KR20070093758A - Isolation, purification and availability of lunasin peptide as a prevention and curing of skin aging and allergy, anticancer and highly functional foods from soybean and barley - Google Patents

Abstract

A method for isolating and purifying lunasin peptide from soybean and barley is provided to obtain the desired product, which is pharmaceutically stable, has the cancer inhibitory activity, is able to prevent and inhibit skin cancer and is applied as a light blocking agent, a stabilized preventive medicine not causing allergy, an anti-cancer agent and a highly-functional food. A method comprises the steps of: (a) after extracting seeds of soybean and barley with a solution consisting of a secondary distilled water including a protease inhibitor, sodium acetate and sodium phosphate buffer solution, centrifuging it; (b) after dialyzing the supernatant obtained from the step(a) with distilled water to remove protein having a molecular weight of less than 3,000 Da therefrom, lyophilizing it to obtain a crude extract; and (c) after primarily purifying the crude extract using an ion exchange column chromatography and secondarily purifying it with a high speed liquid chromatography, thirdly purifying it with an immuno-affinity column. The composition for preventing and curing skin aging, allergy and atopy comprises the lunasin peptide as an effective ingredient.

Description

Isolation, Purification and Availability of Lunasin Peptides as a Prevention and Curing of Skin Aging, Isolation, Purification and Availability of Soybean and Barley and Allergy, Anticancer and highly functional Foods from Soybean and Barley}

Figure 1 is an electrophoresis picture for the isolation and identification of lunasin peptide from various legumes,

Figure 2. Electrophoresis picture for the isolation and identification of various kinds of barley varieties and crops lunasin peptide (lunasin peptide),

Figure 3 is a photograph of the separation and purification of lunasin peptide (lunasin peptide) by using ion exchange column chromatography from soybean and barley extract crude protein,

Figure 4 is a photograph showing the identification of lunasin peptides by Malditope (MALDI TOF) from purified crude extracts of soybeans and barley,

Figure 5 is a correlation graph of concentrations by Western blot of peptides by synthetic lunacin concentration, in order to quantify the lunacin peptide content according to the contrast of the band by Western blot,

Figure 6. Inhibition activity of lunasin peptide (lunasin peptide) purified from soybean and barley protein by IPTG-induced ras -oncogene stably transfected 2-12 cells (A) and lunasin (lunasin) concentration (B) ) Is a graph of.

Figure 7 is a photograph showing the inhibition of histone H3 in cells upon treatment of lunasin peptide purified from soybean and barley,

8. HPLC graph of the isolation of lunasin peptide in rat blood using HPLC after rat 28-day feeding of lunasin peptide-rich crude protein extract from soybean and barley. ,

9. Graph of identification of lunasin peptde from rat blood and liver after rat 28-day feeding of lunasin peptide-rich crude protein extract from soybeans and barley.

Figure 10. Inhibition of foci formation of lunasin peptde extracted from rat liver after rat 28-day feeding of lunasin peptide-rich crude protein extract from soybeans and barley Is a graph about.

Figure 11 is a photograph (A) of lunasin peptide-rich crude protein powder from soybean and barley, and a photograph of a lunasin peptide western blot of powdered lunasin peptide.

12. Pictures of lunasin peptide-rich crude protein from soybeans and barley.

The present invention is to isolate and purify the lunasin peptide (Lunasin peptide) from soybean and barley seed protein, the production mechanism of the lunasin peptide (lunasin peptide), cancer cell proliferation inhibitory, histone acetylation (Histone acetylation) inhibition and mouse The present invention relates to the usefulness of accumulation in tissues after feeding and inhibiting the proliferation of skin cancer cells after feeding to mice and rats.

Soybeans contain phytochemicals such as various protein inhibitors, phytate, phytosterols, saponins, and isoflavones, which are predicted to have anticancer activity. The most effective anti-cancer substance in the field is recognized as a Bowman-Brik inhibitor (BBI), and the inventors of the present invention showed that lunasin peptides (lunasin peptide) in cancer-induced cells have higher anticancer activity than BBI. Have been identified (Jeong et al, 2005).

