KR102054451B1 - Composition for Treatment of Pancreatic Cancer and Functional Food Comprising Extract of Gleditsiae Semen - Google Patents

Abstract

The present invention relates to a novel use of the extract of flakes, and to a therapeutic composition and functional food containing the extract of flakes as an active ingredient exhibiting excellent prophylactic or therapeutic efficacy against pancreatic cancer. Extract, which is a fragment of the present invention, has an effect of inhibiting the growth of pancreatic cancer cells and inducing apoptosis, and thus can be effectively used for the treatment and prevention of pancreatic cancer.

Description

Composition for Treatment of Pancreatic Cancer and Functional Food Comprising Extract of Gleditsiae Semen}

The present invention relates to a novel use of extracts which are flakes. Specifically, the present invention relates to a therapeutic composition and a functional food containing the extract as an active ingredient, which is a fragment showing excellent preventive or therapeutic efficacy against pancreatic cancer.

Among the major diseases of modern people, researches on the treatment and diagnosis of cancer are relatively active, mainly in lung cancer, liver cancer, gastric cancer, and the like. However, studies on esophageal cancer, colorectal cancer, and pancreatic cancer with low incidence are relatively low.

The pancreas produces insulin, which helps the body convert glucose into energy, and enzymes that help the body digest food. Pancreatic cancer is a malignant growth of the pancreas, mainly occurring in cells of the pancreatic duct. The disease is the ninth most common form of cancer, but it is the fourth and fifth most common cause of cancer deaths in men and women, respectively. Pancreatic cancer has a five-year survival rate of less than 3%, most of which is always fatal. Pancreatic ductal adenocarcinoma (pancreatic ductal adenocarcinoma) in the pancreatic cancer, which accounts for more than 90% of pancreatic cancer generally refers to pancreatic adenocarcinoma. Pancreatic adenocarcinoma is also called pancreatic adenocarcinoma, pancreatic adenocarcinoma, or pancreatic adenocarcinoma.

 Like many other malignant diseases, pancreatic cancer arises from the accumulation of acquired mutations. Multiple genetic and epigenetic changes, including activation of protocogenes, inactivation of tumor suppressor genes, and malformations of maintenance genes, may result in the development, sustained growth, and metastasis of pancreatic cancer. Related to Accumulated mutations in such genes are known to occur within predictable time during the "PanINs" (Pancreatic Intraepithelial Neoplsia) phase (Hruban et al. (2000) Clin Cancer Res 6: 2969-2972; Kern (2002) Cancer Biol Therapy 1: 607-613; Li et al. (2004) Lancet 363: 1049-1057). K-ras mutations occur about half of PanIN-1. The PanIN-2 stage is represented by additional changes and increases in the rate of K-ras mutations, and the appearance of multiple p16 malformations, and p53 protein family expression, which may indicate the presence of p53 mutations, is sometimes seen in more advanced PanINs. Loss of the tumor suppressor genes, TP53, DPC4 and BRCA2, appears to occur late in the development of pancreatic tumorigenesis, PanIN-3. Specifically, more than 85% of pancreatic adenocarcinomas have K-ras gene point mutations activated in pancreatic cancer development (Li et al. (2004) Lancet 363: 1049-1057; Xiong (2004) Cancer Chem Pharm 54: S69-77). K-ras mutations induce constitutive activation of Ras-Raf-MEK-ERK, an intracellular signaling pathway that induces cell proliferation and confers transgene properties on cells containing point mutations. Cause. Ras mutations are not associated with tumor stage or prognosis, and indicate that the K-ras oncogene may be involved in the onset of carcinogenesis but not in the likelihood or promotion of human pancreatic cancer. One of the major downstream targets of the ras family is phosphoinositol 3 kinase (PI3K). Activation of PI3K is associated with pancreatic cancer resistance to apotosis induced by chemotherapy or molecular drug targeting agents.

