KR101418164B1 - A pharmaceutical composition comprising extract of UV-induced rice for preventing or treating a colon cancer - Google Patents

Abstract

The present invention relates to an ultraviolet-treated rice ( Oryza sativa L. spp japonica cv. The present invention relates to a pharmaceutical composition for the prevention or treatment of colorectal cancer, which contains an extract of Dongjin as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition for prevention or treatment of colorectal cancer, which comprises an extract of rice or a fraction thereof treated with ultraviolet light, or N-benzoyltryptamine ( N- Benzoyltryptamine) or Sakuranetin as an active ingredient, and a health functional food for preventing or ameliorating colon cancer. The rice extract of the present invention or its fractions can inhibit the growth of colon cancer cells by N-benzoyltryptamine or chacarnettin produced by treating ultraviolet rays of the rice with no side effects derived from natural products, And can be widely used for the development of a preventive or therapeutic agent.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating colorectal cancer, which comprises an extract of rice as an active ingredient,

The present invention relates to a pharmaceutical composition for prevention or treatment of colorectal cancer, which comprises an extract of rice ( Oryza sativa L. spp japonica cv. Dongjin ) treated with ultraviolet rays as an active ingredient. More specifically, A pharmaceutical composition for prevention or treatment of colorectal cancer, comprising an extract of rice or a fraction thereof, or N- benzoyltryptamine or sakuranetin isolated therefrom as an active ingredient, and a composition for preventing or improving colon cancer Health functional foods.

As a method for treating colorectal cancer, an operation method is widely used. However, among colorectal cancer patients with locally advanced disease or metastatic disease, prognosis is highly disadvantageous with high incidence and mortality.

On the other hand, as a method of treating colorectal cancer, drug therapy which shows less favorable results than surgery is being tried. 5-FU (5-fluorouracil), an antineoplastic compound, is a major selective agent in the treatment of colorectal cancer and its use has only proven to be marginally effective. Although 5-FU can temporarily reduce colon cancer size, there is little evidence that the patient's survival has been substantially extended or "healed" (based on a 5 year reliever). Although chemotherapy with 5-FU has been used in patients with metastatic disease, only temporary improvement is observed in only 25% or less of such cases, and overall survival is not significantly affected (Harrison ' s Principles of Internal Medicine (10th Edition), McGraw-Hill, New York, p. 1764, 1983). Despite the marginal effects of 5-FU, there were no other drugs or combination therapies that appeared to be more effective (New Eng. J. Med., 312: 1465, 1985).

Considering the very poor 5-year survival rate (about 50% or less) in patients undergoing conventional therapy for colorectal cancer, for example, surgical resection or chemotherapy using 5-FU, Thereafter, it would be very useful to find new ways to effectively treat human malignant colorectal cancer using drugs or compounds, such as assisting in diagnosing or removing the volume of cancer. Since surgery can not remove all of the cancerous tissue, drug therapy will also be very useful for patients with advanced disease, which have spread or spread to various organs.

Currently, the development of anticancer drugs centering on natural products is based on the development of modern cell biology research, which has been used for a long time in folk remedies and the like, as a cancer gene, a cancer suppressor gene, a signal transduction factor, a drug resistance susceptibility factor And screening for cancer target such as factors related to apoptosis, thereby separating and purifying the active substance and determining the structure of the active substance. In particular, rice, a natural material, is an important grain used in stocks in Asia, Africa and Latin America, and is produced in Korea at about 5.3 million tons per year.

In addition, components such as phytic acid, Oryzanol, and GABA, which are the main functional ingredients of rice, have various physiological activities in addition to functions of inhibiting cancer, lowering cholesterol in blood and inhibiting thrombus formation, suppressing fatty liver and enhancing immune function .

Under these circumstances, the present inventors have made intensive efforts to develop a therapeutic agent for colorectal cancer with few toxicity and side effects, and as a result, it has been found that a composition comprising an extract of rice and a fraction thereof as an active ingredient is obtained by treating N-benzoyl The present invention has been accomplished by confirming that the growth of colon cancer cells can be inhibited by tryptamine ( N- benzoyltryptamine) or sakuranetin and can be used for prevention and treatment of colon cancer.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating colorectal cancer, which comprises an extract of rice or a fraction thereof treated with ultraviolet light as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating colorectal cancer, which comprises N- benzoyltryptamine or sakuranetin or a pharmaceutically acceptable salt thereof as an active ingredient .

