WO2002076487A1

WO2002076487A1 - Anticancer agents containing antigenotoxic and immunostimulative peptides produced from the hydrolysate of silkworm cocoon

Info

Publication number: WO2002076487A1
Application number: PCT/KR2002/000418
Authority: WO
Inventors: Yuk-Hyun Joo; Chang-Kee Hyun; Sung-Hee Lee; Deock-Hyoung Cho; Geum-Ju Park
Original assignee: AMINOGEN Co Ltd
Current assignee: AMINOGEN Co Ltd
Priority date: 2001-03-21
Filing date: 2002-03-11
Publication date: 2002-10-03
Anticipated expiration: 2003-09-21
Also published as: KR20020074746A

Abstract

The present invention relates to anticancer agents having peptides produced from silkworm cocoon as an effective component, more specifically to anticancer agents having peptides produced by hydrolysis of fibroin which is obtained by extracting the silk thread or waste silk thread of silkworm cocoon. According to the present invention, the anticancer peptides capable of inhibiting the damage of DNA from foreign carcinogens and improving the immune activity may be produced from the fibroin hydrolyate extracted from the silk thread or waste silk thread of silkworm cocoon. The anticancer peptides of the invention may 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.

Description

ANTICANCER AGENTS CONTAINING ANTIGENOTOXIC AND IMMUNOSTIMULATIVE PEPTIDES PRODUCED FROM THE HYDROLYSATE

OF SILKWORM COCOON

Technical Field

The present invention relates to an anticancer agent comprising peptides of silkworm cocoons as an active ingredient, and more particularly to an anticancer agent comprising, as an active ingredient, peptides obtained by hydrolyzing fibroin extracted from a silk thread or waste silk thread of silkworm cocoons.

Background Art

Silkworms have been raised traditionally in Korea to produce raw silk. With emergence of artificial silk or synthetic fibers, however, competitiveness of silk threads produced from silkworm cocoons was weakened, and also the traditional sericultural industry has been reduced. Recently, such a sericultural industry has pursued to development of hypoglycemic agents utilizing silkworms, production of vegetable worms, health beverages and cosmetics utilizing silk powder, and health foods made from mulberry leaves.

Cocoon threads (silk threads) produced from silkworms are composed of fibroin, a fibrous protein, and sericin, a glue protein, which coats the fibroin part. The protein of cocoon threads has an amino acid composition as follows: 45 % glycine, 30 % alanine, 12 % serine, and 5 % tyrosine. That is, main amino acids account for more than 90 % in the composition of silk. In Japan, with the aim of providing edibility of these proteins, research into how such proteins can be digested into low molecular weight peptides or amino acids by hydrolysis thereof using acids, alkalis, or enzymes, and digested and absorbed into the body in an efficient manner, is underway.

Although silk proteins are already available as food in Japan, the use is still limited. There is a need to determine broad physiological activities of those proteins and develop the use thereof as materials for medications and functional foods. In addition, application ranges of functional foods containing peptides, which are produced through protein hydrolysis, are still limited. Currently, peptide foods having functions of promotion of digestion, promotion of calcium absorption, prevention of osteoporosis, inhibition of alcohol absorption, prevention of hypertension, improvement of lipid metabolism, inhibition of cholesterol absorption, antioxidation and anti-allergenic effects, are commercialized and are being studied, centering around Japan. Further, Korea Pat. Appln. No. 96-0015242 entitled "Therapeutic agent containing silk fibroin for insulin-independent diabetes" discloses a novel use of an aqueous solution of a silk fibroin peptide as a hypoglycemic agent. In particular, physiological activities of hydrolysates of the silk protein known so far include promotion of alcohol metabolism, hypoglycemic effect, decrease of level of blood cholesterol, and anti-dementia effect. However, these activities are not yet systemically studied. Especially, few studies on peptides having an anticancer effect have achieved success, despite increasing demand for development of anticancer agents.