Pancreatic adenocarcinoma is one of the most deadly human malignancies with more than 30,000 deaths annually in the United States alone. At diagnosis these cancers are found in a state where only 10% to 15% can be excised due to the presence of locally advanced disease or distant metastasis. Recently, the most common treatment strategy for advanced pancreatic cancer is gemcitabine, a 2'-deoxycytidine nucleoside analog for intravenous administration that can induce apoptosis of human pancreatic cancer cells and inhibit tumor growth and progression. It is a treatment by. However, despite the best medical or surgical treatment, the results of treatment of patients with pancreatic adenocarcinoma are terrible, and as a group, the median survival of patients with this disease is 21 months or less. Thus, there is a need for clear, new and efficient treatment strategies to combat this deadly disease. As such, pancreatic cancer is a major health issue in developed countries and has a very poor prognosis (Faint et al. (2004) Datamonitor DMHC2045; Garcea et al. (2005) Pancreatology 5: 514-529; Kern et al. (2002). ) Cancer Biol Therapy 1: 607-613; Laheru and Jaffee (2005) Nature Rev Cancer 5: 59-467; Li et al. (2004) Lancet 363: 1049-1057). Despite excessive surgical and medical treatment, the mean life expectancy is about 15-18 months for patients with local disease and 3-6 months for patients with metastatic disease. Nearly 100% of patients with pancreatic cancer experience metastasis and die due to their unrestricted growth and weakening metabolism, and less than 5% of patients who have not undergone resection survive a total of 5 years. Do. In addition, there is a problem that the initial diagnosis is difficult due to the non-specific initial symptoms. Currently, early detection of pancreatic cancer is still under development and is not commercially available, and conventional cancer treatments have little effect on prognosis or disease outcome. Poor prognosis for pancreatic cancer is due to late manifestation, aggressive local invasion, early metastasis and insufficient response to chemotherapy.

The most common symptoms of pancreatic cancer include jaundice, abdominal pain and weight loss, and are not substantially special along with other emergence factors. Thus, diagnosing pancreatic cancer at an early stage of tumor growth is often difficult and requires a situation that includes considerable doubt and extensive diagnostic overhauls, often exploratory surgery. Endoscopic ultrasonography and computed tomography (CT) are the best noninvasive methods currently available for diagnosing pancreatic cancer. However, as well as differentiation of pancreatic cancer from lesioned pancreatitis, reliable detection of small tumors is difficult. Unfortunately, the majority of patients are currently diagnosed at a late stage where the tumor has already invaded the surrounding organs, extending outward and / or extensively metastasized (Gold et al., Crit. Rev. Oncology / Hematology, 39: 147-54 (2001). The disease is late detection is common, and early diagnosis of pancreatic cancer is rare in clinical settings.

Currently available treatment methods for pancreatic cancer do not cure the disease or reach a substantially improved survival time. Surgical resection is the only way to provide survival. However, due to tumor burden, only 10-25% of patients are targeted for 'curative resection'. In these patients undergoing resection treatment, the 5-year survival rate is still on average at around 10%.

Gleditsiae Semen , on the other hand, is a Gledisia japonica Miquel var. koraiensis Nakai). It is a herb used for various folk remedies such as controlling wind and improving bowel function. Thorn is called a sculptor, and it has the effect of sowing, drainage, etc., used in various boils. Overwintering is also possible in central Korea. Gleditsia sinensis Lam. Is a herb used in various folk remedies, such as improving intestinal function in East Asia. Thorn is called a sculptor, and it has the effect of sowing, drainage, etc., used in various boils. Overwintering is also possible in central Korea.

However, nothing has been disclosed or disclosed in this document in connection with Sculptor's pancreatic cancer-related diseases.

Accordingly, the present inventors completed the present invention by confirming that the extract of the sculptor can effectively kill the pancreatic cancer cells during the herbal research on the sculptor.
<Preceding technical literature>
KR 10-2009-0106264 A
KR 10-2010-0004736 A
KR 10-2011-0139086 A

It is an object of the present invention to provide a composition for treating pancreatic 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 pancreatic cancer comprising the active ingredient extracted with sculpting organic solvent. It is preferable that the said organic solvent is ethanol.