It is still another object of the present invention to provide a health functional food for preventing or ameliorating colorectal cancer comprising an extract of rice treated with ultraviolet light or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a health functional food for prevention or improvement of colorectal cancer, which comprises N- benzoyltryptamine or sakuranetin or a pharmaceutically acceptable salt thereof as an active ingredient .

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating colorectal cancer, which comprises an extract of rice or a fraction thereof treated with ultraviolet light as an active ingredient.

The term " rice ( Oryza sativa L. spp japonica cv. Dongjin ) "of the present invention is a perennial herbaceous plant belonging to the family Gramineae and widely distributed throughout Asia. It is about 1 m in height and grows by forming branches in the branches near the roots. When flowers bloom, they stand upright, but when they are ripe, they fall downward. Many minorities run. Flowers bloom around July to August, and they are eliminated by their own moisture.

Studies on rice have been carried out mainly on the improvement of productivity due to breeding development and the increase of resistance to pest diseases in terms of agricultural cultivation. Several steroids, terpenoids and alkaloids have been isolated from rice. Among them, ergosterol peroxide and 7-oxo-stigmasterol have been reported, Has been reported to have a phytotoxic effect.

It is well known that the attack of pathogens in plants induces the production of antibiotic compounds called phytoalexins. In addition to its defense role, phytoalexin has been reported to exhibit health promoting properties. Rice produces diterpenoids and flavonoids phytoalexins in response to pathogenic attacks such as blast infection. In addition, accumulation of phytoalexin in rice is induced by ultraviolet irradiation and effluent treatment. However, almost no studies have been made on the components of the extracts obtained from ultraviolet ray-treated rice leaves, and it has been confirmed by the present inventors that the extracts obtained from ultraviolet ray-treated rice leaves are effective for preventing or treating colon cancer. .

The term "ultraviolet (UV)" in the present invention is a light ray in a wavelength region shorter than the purple color of a visible ray. There are three types of UV: UVA (200 ~ 280nm), UVB (280 ~ 320nm) and UVC (320 ~ 400nm). UVC is almost absorbed by the ozone layer and only UVA and UVB reach the surface.

In the present invention, "UV-irradiation" means a method of killing microorganisms by irradiating ultraviolet rays. Typical examples of the method are disinfection methods using ultraviolet sterilization or the like. Ultraviolet sterilization is an apparatus capable of artificially producing an ultraviolet ray amount of about 50 times or more the effective ultraviolet ray amount in the sun ray. In the present invention, a culture equipped with five 20 W sterilizing lights (254 nm in the above UVA wavelength, 7.5 W output) In the box, the ultraviolet rays were irradiated to the leaves of rice to induce accumulation of low molecular weight compounds.

The term "rice extract" of the present invention means an extract obtained by extracting rice. For the purpose of the present invention, the rice extract is selected from the group consisting of an alcohol having 1 to 4 carbon atoms and a mixed solvent thereof , Preferably methanol, ethanol or butanol. In addition, in the present invention, the above extract may contain any one of the extract obtained by the extraction treatment, the diluted solution or the concentrate of the extract, the dried product obtained by drying the extract, or the adjusted product or the purified product.

In one embodiment of the present invention, rice leaves were collected by pulverizing collected rice leaves with liquid nitrogen and then extracting the pulverized material with a 70% (v / v) methanol aqueous solution.

The term "fraction " of the present invention means a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents. In the present invention, the rice extract may be obtained by fractionating the rice extract with a solvent fractionation method using a solvent such as ethanol, n-hexane, ethyl acetate or water, and it includes both a polar solvent fraction and a non-polar solvent fraction. Fractions and water fractions can be used. It may also be a fraction by column chromatography.

In the present invention, the solvent fraction of the rice extract can be obtained by suspending the rice extract in water and then fractionating using a non-polar solvent such as ethyl acetate to obtain a polar solvent fraction and a non-polar solvent fraction, respectively.

In one embodiment of the present invention, a fraction of rice extract was obtained by a method using a 50% (v / v) ethyl acetate aqueous solution in order to separate the liquid methanol extract from the rice extract.

