KR20160059536A - A Pharmaceutical composition comprising mixed herbal extract for preventing or treating pancreatic cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising mixed herbal extract for preventing or treating pancreatic cancer. More particularly, the present invention relates to a pharmaceutical composition comprising mixed extract of Meliae fructus, Cinnamomum cassia Presl and Sparganii Rhizoma for preventing or treating pancreatic cancer. The composition comprises mixed extract of Meliae fructus, Cinnamomum cassia Presl and Sparganii Rhizoma as an active ingredient, and the mixed extract induces apoptosis of pancreatic cancer cells, inhibits expression of multi-drug resistance-related genes and self renewal- and pluripotency-related genes and suppresses migration activity, and thus can be useful as an active ingredient for a composition for preventing, improving or treating pancreatic cancer.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating pancreatic cancer,

The present invention relates to a pharmaceutical composition for preventing or treating pancreatic cancer, which comprises a mixed herbal medicine extract, and more particularly, to a pharmaceutical composition for preventing or treating pancreatic cancer, which comprises a celecoxib, broiler,

Among the major diseases of modern people, studies on the methods of treatment and diagnosis of cancer are progressing relatively actively, focusing on lung cancer, liver cancer, stomach cancer which have high incidence. However, studies on esophageal cancer, colorectal cancer, and pancreatic cancer, which have low incidence, are relatively poor.

Pancreatic cancer is a pancreatic tumor located in the back of the stomach, mainly in the cells of the pancreatic duct. The pancreas is at the back of the stomach, in the middle of the body, and is about 20 cm long. It is surrounded by organs such as the stomach, duodenum, small intestine, large intestine, liver, gallbladder, spleen, etc., and it is difficult to find cancer and to resect it because of such a position. It is known that early diagnosis such as pancreatic cancer is difficult, and cancer that can not distinguish its symptoms is significantly higher than other cancers. The 5 year survival rate of pancreatic cancer is less than 5% when diagnosed.

As a treatment for pancreatic cancer, chemotherapy and radiotherapy are used to improve survival rate rather than resection. Recently, the most common treatment strategy for advanced pancreatic cancer is gemcitabine, an intravenous 2'-deoxycytidine neuroleoside analogue that can induce apoptosis of human pancreatic cancer cells and inhibit tumor growth and progression, Lt; / RTI > However, despite the best medical or surgical treatment, the outcome of patients with pancreatic cancer (pancreatic duct adenocarcinoma) is dismal and, as a group, the median survival of patients with this disease is less than 21 months.

As described above, there is a need to develop a drug that can effectively treat pancreatic cancer, and studies have been conducted (Korean Patent Publication No. 10-2013-0023173).

DISCLOSURE OF THE INVENTION The present invention has been made in order to solve the above problems, and the present inventors have made efforts to find out an effective herbal extract material for prevention, improvement or treatment of pancreatic cancer. As a result, And that the present invention has been completed on the basis thereof.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating pancreatic cancer, which comprises a celadon, broiler,

It is still another object of the present invention to provide a health food composition for preventing or ameliorating pancreatic cancer, which comprises a celadon, a broiler, and a mixed extract of Samsun.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating pancreatic cancer, which comprises a celadon, a broiler,

In one embodiment of the present invention, the extract may be extracted with a solvent selected from the group consisting of water, an alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof.

In another embodiment of the present invention, the extract may induce apoptosis of pancreatic cancer cells.

The present invention provides a health food composition for prevention or amelioration of pancreatic cancer, which comprises a celadon, a broiler, and a threonine mixed extract.

The present invention provides a method for preventing or treating pancreatic cancer comprising the step of administering to a subject a celecoxib, broiler, and thyme mixed extract.

The present invention provides a prophylactic or therapeutic use of pancreatic cancer in celadon, broiler, and thyme mixed extracts.

The composition according to the present invention includes an extract of Chennai, broiler, and thyme as an active ingredient, and the mixed extract not only induces apoptosis of pancreatic cancer cells, but also induces apoptosis of multi-drug resistance- inhibit self-renewal and pluripotency-related gene expression and inhibit migration activity, and thus are expected to be useful as an active ingredient of a composition for preventing, ameliorating or treating pancreatic cancer.

