KR20180126936A - Composition for inducing apoptosis of cancer cell containing extract of chestnut inner shell - Google Patents

Abstract

The present invention relates to a composition for inducing the apoptosis of cancer cells containing an extract of the chestnut inner skin, and an anti-cancer use thereof. According to the present invention, the composition generates reactive oxygen species (ROS) in cancer cells for inducing the apoptosis, thereby exhibiting an excellent anticancer effect.

Description

Technical Field [0001] The present invention relates to a composition for inducing apoptosis of cancer cells,

The present invention relates to a composition for inducing apoptosis of cancer cells comprising chest roentgenic skin extract and its anticancer use.

The worldwide incidence of cancer and mortality is increasing year by year. Cancer treatment methods mainly include anticancer drug administration, radiation therapy, and surgical operation. Among them, administration of chemotherapeutic agent or radiation therapy is a treatment method through the apoptosis of cancer cells. In addition to cancer cells, cytotoxicity is also observed in normal cells. As a result, there is a growing interest in alternative medicine and natural medicine to minimize side effects. In addition, there is a growing interest in not only cancer treatment agents but also cancer treatment adjuvants and cancer prevention, and development of natural materials having anticancer activities in addition to synthetic materials has been continuously studied. Many of these natural medicinal materials are derived from plants, and various kinds of ingredients such as alkaloids and flavonoids are present in plants, and various anticancer agents are being developed using their high toxicity and low side effects.

Julie is the chestnut of the night, known to cleanse the skin and prevent aging. In addition, as the effects of antiallergic effect, antioxidant activity, and anti-obesity effect are revealed, many studies are being conducted for various applications for anti-atopic, antioxidant, anti-inflammatory and anti-amnestic use

Korean Patent No. 10-1206543

Accordingly, the inventors of the present invention have discovered that chestnut endothelium extract induces apoptosis by generating ROS in cancer cells, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for inducing apoptosis of cancer cells comprising chestnut endothelial extract, an anticancer composition comprising the same, and a health functional food for cancer.

In order to accomplish the above object, the present invention provides a composition for inducing apoptosis of cancer cells comprising chest roentgenic extract.

The present invention also provides an anticancer composition comprising a composition for inducing apoptosis of cancer cells and a health functional food for cancer.

In one embodiment of the present invention, the extract may be extracted with water, an organic solvent or a combination thereof, and the organic solvent may include, for example, an alcohol having 1 to 4 carbon atoms. The alcohol may be ethanol or methanol.

In one embodiment of the present invention, the chestnut endothelial extract has a concentration of 10 to 500 μg / ml, for example, 10 to 400 μg / ml, 10 to 300 μg / ml, 20 to 500 μg / ml, 500 [mu] g / ml.

In one embodiment of the present invention, the chestnut endothelial extract may induce apoptosis by generating ROS (reactive oxgen species) in the cancer cells.

The chestnut root extract of the present invention induces apoptosis by generating ROS (reactive oxgen species) in cancer cells, thereby exhibiting the preventive or therapeutic effect of cancer. The chestnut root extract is a natural medicine and exhibits high cancer cell toxicity and low side effects. Therefore, it can be variously commercialized as a pharmaceutical composition for anticancer cancer and a health food. Therefore, it can contribute to the increase of income of farmers by high value added of chestnut byproducts.

1 is a schematic view showing a process for producing chestnut hot-water extract and ethanol extract according to an embodiment of the present invention.
FIG. 2 is a graph showing the inhibitory effects of chestnut hot-water extract (CI-W) and ethanol extract (CI-E) on cancer cell proliferation according to an embodiment of the present invention.
FIG. 3 is an image showing the cancer cell proliferation inhibitory effect of chestnut hot-water extract (CI-W) and ethanol extract (CI-E) according to an embodiment of the present invention.
4 is a graph showing ROS incidence rates in cancer cells of chestnut hot-water extract (CI-W) and ethanol extract (CI-E) according to an embodiment of the present invention.
FIG. 5 is an image showing the morphological intracellular ROS incidence rate of chestnut hot-water extract (CI-W) and ethanol extract (CI-E) according to an embodiment of the present invention.
6 is an image showing the result of nuclear morphological observation of chestnut hot-water extract (CI-W) and ethanol extract (CI-E) according to an embodiment of the present invention.
FIG. 7 is a Western blotting result showing a cell death mechanism by chestnut endophytic hot-water extract (CI-W) and ethanol extract (CI-E) according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed description of known techniques well known to those skilled in the art may be omitted. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear. Also, terminologies used herein are terms used to properly represent preferred embodiments of the present invention, which may vary depending on the user, intent of the operator, or custom in the field to which the present invention belongs.

