KR20020074746A - Anticancer agents containing antigenotoxic and immunostimulation peptides produced from the hydrolyate of silkworm cocoon - Google Patents

Abstract

PURPOSE: Provided are anticancer agents having peptides produced by hydrolysis of fibroin which is obtained by extracting silk thread or waste silk thread of silkworm cocoon as a main component. These peptides can be applied to medicine and functional food having anticancer activity, so that it can contribute to the development of medicines useful for prevention and treatment of cancer. CONSTITUTION: These anticancer agents contain peptides produced from silkworm cocoon, wherein the peptides are prepared by the following process consisting of: treating silk thread or waste silk thread of silkworm cocoon with 1 to 10% sodium carbonate or sodium bicarbonate to remove sericin, and treating the obtained fibroin with acid such as hydrochloric acid or oxalic acid or protease.

Description

Anticancer agents containing antigenotoxic and immunostimulation peptides produced from the cocoon protein produced from the hydrolyate of silkworm cocoon}

The present invention relates to an anticancer agent comprising a cocoon peptide as an active ingredient, and more particularly, to an anticancer agent using a peptide obtained by hydrolyzing fibroin extracted from silkworm silk thread or lung dog silk.

Traditionally, in Korea, silkworms have been produced using silkworms, but silk yarn produced from silkworm cocoons has been undermined due to the emergence of artificial dogs or synthetic fibers, and the traditional silkworm industry is shrinking. In recent years, the Yangjam industry has been progressing toward the development of hypoglycemic agents using silkworms, the production of Cordyceps sinensis, the development of health drinks and cosmetics using silk powder, and the development of health foods based on mulberry leaves.

The silkworm cocoon (silk) produced from silkworms is composed of fibroin, a fibrous protein, and sericin, a rubbery protein surrounding it.The amino acid content is 45% glycine, 30% alanine, 12% serine and tyrosine. Major amino acids account for more than 90%, such as 5%. In Japan, studies have been conducted to make the protein edible by digesting the protein into a low molecular peptide or amino acid form through acid, alkali or enzymatic hydrolysis to facilitate digestion and absorption.

Silk protein has already been put into edible use in Japan, but is still in limited use, and it is required to develop its physiological activity as a pharmaceutical material and a functional food material. In particular, the scope of application of functional peptide foods produced through hydrolysis of proteins is still limited. Currently, peptide foods with promoting digestive absorption, promoting calcium absorption, preventing osteoporosis, inhibiting alcohol absorption, preventing high blood pressure, improving lipid metabolism, suppressing cholesterol absorption, antioxidant function and anti-allergic function, have been commercialized and studied in Japan. Patent Application No. 96-0015242, "Insulin-independent diabetes therapeutics containing shoulder fibroin," discloses a novel use of an aqueous solution of peptide fibroin as a hypoglycemic agent. The physiological activities of silk protein hydrolysates known to date include alcohol metabolism promotion, hypoglycemic effect, blood cholesterol lowering effect and antidementia, but these activities have not been systematically studied. As far as the necessity of the development is concerned, research is not progressed yet.

On the other hand, cecropins isolated from hemolymph of silkworm moth and dolastatins isolated from marine cells have anti-cancer functions, and peptides derived from milk proteins such as casein and lactalbumin It has been reported that they have a function of enhancing phagocytosis by phagocytes and regulating differentiation of lymphocytes, and immunomodulatory functional peptides have been produced from soy protein. In addition, cytotoxicity has been confirmed from peptides obtained from hydrolyzates of soy sauce, soybean paste, cheese and soy protein, which are traditional foods in Korea.

However, there have been no reports of anti-cancer peptide components that suppress DNA damage by cancer-causing agents and increase immune activity.

The inventors of the present invention have been developing anticancer drugs that can suppress DNA damage caused by a cancer-causing substance and exhibit increased immune activity, while hydrolysates of fibroin, a silk protein extracted from silkworm cocoon, may be used to repair gene damage from an external carcinogen. The present invention has been completed by finding out that it has an inhibitory effect and an immunosuppressive effect of enhancing the cancer cell proliferation inhibitory activity of macrophages.

Accordingly, it is an object of the present invention to provide an anticancer agent comprising an anticancer peptide that inhibits DNA damage caused by a carcinogen and has an effect of increasing immune activity.

Another object of the present invention is to provide a processed food to which the anticancer peptide is added.

