KR20100041020A - A method for preparing the derivative of rhein which have a boosting anticancer activity from sennoside a using irradiation - Google Patents

Abstract

PURPOSE: A method for preparing a rhein derivative from sennoside A is provided to enhance anticancer effect by apoptosis and use as an anticancer drug. CONSTITUTION: A method for preparing a rhein derivative from sennoside having enhanced anticancer effect comprises: a step of adding radical generating agent to a solution containing sennoside A; and a step of irradiating radiation to the solution. The solution containing sennoside A is a solution in which sennoside A is dissolved or plant-pulverized liquid and extract containing the sennoside A as a main ingredient. The radical generating agent is hydrogen peroxide, acetyl hydroperoxide or ascorbic acid. The radiation is gamma ray, electronic beam, UV or X ray. A pharmaceutical composition for preventing and treating cancer contains the rhein derivative as an active ingredient.

Description

Method for preparing the derivative of rhein which have a boosting anticancer activity from sennoside A using irradiation}

The present invention relates to a method for preparing an anti-cancer effect-enhanced derivative from sennoside A 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 medical device sterilization and food research purposes.

Studies on the components of Rheum palmatum L. have been conducted until recently, and the effects of sennoside A on lowering action, prevention of gastrointestinal mucosal damage, and aloe-emodin , Antibacterial effects of emodin and rhein have been reported (中華 本草 編纂 委, 上海 科學 技術 出版社, Vol. 2, pp.710, 715-716, 1999). Among them, sennoside A is an anthraquinone-based compound that is metabolized by enterobacteriaceae as a main component of rhubarb and senna leaves and has a lowering action and anticancer effect. Sennoside A is produced by cleaving two glucoses from sennoside A glycosides by β-glucosidase such as Streptococcus , followed by NAD (P) H-dependent flavin enzymes (reducing agents) such as Peptostreptococcus. Reduced flavin is non-enzymatically separated from the CC bonds to produce rheinanthrone. Lane anthrone is easy to oxidize, and becomes lane in aerobic conditions. Thus produced compound inhibits the ingestion of glucose in cancer cells to inhibit cancer cell proliferation and to change the function of the cell membrane to induce anti-cancer effect. 1 shows a process of generating a lane from sennoside A using an enzyme and an intestinal microorganism. This method is disadvantageous in that the yield is not high, the method is complicated, and expensive.

Accordingly, the present inventors added hydrogen peroxide to sennoside A dissolved in methanol, and irradiated with radiation to induce the conversion to the lane-8-β-D-glucoside structure, thereby generating a lane derivative in a high yield. The present invention was completed by confirming that the anticancer effect is higher than that of Lane or Aloe-Emodine.

It is an object of the present invention to provide a method for preparing a solution containing a lane derivative having enhanced anticancer effect by irradiating sennoside A with radiation.

Another object of the present invention is to provide a rain derivative-containing solution having improved anticancer effect prepared by the above method.

It is another object of the present invention to provide a pharmaceutical composition for preventing and treating cancer, which contains a lane derivative-containing solution having an enhanced anticancer effect as an active ingredient.

Another object of the present invention to provide a health functional food for cancer prevention and improvement containing the rain derivative-containing solution with enhanced anticancer effect as an active ingredient.

In order to achieve the above object, the present invention

1) adding a radical generator to a solution comprising sennoside A; And,

2) It provides a method for producing a rain derivative-containing solution enhanced anticancer effect comprising the step of irradiating the treatment solution of step 1).

In addition, the present invention provides a rain derivative-containing solution prepared by the above method, the anticancer effect enhanced.

In addition, the present invention provides a pharmaceutical composition for preventing and treating cancer, which comprises a rain derivative-containing solution having an anticancer effect enhanced by the above method as an active ingredient.

In addition, the present invention provides a health functional food for cancer prevention and improvement, which is prepared by the above method, containing an anti-cancer effect-enhanced rain derivative-containing solution as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention

1) adding a radical generator to a solution comprising sennoside A; And,

2) It provides a method for producing a rain derivative-containing solution enhanced anticancer effect comprising the step of irradiating the treatment solution of step 1).

In a specific embodiment of the present invention, as a result of irradiation with hydrogen peroxide at a concentration of 0.05% to the sennoside A solution dissolved in 50% methanol, the content of the lane derivative was increased (see FIG. 3). In addition, the sennoside A itself has no anti-cancer effect, but in the lane derivative produced by converting the sennoside A by the present invention showed a clear anti-cancer effect (see Figure 4), the anti-cancer effect than the conventional lane derivative standard It was confirmed that increased (see FIG. 5). Therefore, the method of the present invention can be usefully used for the preparation of a lane derivative having enhanced anticancer effect from sennoside A.