The term "column chromatography fraction" in the present invention means a fraction obtained by column chromatography of the fraction of rice extract. Column chromatography can use any known column capable of separating fractions containing components having the effect of preventing or treating colorectal cancer from rice. Preferably, the rice paddy extract is fractionated with 70% (v / v) methanol aqueous solution, and the water-soluble fraction obtained is re-fractionated by adding the same amount of ethyl acetate to obtain a water-soluble fraction and an ethyl acetate-soluble fraction. The ethyl acetate soluble extracts obtained by concentrating the acetate soluble fraction under reduced pressure were purified by reversed phase HPLC column chromatography using a 3% aqueous acetic acid solution and a 25-60% acetonitrile mixed solvent using a Sunfire C ₁₈ column, To obtain four fractions, followed by concentration under reduced pressure to obtain a column chromatography fraction.

In the present invention, four column chromatographic fractions separated from the ethyl acetate extract isolated from rice leaves treated with ultraviolet light were added to HCT-116 cells at different concentrations (10, 20, 40, 60, 80, 100 μg / mL ) For 24 hours to determine cell viability by MTT assay. As a result, fraction 3 and fraction 4 inhibited the growth of HCT-116 cells in a dose-dependent manner, and the most remarkable growth inhibition was observed in cells treated with fraction 4 (Fig. 2B). Therefore, it was confirmed that the rice extract of the present invention and its fractions can inhibit the growth of colon cancer cells, and thus are suitable for prevention or treatment of colon cancer.

In the present invention, "colon cancer" refers to a malignant tumor consisting of cancer cells in the large intestine, which can be divided into genetic factors and diets. Even though the food itself does not contain any direct carcinogens, it is possible that carcinogens may form during the process of ingesting and absorbing the food, which is caused by the enzymes produced by intestinal bacteria during the metabolism of the dietary components Can be converted to carcinogens.

For the purpose of the present invention, the colorectal cancer is a disease which is an object of prevention or treatment by the rice extract treated with the ultraviolet rays of the present invention or a fraction thereof. In one embodiment of the present invention, the colon cancer cell line HCT-116 cell Through cytotoxicity experiments, the effect was confirmed.

The term "prevent" in the present invention means all the actions of inhibiting or delaying the onset of the disease upon administration of the composition, and "treatment" means any action that alleviates or alleviates symptoms of the disease do.

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 type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for more effective prevention or treatment, the rice extract or its fraction treated with the ultraviolet rays of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day, The administration may be carried out once a day or divided into several times.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock,

≪ / RTI > All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The pharmaceutical composition for the prevention or treatment of colorectal cancer, which comprises the rice extract treated with the ultraviolet rays of the present invention or a fraction thereof as an active ingredient, further comprises an appropriate carrier, excipient or diluent commonly used in the production of a pharmaceutical composition can do. Preferably, the composition is in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze- The composition may have any one of formulations selected from the group consisting of ointments, creams, patches, cataplasms, pastes, sprays, skin emulsions, skin suspensions, transdermal patches, drug-containing dressings and suppositories, It may, but is not limited to, various forms of parenteral administration.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating colorectal cancer, which comprises N- benzoyltryptamine or sakuranetin as an active ingredient.

In the present invention, "N - benzoyltryptamine" is a compound represented by the following formula (2), which is a kind of flavonoids and is involved in the depression of the central nervous system. N-benzoyltryptamine is a phytoalexin identified in rice leaves infected with Magnaporthe grisea , a causative organism of rice blast fungus, and plays an important role in plant defense and antibiotics against various pathogens.

(2)

In the present invention, "sakuranetin" is a compound represented by the following formula (3), which is a kind of flavonoid. The flavonoids generally function as phytoalexins and have the function of protecting plants against stresses such as ultraviolet radiation etc. Sakuranetin also has an allergen-preventing effect and sterilizing action against Helicobacter pylori , It acts on differentiation.

(3)

However, it has not yet been known that N-benzoyltryptamine represented by the above-mentioned formula (2) and chucrenetin represented by the formula (3) are effective for the prevention or treatment of colon cancer.

In one embodiment of the present invention, three compounds having specific peaks were isolated from the fractions isolated from the extract of rice treated with ultraviolet light. Among them, Compound 1 has no cytotoxicity against colon cancer, And 3 showed cytotoxicity against colorectal cancer. Thus, it was confirmed that the N-benzoyltryptamine and the Sakuranetine were effective ingredients capable of preventing and treating colon cancer.

Therefore, the N-benzoyltryptamine and the sacratinet may be extracted from rice treated with ultraviolet rays, and the ultraviolet-treated rice extract or its fraction described above may be one containing N-benzoyltryptamine or saccharin .

