TECHNICAL FIELD The present invention relates to a polypeptide extract derived from detoxified swellfish and a composition for prevention or treatment of cancer containing the polypeptide extract as an effective ingredient.

BACKGROUND ART Cancer is a disease caused by abnormal proliferation of cells. That is, cancer is a disease caused by abnormally continued cell division due to a disorder of normal cell division regulatory mechanism preventing unnecessary cell division in normal cells. Abnormally proliferated cells form a cell group invading peripheral tissues or internal organs or even spread to distant tissues or organs from an originally affected site (metastasis), thereby leading to loss of normal function of an individual, resulting in death. It is known that about 80% of cancers are caused by lifestyle or environmental factors. Studies about various chemical, physical, and biological carcinogens have been actively done, and thus, substances for prevention or treatment of cancers have been reported. Swellfish is useful as a low-calorie, high-protein, low-fat diet food containing various types of minerals and vitamins. Korean Patent Laid-Open Publication No. 2002-91641 discloses a pharmaceutical composition for treatment of cancer, such as breast cancer, gastric cancer, lung cancer, liver cancer, and ovarian cancer, using an aqueous extract of swellfish eggs. However, since the aqueous extract of the pharmaceutical composition is derived from swellfish eggs, large-scale extraction is difficult. Furthermore, the aqueous extract contains swellfish poison considering the fact that the swellfish poison has cell division preventive activity. That is, this patent document discloses a method of preparing an aqueous extract with anticancer activity from a poison-containing part of swellfish, i.e., the testicles, the ovary, bowels and intestines, the spleen, the eyeball, and blood. Thus, it is not thought that those of ordinary skill in the art can easily predict that an aqueous extract with anticancer activity can be obtained from a non-toxic part of swellfish. While searching for a natural extract useful for the prevention or treatment of cancer, the present inventors surprisingly found that an aqueous extract derived from detoxified swellfish could effectively inhibit the proliferation of cancer cells and even induce the suicide of cancer cells, and thus completed the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 through 5 are views illustrating cell viability of cancer cell lines, SW480, TE2, SNU-668, AGS, and HCE4, treated with an aqueous extract of detoxified swellfish of the present invention; FIGS. 6 and 7 are views illustrating MTT assay results for HCE4 cell line treated with a 3,000 Da or less fraction and a 1 ,000 Da or less fraction, respectively, which are derived from a swellfish extract; FIG. 8 is a view illustrating MTT assay result for a swellfish extract-derived 3,000 Da or less fraction treated with proteinase K; FIG. 9 is a view illustrating Fast Protein Liquid Chromatography (FPLC) assay result for a swellfish extract-derived 3,000 Da or less fraction; and FIGS. 10 through 14 are views illustrating MTT assay results for HCE4 cell line treated with respective FPLC fractions.

DETAILED DESCRIPTION OF THE INVENTION Technical Goal of the Invention The present invention provides an extract useful for the prevention or treatment of cancer.