In the above method, the solution comprising sennoside A in step 1) is a pulverized plant, for example rhubarb or senna leaf, which is known to have dissolved the sennoside A single substance in a solvent or contains sennoside A as a main component. Liquids or extracts. The pulverized liquid indicates that the powder of dried rhubarb or senna leaves is extracted with water, and the extract liquid means that the powder of dried rhubarb or senna leaves is extracted using a solvent selected from the group consisting of alcohol and aqueous alcohol solution. 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.

Radical generators of step 1) include hydrogen peroxide, peracetic acid or ascorbic acid and the like. It is preferably added in an amount of 0.02 to 0.1 parts by weight based on the Sennoside A solution, and 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 1-10 kGy, more preferably 1-5 kGy, and most preferably 1 kGy. The range of the radiation dose was set on the basis that the lane derivatives generated from Senosides A were less than decomposed and disappeared, and the ginsenosides could be changed to show an enhanced anticancer effect.

In addition, the present invention provides a rain derivative-containing solution prepared by the above method, the anticancer effect enhanced.

In a specific embodiment of the present invention according to the method of the present invention it was possible to produce a lane derivative generated from sennoside A with high efficiency (see Figure 3). In addition, the sennoside A itself has no anticancer effect, but in the lane derivative produced by converting the sennoside A by the present invention showed a clear anticancer effect (see Figure 4), the anticancer effect than the conventional lane derivative standard It was confirmed that the increase (see Fig. 5). Accordingly, it was confirmed that the lane derivative having enhanced anticancer effect from the sennoside A of the present invention can be produced with high efficiency, and the anticancer effect of the lane derivative is superior to the existing lane standard.

In addition, the present invention provides a pharmaceutical composition for preventing and treating cancer, which comprises a rain derivative-containing solution having an anticancer effect enhanced by the above method as an active ingredient.

In a specific embodiment of the present invention according to the method of the present invention it was possible to produce a lane derivative generated from sennoside A with high efficiency (see Figure 3). In addition, the sennoside A itself has no anticancer effect, but in the lane derivative produced by converting the sennoside A by the present invention showed a clear anticancer effect (see Figure 4), the anticancer effect than the conventional lane derivative standard It was confirmed that the increase (see Fig. 5). Accordingly, the solution containing the lane derivative having enhanced anticancer effect generated from sennoside A of the present invention may 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 therefore, liver cancer, gastric cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, anal muscle 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 further contain one or more active ingredients exhibiting the same or similar functions in addition to the lane derivative-containing solution of the present invention.

The pharmaceutical composition of the present invention comprises 0.0001 to 10 parts by weight, preferably 0.001 to 1 part by weight, of the rain derivative-containing solution having 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. Administration may be administered once a day or may be divided several times.

In addition, the present invention provides a health functional food for cancer prevention and improvement, which is prepared by the above method, containing an anti-cancer effect-enhanced rain derivative-containing solution as an active ingredient.

The rain derivative-containing solution of the present invention may be added to a food as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement). Generally, in the preparation of food or beverages, the composition of the present invention is added in an amount of 0.2 to 20 parts by weight, preferably 0.24 to 10 parts by weight, based on the raw materials. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

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, 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 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, it is possible to produce a lane derivative in a high yield using a radiation technique without a complicated treatment process, and the anticancer effect caused by apoptosis is enhanced, it can be usefully used as an anticancer agent.

Hereinafter, the present invention will be described in detail by 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> Sennoside  A treatment

Dissolve 1 mg / ml of commercially available Senoside A (Sigma Aldrich, USA) in 50% methanol at a concentration of 1000 ppm, add hydrogen peroxide at a concentration of 0.05%, and place it in a container. Korea Atomic Energy Research Institute 1-5 at room temperature using a 100,000 source cobalt-60 gamma irradiation facility (Point source AECL, IR-79, MDS Nordion International Co. Ltd., Ottawa, ON, Canada) KGy was investigated and the error of total absorbed dose was ± 0.2 Gy.

< Experimental Example  1> Sennoside  Determination of Rain Content in Solution A

The sennoside A solution 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 of analyzing the chromatograph of the sennoside A and the lane standard of FIG. 2, it was confirmed that the content of the lane derivative was increased by the treatment according to the method of the present invention as shown in FIG.

