KR101473903B1 - Method for improving anti-cancer effect of aloe emodin by irradiation and Composition for treatment or prevention of cancer comprising irradiated aloe emodin - Google Patents

Abstract

The present invention relates to a method for enhancing anti-cancer activity of aloe emodin and to a composition for preventing, treating, or alleviating cancer, including aloe emodin having anti-cancer activity enhanced by the same method. More specifically, the present invention relates to a method for enhancing the anti-cancer activity of aloe emodin by irradiation. According to the present invention, the anti-cancer activity of aloe emodin is enhanced by an irradiation technology without complicated processes. Therefore, aloe emodin can be advantageously used in various fields such as anti-cancer agents or health foods.

Description

[0001] The present invention relates to a method for enhancing the anticancer activity of aloe modine by irradiation with radiation and a composition for preventing or treating cancer comprising, as an active ingredient, radiation-irradiated aloe emmidine as an active ingredient. prevention of cancer with irradiated aloe emodin}

The present invention relates to a method for promoting the anticancer activity of aloe emmidine and a composition for preventing, treating or improving cancer, which comprises aloe emodine as an active ingredient having enhanced anticancer activity by the above method.

Aloe emodin (1.8-dihydroxy-3-hydroxymethyl-9,10-anthracenedione) is an anthraquinone-based polyphenol compound mainly found in gels, sap, or leaves of aloe vera. (Food Chemistry 2000, 70, 437441. U.S. Patent No. 4670265A; CANCER RESEARCH 60, 28002804, June 1, 2006), which has been reported on various antioxidative, antiviral, 2000); as well as 1,8-dihydroxy-3- (hydroxymethyl) -anthraquinone, specifically inhibits the growth of human neuroectodermal tumor cells and exhibits antitumor effect in animal models. (Pecere, T., et al., Aloe-emodin is a new type of anticancer agent with selective activity against neuroectodermal tumors. Cancer Res., 11: 2000; Pharmacology & Toxicology 2000, 87, 229233). In particular, many studies have been conducted on the anticancer activity of aloe immunone (Leukemia Research February 2002, Volume 24, Issue 1, pp 17-22; American Journal of Dermatopathology, Volume 27, Issue 9, Pages 831-840, September 2003 ; Oral Oncology Volume 43, Issue 9, October 2007, Pages 905910). Since aloe emodine having various effects is a natural substance, if the anticancer activity of aloe immunine is further increased, it is expected that it can be used as an anticancer drug having a small side effect and excellent drug effect.

On the other hand, the radiation irradiation technique is to irradiate the radiation emitted from the radiation source such as gamma ray (Co-60 or Cs-137), electron beam, X-ray or the radiation generating device, that is, ionized radiation energy, with 0.01 kGy to 200 kGy irradiation dose It is used to control the germination of food, delays in maturity, parasitism and pest control, and to sterilize corruption and pathogenic microorganisms. In addition, recently, radiation irradiation can reduce toxicity by changing the structure of carcinogenic substances such as nitrosamine and biogenic amine which may occur during food processing and storage, besides the purpose of food sterilization . In addition, several studies have been conducted to reduce allergenic substances in foods using radiation (Seo JH, et al., J Food Prot 67: 1463-8, 2004; Lee JW, et al., Radiat Physics Chem 72 : 645-650, 2005). In addition, studies have been reported to increase the activity of a specific substance by irradiation, or to increase the structural change or content of a specific substance (Korean Patent No. 2009-0043706, Korean Patent No. 2008-0046311 Etc.).

Accordingly, the present inventors have made intensive efforts to enhance the anticancer activity of aloe emmidine, and as a result, they have confirmed that the anti-cancer effect of aloe emodine irradiated with radiation is much higher than that of aloe emodine not irradiated with radiation, thereby completing the present invention .

Korean Published Patent Application No. 1995-001676 (published on July 20, 1995) Korean Patent Publication No. 2012-0044583 (Publication date: May 08, 2012)

CANCER RESEARCH 60, 28002804, June 1, 2000

One object of the present invention is to provide a method for promoting the anticancer activity of aloe immunone.