Therefore, the definition of these terms should be based on the contents throughout this specification. Throughout the specification, when an element is referred to as "comprising ", it means that it can include other elements as well, without excluding other elements unless specifically stated otherwise.

The present invention relates to a composition for inducing apoptosis of cancer cells containing chest roentgenic peel extract, an anticancer composition comprising the same, and a health functional food for cancer.

The chestnut endothelial extract may be prepared by extraction with organic solvent such as water or an alcohol having 1 to 4 carbon atoms, for example, by hot water extraction or ethanol extraction by the process as shown in FIG. Specifically, the extract using hot water is prepared by (1) separating the night in half and drying with hot air; (2) separating the dried chestnut into a fruit and a bark, and separating the bark into an endothelium and a sheath; And (3) refluxing the endothelium of step (2) with hot water. The extract using an organic solvent can be prepared by (1) separating the chestnuts in half and drying with hot air; (2) separating the dried chestnut into a fruit and a bark, and separating the bark into an endothelium and a sheath; (3) extracting the endothelium of step (2) by immersing it in an organic solvent.

The chestnut endothelial extract may be contained at a concentration of 10 to 500 μg / ml, for example, 10 to 400 μg / ml, 10 to 300 μg / ml, 20 to 500 μg / ml or 50 to 500 μg / ml Specifically, 25 to 250 占 퐂 / ml or 50 to 250 占 퐂 / ml.

The chestnut endothelial extract may induce apoptosis mechanism by inducing ROS (reactive oxgen species) in the cancer cells to inhibit cancer cell growth. Specifically, the expression of Bcl-2 protein, which has the function of inhibiting apoptosis as an anti-apoptotic molecule during apoptosis, decreased as the concentration of chestnut endothelium extract increased, leading to apoptotic cell death Induced expression of Bax protein increases with increasing concentration, and can induce apoptosis of cancer cells.

The anticancer composition comprising chestnut root extract according to the present invention as an active ingredient may contain a pharmaceutically effective amount of chestnut endothelium extract alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents.

The "pharmaceutically effective amount" as used herein refers to an amount sufficient for the physiologically active component to be administered to an animal or a human to exhibit a desired physiological or pharmacological activity. However, the pharmaceutically effective amount may be appropriately changed depending on the severity of symptoms, the age, body weight, health condition, sex, administration route and treatment period of the patient.

The term "pharmaceutically acceptable" as used herein means physiologically acceptable and does not generally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to a human. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

The compositions of the present invention may also be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal, and may be formulated as a variety of compositions for oral or parenteral administration . ≪ / RTI > The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

The composition according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscular, and the dose of the active ingredient may be varied depending on various factors such as route of administration, age, sex, And the like. In addition, the composition of the present invention may be administered in combination with a known compound capable of raising the desired effect.

Furthermore, the composition according to the present invention can be used not only as a pharmaceutical composition as described above, but also as a health functional food. For example, it can be easily used as a raw material for food, an ingredient, a food additive, a functional food or a beverage.

The term " food " means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be eaten directly through a certain degree of processing, , Food additives, functional foods and beverages.

Foods to which the food composition can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, and functional foods. In addition, it is also possible to use special nutritive foods (eg crude oil, milk, infant food, etc.), meat products, fish products, tofu, jelly, noodles (eg ramen, (Such as fermented milk, cheese, etc.), processed foods, kimchi, pickled foods (various kinds of kimchi, etc.), sauces, confectionery (eg, snacks), candies, chocolate, gums, ice cream, But not limited to, beverages (e.g., fruit drinks, vegetable beverages, beverages, fermented beverages, etc.), natural seasonings (e.g., ramen soup, etc.). The food, beverage or food additive may be prepared by a conventional production method.

The term " functional food " or " health functional food " refers to a food group imparted with added value to function or express the function of the food to a specific purpose by physical, biochemical or biotechnological techniques, Defensive rhythm control, prevention and restoration of disease, and the like, and it can be a health functional food. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

The health supplements may be in the form of powders, granules, tablets, capsules or drinks, although not limited thereto.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for illustrating the present invention specifically, and the scope of the present invention is not limited to these examples.