< Experimental Example  2> Measured enhanced anticancer effect

PC-3 cells (Korea Cell Line Bank) or HepG2 cells (Korea Cell Line Bank), which are human prostate cancer cells, were cultured at 1 × 10 ⁴ cells / well in a 24-well plate, followed by Lane standard (Sigma, USA), Senoside A, the lane derivatives prepared in Example 1, and sennoside A exposed to the radiation according to the method of Example 1 without the addition of the hydrogen peroxide and the concentration of 0 to 24 μM and then 10% FBS was added Incubated in a 37% incubator, 5% CO ₂ in RPMI1640 medium. 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 4, the sennoside A and the material irradiated only to the sennoside A had little cytotoxicity, but in the rain derivative prepared from sennoside A by the method of the present invention has a pronounced anticancer effect I could confirm it.

In addition, as shown in Figure 5 lane derivatives prepared by the method of the present invention showed a higher cytotoxicity compared to the lane standard.

Preparation Example 1 Preparation of Pharmaceutical Formulation

1. Preparation of powder

2 g of a lane derivative-containing solution of the present invention

1 g lactose

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

2. Preparation of Tablets

100 mg of a lane derivative-containing solution of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

3. Preparation of Capsule

100 mg of a lane derivative-containing solution of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg 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

1 g of a lane derivative-containing solution of the present invention

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

150 mg of a lane derivative-containing solution of the present invention

Soybean Extract 50mg

Glucose 200 mg

Starch 600 mg

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 containing the lane derivative-containing solution of the present invention were prepared as follows.

1. Preparation of flour food

0.5 to 5.0 parts by weight of the lane derivative-containing solution of the present invention was added to flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.

2. Manufacturing of Dairy Products

5-10 parts by weight of the lane derivative-containing solution of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

Preparation Example 3 Preparation of Beverage

1000 mg of a lane derivative-containing solution 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 sterilized and stored in the refrigerator after 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 a process of conversion from sennoside A to a lane derivative.

FIG. 2 is a chromatograph of sennoside A and lane standards (Junko Koyama et al ., Journal of Chromatography A 1145: 183-189, 2007):

a: chromatography graph; And,

b: Name of standard by peak.

3 is a diagram showing the results of conversion from sennoside A to lane derivatives by radiation treatment:

a: before radiation treatment; And,

b: after radiation treatment.

Figure 4 is a diagram showing the anticancer effect of the sennoside A standard and the lane derivative prepared by the method of the present invention in liver cancer cells and prostate cancer cells (IRSH: sennoside A + 0.05% hydrogen peroxide + 1 kGy, IRS: sennoside A + 1 kGy):

a: liver cancer cell; And,

b: prostate cancer cells.

5 is a diagram comparing the anticancer effect of the lane derivative of the present invention and the existing lane standard.

Claims (15)

1) adding a radical generator to a solution comprising sennoside A; And, 2) A method for preparing a rain derivative-containing solution having enhanced anticancer effect, comprising irradiating the treatment solution of step 1) with radiation. The solution of claim 1, wherein the solution containing sennoside A is selected from the group consisting of dissolving a sennoside A single substance in a solvent and a pulverized solution and an extract of a plant known to contain sennoside A as a main component. How to. The method of claim 2, wherein the pulverized liquid, characterized in that the dried powder of rhubarb or senna leaves extracted with water. The method of claim 2, wherein the extract is characterized in that the dried powder of rhubarb or senna leaves extracted with an alcohol or an 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 of claim 1, wherein the radical generator is added in an amount of 0.02 to 0.1 parts by weight based on the solution comprising sennoside A. 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. The method of claim 1, wherein the radiation is irradiated at a dose of 1-10 kGy. The method of claim 1, wherein the radiation is irradiated at a dose of 1-5 kGy. The method of claim 1 wherein the radiation is irradiated at a dose of 1 kGy. A rain derivative-containing solution prepared by the method of claim 1, wherein the anticancer effect is enhanced. The method of claim 11, wherein the cancer is 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, anal muscle cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, Pharmaceutical composition, characterized in that selected from the group consisting of 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. A pharmaceutical composition for preventing and treating cancer, which is prepared by the method of claim 1, comprising a rain derivative-containing solution having an enhanced anticancer effect as an active ingredient. The method of claim 13, wherein the cancer is 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, anal muscle cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, Pharmaceutical composition, characterized in that selected from the group consisting of 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. A health functional food for preventing and improving cancer prepared by the method of claim 1, comprising a rain derivative-containing solution having an enhanced anticancer effect as an active ingredient.

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