Another object of the present invention is to provide aloe immunogens with enhanced anticancer activity.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer and a food composition for preventing or ameliorating cancer, which comprises aloe emodine as an active ingredient having enhanced anticancer activity.

In order to solve the above-mentioned object, the present invention provides a method for enhancing the anticancer activity of aloe immunone using radiation.

In addition, the present invention provides aloe emodine in which the anticancer activity is enhanced by the method of enhancing the anticancer activity of the aloe emmine.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer and a food composition for preventing or improving cancer, which comprises aloe emodine having an increased anticancer activity by the method for promoting the anticancer activity of the above-mentioned aloe emmidine as an active ingredient.

Hereinafter, the present invention will be described in detail.

An aspect of the present invention relates to a method of promoting the anticancer activity of aloe immunone comprising irradiating aloe emmidine with radiation.

In the method for promoting the anticancer activity of aloe emmine, the radiation may be gamma ray, electron beam, UV, or X ray. Preferably, the radiation may be gamma ray, but is not limited thereto.

The gamma ray may be emitted from any one radioisotope selected from the group consisting of cobalt (Co) -60, krypton (Kr) -85, strontium (Sr) -90, and cesium The gamma ray may be cobalt (Co) -60, but is not limited thereto.

In order to enhance the anticancer activity of the aloe emmine of the present invention, it is preferable that the radiation is irradiated at a dose of 10 kGy or more. In one embodiment of the present invention, when the radiation was irradiated at a dose of less than 10 kGy, the effect of increasing the anticancer activity of aloe modine was insufficient. More preferably, the dose of radiation may be 10 kGy or more and 150 kGy or less, but is not limited thereto. When the dose of radiation exceeded 150 kGy, there was almost no synergistic effect of anticancer activity due to dose increase. That is, the preferable range of the dose of radiation is set based on the ability of aloe emmidine to exhibit the enhanced anticancer effect. In one embodiment of the present invention, when comparing the toxicity of aloe-emmound irradiated with radiation and aloe-emodine-free irradiated non-irradiated cancer cells (AGS), the greater the dose of radiation irradiated to aloeemodine Showed high toxicity to gastric cancer cell line. In particular, when each aloe emmidine was treated at a concentration of 25 μM or more, the toxicity of aloe emmine irradiated with a dose of 10 kGy, which is the minimum dose compared to the unirradiated aloe emodine, was increased by about 20% (Fig. 2). In addition, when treated with aloe-emodine irradiated with normal cells and not irradiated with aloe-emodine, the survival of normal cells was almost not different, and the irradiation of the normal cell with toxicity of aloe etmodine (Fig. 1 (a)).

Another aspect of the present invention relates to aloe emmine prepared by the method. The method for enhancing the anticancer activity of the present invention can be usefully used for aloe etmodine for enhancing anticancer activity. In a specific example of the present invention, aloe emodine in the form of a commercially available powder was dissolved in an organic solvent, and then irradiated with radiation according to the above-mentioned method to produce aloe emmidine having enhanced anticancer activity. In one embodiment of the present invention, the toxicity of aloe emmidine to cancer cells prepared according to the above method was significantly different from that of unirradiated aloe emmine, and was increased depending on the irradiation doses (Figs. 1 and 2 ). In relation to the cancer cell toxicity, it was confirmed that the irradiated aloe emmidine further induces apoptosis of cancer cells, and it was confirmed that the toxicity to cancer cells is due to apoptosis (FIG. 3). In addition, when the change of the secondary metabolite of aloe modine prepared according to the method of enhancing the anticancer activity of aloe modine of the present invention was analyzed, it was confirmed that the secondary metabolites, (Fig. 4). This suggests that irradiation affects the anticancer activity of aloe immunone by changing the structure and binding pattern of aloe emmine. As a result, it is expected that the aloe emmine of the present invention has an enhanced anticancer effect according to such effects.