Figure 1a shows the result of separation of the fibroin hydrolyzate using gel filtration chromatography using Sephadex G-25.

Figure 1b shows the result of separation of the peptide fraction corresponding to the third peak in Figure 1a by gel filtration chromatography using Sephadex G-15.

Figure 2 is a graph showing the change in the amount of nitric oxide produced when treated with fibroin hydrolyzate in mouse intraperitoneal macrophages.

In order to achieve the above object, the present invention provides an anticancer agent comprising a peptide obtained by treating fibroin extracted from silkworm silkworm silk thread or lung silk thread with acid or protease.

The present invention also provides a processed food comprising a peptide obtained by treating fibroin extracted from silkworm silk thread or lung silk thread with acid or protease in powder or other edible form.

Hereinafter, the present invention will be described in detail.

In order to manufacture an anticancer agent containing the fibroin hydrolyzate of the present invention as an active ingredient, it is necessary to first remove sericin protein from silkworm silk thread or lung silk thread composed of fibroin and sericin protein. In order to remove the sericin, it is preferable to remove sodium carbonate and sodium bicarbonate by treating. Specifically, in the present invention, sericin is removed by treating sodium carbonate at a concentration of 1-10%.

In addition, hydrolyzate was prepared by treating acid or protease with fibroin from which the sericin was removed.

In preparing a hydrolyzate using an acid, the acid may be hydrochloric acid or oxalic acid, but hydrochloric acid is preferably used. In addition, more specifically, it is preferable to use a concentration of hydrochloric acid is 1-5N, after heating for 4-8 hours at a temperature of 90-100 ℃, neutralized to pH 7.0-7.4 using sodium hydroxide. Preference is given to preparing the hydrolyzate. Various materials may be used for decolorization and deodorization of the hydrolyzate, and specifically, activated carbon was used in the present invention. The capacity of the activated carbon is preferably 3-10% of the total solution capacity.

In addition, in the preparation of the hydrolyzate using proteolytic enzymes, the proteolytic enzymes may be used bacteria and protein-derived proteolytic enzymes derived from industrial, specifically, in the present invention trypsin, pepsin, alkalase And nutraceases were used. In treating the protease with fibroin, each concentration is preferably 0.1-5%.

In one embodiment of the present invention, the active peptide fraction was isolated from the hydrolyzate. For the separation of the peptide fraction, a general method known in the art may be used, but in the present invention, the peptide fraction is separated by chromatography, and specifically, gel filtration chromatography using Sephadex G-25. Was separated using. Preferably, the process of obtaining a peptide fraction using Sephadex G-15 from the peptide fraction separated by the chromatography is further performed, and the peptide fraction having a molecular weight in the range of 500-1000 with increased activity compared to the hydrolyzate stock solution. Could get

In another embodiment of the present invention it was confirmed that the hydrolyzate isolated from the fibroin inhibits DNA damage caused by carcinogens. Various carcinogens may be used as a factor for inducing DNA damage. Specifically, in the present invention, MNNG (N-methyl-N'-nitro-N-notrosoguanidine), which is a direct carcinogen, was used. In order to measure the effect of inhibiting DNA damage by the hydrolyzate of the present invention, the Ames test and the SOS Chromotest may be generally used, but the Comet test is used in the present invention. Both methods require the use of Salmonella and Escherichia coli, which may give inaccurate results due to differences in cell biological characteristics between microorganisms and animal cells in measuring inhibition of DNA damage to animal cells. On the other hand, when measuring the degree of DNA damage inhibition by the Comet test used in the present invention, there is an advantage that it is possible to search for a physiologically active substance that inhibits DNA damage more reliably.

The Comet test is a method for specifying DNA damage and repair of various mammalian cells, and can identify DNA damage that cannot be observed by conventional methods. Therefore, it is a method that can be applied to various fields such as the search for various substances related to carcinogenesis, the field of genetic toxicology related to DNA damage and repair, the monitoring of environmental pollution and the cancer prevention effect of lactic acid bacteria.

In another embodiment of the present invention, the effect of increasing immune activity by fibroin hydrolyzate was measured. In order to measure the effect of increasing immune activity, a general method known in the art may be used, but specifically, in the present invention, a reactive nitrogen species, which is a representative chemical which is generated from macrophage and shows the final effect of immune action, for example , Was used to measure the degree to which nitric oxide is produced by the hydrolyzate. Increasing the production of reactive nitrogen species directly means increased production of tumor necrosis factor (TNF), thus confirming the increase in the production of reactive nitrogen species.