Lunasin is a small subunit peptide of 2S albumin encoded by Gm2S-1, a cotyledon-specific cDNA of soybeans (Galvez and Lumen). , 1999), first discovered from soybean by Odani et al. (1972) and sequenced. Lunasin consists of 43 amino acids and has 8 poly asprtic acids at the C terminus (Odani et al., 1987) adhesion motif Arg-Gly-Asp (RGD) and Heryx. It is characterized by having a (helix) structure, and the RGD motif is easily attached to tumor cells (Ruoslahti and Pierschbacher, 1986).

Lunasin peptide (lunasin peptide) binds to the chromatin of the cell (binatin) and inhibits mitosis by killing the cell has been suggested to show anti-cancer activity, when administered lunasin (lunasin) peptide from the outside Carcinogen treatment has been reported to block the conversion of rat fibroblasts into cancer cells (Galvez and Lumen, 1999).

Synthetic lunasin inhibited histone acetylation in MCF-7 and C3H cells and foci formation in cancer-induced C3H cells (Galvez, et al., 2001).

Synthetic lunasin has been shown to inhibit skin cancer tumor generation using the SENCER mouse skin tumor model (Galvez and Lumen, 2001).

Currently, domestic research on lunasin is limited to professors de lumen of UC Berkeley in the United States and researchers in Korea.

A study on the biosynthesis of lunasin is reported by Galvez (1997) that the Gm2S-1 gene is the first to appear three weeks after flowering and completely disappears at seven weeks. Jeong et al. (2001, 2002) reported that the expression of lunasin peptides from soybean and barley began at 5 weeks after flowering and reached their maximum after 8 weeks of full maturation.

In our first joint study, lunasin isolated from soybeans and barley inhibited in vitro histone acetylation and colony formation in cancer-induced 2-12 cells. Inhibition was reported (Jeong, 2003).

The importance and research needs of the effective utilization of lunasin, a soy peptide with anticancer activity, has emerged.

Recently, as interest in functional foods has increased in Korea, soybean consumption has increased, and attention has been focused on industries using the same. Isoflavones are purified through the preparation of hides generated during the tofu manufacturing process, which is used as medical products and cosmetics. However, recent studies have reported that genistein, a type of isoflavone, inhibits the activity of tamoxifen, a breast cancer drug (Young, 2002). It is costing you.

Accordingly, the present invention has in mind the following technical problems.

1) To develop a high-efficiency separation technology of lunasin peptide from soybeans, and to provide a basic technology to separate lunasin high content from medicinal plants as well as other crops and to use it as a cancer prevention agent.

2) The establishment of a method of separating lunasin through the present invention can develop a product such as functional cosmetics by purifying lunasin peptide through the preparation of tofu hides in Korea.

3) Determination of the degree of degradation of lunasin in seed germination enables the development of functional germination in recent years by methods that can delay or inhibit lunasin peptides.

4) In order to meet the consumer's desire to purchase in the future, it is a product with medicinal functions with clear efficacy, and the nature of nutraceuticals is required, so the identified lunasin peptide is more effective than the current dietary supplements and supplements. It is expected that more functional products, which are closer to drugs, can be produced. In particular, lunasin peptides have material properties that can improve the quality of general foods, fortified foods, and specialty foods as functional additives.

5) Isolation of lunasin peptide from soybeans enables the production of differentiated products such as Bauman Brick Inhibitor (BBI), isoflavon, and other known anticancer drugs, and its functional utility can be recognized as an anticancer agent.

6) To date, BBI and isoflavon products as anticancer agents have a possibility of being mixed with lunasin peptide in production, thus allowing pure purification of these products.