In one embodiment of the present invention, N-benzoyltryptamine or chuceretin was cytotoxic to HCT-116 cells by MTT assay of compounds isolated from rice leaves treated with ultraviolet light. Therefore, it has been found that the N-benzoyltryptamine or chacenetin has an anticancer effect on colon cancer and that the ultraviolet irradiated rice leaf is a valuable source for the therapeutic substance.

The term "pharmaceutically acceptable salt " as used herein means a salt which can be used pharmacologically in a salt in which a cation and an anion are bound by an electrostatic attractive force. Usually, Salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), an aluminum salt and the like; Examples of salts with organic bases include salts with organic bases such as triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, And the like; The salt with inorganic acid may be a salt with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like; Salts with organic acids may be salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like; Salts with basic amino acids may be salts with arginine, lysine, ornithine and the like; The salt with an acidic amino acid may be a salt with aspartic acid, glutamic acid and the like.

In another aspect, the present invention provides a health functional food for preventing or ameliorating colorectal cancer comprising the extract of rice leaf treated with ultraviolet rays or a fraction thereof as an active ingredient.

In another aspect, the present invention provides a health functional food for preventing or ameliorating colorectal cancer, comprising the N-benzoyltryptamine or the saccharin or a pharmaceutically acceptable salt thereof as an active ingredient.

Rice extracts, fractions, N-benzoyltryptamine or chucretin and compositions and the like treated with ultraviolet rays are as described above.

The term " health functional food "used in the present invention refers to foods prepared and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods. It is meant to be ingested for the purpose of obtaining a beneficial effect for health use such as controlling nutrients or physiological action. The health functional food may be used in the same meaning as the functional food or the health food.

In the present invention, the term "functional food " means a food having a high medical effect, processed in such a manner that the biocontrol function is efficiently displayed in addition to the nutritional supply. The term "health food" refers to a food having an active health promotion or promotion effect as compared with a general food, and "health supplement food" The food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, rings and the like in order to obtain effects useful for improving and recovering liver function.

As a specific example of such a functional food, it is possible to produce a processed food having good storage properties while utilizing the characteristics of agricultural products, livestock products or aquatic products by using the above composition. The health functional food comprising the composition is not particularly limited, but preferably is margarine, fat continuous or water continuous or bicontinuous spread, fat reduced spread, chocolate, chocolate coating , Chocolate fillings or bakery confectionery, ice cream, ice cream coatings, ice cream inclusions, dressings, mayonnaise, cheese, cream substitute, dried soup, drinks, cereal bars, sauces, snack bars, dairy products, Can be used.

In another aspect, the present invention provides a method for treating colon cancer, comprising administering the pharmaceutical composition to a subject suspected of having colon cancer.

The above composition, colon cancer and treatment are as described above.

Such treatment methods specifically include administering the composition in a pharmaceutically effective amount into a subject suspected of having colon cancer. The term refers to whole mammals including dogs, cows, horses, rabbits, mice, rats, chickens or humans, but the mammal of the present invention is not limited by the above examples. The pharmaceutical compositions may be administered by non-oral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal routes, and for localized immunosuppressive treatment, by a suitable method, including, if necessary, by intralesional administration. Non-oral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Preferred modes of administration are intravenous, subcutaneous, intradermal, intramuscular, and drip.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. Since the extract of rice leaf treated with ultraviolet rays or the fraction thereof of the present invention exhibits a high growth inhibitory effect on cancer cells, the pharmaceutical composition comprising the extract of rice leaf treated with the ultraviolet ray or a fraction thereof as an active ingredient can be used as a colorectal cancer By administering to suspect individuals, colon cancer can be treated by preventing the development or progression of colon cancer.

The rice extract of the present invention or a fraction thereof is obtained by treating N- benzoyltryptamine ( N- benzoyltryptamine) or sakuranetin produced by treating ultraviolet rays of rice with the growth of colon cancer cells It can be widely used for the prevention or treatment of colon cancer.