[Example]

Manufacturing example  1. Preparation of chestnut root extract

The chestnut was cut in half and then the endothelium was separated by hot air drying at 40 ° C for two days. The extract was subjected to reflux 3 times for 1 hour at 55 캜 for 3 hours with 1 L of distilled water per 100 g of chestnut fat to obtain a hot water extract. The ethanol extracts were extracted by immersing in 1 L of 75% ethanol per 100 g of chestnut endothelium at room temperature for 7 days. Each hot water and ethanol extract was concentrated under reduced pressure at 60 ° C or less using a rotary evaporator (Eyela, Japan), and the concentrate was lyophilized at -80 to -110 ° C. The final yield was 12.97% of chestnut hot water extract and 15.98% of chestnut ethanol extract. The dried hot water and ethanol extracts were diluted to a concentration of 10 mg / mL using DMSO (dimethyl sulfoxide, Sigma-Aldrich) and DMEM (Dulbecco's modified Eagle's medium, WeLGENE Inc.) , 100, and 250 μg / mL to prepare an anticancer composition.

Example  1. Evaluation of cell proliferation inhibition on liver cancer cell line

Hepatocellular carcinoma cells were purchased from HepG2 (human liver cancer cells) in Korea Cell Line Bank. The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU / mL) at 37 ° C in a humidified environment with a 5.0% carbon dioxide incubator.

Cell death was analyzed by MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma-Aldrich). Cancer cells were inoculated at a density of 5 × 10 ⁴ cells / well in tissue culture plates with 96 wells. The endothelial extracts at night for 24 and 48 hours were inoculated at various concentrations (10, 25, 50, 100, 250 μg / mL). MTT was then treated at a concentration of 2.0 mg / mL and incubated again for 1 hour. After 1 hour, the medium was removed, and DMSO was added to dissolve all of the generated formazan. Then, the plate was transferred to a 96-well plate and absorbance was measured at 450 nm using an ELISA reader. Data were presented as means ± SD (SD) and all experiments were repeated three times. The data obtained are analyzed using t- test to measure the significance test, ^* P <0.05, are shown in ^** P <0.01 and ^* P <0.001-in, and also considered to be statistically significant in two cases. As shown in FIG. 2, each of the extracts was found to be involved in the inhibition of cancer cell proliferation in a concentration and time-dependent manner. In the case of HepG2 cells treated with 25 ~ 250 μg / mL of chestnut extract for 24 hours, the cell viability was inhibited by 64% at 250 μg / mL for hot water extract and 71% at 250 μg / mL for ethanol extract Respectively. In particular, after 48 hours, the inhibitory activity was higher than that of 24 hours.

Example  2. Proliferation inhibition observation of morphological cancer cells

The cancer cells were divided into 5 × 10 ⁴ cells / well in a tissue culture plate having 96 wells, left for 24 hours, and treated at various concentrations (50, 100, 250 μg / mL) for 48 hours . After the treatment time, the killed cells were observed with a microscope (CKX41, Olympus, Tokyo, Japan) and imaged with a digital image camera (eXcope T500, Olympus, Tokyo, Japan). The results are shown in Fig. As can be seen from Fig. 3, it was visually confirmed that chestnut endothelial extracts inhibited cancer cell proliferation in a concentration- and time-dependent manner.

Example  3. Intracellular ROS  Incidence rate

In the presence of peroxides associated with hydrogen peroxide in the cell, DCF-DA, a non-mineralized substance, was oxidized by ROS to show green light, and ROS was measured by DCF-DA. First, hepatocarcinoma cells were seeded at a density of 5 × 10 ⁴ cells / well on a 96-well black plate, followed by culturing for 24 hours. Twenty-four hours later, the endothelial extracts were treated at a concentration of 50 μg / mL and 250 μg / mL for 24 hours and 48 hours. The plate treated with the sample was treated with 10 μmol / mL DCF-DA (Dichlorofluorescin diacetate, Sigma-Aldrich) dissolved in PBS (Phosphate buffer, Sigma-Aldrich) and reacted for 30 minutes. The reacted plate was washed twice with PBS and fluorescence was measured at Ex: 485 nm and Em: 528 nm. The data of each concentration group were calculated based on the control group as 1, and the data were presented as the mean ± standard deviation (SD). All experiments were repeated three times. ^* P <0.05, ^** P <0.01 and ^* P <0.001 were considered statistically significant. As can be seen from FIG. 4, the ROS production was increased in a time and concentration-dependent manner when each endothelial extract was treated with respect to the ROS production rate of the control group. That is, it was confirmed that cancer cells were inhibited by the proliferation of ROS. Especially, ethanol extract showed high concentration - dependent ROS concentration for 48 hrs treatment, indicating that it is more effective in inhibiting cancer cell growth than hot - water extract.