7) It can be used as cosmetics and pharmaceutical raw materials due to mass production of lunasin.

An object of the present invention is to provide a method for isolating and purifying lunasin peptide (Lunasin peptide) from the seed protein of soybean and barley, and to inhibit the cancer cell proliferation, histone acetylation Anti-cancer, skin anti-aging, light allergy, skin allergy and high functional fortified foods as a prophylactic drug by inhibiting (Histone acetylation) and accumulating in tissues after feeding and inhibiting proliferation of skin cancer cells. And for use as a raw material used as a food additive.

The present invention relates to a method for isolating and purifying lunasin peptides from soybean and barley seed proteins, and further inhibiting cancer cell proliferation, inhibiting histone acetylation and feeding to rats. Post-tissue accumulation and lunasin content changes during germination were studied.

That is, the present invention extracts and separates water-soluble proteins using solvents such as water, sodium phosphate and sodium chloride using soybean and barley seeds grown in Korea, and freeze-dried to remove ions. Exchange chromatography, high performance liquid chromatography. Lunasin was isolated and purified by methods such as immuno-chromatography, and then fed to anticancer cells and rat oral rats to confirm their anticancer activity.

More specifically, in the present invention, the seeds of soybeans and barley were added to the secondary distilled water, sodium acetate and sodium phosphate complete layer containing fresh furotease inhibitor, extracted at 4 ° C. for 24 hours, and centrifuged to take a supernatant. Dialysis with distilled water to remove proteins below 3,000 Da, freeze-dried to obtain crude extract, and then the crude extract was first purified by ion exchange column chromatography, second purified by high-performance liquid chromatography, and then immuno-affinity column The present invention relates to a method for isolating and purifying lunacin peptides, characterized by tertiary purification, and for photoblocking, skin aging, atopy prevention and prevention agents, and blood, liver and kidney in skin diseases. , Lung, skin cancer treatment agent, and functional foods having the treatment and prevention of blood, liver, kidney, lung, skin cancer.

1) Separation of lunasin peptide : systematization of lunasin separation and purification according to extraction solvent.

-Adjustment of extraction solvent (Separation due to the use of secondary distilled water (DDH ₂ O), sodium acetate, protease inhibitor, pH change, etc.)

To extract lunasin, the seed samples of soybeans and barley were ground using a grinder, and then 1 L of each extraction solvent was added and extracted at 4 ° C. for 24 hours, followed by removal of the insoluble material with 7 layers of gauze. The supernatant is taken by centrifugation for 30 minutes at 4 ℃, 14,000 rpm. Extraction solvents include distilled water (DDH ₂ O) with Fresh Protase Inhibitor (Sigma), 0.1 M Sodium Acetate (pH 3.85), 0.1 M Sodium phosphate (pH) 7.0) Use a buffer. The extract removes proteins with a molecular weight of less than 3,000 Da through dialysis. Dialysis is carried out for 24 hours while changing the distilled water every 6 hours in 5 L of secondary distilled water after the extract (membrane) (MWCO 3500), lyophilized to obtain a lunasin (lunasin) crude extract.

2) Purification : (ion-exchange column chromatography: use of resin by type, establishment of purification conditions according to NaCl concentration, crude using crude HPLC and MPLC (Medium Pressure Preparative Liquid Chromatograph) lunasin) separation and purification).

Ion-exchange column chromatography is carried out as a primary method for purifying lunasin from lunasin crude extraction. The stationary phase is placed in 100 g of Biogel resin AG1-X4 and mesh size 100-200 (Bio-Rad Labs) in 1 L of 0.1 M sodium phosphate buffer (ph 7.0). After stabilizing at 4 ° C. for 4 hours, the column (12 cm in diameter, 200 cm in height) is filled. 10 g of Lunasin crude extract is dissolved in 30 ml of 0.1 M sodium phosphate buffer (ph 7.0), loaded and then separated. The mobile phase is prepared in such a manner that the concentration of NaCl in 0.1 M sodium phosphate buffer (ph 7.0) is 0 M, 0.1 M 0.3 M, 0.5 M, 0.7 M, and 1 M, followed by sequential fractionation.