Meanwhile, many functional peptides have been reported. Cecropins and dolastatins have anticancer effects, cecropins being isolated from hemolymph of silk moth and dolastatins being isolated from marine cells. Milk protein-derived peptides such as casein and lactalbumin improve phagocytosis by phagocytes and regulate differentiation of lymphocytes. There is also a report which presents a functional peptide from a soybean protein which regulates immune reactions. Peptides obtained from hydrolysates of Korean traditional foods such as soy sauce and beanpaste, cheese and soybean proteins are reported to have cytotoxicity against tumor cells in vitro. However, there are no reports about anticancer peptide components exhibiting effects of inhibition of DNA damage caused by carcinogens, and improvement of immune activity.

Disclosure of the Invention

The present inventors have conducted research to develop an anticancer agent capable of inhibiting DNA damage caused by carcinogens and improving immune activity. The inventors found that hydrolysates of a silk protein, fibroin, extracted from silkworm cocoons have effects of inhibiting DNA damage caused by a foreign carcinogen, and improvement of immune activity by enhancing a macrophage activity, thereby inhibiting proliferation of tumor cells. Accordingly, it is an object of the present invention to provide an anticancer agent comprising, as an active ingredient, anticancer peptides having effects of inhibition of DNA damage caused by carcinogens and improvement of immune activity.

It is another object of the present invention to provide a processed food prepared by adding the anticancer peptides to the food. In accordance with one aspect of the present invention, the above and other objects can be accomplished by the provision of an anticancer agent comprising, as an active ingredient, peptides obtained by hydrolyzing fibroin extracted from a silk thread or waste silk thread of silkworm cocoons, using an acid or a protease.

In accordance with another aspect of the present invention, there is provided a processed food prepared by adding anticancer peptides in powder or other edible formulations, the peptides being obtained by hydrolyzing fibroin extracted from a silk thread or waste silk thread of silkworm cocoons, using an acid or a protease.

Hereinafter, the invention is described in detail.

To prepare an anticancer agent comprising a fibroin hydrolysate as an active ingredient according to the invention, it is necessary to first remove a sericin protein from a silk thread or waste silk thread of silkworm cocoons, which is composed of fibroin and sericin protein. It is preferable that sericin is removed by treating cocoons with sodium carbonate or sodium bicarbonate. Especially, sodium carbonate was used herein at a concentration of 1 to 10 % to remove sericin. After removing sericin, fibroin was treated with an acid or protease, preparing a hydrolysate.

In preparing a hydrolysate using an acid, the acid may be oxalic acid or HC1, and HC1 is more preferable. The concentration of HC1 is preferably 1 to 5 N. More particularly, after treating with HC1, fibroin is heated at 90 to 100^°C for 4 to 8 hrs, followed by neutralization with sodium hydroxide, adjusting to pH 7.0 to 7.4, thus preparing a hydrolysate. To decolorize or deodorize the hydrolysate, various substances may be used. Especially, an active carbon was used herein. The amount of active carbon is preferably 3 to 10 % relative to a total amount of the solution.

In preparing a hydrolysate using a protease, the protease may be bacteria- derived proteases and bacteria-derived proteases for use in industry. Especially, the proteases used herein are trypsin, pepsin, Alcalase, and Neutrase. In treating fibroin with the above proteases, each concentration of the enzymes is preferably 0.1 to 5 %.

In one embodiment of the invention, an active peptide fraction was isolated from the hydrolysate. The peptide fraction may be isolated using any common methods known in the art. In the invention, the peptide fraction was isolated using chromatography, and especially, gel filtration chromatography on Sephadex G-25. It is preferable that a step of obtaining a peptide fraction using Sephadex G-15 from the peptide fraction isolated using the above chromatography is further conducted. In such a way, a peptide fraction with molecular weights between 500 and 1000, exhibiting an increased activity in terms of inhibition of DNA damage, compared to a crude hydrolysate, could be obtained. In another embodiment of the invention, it was found that the hydrolysate isolated from fibroin inhibits DNA damage by a carcinogen. Various carcinogens may be used to induce DNA damage. The carcinogen used herein is a direct acting carcinogen, MNNG (N-methyl-N'-nitro-N-nitrosoguanidine). Inhibition effects of DNA damage by hydrolysate according to the present invention may be measured by performing Ames test and SOS chromotest. The test used herein is Comet test. The two former tests mentioned above employ Salmonella and E. coli. For this reason, they may produce inaccurate results due to different biological characteristics between microorganisms and animal cells, upon measurement of inhibition of DNA damage with respect to the animal cells. On the other hand, as for the Comet test used herein, it has an advantage in that physiologically active substances inhibiting DNA damage can be screened more accurately.

