KR101184388B1 - A method for boosting anticancer activity in red ginseng extracts using irradiation - Google Patents

Abstract

The present invention relates to a method for enhancing the anticancer effect of red ginseng (Red ginseng) liquid using radiation, and specifically, to extract the red ginseng concentrate in methanol, and to a method of enhancing anticancer effect by adding hydrogen peroxide and irradiating gamma rays. Therefore, the ginsenoside content in the red ginseng solution was increased by the irradiation technique without complicated treatment process, and the anticancer effect caused by apoptosis was enhanced, which can be usefully used as an anticancer agent.

Red ginseng, ginsenoside, radiation, anticancer effect

Description

A method for boosting anticancer activity in red ginseng extracts using irradiation}

The present invention relates to a method for enhancing the anticancer effect of red ginseng solution using radiation.

Food irradiation technology is commonly used in Korea for sterilization purposes, and various effects such as physical property improvement and impurities removal have been verified. Gamma-irradiation is a beneficial and effective technique that can completely kill deadly infectious diseases, parasites and pests by irradiating gamma rays, short-wavelength light from cobalt-60 radioisotopes, on food and medical supplies. Gamma rays are wavelengths that have a high penetration enough to penetrate a few centimeters of concrete, so that sterile products can be produced even when the food and health care products are completely sealed. Gamma rays emitted from cobalt-60 are more permeable to charged particles, such as electrons, and thus are suitable for irradiation inside liquids or solids. Cobalt-60 sources are used for sterilization of medical devices and food research.

Red ginseng (Red Ginseng) is a ginseng that has been steamed for 6 years and dried to be less than 14% of water, and the browning reaction is promoted during the manufacturing process. Red ginseng prevents the contamination of bacteria, mold and microorganisms by reducing moisture through processing and drying, and it is easy to store and transport by reducing volume and weight. Saponin quantitative method and quality control, which is the representative active ingredient of ginseng, prevented the degradation of saponin during the processing process, and saponin, maltol, ginsenoside Rh2 and other active ingredients were additionally produced. To date, numerous studies have been conducted at home and abroad on the ingredients and pharmacological effects of red ginseng, especially red ginseng's antioxidant activity 2000).

Ginsenosides present in red ginseng are known to exhibit anti-cancer, anti-inflammatory and immune system enhancement effects. The ginsenosides have increased functionality as their structure is changed by physical, chemical, and biological treatments. As a method for improving the functionality of red ginseng, a method of preparing fermented red ginseng using microorganisms (Korean Patent No. 618171 or 85679), and a method of chemically treating saponin of red ginseng and separating it by chromatography (Korean Patent No. 316567) ), A method of preparing ginsenoside Rg3 or Rh2 by reacting saponin with an enzyme (Korean Patent No. 485936), but such a method has a high yield, a complicated method, and a costly disadvantage. In addition, the existing methods are mainly related to the enhancement of components related to immunity or anti-inflammatory, and there are not many reports on how to produce substances having anti-cancer effects.

Thus, the present inventors dissolved red ginseng concentrate in methanol, and added hydrogen peroxide and irradiated to induce a change in the composition, the content of ginsenosides having anticancer effect increased the red ginseng concentrate, caused by apoptosis Enhancement of anticancer effect and confirming that ROS is generated during the anticancer action has completed the present invention.

It is an object of the present invention to provide a method for producing red ginseng liquid with improved anticancer effect by irradiation with red ginseng liquid.

Another object of the present invention is to provide an anti-cancer effect enhanced red ginseng liquid prepared by the above method.

Another object of the present invention to provide a pharmaceutical composition for preventing and treating cancer containing the red ginseng liquid with the improved anticancer effect as an active ingredient.

Another object of the present invention to provide a health functional food for cancer prevention and improvement containing red ginseng liquid with enhanced anticancer effect as an active ingredient.

In order to achieve the above object,

1) adding a radical generator to the red ginseng solution; And

2) It provides a method for producing red ginseng solution enhanced anticancer effect comprising the step of irradiating the treatment solution of step 1).

In addition, the present invention provides an anti-cancer effect enhanced red ginseng liquid prepared by the above method.

In addition, the present invention provides a pharmaceutical composition for preventing and treating cancer containing red ginseng as an active ingredient, the anti-cancer effect is enhanced.

In addition, the present invention provides a health functional food for cancer prevention and improvement containing red ginseng liquid with enhanced anticancer effect as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention

1) adding a radical generator to the red ginseng solution; And

2) It provides a method for producing red ginseng solution enhanced anticancer effect comprising the step of irradiating the treatment solution of step 1).