Fig. 1 is a graph showing phenolic compound contents and toxicity of rice extract to cancer cells. Fig. (A) A graph showing the contents of all phenols and flavonoids in the leaves of rice plants treated or not treated with ultraviolet rays. Glycans and quercetin were used as standards to measure phenolic and flavonoid contents, respectively. DW; Dry weight (B) A graph showing the cytotoxic effect of ethyl acetate extract extracted from rice leaves treated with ultraviolet light or untreated on human colon cancer cells HCT-116. Cell viability was calculated as the percentage of viable cells in the rice extract group without or with ultraviolet light for the control (DMSO: ethyl acetate: 1: 1). Data were expressed as mean ± standard deviation after three independent experiments. The asterisk indicates the significant difference between the untreated control and each UV treated specimen (t-test; * p <0.05, ** p <0.01, *** p <0.001).
Fig. 2 is a graph showing changes in composition by ultraviolet treatment and cytotoxicity on cancer cells of HPLC fraction obtained from a sample treated with ultraviolet rays. Fig. (A) HPLC analysis of the ethyl acetate extract of the UV-treated control and UV-treated specimens. (B) Cytotoxic effects of HPLC fractions from ethyl acetate extracts of rice leaves treated with ultraviolet light on HCT-116 cells (Fr. Data were expressed as mean ± standard deviation after three independent experiments. The asterisk indicates the significant difference between the untreated control and each UV treated specimen (t-test; * p <0.05, ** p <0.01, *** p <0.001).
Fig. 3 is a diagram showing the chemical structure of a compound separated from rice leaves treated with ultraviolet rays. Fig.
4 is a graph showing the cytotoxic effect of HTC-116 cells on compounds isolated from ultraviolet-treated rice leaves. (A) The dose-dependent cytotoxic effect of isolated compounds on HTC-116 cells. Data were expressed as mean ± standard deviation after three independent experiments. The asterisk indicates the significant difference between the untreated control and each UV treated specimen (t-test; * p <0.05, ** p <0.01, *** p <0.001). (B) The ID ₅₀ values of the separated compounds were calculated from the experiment presented in the upper panel (A).

Hereinafter, the present invention will be described in more detail with reference to examples. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example 1: Preparation of ultraviolet-treated rice leaf extract and fractions

Example 1-1. Preparation of ultraviolet-treated rice leaf extract

For UV treatment, rice ( Oryza sativa L. spp japonica cv. Dongjin) was cultivated in a greenhouse maintained at 28 ° C during the day and 20 ° C at night for 8 weeks, and 5 20 W germicidal lamps (maximum emission 254 nm , Ultraviolet ray output 7.5 W, Sankyo Denki Co. J), and irradiated for 2 hours.

The rice irradiated with ultraviolet light was transferred to a greenhouse, and after 2 days, rice leaves were collected. The collected rice leaves were pulverized in liquid nitrogen and then extracted in 70% (v / v) methanol (MeOH) - aqueous solution for 1 hour.

The content of all phenol compounds in the liquid methanol extract was measured by a known method using gallic acid as a reference material. As a result of the measurement, the content of the phenolic compounds in the group treated with ultraviolet light was remarkably increased to 26.7% as compared with the untreated control group (A in FIG. 1).

Examples 1-2. Preparation of ultraviolet light treated rice leaf fraction

In order to isolate the methanol extract from the rice leaves treated with the ultraviolet ray, the ethyl acetate fraction was further separated by using 50% (v / v) ethyl acetate (EtOAc) aqueous solution. Lt; / RTI &gt; The dried residue was resuspended in a small amount of methanol, and then a known calorimetric method was performed using quercetin as a flavonoid reference material in order to measure the flavonoid content.

In the ultraviolet treated rice leaves, the flavonoid content was increased up to 82.4% compared to the untreated control (Fig. 1 A). According to the results of this experiment, it was confirmed that the production of phenol compounds including flavonoids was induced in the leaves of UV treated rice.

Example 2 Isolation and Cytotoxic Effect of UV-Treated Rice Leaf Extract

Example 2-1. Cell culture

HCT-116 cells obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) were cultured in RPMI containing 10% fetal calf serum (FBS, Thermo Scientific Hyclone) and 1% penicillin / streptomycin -1640 medium (Thermo Scientific Hyclone, Waltham, Mass.) In a humidified incubator maintained at 37 ° C, 5% CO ₂ .

Example 2-2. Cytotoxicity test

The cytotoxicity of the ethyl acetate extract was determined by colorimetric assay (Yoo et al., 2012) using MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide, Sigma, (Yoo et al., 2012).

HCT-116 cells were plated in 96-well plates at 1 × 10 ⁴ cells / well and cultured in 100 μl RPMI-1640 medium supplemented with 10% FBS.