The Comet test is a method for characterizing DNA damage and repair thereof in various mammalian cells. Using this test, it is possible to detect DNA damage which can't be detected by a conventional method. Thus, this method is applicable to various fields including screening of various substances involved in carcinogenesis, genetic toxicology associated with DNA damage and repair thereof, monitoring of environmental pollution, and determination of a cancer prevention effect by Lactobacillus .

In still another embodiment of the invention, effects of the fibroin hydrolysate for improving immune activity were measured. A common method known in the art may be used for measuring improvement effect of immune activity. Especially, the method used herein is to measure reactive nitrogen species, for example, nitric oxide, produced by macrophages upon treatment of the hydrolysate, the reactive nitrogen species being representative chemical substances involved in immune responses. The increase in production of the reactive nitrogen species directly reflects the increase of TNF (Tumor Necrosis Factor) production. Accordingly, detection of increased production of the reactive nitrogen species indicates an effect of improved immune activity of the hydrolysate.

Macrophages responsible for a first line of immune surveillance in the body are known to play a significant role in a defense system against tumors. In controlling tumors, activated macrophages selectively recognize and eliminate tumor cells through a direct contact with the cells. Such activated macrophages secret various cytokines, nitric oxide and hydrogen peroxide. In the invention, secretion of nitric oxide by macrophages, induced by the hydrolysate, was measured. As a result, amounts of nitric oxide were increased by peptide components of the hydrolysate, resulting in improving immune activity, which is attributable to the hydrolysate, as shown in Fig. 2.

Thus, from these results, it could be found that the hydrolysate of the invention exhibits the effects as anticancer substances.

The invention is also directed to a processed food prepared by adding anticancer peptides in powder or other edible formulations, the peptides being obtained by hydrolyzing fibroin extracted from a silk thread or waste silk thread of silkworm cocoons, using an acid or a protease.

Since the fibroin hydrolysate according to the invention exhibits an anticancer effect, it is possible to produce functional foods containing the hydrolysate. Ingestion of such foods can inhibit DNA damage caused by a foreign carcinogen, and improve immune activity. If the fibroin hydrolysate containing peptides is added to foods in an edible formulation, the formulation is not limited. But a powder form of the hydrolysate is preferable for addition to processed foods. Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: Fig. la is a result of gel filtration chromatography using Sephadex G-25 with respect to a fibroin hydrolysate;

Fig. lb is a result of gel filtration chromatography using Sephadex G-15 with respect to a peptide fraction corresponding to a third peak of Fig. la; and

Fig. 2 is a graph showing a change in amounts of nitric oxide produced upon treatment of a fibroin hydrolysate to intraperitoneal macrophages of mouse.

Best Mode for Carrying Out the Invention

Hereinafter, the present invention will be described in detail, in conjunction with various examples.

These examples are provided only for illustrative purposes, and the present invention is not to be construed as being limited to those examples.

Example 1

Preparation of hydrolysate from silkworm cocoon

1- 1 : Isolation of silk thread consisting of fibroin from silkworm cocoon

Selected cocoons were treated with 10 % sodium carbonate (Na₂CO₃) and heated for 1 hr. The resulting solution was filtered, removing solubilized sericin, thereby obtaining a silk thread consisting of fibroin.