   The first purified extract by ion-exchange chromatography is second purified using HPLC. The stationary phase uses a Jupiter 10 μ proteo 90 A column (250 × 10 mm, Phenomenex), and the mobile phase uses acetonitrile and water (ACN: H 2 O = 3: 7). Separation peaks are identified at 254 nm using a UV detector. The lunasin fraction fractionates the peak at the same position as the synthetic lunasin. Subsequently, pure lunasin was purified using an immunoaffinity column.

3) from seeds Crude protein In extract Lunasin  Sympathy.

Samples for analysis were ground soybean and barley seeds (150 g) using liquid nitrogen, and then 100 ml of 0.1 M phosphate buffer (pH 7.0) was extracted for 24 hours at 4 ° C, followed by 7-ply gauze. After removing the insoluble matter, the supernatant was centrifuged for 30 minutes at 4 ℃, 14000 rpm to take a supernatant.

Protein separation of soybean and barley was performed by SDS-PAGE (SDS-polyacrylamide gel electrophoresis). During SDS-PAGE, the resolving gel was finally prepared to 0.1% SDS, 15% acrylamide concentration, and the stacking gel was 0.1% SDS, 4% acrylamide. The concentration was prepared. Loading sample was prepared by mixing the protein extracted from the pepper seeds with Laemmil buffer 1: 2 and using a sample cut at 100 ° C. for 5 minutes and injecting 25 ㎍ of protein per lane, followed by electrophoresis. The time voltage was carried out at 150 V for 1 hour. After electrophoresis, the gel is shacked for 3 hours with a staining solution (1 g of Coomassie brilliant blue / 450 ml of methanol + 100 ml of acetic acid + After staining in 450 ml of DH ₂ O, the bands were identified by destaining in a destaining solution (methanol 100 ml + acetic acid 100 ml + DH ₂ O 800 ml). The gel was electrophoresed for identification of lunasin peptide and transferred to PVDF membrane (Sigma, USA) for 1 hour and 40 minutes at 100 V. Protein-transferred PVDF membranes were rinsed with methanol, blocked with Blotto A (5% nonfat milk and 1% Tween 20 in saline) for 1 hour 30 minutes, and then TBS-T solution (1% Tween 20). in tris-buffered saline) and washed three times. The washed PVDF membrane was added to the solution diluted with Blotto B (3% nonfat milk and 1% Tween 20 in saline) and the primary antibody Zymed R1 in 5000: 1 for 1 hour, and then shaken again. TBS-T The solution was washed three times. Anti-rabbit antibody (Santacruze Biotec. SC2030) was used as a secondary antibody, and diluted with Blotto B 1: 5000 and blocked for 1 hour. After washing three times with TBS-T solution, it was reacted with ECL reagent (Amersham) and then exposed to X-ray film (X-ray film).

4) To Malditop (MALDI TOF)  by mass  Mass mapping and Sequence.

Purified protein eluted with 0.7 M NaCl sodium phosphate buffer (0.1 M, pH 7.0) on ion exchange chromatography in a protein structure laboratory at Davis, University of California Mass mapping and sequencing by mass mapping and sequence by MALDI TOF.

5) Lunasin Of lunasin peptide  Usefulness.