The macrophages responsible for the first line in the immune surveillance in vivo is known as an important defense against tumors. In controlling tumors, activated macrophages recognize and remove only tumor cells by direct intercellular contact. The activated macrophages secrete various cytokines, nitric oxides, and hydrogen peroxides. In the present invention, the degree of Nantric oxide secretion was measured from the macrophages by the hydrolyzate of these substances. As a result, as shown in FIG. 2, the amount of the nitric oxide was increased by the peptide component of the hydrolyzate. As a result, the effect of increasing the immune activity of the hydrolyzate was confirmed.

Therefore, it was confirmed from the above two results that the fibroin hydrolyzate of the present invention can show an effect as an anticancer substance.

In order to achieve the other object of the present invention, the present invention provides a processed food comprising a peptide obtained by treating fibroin extracted from silkworm silkworm silk thread or lung silk thread with acid or protease in edible form.

Fibroin hydrolyzate according to the present invention exhibits an anticancer effect, so if a functional food containing the same is prepared and consumed, it is possible to suppress DNA damage caused by an external carcinogen and increase immune activity. Peptides of the fibroin hydrolyzate, if added in a edible form is not particularly limited in its form, but is preferably prepared in powder form and added to the processed food.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Hydrolyzate from Cocoon

1-1) Separation of Fibroin Silk from Cocoon

Selected Silkworm cocoon with 10% sodium hydroxide (Na₂CO₃) The solution and heated for 1 hour, The solution Filtration removed the dissolved sericin to obtain silk with only fibroin remaining.

1-2) Preparation of Acid Hydrolysates

Hydrochloric acid at a concentration of 2N was added to the fibroin silk obtained in Example 1-1) at 80 times the weight of the fibroin silk weight, and the fibroin was hydrolyzed by heating at 100 ° C. for 48 hours. The prepared hydrolyzate showed a dark dark brown color, and was neutralized by adding 2N sodium hydroxide solution to a pH of 7.4. Activated carbon was added to 6% of the total solution capacity to the neutralized hydrolyzate, and stirred for 60 minutes to remove various non-hazardous substances and off-flavor generated during the process, and then filtered to obtain a transparent liquid hydrolyzate. The hydrolyzate solution was dialyzed with distilled water for 1 day using a dialysis membrane to remove salts.

1-3) Preparation of Enzymatic Hydrolysates

Fibroin silk obtained in Example 1-1) was digested into peptides of various sizes using protease.

The fibroin silk was added to a 30% concentration of CaCl ₂ solution and heated at 90 ° C. for 30 minutes to dissolve. The dissolved fibroin solution was put in a dialysis membrane and dialyzed with distilled water for 3 days, and trypsin (Sigma) and pepsin (Sigma) were at 37 ° C. (pH of 7.5 and 2.0, respectively) and Alkalase (Novo Nordisk) was 55 ° C. ( pH 7.5), Novor Nordisk was reacted at 45 ° C. (pH 6.2), but 3 ml of sample was taken every 1 hour and heat treated at 100 ° C. for 10 minutes to stop the reaction for 8 hours. At this time, each enzyme was added to a concentration of 0.1-5%.

Thereafter, the hydrolyzate was centrifuged at 13,000 rpm for 15 minutes to obtain a supernatant, and the supernatant was ultrafiltered using a centrifugal ultrafiltration kit (Vivaspin20, Satorius, Germany), and the anticancer effect was measured for the filtrate. It was. On the other hand, the produced peptides were quantified using the Lowry method for these filtrates. Peptide production yield, ie Degree of Hydrolysis (DH), was calculated from the difference between this quantitative value and the protein concentration before hydrolysis. Enzymes and enzyme concentrations were determined by comparing the maximum hydrolysis conditions where the degree of hydrolysis no longer increases with reaction time. As a result, trypsin was treated at 1% for 4 hours, alkalase was added at 0.5% for 2 hours, neutrases were added at 2% for 2 hours, pepsin was treated at 5% for 4 hours. In case of performing the maximum hydrolysis effect.