1-2: Preparation of acid hydrolysate

One part of the cocoon consisting of fibroin obtained as in Example 1-1 was added with 80 parts by weight of 2 N HC1, and heated at 100^°C for 48 firs, thus hydrolyzing fibroin. The hydrolysate thus prepared was dark brown. Using a 2 N sodium hydroxide solution, the hydrolysate was neutralized, adjusting to pH 7.4. The neutralized hydrolysate was then added with active carbon in an amount of 6 % relative to a total amount of the solution. The solution was stirred for 60 min to remove diverse non-dissolved substances and abnormal odor generated in the process. The solution was then filtered, obtaining a clear liquid hydrolysate. The hydrolysate solution was finally dialyzed with distilled water for 1 day, using a dialysis membrane, thereby removing salts.

1- 3: Preparation of enzymatic hydrolysate The silk thread consisting of fibroin obtained as in Example 1-1 was digested into a variety of sizes of peptides by using protease.

First, the silk thread consisting of fibroin was added with a 30 % CaCl₂ solution and heated at 90 ^°C for 30 min to solubilize it. The solubilized fibroin was then dialyzed with distilled water for 3 days. After dialysis, the resulting solution was treated with trypsin (Sigma) at 37 ^°C (pH7.5), pepsin (Sigma) at 37 ^°C (pH 2.0),

Alcalase (Novo Nordisk) at 55 ^°C (pH 7.5), or Neutrase (Nove Nordisk) at 45 ^°C (pH 6.2). In a course of such reaction, 3 ml of samples were collected every 1 hr, and heated at 100^°C for 10 min to stop the enzyme reaction. The enzyme reaction lasted for 8 l rs. Each enzyme was added at a concentration of 0.1 to 5 %.

The hydrolysate thus prepared was centrifuged at 13,000 rpm for 15 min. The supernatant was collected and subjected to ultrafiltration using a centrifugal ultrafiltration kit (Vivaspin 20, Satorius, Germany). The filtrate was assayed for an anticancer effect. Meanwhile, with respect to the filtrates prepared thus, peptide quantification was performed by the Lowry method. Based on differences between the measurements of the peptide and the concentrations of proteins measured before hydrolysis, peptide yields, that is, degree of hydrolysis (DH), were calculated. Concentration and reaction time of enzymes were determined by finding the maximal hydrolysis conditions where DH is not further increased with increased reaction time. The results showed that the maximal hydrolysis effects are exhibited by treating the hydrolysate with 1 % trypsin for 4 hrs, 0.5 % Alcalase for 2 hrs, 2 % Neutrase for 2 hrs, and 5 % pepsin for 4 hrs.

Example 2

Treatment of hydrolysate with direct acting carcinogen (MNNG)

2- 1 : Animal cell culture

The cell line used herein was a normal mouse embryo 3T3 cell line (ATCC CCL No. 163), provided from the Korean Collection for Type Cultures (KCTC). As for a cell culture medium, DMEM (Gibco, USA) was supplemented with 10 % heat- inactivated fetal calf serum (Hyclon, USA), 100 unit/ml of 10 % penicillin G (Sigma) and 100 mg/ml of streptomycin sulfate (Sigma, USA). The 3T3 cell line was maintained on a 10 cm round culture dish (Falcon, USA) containing the DMEM medium at 37 ^°C, and 5 % CO₂ in a humidified incubator (Labline instruments, USA). The cells were subcultured every 3 days.

2-2: Treatment of hydrolysate with direct acting carcinogen

The direct acting carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine was diluted in HBSS (10 mM, pH 7.4) to a concentration of 100 g/ml. Each of the hydrolysates obtained as in Example 1 was also dissolved in HBSS (10 mM, pH 7.4) to concentrations of 10 mg/ml, 5 mg/ml, 2 mg/ml, and 1 mg/ml, respectively. To the hydrolysates with varying concentrations prepared above, 10 μi of MNNG dissolved in HBSS in the above way was added, giving a final concentration of 1 g/ml MNNG. The samples were pre-incubated for 30 min. As a negative control, HBSS only was used. As a positive control, HBSS containing MNNG at a final concentration of 1 zg/ml was used. After pre-incubation, each sample was added to the 3T3 cells, which had been cultured to a density of 1 x 10⁵ cells/plate on 3 cm diameter round culture dishes (Falcon, USA) at 37^°C, and 5 % CO₂ in the humidified incubator, followed by incubation for 30 min.