The present invention also provides a functional food for preventing pancreatic cancer containing ethanol extract as an active ingredient, which is a fragment containing a food supplement acceptable food supplement.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for the prevention and treatment of pancreatic cancer containing the ethanol extract as a fragment as an active ingredient. The compositions of the present invention comprise extracts that are flakes as active ingredients and may further comprise pharmaceutically acceptable carriers or diluents.

In the composition for preventing and treating pancreatic cancer of the present invention, the ethanol extract is preferably extracted for 24 hours at 50 ℃, wherein the ethanol extract is more preferably dried and concentrated at 45 ℃ reduced pressure conditions, the ethanol is Most preferred is 95%.

In addition, in the composition for preventing and treating pancreatic cancer of the present invention, the pancreatic cancer is preferably pancreatic ductal adenocarcinoma.

The extract which is the fragment of the present invention is possible at any site of the sculptor, but is preferably extracted from the leaf. 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 flake dry or powder and 10 to 100 hours, preferably 15, at a temperature of 20 to 100 ° C., preferably 40 to 60 ° C. To 40 hours, more preferably, the extract for 24 hours and then filtered to prepare the ethanol extract of the sculpting. 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 pancreatic 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 extract, which is a fragment of the present invention can be administered orally during clinical administration and can be used in the form of general pharmaceutical formulations, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants And diluents such as surfactants or excipients. 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, Minthae Herba, Dioscoreae Rhizoma, Polyporus, Polygoni multiflori Radix, Gusi (Allii tube) Semen, Cassiae Semen, Lycii Fructus, Araliae cordatae Radix, Eucommiae Cortex, Heyotis Herba, Saururus Herba, Artemisiaecapillaris Herba, Anemarrhenae 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 prior to use, sterile liquid carriers such as water for injection 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 Sculptor killed 99.7% of Panc-1 pancreatic cancer cells at 100 μg / ml and IC ₅₀ (half maximal inhibitory concentration) was 24.2 μg / ml. The above results demonstrate that the extract of the present invention has excellent killing activity of Panc-1 pancreatic cancer cells and further has pancreatic cancer treatment and prophylactic activity.

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

As used herein, the term "treatment" means any action that improves or beneficially alters the symptoms of pancreatic cancer by administration of the composition.

Extract which is a flake in the present invention can 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 may be subjected to 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 pancreatic 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, organic additives such as vitamins B1, B2, B6, C, E, niacin, carnitine, betaine, folate pantothenic acid, biotin, zinc, iron, calcium, chromium, magnesium, mixtures thereof It can be included as. In addition, the composition may include a substance having a therapeutic activity against pancreatic cancer, used alone or previously used.

As used herein, the term "patient" refers to humans and horses, sheep, pigs, goats, camels, who have diseases caused by pancreatic cancer and its direct and indirect causes, and whose symptoms may be improved by administering the composition of the present invention. Means antelope, dog and other animals. By administering to a patient a composition comprising an extract which is a fragment of the present invention, the above-mentioned pancreatic cancer can be effectively prevented and treated. The composition of the present invention can be administered in parallel with existing pancreatic cancer therapeutics.

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 composition of the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. It can 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 sex, age, health condition, diet, time of administration, method of administration, drug mixture, the condition of the patient, and the extent of the onset of neurological disease.

The present invention also provides a functional food for preventing pancreatic cancer containing ethanol extract as an active ingredient, which is a fragment containing a food acceptable additive.

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, gums, ice cream, Various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, etc., and includes all the fruit 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, as a result of injecting the extract of the present invention into Panc-1 pancreatic cancer cells, it was confirmed that Panc-1 cells are killed (see Fig. 1 ). Therefore, it can be seen that the extract of the fragment has a pancreatic cancer treatment effect. Thus, in the present invention, the present invention was completed by preparing a cosmetic composition for preventing pancreatic cancer, which contains the extract of the flakes as an active ingredient (see Preparation Example 2 ).