Example 2 Direct Carcinogen (MNNG) Treatment of Hydrolyzate

2-1) Animal Cell Line Culture

The cell line used in the present invention was used as a normal 3T3 cell line (ATCC CCL No. 163) of mouse embryo origin, obtained from the Biotechnology Research Institute Gene Bank. Non-heated bovine serum (Hyclon, USA), 10 units of penicillin G (Sigma) and 100 mg / ml of streptomycin sulfate (Sigma) were added to DMEM medium (Gibco) and used as cell culture medium. The 3T3 cell line was incubated in a wet incubator (Labline instruments, USA) at 37 ° C., 5% CO ₂ conditions on a 10 cm round culture dish (Falcon, USA) containing the DMEM medium. Subcultures were performed once every three days.

2-2) Treatment of carcinogens directly with hydrolyzate

Direct carcinogen MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) was diluted in HBSS (10 mM, pH 7.4) buffer solution at a concentration of 100 μg / ml, and the hydrolyzates obtained in Example 1 were each It was dissolved in HBSS (10 mM, pH 7.4) buffer solution at concentrations of 10 mg / ml, 5 mg / ml, 2 mg / ml and 1 mg / ml. 10 μl of the MNNG dissolved in HBSS buffer solution was added to the hydrolysates dissolved at different concentrations, and then pre-incubated for 30 minutes. In this case, HBSS buffer solution was used as a negative control, and HBSS buffer solution adjusted to a concentration of 1 μg / ml of MNNG was used as a positive control. After preculture, each of the samples was added to a 3T3 cell line incubated with a wet incubator at 37 ° C., 5% CO ₂ at a concentration of 1 × 10 ⁵ cells / plate in a round tissue culture dish (Falcon, USA) of 3 cm diameter and The reaction was carried out for 30 minutes.

Example 3 DNA Damage Inhibition Measurement by Hydrolysate

3-1) Preparation of pre-coated agarose gel slide

NMA (normal melting point agarose, Sigma, USA) was added to the PBS buffer to a concentration of 0.5%, and then heated in a microwave oven, and maintained at 60 ℃ in a constant temperature bath again. 35 μl of the NMA solution was applied to the frosted surface of a fully frosted slide glass (ERIE Scientific, USA) and dried, and then 75 μl of the NMA was added using a cover glass (24 × 50 mm, Superior, Germany). It was applied uniformly to the slide glass and placed on a flat stainless box containing ice to harden. The pre-coated agarose gel slides were stored at 4 ° C. in a damp slide holder.

3-2) Cell Preparation

The 3T3 cell line was dispensed at 3 × 10 ⁴ cells / plate in a round 3 cm diameter tissue culture dish at each passage and incubated for 72 hours. After 4 days, washed twice with PBS buffer solution without Ca ²⁺ and Mg ²⁺ , and prepared in Example 2-2, 10 mg / ml, 5 mg / ml, 2 mg / ml and 1 mg / ml. After the concentration of the hydrolyzate and the mixture of MNNG and the positive control HBSS solution containing MNNG, negative control HBSS solution containing no MNNG, respectively, and then incubated for 30 minutes at 37 ℃ at a speed of 25rpm in a shaking thermostat. Immediately after incubation, the cells were washed three times with PBS buffer solution, and treated with 100 μl of proteinase K (Sigma, USA) solution to completely separate cells from the tissue culture dish. To this was added 1 ml of cell culture and the cells were suspended and transferred to an Eppendorf tube.

The cell suspension was centrifuged at 1,000 rpm for 5 minutes, the supernatant was discarded, and then 300 µl of medium was resuspended. Of this, 30 μl of cell suspension was centrifuged again and a precipitate was obtained.

3-3) Comet test

The cell precipitates obtained by treatment of the hydrolyzate of different concentrations of Example 3-2 with MNNG and the positive and negative controls, respectively, were suspended in 75 μl of 0.75% LMA solution maintained at 45 ° C. in a constant temperature water bath. The micropipette was transferred onto the glass prepared in 3-1). The coverslips were used to cover the cell suspension evenly and then placed on an ice-filled flat stainless steel box for another 10 minutes. After 5 minutes, the coverslips were removed and 75 μl of 0.75% LMA solution was again dropped on the slide glass to which 3T3 cells were applied, the coverslips were covered, and again placed on an ice-filled flat stainless steel box for another 10 minutes. Thereafter, the coverslips were peeled off and soaked in the refrigerated alkaline lysis buffer for 1 hour to dissolve all the components in the cell except the nucleolus.