After culturing at 37 ° C for 24 hours, the cells were treated with ethylacetate extract (40, 60 ug / ml) extracted from leaves of untreated or untreated rice and cultured in RPMI-1640 serum-free medium.

After incubating the cells for 24 hours, 50 μl MTT (5 mg / ml in PBS) was added to each well and further incubated at 37 ° C for 2 hours.

The medium was removed and the cells were treated with 100 μl DMSO for 5 minutes and then absorbance was measured at 550 nm using a microplate (BIO-TEK, Winooski, VT).

Cell viability was calculated as the percentage of viable cells in a control group (1: 1 mixture of DMSO and EtOH) with a group of extracts of rice treated or untreated with ultraviolet light.

The cell viability was measured using the following equation:

[Equation 1]

Cell viability (%) = [OD (compound treated cells) -OD (white cells) / OD (control cells) -OD

Similar to the increase in phenolic compound and flavonoid content in response to ultraviolet light treatment, the results of MTT measurement showed that stronger cells were found in HCT-116 cells compared with the control group not treated with ethylacetate extract extracted from ultraviolet- (FIG. 1, B). These results suggest that the extract of rice treated with ultraviolet light may be effective for the prevention and treatment of colorectal cancer by killing colon cancer cells.

Example  3: From ultraviolet light treated rice leaf extract HPLC Fraction  Isolation and cytotoxic effect

Example 3-1. Isolation of HPLC fractions

To confirm the chemical structure change by ultraviolet treatment, ethylacetate extracts from rice leaves treated with UV or not treated with UV light were treated with 3% ethanol by reverse phase HPLC on a Sunfire C18 column (Waters, Milford, MA) (v / v) acetic acid-aqueous solution at 280 nm for 25 minutes in a linear gradient of 25-60% acetonitrile.

The major chemical structural changes in response to UV treatment were observed between 15 and 25 minutes of retention time of HPLC chromatography (Fig. 2A).

Ethyl acetate extracts from rice leaves treated with ultraviolet light were separated into four fractions (Fr. I - IV), and cytotoxic effects of each of the HLPC fractions were confirmed (FIG. 2A).

Example 3-2. To confirm cytotoxic effect

HCT-116 cells were treated with different concentrations (10, 20, 40, 60, 80 and 100 μg / mL) of each HPLC fraction for 24 hours to confirm cell viability by MTT method (FIG.

Fraction 1 and Fraction 2 did not affect the viability of HCT-116 cells, while Fraction 3 and Fraction 4 inhibited the growth of HCT-116 cells in a dose-dependent manner. The most significant inhibition was observed in cells treated with fraction 4.

From the results of the experiment, it was confirmed that the compounds showing cytotoxicity to HCT-116 cells were mainly contained in fraction 3 and fraction 4. These results are as shown in Fig. 2 (A). The experimental results are consistent with those in which the major chemical structural changes of the ethyl acetate extract from rice leaves treated with ultraviolet light were observed in fraction 3 and fraction 4.

Example 4: Structural analysis of compounds obtained from ultraviolet-treated rice leaves

In order to isolate and identify cytotoxic substances from ultraviolet light treated samples, three peak compounds (1-3), which were predominantly induced by the reaction of ultraviolet light treatment, were collected from HPLC columns. The isolated compounds were identified using 1 and 2-dimensional NMR spectroscopy including ¹ H, ¹³ C, ¹ H- ¹ H correlation spectroscopy (COZY) and binuclear multiple bond correlation (HMBC) spectroscopy, and positive ESI MS.

Example 4-1. N-trans-cinnamoyltyramine &lt; / RTI &gt;

First, typical proton signals of cinnamoyl and tyramy moieties were observed in the ¹ H and ¹ H- ¹ H COZY NMR spectra of compound 1.

The two olefin protons signals at δ 7.51 (H-7 ') and 6.56 (H-8') are related to the 1-substituted benzene ring leading to confirmation as a cinnamoyl moiety, and δ 2.75 (H-7) It was observed in the ¹ H- ¹ H COZY spectrum that the two methine proton signals at 3.47 (H-8) are related to 1,4-2-substituted benzene rings for the tilami moiety.

The ¹ H and ¹³ C NMR data of Compound 1 presented in Table 1 are consistent with those for the known N-trans- cinnamoyltyramine (Yang et al., 2011).