Example 3

Measurement of inhibition of DNA damage by hydrolysate 3- 1 : Preparation of pre-coated agarose gel slide

NMA (Normal Melting Point Agarose, Sigma, USA) was added to PBS to a concentration of 0.5 %, heated using a microwave, and placed in a water bath maintained at 60 ^°C . 35 μl of the NMA solution was applied onto a frosted area of a fully frosted slide glass (ERIE Scientific, USA), followed by drying. The slide glass was again smoothly applied with 75 μl of the NMA solution, using a cover glass (24 x 50 mm, Superior, Germany), and the agarose layer was hardened by placing the slide glass on ice in a flat stainless steel tray. The pre-coated agarose gel slide was transferred to a humidified slide box and stored at 4 ^°C .

3-2: Cell preparation

The 3T3 cells were cultured for 72 hrs, while being subcultured by placing 3 x 10⁴ cells/plate on a 3 cm round culture dish. After 4 days, the cells were washed twice with Ca²⁺, Mg²⁺-free PBS. The cells were then treated with a mixture of MNNG and the hydrolysate, the hydrolysate being at a concentration of 10 mg/ml, 5 mg/ml, 2 mg/ml, or 1 mg/ml, prepared as in Example 2-2. The cells treated with HBSS containing MNNG served as a positive control, while the cells treated with HBSS only served as a negative control. The cells thus treated were incubated at 37^°C for 30 min at a speed of 25 rpm in a shaking incubator. After incubation, the cells were immediately washed three times with PBS. With a 100 μl of proteinase K (Sigma, USA) solution, the cells were completely detached from the tissue culture dish. The cells were resuspended in a 1 ml culture medium and transferred to an Eppendorf tube. The cell suspension was centrifuged at 1,000 rpm for 5 min, and the supernatant was discarded. The cell pellet was resuspended in a 300 μl of medium. From this, 30 μi was again centrifuged, obtaining a cell pellet. 3-3: Comet test

The cell pellet obtained as in Example 3-2, which had been treated with a mixture of MNNG and varying concentrations of the hydrolysate, HBSS containing MNNG, or HBSS only, were suspended in 75 μl of a 0.75 % LMA (Low Melting Point Agarose) solution which had been maintained at 45 °C in a water bath. The cell suspension mixed with LMA was transferred onto the coated slide prepared as in Example 3-1, using a pipet. The slide was covered with a coverslip to ensure that the cell suspension was smoothly spread, and placed in a flat stainless steel tray filled with ice, to harden for 10 min. After 5 min, the coverslip was removed, and 75 μl of the 0.75 % LMA solution was again added onto the slide glass which has been applied with the 3T3 cells. The slide was covered with a coverslip and again placed in a flat stainless steel tray filled with ice, to harden for 10 min. The coverslip was then slid off, and the slide was immersed in a cold alkaline lysing solution for 1 hr, thereby lysing all intracellular components except nuclear bodies. All slides immersed in the lysis buffer for 1 hr were then laid on a horizontal electrophoresis reservoir. The reservoir was filled with an electrophoresis buffer until the liquid level reached 0.7 cm above a surface of the slide, followed by letting stand for 20 min. Electrophoresis was then conducted at 25V/300mA for 20 min. After electrophoresis, each slide was washed with a neutralization buffer. The slide was stained with 80 μl of YOYO-1 (Molecular Probes, USA), a fluorescent dye, at a concentration of 10 g/ml to examine the degree of DNA damage in the 3T3 cells treated with a mixture of MNNG and varying concentrations of the hydrolysate, HBSS containing MNNG, or HBSS only, as in Example 3-2. The stained slide was observed at a magnification of 250X, using a fluorescent microscope (CSB-FEI, China) with a 200 W mercury lamp (Osram, Germany). Individual images of the region of the nucleus captured through a CCD camera (COHU, USA) were analyzed using a comet image analyzing system (Perceptive instrument, UK) installed in a computer. Degrees of DNA damage in individual nuclei caused by the mutagen and inhibition of such DNA damage by the peptides were estimated, in terms of tail length and tail moment of the comet images with respect to 101 nuclei per slide. The values were subjected to a statistical analysis. The degrees were quantified as mean ± SD values. The results are shown in Table 1.