FIG. 1 shows the results of confirming the expression of CXCR4 gene in the pancreatic cancer cell line (PANC-1) according to the mixed extract treatment of the present invention.
FIG. 2 shows the results of confirming the expression of JAK2 gene in the pancreatic cancer cell line (PANC-1) according to the mixed extract treatment of the present invention.
FIG. 3 shows the results of confirming the expression of XIAP gene in the pancreatic cancer cell line (PANC-1) according to the mixed extract treatment of the present invention.
FIG. 4 shows the results of the protein expression changes of COX-2, Cytochrome c and Caspase-7 in the pancreatic cancer cell line (PANC-1) according to the mixed extract treatment of the present invention.
FIG. 5 shows the results of confirming the expression of ABCG2 gene in the pancreatic cancer cell line (PANC-1) according to the mixed extract treatment of the present invention.
FIG. 6 shows the results of confirming the expression of POU5F1 gene in the pancreatic cancer cell line (PANC-1) according to the mixed extract treatment of the present invention.
FIG. 7 shows the results of confirming the expression of SOX2 gene in the pancreatic cancer cell line (PANC-1) according to the mixed extract treatment of the present invention.
FIG. 8 shows the results of confirming the migration activity change in the pancreatic cancer cell line (PANC-1) according to the mixed extract treatment of the present invention.
FIGS. 9 and 10 are the result of confirming the tumor size and weight change according to the mixed extract treatment of the present invention in the first experiment using the animal model in which the pancreatic cancer cell line (PANC-1) has been transferred.
FIGS. 11 and 12 are the result of confirming the tumor size and weight change according to the mixed extract treatment of the present invention in the second experiment using the animal model in which the pancreatic cancer cell line (PANC-1) has been transferred.
FIG. 13 shows the results of the combined measurements of tumor size and body weight of D + 29 days in the primary and secondary experiments.
FIG. 14 shows the results of H & E staining for the degree of cell necrosis according to the mixed extract treatment of the present invention in an animal model in which pancreatic cancer cell line (PANC-1) was transplanted.

The present invention provides a pharmaceutical composition for preventing or treating pancreatic cancer, which comprises a celadon, a broiler,

As used herein, the term "prophylactic " means any action that inhibits or slows the onset of pancreatic cancer by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" means any action that improves or alleviates symptoms of pancreatic cancer by administration of the pharmaceutical composition of the present invention.

In the present invention, the extract can be extracted using conventional solvents known in the art for extracting the extract from natural products, that is, under ordinary temperature and pressure conditions. For example, in the present invention, the extract may be at least one solvent selected from water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof, preferably methanol or ethanol. In addition, the method of extracting the extract can be carried out by various methods such as hot water extraction, cold extraction, reflux extraction, ultrasonic extraction and the like, but is not limited thereto.

The extract thus prepared may be filtered, concentrated or dried to remove the solvent, and may be subjected to both filtration, concentration and drying. For example, the filtration can be performed using a filter paper or a vacuum filter, the concentration can be carried out using a vacuum concentrator, and the lyophilization can be carried out, but the present invention is not limited thereto.

Further, the extract extracted with the solvent may be further fractionated with a solvent selected from the group consisting of hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water, and mixtures thereof. The fractionation temperature may be 4 캜 to 120 캜, but is not limited thereto.

The present invention relates to a method for inducing apoptosis of a pancreatic cancer cell line, a multi-drug resistance-related gene, a self renewal and a pluripotency-related gene, Expression and inhibit migration activity, and thus can be effectively used as an active ingredient of a composition for preventing or treating pancreatic cancer.

In one embodiment of the present invention, a mixed extract of Chennai, broiler, and Taenung was prepared (see Example 1), and the effect of treating the pancreatic cancer with the mixed extract was examined in vitro. As a result, (See Example 2), as well as the ABCG2 gene, a gene related to multi-drug resistance, and self renewal and starch, as well as suppression of CXCR4, JAK2, XIAP gene expression and COX-2 protein expression It was confirmed that the expression of POU5F1 and SOX2 genes related to pluripotency was inhibited (see Example 3).

In another embodiment of the present invention, the efficacy of the above-described mixed extract in treating pancreatic cancer was confirmed to be superior to that of Gemcitabine alone in treating pancreatic cancer alone (See Example 4).

The pharmaceutical composition according to the present invention may contain, in addition to the active ingredient, a pharmaceutically acceptable carrier. Herein, pharmaceutically acceptable carriers are those conventionally used at the time of formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like may be further included.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may vary depending on the condition and the weight of the patient, The mode of administration, the route of administration, and the time, but may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts.

In another aspect of the present invention, the present invention provides a method of preventing or treating pancreatic cancer comprising the step of administering to a subject a celecoxib, broiler and Thunb's mixed extract.

