KR20130022394A

KR20130022394A - Composition for treatment of pancreatic cancer and functional food comprising extract of caryophylli cortex

Info

Publication number: KR20130022394A
Application number: KR1020120093456A
Authority: KR
Inventors: 황성연; 정경채
Original assignee: 주식회사한국전통의학연구소; 정경채; 황성연
Priority date: 2011-08-26
Filing date: 2012-08-27
Publication date: 2013-03-06

Abstract

PURPOSE: A composition and a health functional food containing a Caryophylli cortex extract are provided to induce pancreatic cancer apoptosis and to effectively prevent and treat pancreatic cancer. CONSTITUTION: A composition and a health functional food for treating pancreatic cancer contain a Caryophylli cortex extract which is prepared using an organic solvent at 50 deg. C. for 24 hours. The organic solvent is ethanol. The extract is dried and concentrated at 45 deg. C. The composition contains a pharmaceutically acceptable carrier or diluents. The pancreatic cancer is pancreatic ductal adenocarcinoma. [Reference numerals] (AA) Relative cellular viability

Description

Composition for Treatment of Pancreatic Cancer and Functional Food Comprising Extract of Caryophylli Cortex}

The present invention relates to a novel use of clove bark extract. Specifically, the present invention relates to a therapeutic composition and a functional food containing a clove bark extract as an active ingredient exhibiting excellent prophylactic 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 analogue 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, which weakens 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 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%.

On the other hand, Clove Bark (丁香樹皮, Caryophylli Cortex) is a bark of cloves. Clove tree is a dicotyledonous genus Aspenaceae, distributed in Jeolla-do, north of Gyeongsang-do, and Manchuria, and grows at the foot of the mountain. It grows to about 3 m and has many branches and has a trunk. The leaves are opposite, oval or upside down, or almost round, with flat edges. Flowers bloom in May, run on inflorescences, and spikes hang on old branches. Fruits are capsules, obtuse, elliptical, with a cortex. When one flower is gathered from the side, it is seen as a '丁' and the flower has a fragrance, so it is called a clove tree. Buds are cloves, roots are cloves, barks are cloves, resins are cloves, the fruits are the mother cloves, and the oil from the distilled oil is called cloves. Clove bark treats severe abdominal pain, nursing limbs, and toothache.

However, nothing has been disclosed or disclosed in this document with regard to pancreatic cancer-related diseases of cloves bark.

Accordingly, the present inventors completed the present invention by confirming that the extract of the clove bark can effectively kill pancreatic cancer cells during the herbal research on the clove bark.

It is an object of the present invention to provide a composition for treating pancreatic cancer and a functional food containing the extract of clove bark 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 an active ingredient extracted from cloves bark with an organic solvent. It is preferable that the said organic solvent is ethanol.

In addition, the present invention provides a functional food for preventing pancreatic cancer, which comprises a clove bark ethanol extract containing a food supplement acceptable food supplement 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 pancreatic cancer, containing clove bark ethanol extract as an active ingredient. The composition of the present invention comprises a clove bark extract as an active ingredient, and may further include a pharmaceutically acceptable carrier or diluent.