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

1 is a result of Alamar Blue analysis to determine the effect of the introduction of sculptor in panc-1 cells, pancreatic cancer cells of human pancreatic cancer cells, the growth of pancreatic cancer cells, X-axis is the concentration of the extract sculpted, Y-axis is surviving human pancreatic cancer The viability of the cells is shown.
Figure 2 is the EdU assay results to determine whether the introduction of the fragment obtained in Example 1 in panc-1 cells, a human pancreatic cancer cell line stops the cell cycle and inhibits proliferation of pancreatic cancer cells.
Figure 3 is a reporter assay to determine the effect of the introduction of the fragment obtained in Example 1 on pancreatic cancer cell line panc-1 cells on the c-Myc cell signal of pancreatic cancer cells in the activity of c-Myc cell signal The result of comparative analysis.
Figure 4 is a reporter assay to determine the effect of the introduction of the fragment obtained in Example 1 on the pancreatic cancer cell line panc-1 cells in the pancreatic cancer cells AP-1 cell signal activity in the panc-1 cells The result of comparative analysis.
5 is a reporter assay to determine the effect of the introduction of the fragment obtained in Example 1 on pancreatic cancer cell line panc-1 cells on the NF-κB cell signal of pancreatic cancer cells in the activity of NF-κB cell signal The result of comparative analysis.

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 Flakes Extract

3 kg of flakes (made in China) purchased from Seoul medicinal herb were dried and ground for 5 days at the shade and room temperature. The ground flakes were immersed 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 at 45 ° C. to obtain 419 g of the total extract, which was stored at −20 ° C.

< Example  2> Effect of Carving Extract on Pancreatic Cancer Cell Growth

In order to determine the effect of the extract extracted from Example 1 on the growth of pancreatic cancer cells, Panc-1 cells, which are human pancreatic cancer cells, were treated with ethanol extract of the sculptor 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 Pancreatic Cancer Cell Lines

Panc-1 cells, a pancreatic cancer cell line used in the present invention, were distributed from the Korean Cell Line Bank (KCLB) and used for experiments. Specifically, Panc-1 pancreatic cancer cells are DMEM (Dulbeco's Modified Eagle's) containing 10% FBS (fetal bovine serum, fetal bovine serum) (Welgene) and 25 mM HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) Medium) was passaged in medium.

<2-2> Panc Inhibition of Cell Growth in -1 Pancreatic Cancer Cells

The extract extracted in Example 1 was confirmed the effect of inhibiting the growth of Panc-1 cells, which are pancreatic cancer cells. Specifically, 4.5 x 10 ³ Panc-1 pancreatic cancer cells per well were seeded in a 96 well plate, cultured for 24 hours, and then the fragments 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). 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 uniformly react the cells of 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. Survival is shown in FIG.

Carving Concentration Pancreatic cancer cell survival rate (average) Standard Deviation 0 μg / ml 1.000 0.000 3.125 μg / ml 0.975 0.029 6.25 μg / ml 0.965 0.059 12.5 μg / ml 0.938 0.040 25 μg / ml 0.470 0.036 50 μg / ml 0.003 0.001 100 μg / ml 0.003 0.001

As a result, as shown in Table 1 and Figure 1, the higher the concentration of the sculptor treatment, the growth of pancreatic cancer cells was reduced, from which it can be seen that the sculptor has a pancreatic cancer treatment effect. Ie 2.5% at 3.125 μg / ml, 3.5% at 6.25 μg / ml, 6.2% at 12.5 μg / ml, 53.0% at 25 μg / ml, 99.7% at 50 μg / ml, 99.7% at 100 μg / ml Pancreatic cancer cells were killed. In addition, the IC ₅₀ (half maximal inhibitory concentration) was determined to be 24.2 μg / ml. Table 1 describes the relative cell numbers of pancreatic cancer cells after 48 hours according to the treatment concentrations of the respective fragments based on the number of survival rates of the pancreatic cancer cells of the control group that was not treated with fragments. As such, the extract, a fragment of the present invention, has excellent Panc-1 pancreatic cancer cell killing activity and further demonstrates pancreatic cancer treatment and prophylactic activity.