The term "individual" as used herein refers to a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, cat, It means mammals.

As another aspect of the present invention, the present invention provides a health food composition for preventing or ameliorating pancreatic cancer, which comprises Tian Chen, broiler chick, and Chen Xiang mixed extract as an active ingredient.

In the functional food composition of the present invention, the active ingredient may be directly added to the food or may be used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range.

The functional food composition of the present invention has no particular limitation on the ingredients other than those containing the active ingredient as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary drinks . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above . The ratio of the above-mentioned natural carbohydrate can be appropriately determined by a person skilled in the art.

In addition to the above, the functional food composition of the present invention may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. These components may be used independently or in combination. The ratios of these additives can also be appropriately selected by those skilled in the art.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Preparation of mixed extract

(Hereinafter referred to as " H3 ") were prepared by the following method.

First of all, Tianjin, broiler, and Samson were purchased from Omni Hub. (W / V) of 40% ethanol and extracted at 90 to 100 ° C for 3 hours. Then, the extract was filtered using a microfilter of 11 μm in size , And the residue was subjected to secondary extraction under the same conditions. The filtrate was concentrated under reduced pressure at 60 ° C or lower and then dried to obtain an ethanol extract (1551.6 g).

Example 2. Confirmation of the ability of mixed extract to induce apoptosis of pancreatic cancer cell line

2-1. Identification using quantitative real-time PCR

In this example, RNA was isolated from cells using RNAeasy (Qiagen, USA), and first strand cDNA was synthesized according to Reverse Transcription System (promega, USA). Real-time PCR was performed using TaqMan Gene expression master mix (Applied Biosystems, USA) and TaqMan Gene expression assay (Applied biosystems). GAPDH was used as an internal control. Real-time PCR was performed using CFX-96 (Bio-Rad, USA). The PCR was performed at 95 ° C. for 10 minutes and then at 50 ° C. for 1 hour at 90 ° C. (15 seconds) Respectively. Each sample was verified with a triplicate. Quantification was normalized through GAPDH expression, a house keeping gene.

After the combined treatment of H3 alone, Gemcitabine alone, and H3 and Gemcitabine, pancreatic cancer cell line (PANC-1) was treated with quantitative real-time PCR and CXCR4, JAK2, and XIAP gene expression, and the results are shown in Figs. 1 to 3, respectively.

As shown in Figs. 1 to 3, when H3 was treated, it was confirmed that CXCR4, JAK2 and XIAP gene expression was suppressed.

More specifically, in the case of CXCR4, the expression level of CXCR4 was reduced by about 57% when H3 alone was treated, and the expression level of CXCR4 was reduced by about 69% when the combination of H3 and gemcitabine was administered ( 1). In addition, in the case of JAK, the gene expression level of JAK2 was reduced by about 56% when the combination of H3 and gemcitabine was applied (see FIG. 2). Furthermore, in the case of XIAP, the degree of gene expression of XIAP was most effectively inhibited by about 80% when the combination of H3 and gemcitabine was applied (see FIG. 3).

2-2. Confirmation with western blot

The expression of COX-2, a major mediator of inflammation, and cytochrome c and caspase-7, which are known to be involved in apoptosis, were examined by western blotting, and the results are shown in FIG. At this time, β-actin was used as an internal control.

As shown in FIG. 4, it was confirmed that the degree of COX-2 protein expression was significantly reduced in the case of H3 alone treatment. In addition, the degree of protein expression of Cytochrome c was significantly increased in the case of H3 alone compared to the other treatment groups.

Example 3 Confirmation of the Effect of Mixed Extracts in vitro in the Treatment of Pancreatic Cancer

3-1. Confirming the inhibition of gene expression related to multi-drug resistance

(PANC-1) was treated with H3 alone, gemcitabine alone, and H3 and gemcitabine, followed by quantitative real-time PCR to measure the drug resistance ), And the results are shown in Fig. 5.

As shown in FIG. 5, it was confirmed that the degree of gene expression of ABCG2 was remarkably inhibited to about 58% when H3 and gemcitabine were combined.

3-2. Identification of self renewal and inhibition of gene expression related to pluripotency

(PANC-1) were treated with H3 alone, gemcitabine alone, H3 and gemcitabine, followed by quantitative real-time PCR for self renewal and starch Changes in POU5F1 and SOX2 gene expression associated with pluripotency were confirmed, and the results are shown in FIGS. 6 and 7, respectively. At this time, Housekeeping gene GAPDH was used as an internal control.