-2-12 cells Colony  Formation inhibition

The cells used in the experiment is a tumor causing genes Ha-Ras on Kogen in (Ha- r as oncogene) (valine mutation at codon 12) the NIH 3T3 2-12 6-well plate (well plate) in the cell line are made to switch to the cell It was used for colony formation inhibition experiment. The degree of cell culture and inhibition of colony formation was determined by the method of Jeong et al. (2002). 2-12 transgenic cell lines have strong metastases including high Ha- r as oncogenes and independent adherence by induction of isopropyl β- D- thiogalactoside (IPTG, Sigma) Properties (Liu et al., 1992; Liu et al., 1998). Cell lines were cultured on a regular basis and the culture conditions were maintained at 5% CO ₂ and 37 ℃. DMEM (Dulbecco ^, Modified Eagle medium) containing 10% calf serum was used as the culture. 120 μl of 5% agar was diluted in 880 μl of culture medium per well, and 1 ml of the 0.6% base agar layer was injected and left at room temperature for 15 minutes. The upper agar layer was injected with 550 μl of 0.6% agar, 330 μl of culture, 20 μl of 1 M IPTG and 100 μl of 2-12 cells (40,000 cells / ml) at 1 ml each well. Cells were cultured in a CO ₂ incubator for 14 days and observations of the colonies formed were performed under an optical microscope (× 40). The treated sample was treated with purified lunasin peptide at various concentrations.

Cores in animal cells Histone Acetylation (Core Histone Acetylation Suppression.

Human breast cancer cells MCF-7 (ATCC) were grown in an incubator at 37 ° C., 5% CO 2 under DMEM + 10% FBS medium. 10 mM soybean lunasin purified by immunoaffinity column chromatography was incubated for 24 hours in 5 mM sodium butyrate, a histone deacetylase inhibitor. Acid extraction was performed to investigate the degree of inhibition of core histone acetylation by lunasin (lunasin) by SDS-PAGE and Western blot.

  - In animal tissue Lunasin Of lunasin peptide  Inhibition of accumulation and foci formation.

Various plant seeds containing lunasin peptide were extracted with water and lyophilized and fed to 5 week old rats by feeding 8.7 g per day for 4 weeks, and rats after 4 weeks. Tissues and organs were extracted and separated to confirm the accumulation of lunasin by SDS-PAGE and Western blot.

In order to investigate the anticancer activity of lunasin peptide accumulated in animal tissues, blood and liver of rats wetted for 4 weeks were extracted and purified and the degree of inhibition of foci formation was investigated.

Soybean and barley seed proteins were extracted with a solvent (water, sodium acetate, 0.1 M NaCl) to identify 4.8 KD of lunasin peptide by SDS-PAGE and Western blot.

For the purification of lunasin peptides, pure and large amounts of lunasin peptides were obtained when eluted with 0.7 M NaCl phosphate buffer under ion exchange column chromatography. . The sequence of Lunasin Peptide in barley and soybean was composed of 43 amino acids.

Purified lunasin peptide inhibited ras -oncogene and core H3-histone acetylation, and when lunasin-containing protein extracted from soybeans and barley was fed to rats for 4 weeks , Lunasin peptides were accumulated from blood, liver, lung and heart of rats, and lunasin peptides extracted from these livers and blood were extracted during foci formation inhibition experiments. Cancer cell formation was inhibited.

6) Lunasin Peptide ( lunasin  peptide) high Inclusion  Preparation of Containing Formulations.

  - Lunasin Peptide ( lunasin  peptide) Preparation of high content dietary powders.

After washing the beans in cold water for about 4 hours, air-dried, roughly crushed to about 1/15 size of beans, put 200 liters of water in 10 kg soy flour, shake for 48 hours at 4 ℃, and then hold for 48 hours. . The supernatant was centrifuged at 20,000 rpm for 30 minutes, and then the lipids, insolubles and precipitates in the suspended layer were removed and then lyophilized.

- Lunasin Peptide ( lunasin  peptide) high content tablet preparation.

After freezing the dried material prepared with lunacin peptide high-density powder medicine through the ion-exchange chromatography, the extract dissolved in 10 times the distilled water (DDH ₂ O) of capacity was dialyzed. ) To remove proteins with molecular weight less than 3k Da. Dialysis was carried out for 24 hours while the extract (membrane) (MWCO 3000) in distilled water every 6 hours in distilled water and then freeze-dried to obtain a powder, and then made a tablet by granulation and tableting process.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited to the following Examples.

