, black shaft extract 0.2 ~ 0.5 kg And biscuits were prepared using conventional methods in amounts.

<2-5> Preparation of Ice Cream

Milk fat 10.0%, nonfat milk solids 10.8%, sugar 12.0%, starch syrup 3.0%, emulsifying stabilizer (span, span) 0.5%, fragrance (strawberry) 0.15%, water 63.31 ~ 62.91%, black shaft extract 0.24 ~ 0.64% Ice cream was prepared using conventional methods in terms of composition and content.

<2-6> manufacturing of chocolate

With the above composition and content of sugar 34.36 to 34.76%, cocoa butter 34%, cocoa mass 15%, cocoa powder 15%, lecithin 0.5%, vanilla flavor 0.5%, black shaft extract 0.24 to 0.64% Was prepared.

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)

Kidney cancer prevention and treatment composition comprising the active ingredient extracted black axis with an organic solvent. The method of claim 1, wherein the organic solvent is a composition for preventing and treating kidney cancer, characterized in that ethanol. According to claim 2, wherein the active ingredient extracted with ethanol is a composition for preventing and treating kidney cancer, characterized in that extracted for 24 hours at 50 ℃. According to claim 3, wherein the ethanol extract is a composition for preventing and treating kidney cancer, characterized in that the drying and concentrated under reduced pressure conditions 45 ℃. The composition for preventing and treating kidney cancer according to any one of claims 1 to 4, wherein the composition comprises a pharmaceutically acceptable carrier or diluent. The composition for preventing and treating kidney cancer according to any one of claims 1 to 4, wherein the kidney cancer is kind cell cell cancer. Functional food for the prevention of kidney cancer, which contains a black shaft extract containing a food supplement acceptable food supplement as an active ingredient.

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