Table 1 : Results of inhibition effect of DNA damage by fibroin hydrolysate to

MNNG

Positive Control 142.04 ±24.18 30.20 ±12.10 lOmg 49.60 ±21.38 3.34 ±3.98

5mg/m£ 78.60 ±34.31 4.18 ±3.0

2mg/m^ 110.63 ±26.95 9.69 ±7.36

I gM 117.14 ±31.63 11.80 ±6.43

As shown in Table 1, upon treatment with fibroin hydrolysates at 10 mg/ml, 5 mg/ml and 2 mg/ml, significant inhibition effects on DNA damage were seen.

Moreover, to elucidate a mechanism of fibroin hydrolysate action for inhibiting DNA damage, the hydrolysate of a concentration of 4 mg/ml was mixed with MNNG, followed by pre-incubation. The hydrolysate was thus reacted with MNNG, and the mixture was added to 3T3 cells. Degrees of inhibition of DNA damage were examined. The results are shown in Table 2.

Table 2: Results of elucidating an inhibition mechanism of DNA damage by fibroin hydrolysate

Comet Parameter Conditions of Tail length Tail moment hydrolysate treatment (mean ± SD) (mean ± SD)

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 Table2, the sample 1 refers to cells with a certain degree of DNA damage where the hydrolysate and MNNG were pre-incubated, allowing a reaction therebetween, and then the mixture was added to the 3T3 cells. The sample 2 refers to cells with a certain degree of DNA damage where the hydrolysate and MNNG were separately added to the 3T3 cells, without pre-incubation thereof. Each of the samples 3 and 4 refers to cells with a certain degree of DNA damage where the hydrolysate was first pre-incubated with the 3T3 cells for 30 min, allowing a reaction between the peptides and cells to occur. As for the sample 3, the hydrolysate added to the cells was washed three times with PBS, and then the cells were incubated with MNNG for 30 min, that is, without the hydrolysate. As for the sample 4, without washing off the hydrolysate, the cells were further incubated with MNNG for 30 min, with the hydrolysate. As shown in Table 2, no effect of inhibition of DNA damage was seen in the sample 2. These results infer that the peptide components of the hydrolysate bind to MNNG, preventing MNNG from acting as a DNA alkylating agent. Thus, on the basis of such a mechanism, DNA damage is inhibited.

In addition, the samples 3 and 4 exhibited reduced DNA damage. Consequently, it can be inferred that the mechanism by which the hydrolysate inhibits DNA damage is by reacting with the carcinogen and cells, thereby functioning to inhibit DNA damage.

Example 4

Isolation of active peptide fraction from hydrolysate and measurement of degree of inhibition of DNA damage

Each of the hydrolysates obtained as in Example 1 was subjected to gel filtration chromatography, and fractionated according to molecular weights of peptides. With respect to respective peaks for the hydrolysate, the degrees of inhibition of DNA damage were measured. Gel filtration chromatography on Sephadex G-25 showed three peaks, having molecular weights between 1,000 and 5,000, as shown in Fig. la. The peptides corresponding to respective peaks were separated and each peptide fraction was lyophilized. Using the Comet assay described above, the degrees of inhibition of DNA damage versus a carcinogen were measured. Among those fractions of the hydrolysate, the fraction of a third peak, whose activity had been confirmed, was refractionated using Sephadex G-15. The chromatography showed again three peaks. Among those fractions, the peptides corresponding to a third peak were subjected to the

Comet test. The comet test showed that the activity for inhibition of DNA damage by the peptides of the third peak is 1.5 times higher than that of the crude hydrolysate, as shown in Fig. lb.