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 clove bark extract of the present invention is possible at any part of the clove bark, but is preferably extracted from the leaves. 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 clove bark or powder, and 10 to 100 hours, preferably 15 at a temperature of 20 to 100 ℃, preferably 40 to 60 ℃ To 40 hours, more preferably, the extract for 24 hours and then filtered to prepare an ethanol extract of the clove bark. 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 preparation of the clove bark 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 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 Flosbour, Windproof (Ledei) Radix, Ginger (Zingiberis Rhizoma crudus), Natrii sulfas, Large (Zizyphi Fructus), Salviae Radix, Mautan Radicis Cortex, Rehmanniae Radix, Mint Herba, Dioscoreae Rhizoma, Polyporus, Polygoni multiflori Radix, Guja tuberosi Semen, Cassae Semen, Lycii Fructus, Araliae cordatae Radix, Eucommiae Cortex, Hedyotis Herba, Saururus Herba, Injin (Artemisiaecapillaris Herba) Anemarrhenae Rhizoma, Carthami Flos, Astragali Radix, Lycopodium, Ginkgonis Folium, Polygonati Rhizoma, Nelumbinis Semen, Keel (Fossilia ossis Mastosyc) Cortex, Achyranthis Radix, Rehmanniae Radix preparata, Perillae Semen, Thujae Semen, Malt (Hordei Fructus germinatus), Cuscutae Semen, Morindae Radix, Sea Song (Pini) koraiensis Radix) may 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 extract of the clove bark killed 59.6% of Panc-1 pancreatic cancer cells at 100 μg / ml and the IC ₅₀ (half maximal inhibitory concentration) was 89.3 μg / ml. The above results demonstrate that the clove bark 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.

Clove bark extract in the present invention can be extracted 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 post-concentration drying step includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, vacuum 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 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 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 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 the patient a composition comprising the clove bark extract of the present invention, it is possible to effectively prevent and treat the above-mentioned pancreatic cancer. 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 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, 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.

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 clove bark 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 Clove bark extract of the present invention was injected into Panc-1 pancreatic cancer cells, it was confirmed that Panc-1 cells were killed (see FIG. 1 ). Thus, it can be seen that the extract of the clove bark has a pancreatic cancer treatment effect. Thus, in the present invention, the present invention was completed by preparing a functional food for preventing pancreatic cancer containing the extract of clove bark as an active ingredient (see Preparation Example 2 ).

As described above, the clove bark 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 clove bark on the growth of pancreatic cancer cells in Panc-1 cells, which are human pancreatic cancer cells, wherein X-axis is the concentration of clove bark extract, Y-axis is surviving human pancreatic cancer The viability of the cells is shown.
2 is an EdU assay result to determine whether the introduction of the clove bark 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.
3 to 6 is a Western blot method to examine the effect of the introduction of the clove bark obtained in Example 1 in the pancreatic cancer cell line human pancreatic cancer cell lines to promote the proliferation of pancreatic cancer cells, c-myc, This is the result of quantitative changes of ERK, Ras, and β-actin proteins.

Hereinafter, the present invention will be described in more detail based on the following examples. It should be noted, however, that the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The present invention is not limited to the following examples. Will be apparent to those skilled in the art to which the present invention pertains.

< Example 1> Preparation of Clove Bark Extract

3 kg of Clove Bark (Chinese) purchased from Seoul Herbal Medicines was dried and ground for 5 days at the shade and room temperature. The ground cloves 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 289 g of the total extract, which was stored at −20 ° C.

< Example 2> Effect of Clove Bark Extract on the Growth of Pancreatic Cancer Cells

In order to determine the effect of the extract of Clove Bark extracted in Example 1 on the growth of pancreatic cancer cells, Panc-1 cells, which are human pancreatic cancer cells, were treated with ethanol extract of Clove bark 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

Clove bark extract extracted in Example 1 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 in a 96 well plate (seeding) and incubated for 24 hours, the clove bark 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 evenly 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 irradiated light at a wavelength of 544 nm. Survival is shown in FIG.

Clove Bark Concentration Pancreatic cancer cell survival rate (average) Standard Deviation 0 μg / ml 1,000 0.000 3.125 μg / ml 0.981 0.016 6.25 μg / ml 0.968 0.025 12.5 μg / ml 0.956 0.016 25 μg / ml 0.970 0.010 50 [mu] g / ml 0.883 0.049 100 μg / ml 0.404 0.041