Example  4. Morphological Intracellular ROS  Incidence rate

HepG2 cells were plated in 6-well plates at 5 x 10 ⁴ cells / well and cultured for 24 hours. Twenty-four hours later, the endothelial extracts were treated at a concentration of 50 μg / mL and 250 μg / mL for 48 hours. The cells were then suspended using Trpsin-EDTA (WelGENE Inc.), placed in microtube, and centrifuged at 10,000 rpm, 4 ° C for 20 minutes. The supernatant was removed and treated with 10 μmol / mL DCF-DA (Dichlorofluorescin diacetate, Sigma-Aldrich) dissolved in PBS (Phosphate buffer, Sigma-Aldrich) for 30 min. The reacted microtube was washed twice with PBS, dropped on a cover glass, and then measured with a fluorescence microscope (Axioskop 40, Carl Zeiss, Germany). The results are shown in FIG. As can be seen from FIG. 5, the ROS production of the control group hardly appeared, but it was clearly observed that the treatment with the endothelium extract of each chestnut increased in a concentration-dependent manner.

Example  5. Nuclear morphological observation

HepG2 cells were plated at 6 × 10 ⁴ cells / well in a 6-well plate and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. The chestnut endothelium extract was treated for each concentration and cultured for 48 hours. After incubation, the cells recovered from the wells were washed three times with PBS and stained with Hoechst 33258 (Sigma-Aldrich Co., St. Louis, Mo., USA) for 20 minutes at room temperature. Carl Zeiss, Germany) and the results are shown in FIG. As can be seen from FIG. 6, the nucleus of the control group was constant in shape without damage, while the nucleus of the group treated with the extract was damaged and fragmented, and an apoptotic body, which is one of the characteristics of apoptosis, there was. Therefore, it can be understood that the chestnut endothelial extract is related to ROS-induced apoptosis during the death of cancer cells.

Example  6. Western Blotting  Detection of cell death through

Western blot analysis revealed that the effect of chestnut endothelial extract on the expression of the Bcl-2 family protein, the protein most closely related to apoptosis when treated with HepG2 cells, was confirmed.

HepG2 cells were seeded at a density of 1 × 10 ⁶ cells / well in 10 mm culture dishes and treated with 50 μg / mL and 250 μg / mL of extracts, respectively. All the cultured cells were collected and lysed in ice (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 50 mM NAF, 30 mM Na ₄ P ₂ O ₇ ). The cell lysate and 2 × sample buffer were mixed in the same volume, boiled at 60 ° C for 5 minutes, and subjected to 8% and 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After electrophoresis, the proteins of the gel were transferred to a nitrocellulose membrane and incubated with blocking buffer (2.5%, 5% BSA) for 1 hour at room temperature. Primary antibodies (anti-Bax, anti-Bcl- cleaved caspase-3, anti-β-actin) were diluted and reacted at 4 ° C for one day. Figure 7 shows the immunoreactivity visualized and graphed using an enhanced chemiluminescence kit (ECL kit) after washing with T-TBS for more than 1 hour, washing the secondary anti-rabbit IgG conjugated HRP at 4 ° C for more than 30 minutes . As shown in FIG. 7, the expression of Bcl-2 protein having the function of inhibiting apoptosis as an anti-apoptotic molecule was decreased as the concentration of chestnut endothelial extract was increased, and pro-apoptotic molecule The expression of inducible Bax protein was increased with increasing concentration. In addition, Cleaved caspase-3 was found to increase in concentration-dependent manner in each extract. These results suggest that caspase-3, which is an active caspase, is induced by Bcl-2 expression and induces apoptosis. It is thought to stimulate endogenous pathway during apoptosis.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (7)

A composition for inducing apoptosis of cancer cells comprising chestnut root extract. The composition of claim 1, wherein the extract is extracted with water, an organic solvent, or combinations thereof. The composition according to claim 2, wherein the organic solvent comprises an alcohol having 1 to 4 carbon atoms. The composition according to claim 1, wherein the chestnut endothelial extract is contained at a concentration of 10 to 500 占 퐂 / ml. The composition according to claim 1, wherein the chestnut root extract produces ROS (reactive oxgen species) in the cancer cells to induce apoptosis. An anticancer composition comprising the composition for inducing apoptosis of cancer cells according to any one of claims 1 to 5 as an active ingredient. A health-functional food for anticancer comprising the composition for inducing apoptosis of cancer cells according to any one of claims 1 to 5.

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