In a specific embodiment of the present invention, after extracting the red ginseng concentrate using 50% methanol, irradiation with red ginseng extract added with hydrogen peroxide at a concentration of 0.05%, it was confirmed that the content of ginsenoside increases (Fig. 1). Reference). However, all ginsenoside compounds were decomposed in the red ginseng extract irradiated with 10 ~ 20 kGy. In addition, it was confirmed that the anti-cancer effect significantly increased when the treatment was adjusted to the radiation dose of 1 to 5 kGy (see Fig. 2). The anticancer effect was confirmed by cytotoxicity by apoptosis (see FIGS. 3 and 4), it was confirmed that reactive oxygen species (ROS) is produced by the anticancer action (see FIG. 5). Therefore, the method of the present invention can be usefully used to enhance the anticancer effect of red ginseng solution.

In the above method, the red ginseng liquid of step 1) is ginsenoside, containing red ginseng pulverized liquid or red ginseng extract, the red ginseng pulverized liquid indicates that the dried red ginseng powder is extracted with water, and the red ginseng extract is dried red ginseng powder or red ginseng concentrate To extract using a solvent selected from the group consisting of alcohol and aqueous alcohol solution, generally red ginseng concentrate means a liquid red ginseng extract. The alcohol is preferably C ₁ to C ₄ lower alcohols, it is preferable to use ethanol or methanol as the lower alcohol, most preferably the solvent is a 50% methanol aqueous solution. The solvent is preferably extracted by adding 0.5 to 2 times the amount of red ginseng concentrate, and more preferably, by adding the same amount.

Radical generators of step 1) include hydrogen peroxide, peracetic acid or ascorbic acid and the like. The red ginseng solution is preferably added in an amount of 0.02 to 0.1 parts by weight, most preferably 0.05 part by weight.

The radiation of step 2) may use either gamma rays, electron beams or X-rays, preferably gamma rays.

The gamma rays are preferably irradiated using gamma rays emitted from radioactive isotopes such as cobalt (Co) -60, krypton (Kr) -85, strontium (Sr) -90, or cesium (Cs) -137. More preferably, but not limited to, emission from Co) -60 radioisotopes. The irradiation dose is preferably 1 to 10 kGy, more preferably 1 to 7 kGy, and most preferably 1 to 5 kGy. The range of irradiation dose is that all ginsenosides in red ginseng are decomposed. It was set as the basis that less than enough to disappear, the ginsenosides can be changed to show an enhanced anticancer effect.

In addition, the present invention provides an anti-cancer effect enhanced red ginseng liquid prepared by the above method.

In a specific embodiment of the present invention, after extracting the red ginseng concentrate using 50% methanol, and irradiating the red ginseng extract added with hydrogen peroxide at a concentration of 0.05%, it was confirmed that the content of ginsenoside increases by irradiation. (See FIG. 1). In addition, it was confirmed that the anti-cancer effect significantly increased when the treatment was adjusted to the irradiation dose of 1 to 5 kGy (see Fig. 2). The anticancer effect was confirmed by cytotoxicity by apoptosis (see FIGS. 3 and 4), it was confirmed that reactive oxygen species (ROS) is produced by the anticancer action (see FIG. 5). Thus, it was confirmed that the anticancer effect of the red ginseng extract of the present invention was enhanced.

In addition, the present invention provides a pharmaceutical composition for preventing and treating cancer containing red ginseng as an active ingredient, the anti-cancer effect is enhanced.

In the specific embodiment of the present invention, the red ginseng extract treated according to the method of the present invention was confirmed that the anticancer effect was increased by the content of ginsenoside (see FIG. 1) and by apoptosis (see FIGS. 3 and 4) (FIG. 2). Reference). In addition, it was confirmed that reactive oxygen species (ROS) are produced by the anticancer action (see FIG. 5). Therefore, the red ginseng solution of the present invention can be usefully used as a pharmaceutical composition for preventing and treating cancer.

The pharmaceutical composition of the present invention exhibits cytotoxicity due to apoptosis and produces ROS, and thus, liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, and anal muscles. Cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma and central nervous system tumor It can be used for the prevention and treatment of cancer diseases.

The pharmaceutical composition of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the red ginseng solution of the present invention.

The pharmaceutical composition of the present invention comprises 0.0001 to 10% by weight, preferably 0.001 to 1% by weight of the red ginseng liquid with enhanced anticancer effect relative to the total weight of the composition.

The pharmaceutical composition according to the present invention may further include conventionally used carriers, excipients, disintegrants, sweeteners, lubricants, flavoring agents and diluents. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The disintegrants include sodium starch glycolate, crospovidone, croscarmellose sodium, alginic acid, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, chitosan, guar gum, low-substituted hydroxypropyl cellulose, magnesium aluminum silicate, and polyacryline Potassium and the like. In addition, the pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, Calcium hydrogen phosphate, lactose, mannitol, malt, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, Aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included 0.1 to 90 parts by weight based on the pharmaceutical composition.