All slides soaked in lysis buffer for 1 hour were placed on a horizontal electrophoresis plate, filled with electrophoretic buffer solution to 0.7 cm above the slide surface, and allowed to stand for 20 minutes. Electrophoresis was performed for 20 minutes at a speed of 25 V / 300 mA, and after electrophoresis, each slide was washed with neutralizing buffer. Then, 10 µg / ml fluorescence staining reagent YOYO-1 to examine the DNA damage level of each 3T3 cell obtained by treating the hydrolyzate with different concentrations of Example 3-2 with MNNG and each of the positive and negative controls. (Molecular Probes, USA) stained with 80ul.

The stained slides were observed at a magnification of 250x using a fluorescence microscope (CSB-FEI, China) attached with a 200W mercury lamp (Osram, Germany), and the shape of each cell nucleus sent through a CCD camera (COHU, USA) was measured. The analysis was performed on a computer equipped with a image analysis system (Perceptive instrument, UK). The degree of DNA damage by mutagen and the inhibition of peptide damage in each cell nucleus is determined by measuring the tail length and the tail moment of the comet image formed on 101 cells per slide. Statistical treatment was quantified by the mean and standard deviation values, and the results are shown in Table 1 below.

Inhibitory Effect of Fibroin Hydrolysates on DNA Damage in Direct Carcinogen MNNG Tail length (mean ± standard deviation) Tail moment (mean ± standard deviation) Negative control 36.12 ± 19.51 1.17 ± 3.36 Positive control 142.04 ± 24.18 30.20 ± 12.10 10mg / ml 49.60 ± 21.38 3.34 ± 3.98 5mg / ml 78.60 ± 34.31 4.18 ± 3.0 2mg / ml 110.63 ± 26.95 9.69 ± 7.36 1mg / ml 117.14 ± 31.63 11.80 ± 6.43

As shown in Table 1, when the fibroin hydrolyzate was treated at a concentration of 10 mg / ㎖, 5 mg / ㎖ and 2 mg / ㎖ showed a clear DNA damage inhibitory effect.

In addition, in order to investigate the mechanism of inhibiting DNA damage of fibroin hydrolyzate, the concentration of the hydrolyzate was 4 mg / ml and pre-cultured with MNNG to induce a reaction between the hydrolyzate and MNNG, and then treated with 3T3 cell line. DNA damage inhibition was measured and the results are shown in Table 2 below.

Investigation of DNA damage inhibition mechanism of fibroin hydrolyzate Tail length (mean ± standard deviation) Tail moment (mean ± standard deviation) Negative control 20.27 ± 9.29 0.55 ± 3.80 Positive control 95.83 ± 18.70 34.50 ± 6.30 Sample 1 71.79 ± 113.58 16.26 ± 5.73 Sample 2 94.47 ± 17.37 33.09 ± 4.42 Sample 3 79.05 ± 15.45 16.42 ± 8.85 Sample 4 79.13 ± 15.42 12.33 ± 5.81

In Table 2, Sample 1 induced the reaction by pre-culture of the hydrolyzate and MNNG, and treated with 3T3 cells to see the effect of inhibiting DNA damage, and Sample 2 treated the 3T3 cells without pre-culture of the hydrolyzate with MNNG. After that, the effect of inhibiting DNA damage is seen. Samples 3 and 4 were first pre-cultured with the hydrolyzate for 3 minutes to a 3T3 cell line, followed by a reaction between the peptide and the cell. Subsequently, in case of sample 3, MNNG was treated for 30 minutes while washing the hydrolyzate treated with cells three times with PBS buffer solution, and in case of sample 4, the hydrolyzate remained without washing step. MNNG was treated for 30 minutes.

As shown in Table 2, the sample 2 did not show a DNA damage inhibitory effect. These results show that the peptide component of the hydrolyzate inhibits DNA damage through a mechanism that binds to MNNG and interferes with its role as an alkylating agent.

In addition, both samples 3 and 4 showed a DNA damage inhibitory effect, as a result it was confirmed that the hydrolyzate inhibits DNA damage by the mechanism through the reaction with the carcinogen and the cell.