Example 1 . Isolation and Identification of Lunasin Petide from Soybean and Barley.

After extracting various varieties of soybeans and barley with water for 24 hours, insoluble matters were removed with 7 layers of gauze, the supernatant was centrifuged at room temperature, and the supernatant was taken and lunasin was obtained by SDS-PAGE and Western blot. ) Is isolated and identified (Figs. 1 and 2), and quantitative graphs were prepared using synthetic lunacin peptides for quantification thereof (Fig. 5). Glycine max only confirmed the presence of a lunasin peptide (lunasin peptide), but not identified in legume seeds such as peanuts, red beans, and the like, but not in the glycine ( Glycine ) genus. The lunasin peptide content between the soybean varieties of Glycine max was 0.045-0.156 mg per g seed (Table 1). Barley was 0.013-0.021 mg (Table 2).

Table 1. Content of lunasin peptide by soybean varieties.

Table 2. Content of lunasin peptide by barley varieties.

Example 2 . Establishment of a method for the purification of lunasin peptide from soybean and barley extracts.

Ion-exchange column chromatography was performed as a primary method for purifying lunasin from lunasin crude extraction. Stationary phase was added to 10 ml of Biogel resin AG1-X4, mesh size 100-200 (Bio-Rad Labs) in 100 ml of 0.1 M sodium phosphate buffer (ph 7.0), and then After stabilization for a period of time, the column (1.2 cm in diameter and 20 cm in height) was filled and 1 g of Lunasin crude extract was dissolved in 3 ml of 0.1 M sodium phosphate buffer (ph 7.0) and then loaded. ) And separated. The mobile phase is prepared in such a manner that the concentration of NaCl in 0.1 M sodium phosphate buffer (pH 7.0) is 0 M, 0.1 M, 0.3 M, 0.5 M, 0.7 M, and 1 M, followed by sequential fractionation.

Thus, when eluted with 0.7 M NaCl sodium phosphate buffer, more than about 99.5% of lunasin peptide (lunasin peptide) attached to the resin in the ion exchange column could be extracted (FIG. 3).

Example 3 . Isolation of Lunasin Peptides by HPLC.

As a result of secondary purification using HPLC for the first purified extract by ion-exchange chromatography (FIG. 9), the stationary phase was a Jupiter 10 μ proteo 90 A column (250 × 10 mm, Phenomenex) and the mobile phase uses acetonitrile and water (ACN: H2O = 3: 7). Separation peaks are identified at 254 nm using a UV detector. The lunasin fraction fractionates the peak at the same position as the synthetic lunasin.

Example 4 Mass mapping and sequence of lunasin peptides by MALDI TOF of purified crude extracts of soybeans and barley.

As a result of confirming that the lunasin peptide is present in the purified material from the soybean and barley extract by mass mapping by MALDI TOF (FIG. 4), 43 amino acids of SKWQHQQDSCRKQLQGVNLTPCEKHIMEKIQGRGDDDDDDDDD It was confirmed that the peptide (peptide) consisting of.

The sequence content is as follows.

Ser Lys Trp Gln His Gln Gln Asp Ser Cys Arg Lys Gln Leu Gln Gly

  1 5 10 15

Val Asn Leu Thr Pro Cys Glu Lys His Ile Met Glu Lys Ile Gln Gly

             20 25 30

Arg Gly Asp Asp Asp Asp Asp Asp Asp Asp Asp

         35 40

Lunasin is an anticancer protein isolated from soybeans in soybean, a small subunit of 2S albumin encoded by Gm2S-1, a cotyledon-specific cDNA (Galvez and Lumen, 1999), Structurally, it has eight poly asprtic acids at the C terminus, attracting attention to its physiological functions in the development of seeds (Earnshaw, 1987), and the inventors Galvez and Lumen (1999). By lunasin (lunasin) binds to the chromatin of the cell (chromatin) and inhibits mitosis, suggesting the anti-cancer activity by killing the cells, the lunasin peptide extracted from the plant (lunasin peptide) ) Is thought to have anticancer activity.