The HMBC spectrum of Compound 1 shows the connectivity between the proton signal at δ 3.47 (H-8) of a portion of thylami and the carbonyl carbon signal at δ 168.8 (C-9 ') of the cinnamoyl moiety, Amino-linkage. &Lt; / RTI &gt;

The molecular weight of Compound 1 was found to be 267.3 from the similar molecular ion peak at m / z 268.3 [M + H] ⁺ in positive ESI MS and this result is consistent with N-trans-cinnamoyltyramine .

Based on these results, it was confirmed that Compound 1 was N-trans-cinnamoyl-tyramine, which was first isolated from rice (Chemical Formula 1 and FIG. 3).

Example 4-2. N-benzoyl tryptamine ( N- Benzoyltryptamine)

^{The 1} H and ¹ H- ¹ H COZY NMR spectra confirmed that the 1-substituted benzene ring and the indole ring were in compound 2. The two methine proton signals at δ 3.06 (H-10) and 3.66 (H-11) were found to be related to the indole ring in the ¹ H- ¹ H COZY spectrum (Fig. 3).

The ¹ H and ¹³ C NMR data of compound 2 (Table 1) are consistent with those for the known N-benzoyl tryptamine (Hifnawy et al., 1977; Peng et al., 2002). By finding the connectivity between the proton signal at the δ 3.66 (H-11) of the tryptamine and the carbonyl carbon signal at δ 170.3 (C-9 ') of the benzoyl moiety in the HMBC spectrum of compound 2, And it is confirmed that they are connected through the coupling. Also, the positive ESI MS of the N-benzoyltryptamine showed [M + H] ⁺ ion at m / z 265.3, consistent with N-benzoyltryptamine. Based on these results, compound 2 was found to be N-benzoyl tryptamine, which was first isolated in Myrtopsis myrtoidea (Hifnawy et al., 1977) (Formula 2 and Figure 3).

Example 4-3. Sakuranetin

Compound 3 showed the same retention time as the flavonoid phytoalexin saccharin standard in HPLC analysis. The molecular weight of the compound was confirmed by positive ESI MS analysis to be 286.3 and ¹ H NMR data (Table 1) are consistent with those for the known Sakuranetine (Ibrahim et al., 2003; Freitas et al., 2008) . Accordingly, Compound 3 was identified as Sakuranetine (Formula 3 and Fig. 3).

Example 5: Cytotoxic effect of compounds obtained from UV-treated rice leaves

The cytotoxic effect of Compound 1, Compound 2 and Compound 3 on HCT-116 cells was confirmed by MTT assay (Fig. 4).

HCT-116 cells were treated with different concentrations (10, 20, 40, 60, 80, 100 μg / mL) of Compound 1, Compound 2 and Compound 3 for 24 hours to determine the survival rate of the treated cells by MTT Respectively. Compound 2 and Compound 3 decreased the survival rate of HCT-116 cells dose-dependently, but did not decrease Compound 1 (Fig. 4A). The IC ₅₀ (50% inhibitory concentration) values of Compound 2 and Compound 3 were 89.2 ± 4.8 and 68.8 ± 5.2 μg / mL, respectively (FIG.

From these results, it can be seen that among the compounds 1, 2 and 3 isolated from rice leaves treated with ultraviolet rays, the active ingredient having cytotoxicity against colon cancer is N-benzoyltryptamine of compound 2 and Sakuranetine of compound 3 Thus, it was confirmed that the compounds of Chemical Formulas 2 and 3 are effective for the prevention and treatment of colon cancer.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (9)

A pharmaceutical composition for the prevention or treatment of colorectal cancer, comprising a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient.
(2)

The composition according to claim 1, wherein the compound is contained in an extract of rice or a fraction thereof treated with ultraviolet light. The composition according to claim 2, wherein the extract is obtained by extracting rice with a solvent selected from the group consisting of water, an alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof. 4. The composition of claim 3, wherein the alcohol is selected from the group consisting of methanol, ethanol and butanol. 3. The composition according to claim 2, wherein the fraction is obtained by fractionating the extract of rice with a solvent selected from the group consisting of ethanol, hexane, ethyl acetate, water and a mixed solvent thereof. A health functional food for preventing or ameliorating colorectal cancer, comprising a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient.
(2)

The health functional food for preventing or ameliorating colorectal cancer according to claim 6, wherein the compound represented by Formula 2 is contained in an extract of rice or a fraction thereof treated with ultraviolet light. delete delete

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