Disclosure of the Invention According to an aspect of the present invention, there is provided a polypeptide extract containing a polypeptide with a molecular weight of 1 ,000 to 3,000 Da, obtained by adding detoxified swellfish to water in a ratio of 50-1 ,500 g:1 L, followed by distillation. In the polypeptide extract of the present invention, the distillation may be performed in a batch distillation tower at a temperature of 100 to 1500C under a pressure of 1 to 5 kgF/cm2 for 2 to 7 hours. According to another aspect of the present invention, there is provided a composition for prevention or treatment of cancer including a polypeptide extract containing a polypeptide with a molecular weight of 1 ,000 to 3,000 Da, obtained by adding detoxified swellfish to water in a ratio of 50-1 ,500 g :1 L, followed by distillation, and a pharmaceutically acceptable additive. In the composition of the present invention, the distillation may be performed in a batch distillation tower at a temperature of 100 to 1500C under a pressure of 1 to 5 kgF/cm2 for 2 to 7 hours. The cancer is not particularly limited, but may be esophageal cancer, gastric cancer, colorectal cancer, uterine cervical cancer, or liver cancer. The composition of the present invention may be in any administration form, but preferably be a formulation selected from the group consisting of a liquid, a suspension, an emulsion, and a syrup. All taxonomically known swellfish species can be used herein. For example, swellfish species belonging to the family Tetraodontidae, Triodontidae, Ostraciidae, Diodontidae, etc. can be used. It is known that 19 swellfish species including 18 species belonging to the family Tetraodontidae, such as Fugu rubripes rubripes, Fugu vermicularis porphyreus, and Fugu xanthopterus, and 1 species belonging to the family Diodontidae are found in Korean waters. Fugu vermicularis porphyreus, Fugu rubripes rubripes, Fugu xanthopterus, etc. contain a highly toxic poison. As used herein, the term "detoxified swellfish" refers to swellfish in which a nerve toxin known as tetrodotoxin contained in the bowels and intestines, genital gland, digestive organs, and skin is removed. Generally, detoxification can be done by removing the fins, gills, and eyes and cutting the abdomen open to remove the internal organs, eggs, and blood, followed by cleaning with running water for one hour. The distillation may be performed by a common distillation method (e.g., simple distillation). Preferably, the distillation may be performed in a batch distillation tower at a temperature of 100 to 1500C under a pressure of 1 to 5 kgF/cm2 for 2 to 7 hours, and more preferably, at a temperature of 125 to 135°C under a pressure of 2.8 to 3.2 kgF/cm2 for 5 to 7 hours, to effectively extract an effective ingredient. The polypeptide extract of the present invention is an aqueous extract obtained by distillation of a mixture containing a large quantity (i.e., 50-1 ,500 g) of detoxified swellfish in 1 L of water. Thus, an effective ingredient contained in swellfish, in particular, protein and peptide can be obtained in the form of a liquid phase. In particular, since an effective ingredient (e.g.: effective peptide, see "study on development of effective peptide ligands, and their information management and applications" in new technology fusion project sponsored by the Ministry of Science and Technology in 2003) contained in swellfish is present in a trace amount, it is difficult to obtain the effective ingredient from a small quantity of swellfish. Thus, in the present invention, a high-concentration effective ingredient is obtained using a large quantity of swellfish. The pharmaceutically acceptable additive contained in the composition of the present invention may be one or more conventional additives selected from a diluent, a disintegrating agent, a binder, a lubricant, and a suspending agent. For example, when the composition of the present invention is formulated into a solid form such as a tablet or a hard capsule, microcrystalline cellulose, fructose, or low-substituted hydroxypropylcellulose can be used as a diluent and sodium glycolate starch or anhydrous calcium monohydrogen phosphate can be used as a disintegrating agent. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, or hydroxypropylcellulose and the lubricant may be magnesium stearate, silicon dioxide, or talc. The suspending agent may be a surfactant (e.g., sorbitan esters or polysorvates) commonly used in the pharmaceutical field. The composition of the present invention can be formulated in various forms including a solid form and a liquid form. Examples of such a formulation include a tablet, a (hard or soft) capsule, a solution, a suspension, an emulsion, and a syrup. Preferably, the composition of the present invention may be a liquid formulation such as a solution, a suspension, an emulsion, and a syrup. The composition of the present invention may also be prepared as functional food. The composition of the present invention can be administered in various methods including oral or parenteral administration. Preferably, the composition of the present invention may be orally administered. The composition of the present invention can be administered to a human subject for the prevention or treatment of cancer in the form of a dosage of 0.1 to 1000 mg/day. However, an adequate dosage is determined depending on age, sex, or the degree of disease severity. An adequate dosage can be easily determined by an ordinary person skilled in the clinical treatment or prevention of patients. The composition of the present invention has a preventive or therapeutic effect on liver cancer, lung cancer, rectal cancer, gastric cancer, breast cancer, pancreatic cancer, skin cancer, uterine cervical cancer, colorectal cancer, esophageal cancer, etc. Preferably, the composition of the present invention exhibits a preventive or therapeutic effect on gastric cancer, colorectal cancer, liver cancer, uterine cervical cancer, and/or esophageal cancer.

Effect of the Invention A composition of the present invention can be effectively used in the prevention or treatment of cancer.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically with reference to the following Examples. The following Examples are for illustrative purposes and are not intended to limit the scope of the invention. Example 1 : Preparation of aqueous extract from swellfish Fugu rubripes rubripes belonging to the family Teraodontidae was detoxified by removing the fins, gills, and eyes, cutting the abdomen open to remove the internal organs, eggs, and blood, followed by cleaning with running water for one hour. 30 kg of the detoxified swellfish were mixed in 20 L of water. The mixture was injected into a cylindrical batch distillation tower (55 L in capacity) and distillation was performed at 130°C and 3 kgF/cm2 for about 6 hours. As a result, 30 L of an aqueous extract including effective ingredients such as protein or peptide was obtained from a top stage of the distillation tower and 20 kg of a solid residue was obtained from a bottom stage of the distillation tower. Anticancer activity of the aqueous extract obtained from the top stage of the distillation tower was evaluated.