As a result, as shown in Table 1 and Figure 1, the higher the treatment concentration of clove bark decreased the growth of pancreatic cancer cells, from which it can be seen that the clove bark has a pancreatic cancer treatment effect. Ie 1.9% at 3.125 μg / ml, 3.2% at 6.25 μg / ml, 4.4% at 12.5 μg / ml, 3.0% at 25 μg / ml, 11.7% at 50 μg / ml, 59.6% at 100 μg / ml Pancreatic cancer cells were killed. In addition, the IC ₅₀ (half maximal inhibitory concentration) was determined to be 89.3 μg / ml. Table 1 describes the relative cell numbers of pancreatic cancer cells after 48 hours according to the concentration of each clove bark on the basis of the survival rate of pancreatic cancer cells of the control group not treated with the clove bark. As such, the clove bark extract 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 Clove Bark Extract Acute toxicity exam

Clove bark 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 in suspension of clove bark extract of Example 1 of the present invention in 0.5% methylcellulose solution, respectively. 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 Clove Bark 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, clove bark was treated by concentration on pancreatic 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 nuclei of newly generated cells after the time point of clove bark treatment are the second one. The third is the sum of these.

As shown in FIG. 2, when the clove bark was treated at each concentration, it can be seen that the number of newly generated cells is smaller than that of the untreated control. As the concentration increased, pancreatic cancer cells increased apoptosis, which resulted in a decrease in total pancreatic cancer cells and a sharp decrease in newly proliferated pancreatic cancer cells. This shows that clove bark has the effect of inhibiting the proliferation of pancreatic cancer cells.

< Example 5> Quantitative change test of signal protein ( Westernblotting , immunoblotting )

Western blot testing was performed to determine the effect of fragmentation 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.

The amount of Ras remains unchanged but the amount of Erk is decreasing, suggesting that the extract of clove bark strongly inhibits its activity. In addition, it can be seen that the amount of the oncogene c-myc is also reduced.

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

The inventors have confirmed that the pancreatic cancer treatment effect of the clove bark extract is excellent through the above embodiment to prepare a pancreatic cancer therapeutic agent containing the clove bark extract as an active ingredient as follows. In addition, the preparation of the following therapeutic agent can be used for the application of not only a therapeutic agent but also a preparation of clove bark food.

<1-1> soft capsule containing clove bark extract ( soft gelatin capsules )(weight%)

Clove Bark 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 Clove Bark Extract (wt%)

Clove Bark Extract 0.2%, Mannitol 0.3%, Saline 9.5%

<1-3> tablets containing the extract of clove bark ( tablet )(weight%)

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

< Manufacturing example 2> Preparation of Functional Foods Containing Clove Bark Extract

The present inventors confirmed that the clove bark extract has excellent pancreatic cancer therapeutic activity through the above embodiment, and prepared a functional food containing the same 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 clove bark extract, and 200 ml of water. And the contents were prepared using conventional methods.

<2-2> Of chewing gum Produce

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

<2-3> Preparation of Candy

Candy was prepared by the conventional method using the composition and content of 50 to 60% of sugar, 39.26 to 49.66% of starch syrup, 0.24 to 0.64% of clove bark extract, and 0.1% of 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 , 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, clove bark 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%, clove bark extract 0.24 ~ 0.64% Ice cream was prepared using conventional methods using the above composition and content.

<2-6> Of chocolate Produce

The 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%, cloves bark extract 0.24 to 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

Pancreatic cancer prevention and treatment composition comprising the active ingredient extracted from the clove bark with an organic solvent.

According to claim 1, wherein the organic solvent is a composition for preventing and treating pancreatic cancer, characterized in that the ethanol.

According to claim 2, wherein the active ingredient extracted with ethanol is a composition for preventing and treating pancreatic 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 pancreatic cancer, characterized in that the dried and concentrated under reduced pressure conditions 45 ℃.

The composition for preventing and treating pancreatic cancer according to any one of claims 1 to 4, wherein the composition comprises a pharmaceutically acceptable carrier or diluent.

The pancreatic cancer prevention and treatment composition according to any one of claims 1 to 4, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

A functional food for preventing pancreatic cancer, comprising a clove bark ethanol extract containing a food supplement acceptable food additive.