Another aspect of the present invention relates to a pharmaceutical composition for preventing and treating cancer, which comprises aloe furamine as an active ingredient, the anticancer activity being enhanced by the above method. In an embodiment of the present invention, the toxicity of each aloe emmidine to cancer cells was examined, and the toxicity of cancer cells to cancer cells was clearly increased compared with the non-irradiated aloe emmine, (See Figs. 1 to 3). Therefore, the aloe emodine irradiated with the radiation of the present invention can be usefully used as a pharmaceutical composition for preventing or treating cancer.

The pharmaceutical composition of the present invention can be used for the treatment of 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 cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, Can be used for the prevention or treatment of various cancer diseases such as vaginal cancer, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, blood cancer, ureter cancer, kidney cell carcinoma, renal pelvic carcinoma and central nervous system tumor.

The pharmaceutical composition of the present invention may contain 0.0001 to 50 parts by weight, preferably 1 to 50 parts by weight of aloe emodine, which is irradiated with the above-mentioned radiation to enhance the anti-cancer effect, based on the total weight of the composition.

The pharmaceutical composition of the present invention may be formulated into a unit dosage form suitable for administration to a patient in vivo according to conventional methods in the pharmaceutical field. Formulations suitable for this purpose include injectable preparations such as injectable solutions or suspensions for parenteral administration, or ready-to-use injectable dry powders which can be used as distilled water for injections at the time of injection; Or a preparation for topical administration such as an ointment. In addition, the pharmaceutical compositions of the present invention may be formulated into tablets, aqueous or oily suspensions, powders or granules, emulsions, hard or soft capsules, syrups or elixirs suitable for oral administration. The preparation of such compositions can be prepared by any method known to those skilled in the art.

The pharmaceutical composition according to the present invention may further contain commonly used carriers, excipients, disintegrants, sweeteners, lubricants, flavors and diluents. 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, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Examples of the disintegrant include sodium starch glycolate, crospovidone, croscarmellose sodium, alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium, chitosan, guar gum, low substituted hydroxypropylcellulose, magnesium aluminum silicate, 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, phosphoric acid Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, stearic acid, stearic acid, sodium carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, hydrogenated calcium, lactose, mannitol, sugar, arabic gum, pregelatinized starch, cornstarch, powdered cellulose, Aluminum, calcium stearate, white sugar, dextrose, sorbitol, talc, and the like. The pharmaceutically acceptable additives according to the present invention may be contained in an amount of 0.1 to 90 parts by weight based on the pharmaceutical composition.

The pharmaceutical composition of the present invention further comprises administering to the patient in need of treatment of cancer at least one selected from the group consisting of antibiotics, alkylating agents, antimetabolites, hormones, immunological agents, interferon agents and other anticancer agents can do. However, the pharmaceutical use and therapeutic pharmaceutical composition of the present invention are not limited to those described above, and other medicaments that can be used in combination are not limited to those described above.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. The administration may be carried out once a day or divided into several times. However, for the desired effect, the pharmaceutical composition of the present invention is preferably administered at a daily dose of 0.0001 to 200 mg / kg, preferably 0.001 to 200 mg / kg. However, the actual dosage of the active ingredient may be determined taking into account various relevant factors such as the amount of cancer cells, the route of administration, the body weight of the patient, age and sex, and so the dose is not limited in any way to the scope of the present invention It is not.

Also, the subject to which the pharmaceutical composition of the present invention can be applied is a vertebrate animal, preferably a mammal, more preferably an experimental animal such as a mouse, a rabbit, a guinea pig, a hamster, a dog or a cat, , And ape-like animals such as gorillas. The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections. In addition, the pharmaceutical composition may be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response modifiers for the prevention and treatment of cancer.

Another aspect of the present invention relates to a food composition for preventing or ameliorating cancer, which comprises aloe emmidine prepared by the above method as an active ingredient. The aloe emodine irradiated with the radiation of the present invention has further increased anticancer activity, and can be used as a health food to help prevent or improve cancer.

The food composition of the present invention is useful as a food composition for the prevention and treatment of liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectum cancer, esophageal cancer, small intestine cancer, Can help prevent or improve a variety of cancer diseases such as vaginal cancer, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, blood cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma and central nervous system tumor have.