In addition, the subject to which the pharmaceutical composition of the present invention can be applied is a vertebrate and preferably a mammal, more preferably an experimental animal such as a rat, rabbit, guinea pig, hamster, dog, cat, and most preferably It is ape-like, such as chimpanzees and gorillas. In addition, the pharmaceutical composition of the present invention can be administered orally or parenterally, and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine epidural injection, cerebrovascular (intracerebroventricular) ) Or by intrathoracic injection. In addition, the pharmaceutical composition may be used alone or in combination with methods using surgery, hormonal therapy, drug treatment and biological response modifiers for the subject for the prevention and treatment of cancer.

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. The administration may be carried out once a day or divided into several times.

In addition, the present invention provides a health functional food for cancer prevention and improvement containing red ginseng liquid with enhanced anticancer effect as an active ingredient.

The red ginseng solution of the present invention may be added to foods as it is or used with other foods or food ingredients, and may be appropriately used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). Generally, in the preparation of food or beverages, the composition of the present invention is added at 0.2 to 20% by weight, preferably 0.24 to 10% by weight relative to the raw materials. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health food of the present invention may contain various flavors or natural carbohydrates as additional ingredients. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is preferably selected in the range of 0.01 to 0.04 parts by weight, preferably about 0.02 to 0.03 parts by weight, per 100 parts by weight of the health food of the present invention.

There is no particular limitation on the kind of the food. Examples of foods to which the substance may be added include drinks, sausages, biscuits, chocolates, candy, snacks, sweets, gums, dairy products including ice cream, various soups, beverages, alcoholic beverages, and vitamin complexes. It includes all of the health foods in the sense.

In addition to the above, the health food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. In addition, the health food of the present invention may contain a flesh for producing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. Although the ratio of such an additive is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention.

According to the method of the present invention, the ginsenoside content in the red ginseng solution was increased by the irradiation technique without complicated treatment process, and the anticancer effect caused by the apoptosis was enhanced, which can be usefully used as an anticancer agent.

Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples.

Example 1 Red ginseng extract treatment

A red ginseng extract prepared by dissolving commercially available red ginseng extract (red ginseng extract) in 50% methanol was prepared. Cobalt-60 gamma irradiation facility of a source of 100,000 Ci sourced in the red ginseng extract (20 mg / ml) in a container by adding hydrogen peroxide at a concentration of 0.05% and contained in the Jeongeup Radiation Research Institute of the Korea Atomic Energy Research Institute Point sources AECL, IR-79, MDS Nordion International Co. Ltd., Ottawa, ON, Canada) were used to investigate 1 to 20 KGy at room temperature, respectively, and the total absorbed dose was 0.2 Gy.

Experimental Example 1 Determination of Ginsenosides Content in Red Ginseng Extract

The red ginseng extract treated in Example 1 under the following conditions was analyzed by chromatography:

1) Instrument: Agillent HPLC system;

2) column: C18;

3) UV: 203 nm;

4) injection amount: 10 μl;

5) Oven temperature: 35 ° C .; And

6) Flow rate: 1.00 ml / min.

As a result, it was confirmed that the content of ginsenosides increased by the treatment according to the method of the present invention as shown in FIG. However, all ginsenoside compounds were decomposed in the red ginseng extract irradiated with 10 ~ 20 kGy.

Experimental Example 2 Measurement of Enhanced Anticancer Effect

PC-3 cells (Korea Cell Line Bank), which are human prostate cancer cells, were cultured in 1 well 10 ⁴ cells / well in a 24-well culture plate, and then the red ginseng extract irradiated with 0, 1 and 5 kGy at a concentration of 0 to 30 ㎍ / ml. After treatment, the cells were incubated in a 37% incubator at 5% CO ₂ in RPMI1640 medium to which 10% FBS was added. After 48 hours, the medium was discarded and filled with RPMI medium containing 100 μg / ml of MTT, followed by incubation for another 3 hours, the medium was removed, and 500 μl of isopropanol was dispensed in each well, and the absorbance was measured at 570 nm.

As a result, as shown in Figure 2 there was little cytotoxicity of the red ginseng concentrate itself (0 kGy), but the red ginseng concentrate irradiated with 1 and 5 kGy of the present invention confirmed a significantly increased anticancer effect.