In the case of the gene of POU5F1, the degree of gene expression of POU5F1 was reduced to about 62% when H3 alone was treated as shown in Fig. 6, and the gene expression of POU5F1 when combined with H3 and gemcitabine Of the total amount of water was suppressed to about 53%.

Further, in the case of the SOX2 gene, as shown in Fig. 7, when H3 and gemcitabine were combined, SOX2 gene expression was most effectively inhibited to about 85%.

3-3. Identify inhibition of migration activity

To analyze the migration activity of pancreatic cancer cell line (PANC-1), CytoSelect 24-Well Wound Healing Assay (Cell Biolabs, USA) was performed. The assay was performed according to the protocol of the manufacturer. More specifically, pancreatic cancer cells (PANC-1) were seeded at 2.5 × 10 ⁵ per well. After the pancreatic cancer cells were cultured overnight, the wound field insertion was removed, . Each sample was examined for wound healing with an optical microscope (CK70, Olympus Imaging Corp., Japan). All experiments triplicate.

After the combination of H3 alone, Gemcitabine alone, and H3 and Gemcitabine in the pancreatic cancer cell line (PANC-1), migration activity known to be related to tumor formation and metastasis was measured using a wound healing assay And the results are shown in Fig.

As shown in FIG. 8, it was confirmed that migration activity was most effectively inhibited when H3 alone was administered, compared with the case of the combination treatment of gemcitabine alone and H3 and gemcitabine.

Example 4. Confirmation of the Effect of Mixed Extracts in Vivo in Treatment of Pancreatic Cancer

4-1. The first experiment

In this experiment, 5-week-old female Balb / c mice were used as experimental animals. Pancreatic cancer cell line (PANC-1) was injected, followed by oral administration of H3 for 6 days from day 0, 2 days after day 2, gemcitabine was administered by intraperitoneal injection every 3-4 days, The experiment was carried out for 31 days in total. The concentrations of H3 (200 mg / kg), Gemcitabine (100 mg / kg) and H3 (100 mg / kg) and Gemcitabine (50 mg / kg) Tumor size and body weight were measured by sacrificing the experimental animals, and the results are shown in FIGS. 9 and 10, respectively.

As shown in FIG. 9 and FIG. 10, tumor size was reduced when each of H3 and gemcitabine alone was treated (see FIG. 9), but it was confirmed that the weight of each treatment group did not show any difference (See Fig. 10).

4-2. Second experiment

Pancreatic cancer cell line (PANC-1) was injected, followed by oral administration of H3 for 6 days from day 0, 2 days after day 2, gemcitabine was administered by intraperitoneal injection every 3-4 days, The experiment was carried out for 29 days. The concentrations of H3 (200 mg / kg), Gemcitabine (100 mg / kg) and H3 (100 mg / kg) and Gemcitabine (50 mg / kg) Tumor size and body weight were measured by sacrificing the experimental animals, and the results are shown in FIGS. 11 and 12, respectively.

As shown in FIG. 11 and FIG. 12, tumor size was most effectively decreased when H3 alone was treated, and even when H3 and gemcitabine were treated in combination, the tumor size was smaller than that of Gemcitabine alone (See FIG. 11), while it was confirmed that the weight of each treatment group did not show any significant difference (see FIG. 12).

In addition, during the above primary and secondary experiments, tumor size and body weight measurement results of D + 29 days were synthesized, and the results are shown in FIG. As shown in Fig. 13, tumor size showed the greatest reduction effect when H3 alone treatment was applied, and it was confirmed that weight gain was the largest when H3 alone treatment was applied to the body weight.

4-3. Confirmation of cell necrosis using H & E staining

The degree of cellular necrosis of tumors extracted after the combined treatment of H3 and gemcitabine in the PANC-1 transferred animal model was confirmed by H & E staining, and the results are shown in FIG.

As shown in Fig. 14, when H3 alone was treated, it was confirmed that cell necrosis significantly progressed compared with other treatment groups.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (4)

A pharmaceutical composition for the prevention or treatment of pancreatic cancer, which comprises a celecoxib, broiler,
The composition according to claim 1, wherein the extract is extracted with a solvent selected from the group consisting of water, an alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof.
The composition of claim 1, wherein the extract induces apoptosis of pancreatic cancer cells.
A composition for preventing or ameliorating pancreatic cancer, comprising a mixture of celecoxib, broiler,

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