Example 5 . Cancer cell proliferation inhibition of lunasin peptide purified from soybean and barley.

Tumor-induced genes, r as oncogene Ha- (valine mutation at codon 12), the 6-well plate with 2 to 12 cell line was created by switching to NIH 3T3 cells (well plate) The results (Fig. 6) to inhibit the colony forming experiments in, Lunasin extracted and purified from soybeans and barley was highly resistant to cancer cell formation to the extent that it was not significantly different from the negative control. This shows that lunasin peptide (lunasin peptide) has a very high anticancer activity. In addition, 100 nM lunasin peptide isolated from soybean and barley inhibited 50% of cancer cells 2-12 cells.

Example 6 Inhibition of histone H3 acetylation of lunasin peptide from soybean and barley.

Purified 10 uM lunasin peptide extracted from soybean and barley was tested for histone H3 acetylation after MCF-7 cells (FIG. 7), MCF-7 cells. Treatment with 10 uM lunasin peptide in inhibited 100% histone H3 acetylation.

Example 7 Accumulation of Lunasin Peptides in Animal Tissue after Rat 28-Day Wet of Lunasin Peptide-Containing Crude Protein Extract from Soybean and Barley.

Crude extraction of soybeans and barley to make lunasin peptides high. Freeze-dried protein was fed to 60 rats of 5 weeks of age by feeding 8.7 g per day for 4 weeks, 4 After weeks, the blood, tissues and organs of rats were extracted with 0.1 M sodium acetate and confirmed the accumulation of lunasin by HPLC, SDS-PAGE and Western blot. (FIG. 7, 8), 17.54 μg lunasin peptide was accumulated in 1 ml blood of rat, and 3.06 μg lunasin peptide was accumulated in 1 g liver (FIG. 9).

Example 8 . Inhibition of foci formation of lunasin peptide purified by rat liver extract after rat 28-day wet of lunasin peptide-rich crude protein extract from soybean and barley.

After 4 weeks of wet lunasin peptide (lunasin peptide) to extract and purify the blood and liver of rats (Rat), as a result of examining the degree of inhibition of foci formation (Fig. 10), lunasin peptide (lunasin peptide) 14.2 foci were formed in the control which was not wetted), but the foci formation was suppressed to about 5.1 in the liver extract treatment.

Example 9 . Production and use of powder containing high leucine peptide from soybeans and barley.

After washing the beans in cold water for about 4 hours, air-dried, roughly crushed to about 1/15 size of beans, put 200 liters of water in 10 kg soy flour, shake for 48 hours at 4 ℃, and then hold for 48 hours. . The supernatant was centrifuged at 20,000 rpm for 30 minutes, and then the lipids, insolubles and sediments of the suspended layer were removed and lyophilized to obtain soy protein containing 580 ug lunacin peptide per gram of powder. ). Drinking method is to dissolve 4 g of powder twice a day in 65 ml of the required route to drink. In view of the above experimental results (Examples 7 and 8), the drinking of powder has the effect of improving liver function and preventing liver cancer due to accumulation of lunacin peptide in blood and liver.

Example 10 . Production and utilization of tablets containing leucine peptide high from soybeans and barley.

After freezing the dried material prepared with lunacin peptide high-density powder medicine through the ion-exchange chromatography, the extract dissolved in 10 times the distilled water (DDH ₂ O) of capacity was dialyzed. ) To remove proteins with molecular weight less than 3k Da. Dialysis is carried out for 24 hours with distilled water changed every 6 hours in secondary distilled water after the extract (membrane) membrane (MWCO 3000) and freeze-dried to obtain a powder, then acceptable carrier (excipient) by granulation process Tablets were made, including (Figure 12).