Experimental Example 3 Apoptosis Confirmation

<3-1> Annexin V / PI Staining

After injecting PC-3 cells 5 -3 10 ⁶ cells into the culture plate, the red ginseng extract irradiated with 0, 1 and 5 kGy was treated at a concentration of 10 μg / ml, and then cultured for 48 hours. Cells were extracted by treatment with trypsin-EDTA, stained with fluorescent Annexin V-FITC / PI (Promega, USA) for 30 minutes and measured using a flow cytometer (Beckman FC500, CA).

As a result, it was confirmed that cytotoxicity was induced by apoptosis as shown in FIG.

<3-2> Nuclear Segment Measurement

Cells treated and cultured according to the method of Example 3-1 were washed twice with PBS, and then the cells were fixed by treating paraformaldehyde solution at room temperature for 30 minutes. After washing three times with PBS, the nuclei of the cells were stained with a fluorescent dye Hoechst33258 (2 μg / ml; Sigma, U.S.A.) for 30 minutes. Washed three times with PBS and observed with a fluorescence microscope (Olympus I X 70, Japan).

As a result, it was confirmed that cytotoxicity was induced by apoptosis as shown in FIG.

Experimental Example 4 Analysis of Reactive Oxygen Species (ROS)

PC-3 cells (2 × 10 ⁴ cells / well) were treated according to the method of Example 3-1, and then cultured for 48 hours. Subsequently, 5 μM of carboxy-H ₂ DCFDA (6-carboxy-2 ', 7'-dichloro-dihydrofluoresceine diacetate, dicarboxymethylester) was added and reacted at 37 ° C. for 30 minutes, and the cells were washed twice with PBS. The cells were removed with trypsin and resuspended in Opti-MEM I medium, and measured at a wavelength of 480 nm to 530 nm with a flow cytometer.

As a result, the production of ROS was observed as shown in FIG.

Preparation Example 1 Preparation of Pharmaceutical Formulation

1. Preparation of powder

2 g of red ginseng extract of the present invention

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

2. Preparation of tablets

Red ginseng extract 100 mg of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

Red ginseng extract 100 mg of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

4. Manufacture of rings

Red ginseng extract of the present invention 1 g

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

5. Manufacture of granules

Red ginseng extract 150 mg of the present invention

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, and then filled in fabric.

Preparation Example 2 Preparation of Food

Foods comprising the lane of the present invention were prepared as follows.

1. Preparation of flour food

0.5 to 5.0 parts by weight of the lane of the present invention was added to the flour, using the mixture to prepare a cake, cookies and crackers to prepare a food for health promotion.

2. Manufacturing of Dairy Products

5 to 10 parts by weight of the lane of the present invention was added to milk, and the milk was used to prepare various dairy products such as butter and ice cream.

Preparation Example 3 Preparation of Beverage

1. Manufacture of health drinks

       1000 mg of lane of the present invention

       Citric acid 1000 mg

       100 g oligosaccharides

       Plum concentrate 2 g

       1 g of taurine

       Add 900 ml of purified water

After mixing the above components according to the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored Used to prepare the healthy beverage composition of the invention. Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and intended use.

1 is a diagram showing the results of measuring ginsaneoid content in red ginseng extract:

a: before radiation treatment; And

b: after radiation treatment.

Figure 2 is a diagram showing the anticancer effect of red ginseng extract enhanced according to the method of the present invention.

Figure 3 is a diagram showing the results of confirming the apoptosis effect of the red ginseng extract treated in the method of the present invention by Annexin V / PI staining method.

4 is a diagram showing the results of confirming the apoptosis effect of the red ginseng extract treated in the method of the present invention by the nuclear segment measurement method.

5 is a view showing the results of confirming the ROS generated during the anticancer action.

Claims (15)

1) adding a radical generator to the red ginseng solution; And 2) A method for producing red ginseng solution having an anticancer effect enhanced by irradiating 1 to 5 kGy of radiation to the treatment solution of step 1). delete The method according to claim 1, wherein the red ginseng liquid is red ginseng grinding liquid or red ginseng extract. The method according to claim 3, wherein the red ginseng pulverized liquid is extracted with dried red ginseng powder. The method of claim 3, wherein the red ginseng extract is a dried red ginseng powder or red ginseng concentrate is extracted with any one selected from the group consisting of alcohol and aqueous alcohol solution. The method of claim 1 wherein the radical generator is selected from the group consisting of hydrogen peroxide, peracetic acid and ascorbic acid. The method according to claim 1, wherein the radical generator is added in an amount of 0.02 to 0.1 parts by weight based on the red ginseng solution. The method of claim 1 wherein the radiation is any one selected from the group consisting of gamma rays, electron beams, UV or X-rays. delete delete delete delete delete delete delete

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