Example 5 Measurement of improvement of immune activity

To determine that the hydrolysate of the silk thread consisting of fibroin improves an immune activity, it was examined whether intraperitoneal macrophages of mouse increase production of nitric oxide upon treatment with the hydrolysate.

5-1 : Culture of macrophage BALB/c mice were injected intraperitoneally with 1 ml of a 1 % solution of thioglycollate. After 4 days, the mice were sacrificed by cervical dislocation. 10 ml of RPMI 1640 was injected to the abdominal cavity, and intraperitoneal cells were collected after flushing three times, followed by centrifuging. The intraperitoneal cells were suspended in RPMI 1640 containing 10 % FBS, and 200 μl each was plated on a 96 well plate. The cell number per well was 2 x 10⁵. The plate was incubated at 37 ^°C under 5 % CO₂. After 2 hr incubation, the cells were washed with phosphate buffered saline (pH 7.4) to remove non-adherent cells from the plate. The adherent macrophages were subject to continued incubation. To each well, the filtered sterile hydrolysate sample was added at a concentration of 0.01 to 10 mg/ml, and interferon-γ was added after 16 hrs, thereby activating macrophages. After 24 hour's incubation, a portion of the culture was collected, and the amount of nitric oxide produced during incubation was measured. The cell culture obtained by treating with 1 μgl l of lipopolysaccharide served as a positive control.

5-2: Measurement of production of nitric oxide

Amounts of nitric oxide, one of reactive nitrogen species produced from macrophages, were measured by quantifying amounts of nitrite accumulated in the culture of the activated macrophages.

Each 50 μl of the supernatant was collected from the macrophage culture and transferred to another 96 well plate. An equal volume of Griess Reagent (0.05 % naphthylethylenediamine dihydrochloride, 0.5 % sulfanilamide, 2.5 % H₃PO₄) was added to each well. After 10 min, absorbance at 550 nm was measured using an

ELISA reader. Sodium nitrite was used as a standard. As shown in Fig. 2, the amounts of produced nitric oxide increased depending on concentrations of the hydrolysate added to the macrophage culture. From these results, it can be inferred that components of the hydrolysate such as amino acids or peptides induce production of nitric oxide in macrophages, thereby increasing immune activity. Industrial Applicability

As apparent from the above description, the present invention provides an anticancer agent comprising peptides obtained from the fibroin hydrolysate which was extracted from silk threads or waste silk threads of silkworm cocoons. The peptides are capable of inhibiting DNA damage caused by foreign carcinogens and improving immune activity. Those peptides of the invention may be applied in the future to produce medications and functional foods exhibiting anticancer effects, thereby contributing to development of medicines for prevention and treatment of cancer.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

What is claimed is:

1. An anticancer agent comprising peptides obtained by treating fibroin with an acid or a protease, as an active ingredient, the fibroin being extracted from a silk thread or waste silk thread of silkworm cocoons.

2. The anticancer agent according to claim 1, wherein the fibroin is obtained by removing sericin from the silk thread or waste silk thread of silkworm cocoons.

3. The anticancer agent according to claim 2, wherein the sericin is removed using sodium carbonate (Na₂CO₃) or sodium bicarbonate (NaHCO₃) at a concentration of l to l0 %.

4. The anticancer agent according to claim 1, wherein the acid is hydrogen chloride or oxalic acid.

5. The anticancer agent according to claim 1, wherein the protease is one or more selected from the group consisting of trypsin, pepsin, Alcalase, and Neutrase.

6. The anticancer agent according to claim 1 or claim 5, wherein the protease is added at a concentration of 0.1 to 5 %.

7. A processed food prepared by adding peptides in powder or other edible formulations, the peptides being obtained by treating fibroin with an acid or a protease, the fibroin being extracted from a silk thread or waste silk thread of silkworm cocoons.