There is no particular limitation on the kind of the food. Examples of foods in which the anti-cancer activity of the present invention can be added as an active ingredient include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gum, Dairy products, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and includes foods in the conventional sense.

In the food composition of the present invention, the aloe momentine having enhanced anticancer activity may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on the total food composition. 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 beverage composition as the food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient, such as ordinary beverages. 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. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention. In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. Although the ratio of such additives is not critical, it is generally selected from the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention, and can be used independently or in combination.

Aloe immunogens irradiated with the present invention have almost no toxicity to general cells as compared to aloe emodine with no irradiation, whereas cancer cells have increased toxicity due to apoptosis, And exhibits a high anticancer effect even in a small amount compared to emmodine. Thus, aloe emodine, which has enhanced anticancer activity according to the method of the present invention and the method of the present invention, can be usefully used for anticancer drugs or health foods.

FIG. 1 is a graph showing the toxicity of irradiated aloe emodine and non-irradiated aloe emodin of the present invention to tumors and non-tumor cells using MTT assay. FIG. (a) is for the tumor cell MSV BALB 3T3, and (b) is for the non-tumor cell BASB 3T3.
FIG. 2 is a graph comparing the toxicity of the irradiated aloe emmidine of the present invention and the non-irradiated aloe emodine to the gastric cancer cell (AGS) strain. FIG.
FIG. 3 shows the results of comparing the ability of aloe-emodine irradiated with radiation and non-irradiated aloe emodin to induce apoptosis using FACS.
4 shows the results of analysis of the change of the secondary metabolite of aloe emmidine after irradiation with the use of HPLC.

Hereinafter, the present invention will be described in more detail by way of examples. The present invention may be embodied in many different forms and is not limited by the embodiments described herein. Also, terms, techniques, and the like used in the present specification are used in the meaning commonly used in the technical field to which the present invention belongs, unless otherwise specified.

<Example 1> Irradiation of Aloe Emmidine

Aloe-amodine was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg / mL, purchased from Sigma, and irradiated with 100,000 Ci of cobalt-60 gamma irradiation 30, 50, 70, 100, and 150 kGy were irradiated at room temperature using a facility (Point source AECL, MDS Nordion International Co. Ltd., Ottawa, On, Canada).

EXPERIMENTAL EXAMPLE 1 Evaluation of Cancer Cell Toxicity of Irradiated and Unirradiated Aloe Emmidine

To evaluate the anticancer activity of irradiated and unirradiated aloe immunogens, MTT assay was performed to evaluate cancer cell toxicity in tumor (MSV BALB 3T3) and non-tumor cells (BALB 3T3) and gastric cancer cells (AGS) Respectively. To assess cytotoxicity, each cell was seeded at 2 × 10 ⁴ cells / well in a 96-well plate, cultured in a CO ₂ incubator for 4 h before cells were completely attached, and irradiated (10, 30 , 50, 70, 100, 150 kGy) and unirradiated (0 kGy) aloe emmine by concentration (6.25, 12.5, 25, 50, 100, 200 uM) and incubated for 24 hours in a CO ₂ incubator . After 24 hours, 10 μL of MTT solution dissolved in PBS at a concentration of 5 mg / mL was added to each well and incubated for 1 hour. Then, the culture supernatant was removed and 100 μL of DMSO solution And the absorbance was observed at 517 nm. The cell survival rate was expressed as 100% of the group without any treatment compared with the group treated with irradiated and unirradiated aloe emmidine. Cell viability was calculated by the following equation.

Cell survival rate = (sample treatment / sample non-treatment) X 100

Experimental Results As can be seen from FIGS. 1 and 2, in the case of irradiated aloe emodine, cancer cell toxicity was observed to be increased depending on radiation dose, and cancer cell toxicity was found to be increased less in non-tumor cells . In particular, in the case of gastric cancer cell line, the increase of cancer cell toxicity of aloe emodine irradiated with 25 uM concentration was remarkable. Specifically, at 10 kGy dose, The toxicity to gastric cancer cells increased by more than 20%. At 150 kGy dose, the survival rate of gastric cancer cells decreased to less than 20% at 50 uM concentration.