Example 2: Evaluation of anticancer activity using SW480, TE2, SNU-668, AGS and HCE4 cells An effect of the detoxified swellfish-derived aqueous extract (hereinafter, simply referred to as "swellfish extract") prepared in Example 1 on cancer cells was evaluated. For this, there were used SW480 (human colorectal cancer cell line, ATCC CCL-228), TE2 (human esophageal cancer cell line; Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Japan; http://www.idac.tohoku.ac.ip/dep/ccr/; Catalog # TKG 0253), SNU-668 (human gastric cancer cell line, Korean Cell Line Bank (KCLB) 00668), AGS (human gastric cancer cell line, KCLB 21739) and HCE4 (human esophageal cancer cell line; Anil K Rustgi University of Pensylvania). Each cancer cell line culture was treated with a swellfish extract of the present invention according to protein concentration determined by the Bradford assay, and then an effect of the swellfish extract on the cancer cell lines were evaluated.

(I) CeII culture In this Example, SW480, HCE4, and TE2 cells were incubated at 370C CO2 incubator in DMEM containing 10 % FBS. SNU-668 and AGS cells were incubated at 370C CO2 incubator in RPMI containing 10 % FBS.

(2) Treatment with swellfish extract On the first day of the test, each cell line culture obtained in the section (1) was added to a 96-well plate at a number of 5 x 103 cells per well. A medium volume in each well was 200 μJL. A swellfish extract used in this Example was obtained in the same manner as in Example 1. On the second day of the test, the swellfish extract obtained in the same manner as in Example 1 was sterilized by filtering thorough a 0.2 μm filter, and added to each well of the 96-well plate with increasing protein concentrations (1 βg, 5 μg, 10 μg, and 20 βg). Each cell culture treated with the swellfish extract was incubated at 370C CQz incubator for 72 hours. At 72 hours after the treatment, 20 μJL of MTT reagent (widely used in determining cell viability) was added to each well, and was incubated at 370C CO2 incubator for 3 hours. After the medium and MTT were removed from each well, 100 ≠ of DMSO was added to each well. MTT present on the bottom of each well was dissolved with DMSO for about 5 minutes and then absorbance was measured at 570 nm using ELISA Reader.

(3) Effect of swellfish extract on cell viability The absorbance measurements of the section (2) are shown in FIGS. 1 through 5. FIGS. 1 through 5 show cell viability for SW480, TE2, SNU-668, AGS, and HCE4 cell lines, respectively. As shown in FIGS. 1 through 5, assuming that the cell viability for a control group was 1 , cell viabilities for SW480, TE2, SNU-668, AGS, and HCE4 cell lines were respectively 0.518, 0.657, 0.798, 0.719, and 0.308 at the extract concentration of 40 βg. That is, the swellfish extract of the present invention exhibited a significant cell death inducing effect relative to the control group. Among the above cancer cell lines, HCE4 cell line experienced the greatest cell death.

Example 3: Separation and characterization of active fraction The test result of Example 2 confirmed that a swellfish extract of the present invention exhibited a cancer cell death inducing effect. In this Example, a swellfish extract of the present invention was fractionated according to a molecular weight, and a cell death inducing effect of each fraction on cancer cells was evaluated.