Example 4 Isolation of Active Peptide Fraction and Measurement of DNA Damage Inhibition

The hydrolyzate obtained in Example 1 was separated according to molecular weight using gel filtration chromatography, and then the degree of DNA damage inhibition was measured for each peak.

The hydrolyzate was separated by gel filtration chromatography using Sephadex G-25. As shown in FIG. 1, it was confirmed that the hydrolyzate was divided into three peaks according to the molecular weight between 1,000 and 5,000. In addition, the peptides corresponding to each peak were separately isolated and lyophilized, and DNA damage inhibition of carcinogen was measured using the Comet test, and the hydrolyzate of the third peak having double activity was identified as Sephadex. As a result of re-separation using G-15, it was confirmed that it was separated into three peaks again, and the Comet test was performed with peptides separated from the third peak. As shown in FIG. It was confirmed that 1.5 times higher than this stock solution.

Example 5 Measurement of Increasing Immune Activity

In order to confirm the effect of increasing the immune activity on the hydrolyzate of fibroin silk, it was examined whether the production of nitric oxide was increased when the hydrolyzate was treated using the mouse intraperitoneal macrophages.

5-1) Culture of Macrophages

Balb / c mice were injected intraperitoneally with 1 ml of 1% thioglycolate. Four days later, mice were sacrificed by cervical dislocation, and intraperitoneal cells were collected by centrifuging the washed solution three times by injecting 10 ml of RPMI 1640 medium into the abdominal cavity. The cells were suspended in RPMI medium containing 10% FBS and aliquoted 200 μl into 96 well plates. The number of cells per well was 2 × 10 ⁵ cells and cultured under conditions of 5% CO ₂ and 37 ° C. After 2 hours, the cells not attached to the plate were removed by washing with PBS (phosphate buffered saline, pH 7.4) buffer, and the attached macrophages were continued to be cultured. Filtrate sterilized hydrolyzate samples were added to each well at a concentration of 0.01-10 mg / ml, and after 16 hours, interferon-γ was added to activate macrophages. After 24 hours, a portion of the culture was taken to measure the amount of nitric oxide generated during the culture. At this time, as a positive control, LPS (lipopolysaccharide) was used after 1 ug / ml treatment.

5-2) Measurement of Formation of Nitric Oxide

The amount of nitric oxide, a reactive nitrogen species generated in macrophages, was measured by quantifying the amount of nitrite accumulated in activated macrophage culture.

50 μl of macrophage culture supernatant was taken and transferred to another 96 well plate and the same amount of grease reagent (Griess, 0.05% naphthylethyenediamine dihydrochloride, 0.5% sulfanilamide, 2.5% H ₃ PO ₄ ) was added and after 10 minutes using an ELISA reader Absorbance at 550 nm was measured. At this time, sodium nitrite was used as a standard material. As shown in Figure 2, as the concentration of the hydrolyzate treated in the macrophage culture medium was confirmed that the amount of nitric oxide production increased. From these results, the nitric oxide production of macrophages was induced by the amino acid or peptide component of the hydrolyzate, thereby confirming the effect of increasing immune activity.

As described above, in the present invention, anticancer peptides having an effect of inhibiting DNA damage caused by an external carcinogen and increasing immune activity could be prepared from a hydrolyzate of fibroin extracted from silkworm silkworm or silkworm silkworm silkworm cocoon. These peptides of the present invention can be applied to medicines and functional foods having anticancer effects in the future, which can contribute to the development of cancer prevention and treatment.

Claims (7)

An anticancer agent comprising a peptide obtained by treating fibroin extracted from silkworm silkworm silk thread or lung silk thread with acid or protease. The anticancer agent according to claim 1, wherein the fibroin is obtained by removing sericin from silkworm silk thread or lung silk thread of cocoon. The anticancer agent according to claim 2, wherein the sericin is removed using sodium carbonate (Na ₂ CO ₃ ) or sodium bicarbonate (NaHCO ₃ ) at a concentration of 1-10%. The anticancer agent according to claim 1, wherein the acid is hydrochloric acid or oxalic acid. The anticancer agent according to claim 1, wherein the protease is at least one selected from the group consisting of trypsin, pepsin, alkalase and neutrases. 6. The anticancer agent according to claim 1 or 5, wherein the protease has a concentration of 0.1-5%. A processed food comprising, in powder form or other edible form, a peptide obtained by treating fibroin extracted from silkworm silkworm or silkworm silkworm with acid or protease.