The purified gave soy protein with high content of 100 mg lunacin peptide per gram. Dosage method is twice a day, take 1 ring per time. In view of the above experimental results (Examples 7, 8), the administration of tablets has the effect of improving liver function and preventing liver cancer due to accumulation of lunacin peptides in blood and liver.

Method for isolating and purifying lunasin peptide (lunasin peptide) from soybean and barley according to the present invention, inhibiting cancer cell proliferation, inhibiting histone acetylation and accumulation in tissues after feeding on rats and cancer cells The present invention relates to the usefulness of inhibiting proliferation, and may be used as a dietary supplement, a skin anti-aging agent, liver cancer prevention and treatment agent by lunasin peptide crude extract of soybean and barley.

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<110> Andong National University Industry-Academic Cooperation          Filgen Biosciences Inc. <120> Isolation, Purification and Availability of Lunasin peptide as a          Prevention and Curing of Skin Aging and Allergy, Anticancer and          highly functional Foods from Soybean and Barley <130> JCK001 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 43 <212> PRT <213> Glycine soja <220> <221> HELIX (222) (23) .. (30) <223> Helix structure <220> <221> PEPTIDE (222) (33) .. (35) <223> RGD motif <300> <301> Jeong, Hyung Jin          Park, Jae Ho          Yi, Lam          Ben O, de Lumen <302> Characterization of Lunasin Isolated from Soybean <303> JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY <304> 51 <305> 27 <306> 7901-7906 <307> 2003-12-31 <308> 0021-8561 <400> 1 Ser Lys Trp Gln His Gln Gln Asp Ser Cys Arg Lys Gln Leu Gln Gly   1 5 10 15 Val Asn Leu Thr Pro Cys Glu Lys His Ile Met Glu Lys Ile Gln Gly              20 25 30 Arg Gly Asp Asp Asp Asp Asp Asp Asp Asp Asp          35 40 <210> 2 <211> 43 <212> PRT Hordeum vulgare <220> <221> HELIX (222) (23) .. (30) <223> Helix structure <220> <221> PEPTIDE (222) (33) .. (35) <223> RGD motif <300> <301> Jeong, Hyung Jin          Yi, Lam          Ben O, de Lumen <302> Barley Lunasin Suppresses ras-induced Colony Formation and          Inhibits Core Histone Acetylation in Mammalian <303> JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY <304> 50 <306> 5903-5908 <307> 2002-09-17 <308> 0021-8561 <400> 2 Ser Lys Trp Gln His Gln Gln Asp Ser Cys Arg Lys Gln Leu Gln Gly   1 5 10 15 Val Asn Leu Thr Pro Cys Glu Lys His Ile Met Glu Lys Ile Gln Gly              20 25 30 Arg Gly Asp Asp Asp Asp Asp Asp Asp Asp Asp          35 40

Claims (4)

Seeds of soybeans and barley were added with secondary distilled water, sodium acetate and sodium phosphate buffer containing fresh furotese inhibitor, extracted at 4 ° C. for 24 hours, centrifuged to obtain a supernatant, and dialyzed with distilled water to obtain proteins of 3,000 Da or less. The crude extract was first purified by ion exchange column chromatography, the second purified by high-performance liquid chromatography, and the third purified by an immuno-affinity column. Method for Isolation and Purification of Lunacin Peptides. Light blocking agent, skin aging, atopy prevention and preventive agent comprising the lunacin peptide prepared according to claim 1 as an active ingredient. Blood, liver, kidney, lung, skin cancer treatment agent comprising the lunacin peptide prepared according to claim 1 as an active ingredient. Functional foods having atopic and anti-topic treatment of blood, liver, kidney, lung, and skin cancer, using crude and high lunasin peptides prepared according to claim 1 as an active ingredient.

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