(1 ) Fractionation of swellfish extract of the present invention by molecular weight and evaluation of cell death inducing effect of extract fractions Cancer cells were treated with the swellfish extract prepared in Example 1 to determine whether the swellfish extract had a cell death inducing effect. First, the swellfish extract was filtered using an ultrafiltrator (Amicon™) with different molecular weight cut-off membranes to obtain a 1 ,000 Da or less fraction and a 3,000 Da or less fraction. Then, each molecular weight fraction of the swellfish extract was lyophilized. 100 mg of each lyophilate was dissolved in 1 ml of a 1X PBS buffer and filtered with a 0.2 m filter. For MTT assay, HCE4 cells (human esophageal cancer cell line: Anil K Rustgi University of Pennsylvania) were incubated at 37 °C CO2 incubator in DMEM containing 10 % FBS. On the first day of the test, the cell line culture obtained in the above was added to a 96-welI plate at a number of 5 x 103 cells per well. A medium volume in each well was 200 μi. On the second day of the test, each molecular weight fraction was added to each well of the 96-well plate with increasing concentrations (0, 1 , 2, 5 and 10 mg/ml). No treatment was done in wells used as a control group. The cell culture treated with each molecular weight fraction of the swellfish extract was incubated at 37 °C CO2 incubator for 72 hours. At 72 hours after the treatment, 20 μi of MTT reagent (widely used in determining cell viability) was added to each well, and each cell culture was incubated at 370C CO2 incubator for 4 hours. After the medium and MTT were removed from each well, 100 μl of DMSO was added to each well. MTT present on the bottom of each well was dissolved with DMSO for about 5 minutes and then absorbance was measured at 570 nm using ELISA Reader. The results are shown in FIGS. 6 and 7. FIGS. 6 and 7 show MTT assay results for HCE4 cell line treated with the 3,000 Da or less fraction and the 1 ,000 Da or less fraction of the swellfish extract. As shown in FIGS. 6 and 7, HCE4 cell line treated with the 3,000 Da or less fraction exhibited a cell death inducing effect but no cell death was observed in HCE4 cell line treated with the 1 ,000 Da or less fraction. This shows that an active ingredient of a swellfish extract of the present invention has a molecular weight of 1 ,000 to 3,000 Da.

(2) Characterization of active fraction by protease treatment It was determined whether an active ingredient of a swellfish extract of the present invention is a peptide. The 3,000 Da or less fraction (100 mg of swellfish extract/1 ml of PBS) of the swellfish extract was treated with 100 μg/ml of proteinase K and then incubated at 37 °C for overnight. MTT assay for the 3,000 Da or less fraction treated with proteinase K was performed in the same manner as above. For MTT assay, the 3,000 Da or less fraction treated with proteinase K was added to each well of the 96-well plate with increasing concentrations (5 and 10 mg/ml). No treatment was done in wells used as a control group. The results are shown in FIG. 8. FIG. 8 shows the MTT assay result for the 3,000 Da or less fraction of the swellfish extract treated with proteinase K. As shown in FIG. 8, the cell death inducing effect of the 3,000 Da or less fraction of the swellfish extract was not observed due to protease treatment. This shows that an active ingredient of a swellfish extract of the present invention is a peptide compound.

(3) Liquid chromatographic fractionation The swellfish extract of Example 1 was dialyzed with a 1 ,000 Da dialysis membrane in a 0.1X PBS buffer overnight to remove a 1 ,000 Da or less fraction from the swellfish extract. Then, a 3,000 Da or less fraction was obtained using an ultrafiltrator and then lyophilized. The resultant powders were dissolved in a solvent A (0.1% TFA/H2O) and further fractionated by liquid chromatography. First, the lyophilate of the 3,000 Da or less fraction was dissolved in a buffer A (0.1 % TFA in water) and Fast Protein Liquid Chromatography (FPLC) was performed. Before FPLC, a mobile solvent was filtered with a 0.4 μm filter, and subjected to degassing for about 30 minutes to remove gases such as dissolved oxygen from the mobile solvent. Deltapak C18 column (19x 300 mm, 15 μm, 100 A) was used as a stationary phase, and the buffer A (0.1 % TFA in water) and a buffer B (0.1 % TFA in CH3CN) were used as developing solvents for gradient elution. The elution was done with a concentration gradient (from 0 to 100%) of the buffer B at a flow rate of 5 ml/min for 65 minutes, and absorbance was measured at 280 nm. FIG. 9 is a chromatogram illustrating FPLC assay result for the lyophilate of the 3,000 Da or less fraction in the buffer A (0.1 % TFA in water). At 11-12 minutes (first fraction), 14-16 minutes (second fraction), 23-25 minutes (third fraction), 25-30 minutes (fourth fraction), and 30-31 minutes (fifth fraction) after the FPLC was initiated, corresponding fractions were collected and MTT assay was performed in the same manner as above. The fractions used for the MTT assay were made into samples with a concentration of 100 mg fraction/1 ml PBS and each sample was treated on HCE4 cells. FIGS. 10 through 14 show MTT assay results for HCE4 cell line treated with the respective fractions. From FIGS. 10 through 14, it is thought that an active ingredient of a swellfish extract of the present invention is present in the third FPLC fraction.