<Experimental Example 2> Confirmation of induction of apoptosis of irradiated and non-irradiated aloe emmidine

As a result of observing cytotoxicity of irradiated and unirradiated aloe emmidine, cytotoxicity was observed to be dependent on irradiation doses. Therefore, in relation to the increase of cancer cell toxicity, we examined whether aloe emmodin - induced cancer cell toxicity induces necrosis or apoptosis of cells. AGS stomach cancer cells were plated at 5 × 10 ⁵ cells / well in a 6-well plate. Cells were cultured in a CO2 incubator for 4 hours until the cells were completely adhered. After 4 hours, aloe steroids with 150 kGy Uncoated (0 kGy) aloe emmidine was treated with concentrations (12.5, 25, 50, 100 uM) and incubated in a CO2 incubator for 12 hours. After 12 hours, annexin 5 FITC and PI were stained and stained cells were counted by flow cytometry.

As a result of the experiment, it was found that the treatment with the irradiated emmodin significantly increased the annexin 5 FITC stained at the time of apoptosis compared with the irradiated emodin (FIG. 3).

<Experimental Example 3> Analysis of the change of secondary metabolites by using HPLC after irradiation

The changes of secondary metabolites produced after irradiation were analyzed using Agilient HPLC (agilent Technological, USA). The column was Capcell PAK C18 UG 120 (4.6 mm x 250, 5 μm), the temperature of which was adjusted to 40 ° C. HPLC was carried out using 100% of water (A) and 100% of acetonitrile solution (B) as mobile phases. HPLC conditions are shown in Table 1 and maintained at a flow rate of 0.5 mL / min for 50 minutes and analyzed at a wavelength of 254 nm.

Mobile phase A: Water B: Acetonitrile Extraction mode Travel time % Mobile phase B 10 80 20 85 25 90 30 100 40 50 50 50

As a result of the experiment, it was observed that the peak was observed at 17 minutes in the case of the radiation-unirradiated aloe emodine, and that the secondary metabolites produced by the irradiation at 18 to 20 minutes increased as the irradiation dose increased (Fig. 4).

An example of a preparation for the composition of the present invention is shown below.

< Formulation example  1> Preparation of pharmaceutical composition

1. Preparation of tablets

20 mg &lt; RTI ID = 0.0 &gt;

Lactose 100 mg

Starch 100 mg

Magnesium stearate qs

The above-mentioned mixed ingredients are mixed and pressed according to a conventional tablet preparation method to prepare tablets.

2. Preparation of capsules

10 mg &lt; RTI ID = 0.0 &gt;

3 mg of crystalline cellulose

Lactose 14.8 mg

0.2 mg of magnesium stearate

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

3. Preparation of Injection

10 mg &lt; RTI ID = 0.0 &gt;

180 mg mannitol

2974 mg of sterile distilled water for injection

26 mg of Na2HPO4.12H2O

(2 ml) per ampoule in accordance with the usual injection method.

< Formulation example  2> Formulation of food composition

1. Manufacture of beverages

500 mg &lt; RTI ID = 0.0 &gt;

Liquid fructose 2 g

100 g of oligosaccharide

Sugar 2 g

Salt 1 g

900 ml of water

The above components are mixed according to a conventional health drink manufacturing method, and the mixture is stirred and heated at 87 DEG C for about 1 hour. Then, the solution thus prepared is filtered and sterilized in a sterilized 2 mL container.

Claims (10)

A method for promoting the anticancer activity of aloe emmidine in an animal other than human, comprising the step of irradiating a gamma ray emitted from cobalt (Co) -60 radioisotope at 6.25-200 uM of aloe emmine with a dose of 10-150 kGy. delete delete delete delete delete A pharmaceutical composition for preventing or treating gastric cancer, which comprises aloe emodine as an active ingredient, the anticancer activity being enhanced by the method of claim 1. delete A food composition for preventing or ameliorating gastric cancer, comprising aloe emodine as an active ingredient having enhanced anticancer activity by the method of claim 1. delete

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