< Example  3> By extract which is flakes Acute Toxicity  exam

Sculpture used in the present invention was widely used as a medicinal herb was determined that there is no problem in the stability, but the oral and intraperitoneal toxicity experiments were carried out to confirm this.

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, each of the extracts of the fragment of Example 1 of the present invention suspended 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 extracts of the fragments 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, the pancreatic cancer cells in culture were treated with fragments at different concentrations, 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 nuclei of newly generated cells after the time of fragmentation treatment are the second one. The third is the sum of these.

As shown in Figure 2, when treated with each concentration of fragment, it can be seen that the number of newly generated cells as compared to the untreated control group. This shows that sculpting has the effect of inhibiting the proliferation of pancreatic cancer cells.

< Example  5> Cell Signaling Test Reporter Assay )

A reporter gene assay was performed to determine the effect of fragmentation on key cell signals that regulate the growth and proliferation of cancer cells. 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 fragments were treated at each concentration. After 24 hours, the expression level of the reporter gene was compared with the non-sculpted group 100% and the non-transduced group 0%.

As a result of confirming the activity of the cell signal, it was confirmed that all the major signals promoting the proliferation of pancreatic cancer cells decreased when the extract was treated with a fragment of 100 ug / ml concentration.

< Preparation Example 1> Preparation of pancreatic cancer therapeutic agent containing the extract of flakes as an active ingredient

The present inventors have confirmed that the pancreatic cancer treatment efficacy of the extract sculpted through the above examples to prepare a pancreatic cancer treatment containing the extract sculpted as an active ingredient as follows. In addition, the preparation of the following therapeutic agents can be used for the application of not only the therapeutic agent but also the preparation of flake food.

Soft capsule containing <1-1> flake extract soft gelatin capsules )(weight%)

Sculpture 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 Flaky Extract (wt%)

Carrot extract 0.2%, mannitol 0.3%, physiological saline 9.5%

<1-3> tablet containing the extract which is a flake ( tablet )(weight%)

Root Extract 35%, Vitamin C 10%, Vitamin D ₃ 0.001%, manganese sulfate 0.1%, crystalline cellulose 25.0%, lactose 17.999%, magnesium stearate 2%

< Production Example  2> Preparation of Functional Foods Containing Extracts of Carvings as Active Ingredients

The present inventors confirmed that the extract extract is excellent in pancreatic cancer treatment activity through the above-mentioned Example to prepare a functional food containing it as an active ingredient as follows.

<2-1> Preparation of Drink

The 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 flake extract, and 200 ml of water. And the contents were prepared using conventional methods.

<2-2> Chewing gum  Produce

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

<2-3> Preparation of Candy

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

<2-4> Biscuit  Produce

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 , 0.003 kg of vitamin B2, 0.16 kg of milk flavor, 71.1 kg of water, whole milk powder 4 kg, substitute milk powder 1 kg, calcium phosphate 0.1 kg, spray salt 1 kg, spray oil 25 kg, flake extract 0.2-0.5 kg Biscuits were prepared using conventional methods in terms of composition and content.

<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%, flake extract 0.24 ~ 0.64% Ice cream was prepared using conventional methods using the above composition and content.

<2-6> Chocolate  Produce

The composition and content of the sugar 34.36 ~ 34.76%, cocoa butter 34%, cocoa mass 15%, cocoa powder 15%, lecithin 0.5%, vanilla flavor 0.5%, flake extract 0.24 ~ 0.64% using conventional methods Chocolate 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)

Pancreatic cancer prevention and treatment composition consisting of ethanol extract of the sculptor as an active ingredient. delete delete delete The composition for preventing and treating pancreatic cancer according to claim 1, wherein the composition comprises a pharmaceutically acceptable carrier or diluent. delete delete

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