KR20120079971A - Anticancer composition comprising enzymatic hydrolysates of crassostrea gigas - Google Patents

Abstract

PURPOSE: An anticancer agent composition containing oyster hydrolysate is provided to be used in a pharmaceutical product and health functional foods without toxicity and side effect. CONSTITUTION: An anticancer agent composition contains peptides isolated from oyster protein hydrolysate as an active ingredient. A method for preparing the oyster protein hydrolysate comprises: a step of dissolving freeze-dried oyster in a buffer solution; a step of adding hydrolase to the solution; a step of heating the hydrolysate in hot water; and a step of filtering, concentrating, and drying the hydrolysate. The buffer solution is a phosphate buffer or glycine-HCl buffer. The protease is flavourzyme, neutrase, papain, pepsin, trypsin, alpha-chyotrypsin, alcalase, or protamex.

Description

Anticancer composition comprising oyster hydrolyzate as active ingredient {Anticancer composition comprising enzymatic hydrolysates of Crassostrea gigas}

The present invention relates to an anticancer composition comprising an oyster hydrolyzate as an active ingredient, and more particularly, to a method for producing an anticancer composition and a oyster protein hydrolyzate comprising a peptide isolated from the oyster protein hydrolyzate and a pharmaceutically acceptable carrier. And a method for separating and purifying peptides from the hydrolyzate and primary structure sequences of oyster protein-derived peptides having anticancer efficacy.

Marine organisms contain chemicals and biological metabolites that cannot be retained and produced by existing terrestrial organisms due to species diversity and specificity of the habitat environment. Factors also exist, attracting attention as materials for new drugs and new physiologically functional substances. Moreover, as the exploration and development of leading materials, which have been actively conducted on land, began to reach their limits, the interests naturally turned to marine life. A large number of pharmacologically active substances have been reported that show anti-cancer, antioxidant, anti-fungal, antimicrobial and antiviral properties from marine organisms, which have been discovered to date by marine advanced countries such as the US, Japan and Europe. The importance of research on the production of food resources from marine resources is also increasing.

In general, shellfish have been used as important food resources since ancient times, due to the regional characteristics of Korea surrounded by the sea on three sides, which have been used as important food resources since the 1990s. As the share of production exceeds 90% and its output increases every year, in addition to the income increase of fish prices and the job creation effect in the fishing villages, it also provides a more hygienic and savory raw material and provides the unique flavor and nutritional characteristics of shellfish. High value-added processing technology that utilizes this technology has become a major concern for fishery development.

In particular, recently, studies on separating and purifying hydrolyzate obtained by hydrolyzing animal and plant proteins using various enzymes have been conducted. These hydrolysates have been found to have antioxidant and blood pressure lowering effects, and have been reported to be used as anti-aging agents for skin aging and hypertension. In addition, protein hydrolysates are used in a variety of other fields such as processed foods, seasonings, shampoos, cosmetics.

However, in Korea, acid hydrolyzate of protein is almost used, and when hydrolyzing protein with acid or alkali, essential amino acids such as tryptophan and cysteine are lost, as well as lignolanine Toxic by-products such as lysinoalanine have been produced, which has raised the issue of the safety of acid hydrolysates of proteins in developed countries, including Japan.

Cancer is caused by the transformation of normal cells by environmental carcinogens and can occur anywhere in the body. Cancer cells generally have low dependence on growth factor signaling and thus do not regulate cell proliferation, inhibit contact with surrounding cells, and lack characteristics of differentiation. In addition, it secretes angiogenic factors and penetrates and metastasizes to surrounding tissues.

Most chemical anticancer drugs work by proliferating. However, due to the difference in the control of proliferation between normal cells and cancer cells, there is no state. Therefore, since there is no selectivity for cancer cells, they are rather toxic to normal cells. Development of new anticancer drugs is urgently needed. Recently, attention has been focused on the development and development of anticancer ingredients from natural products with high efficacy and low toxicity.

Therefore, the present inventors have made diligent efforts to develop anti-cancer agents derived from natural products without toxicity and side effects, confirming the excellent anti-cancer effect of the oyster protein hydrolyzate according to the present invention and completed the present invention.

After all, the main object of the present invention is to provide a natural anticancer composition containing peptide isolated from oyster protein hydrolyzate as an active ingredient, effective without toxicity and side effects.

Still another object of the present invention is to provide a method for preparing oyster protein hydrolyzate and a method for separating and purifying functional peptides having a strong anticancer effect from the hydrolyzate.

In order to achieve the above object, the present invention provides an anticancer composition comprising a peptide isolated from the oyster protein hydrolyzate and a pharmaceutically acceptable carrier and a method for preparing and separating the peptide from the hydrolyzate. It is about.

The present invention provides an anticancer composition comprising a peptide isolated from oyster protein hydrolyzate as an active ingredient.

The oyster protein hydrolyzate is a finely pulverized lyophilized oyster dissolved in a buffer, the protein hydrolase is added to the solution and stirred to proceed the hydrolysis reaction, and then heated in hot water and filtered, concentrated and It is characterized by being manufactured by drying.

In addition, the buffer is a phosphate buffer (glycine-HCl buffer) or glycine-HCl buffer (protease), the proteolytic enzyme Flavorzyme (Neutrase), Papain (Papain), pepsin ( pepsin), Trypsin, Alpha-chymotrypsin, α-Chymotrypsin, Alcalase, and Protamex.

In addition, in the present invention, the hydrolysis step is characterized in that the hydrolysis of the reaction temperature of 25 ~ 70 ℃, reaction time 6 ~ 10 hours in a buffer of pH 1.0 ~ 10.0, preferably the pH of the buffer is 2.0 ~ 8.0, the reaction temperature is heated to 30 ~ 60 ℃ reaction time 8 hours, concentrated with a reduced pressure evaporator is preferably lyophilized.

In addition, the peptide isolated from the oyster protein hydrolyzate comprises the steps of: (1) separating the oyster protein hydrolyzate by molecular weight by TFF (Tangetial Flow Filtration) system and measuring the physiological activity of each fraction; (2) separating the fraction having the most physiological activity among the separated fractions by chromatography; (3) measuring the biological activity of each of the separated fractions; And (4) purifying the fraction having the highest physiological activity among the separated fractions. The method is prepared by the method comprising the steps of: (2) to (4) by repeating steps to separate and purify into a single substance. do.

In addition, in the step (2), the peptide is separated by protein liquid chromatography equipped with an anion exchange column (DEAEFF 16/10), and then equipped with a GPC column (GPC-SB 802.5) for easy separation by size. High performance chromatography, preparative C ₁₈ column (GROM-SIL 120 C ₁₈ , 20 X 250 mm), two analytical C ₁₈ columns (GROM-SIL 120 C ₁₈ , 4.0 X 250 mm) It is characterized by having a strong anticancer activity through a purification process using the high performance chromatography equipped.

In addition, the present invention is characterized by analyzing the amino acid sequence using the PTH analysis column by the Edman degradation method of the separated and purified peptide.

The present invention also provides an anticancer agent and a health functional food containing the composition as an active ingredient.

In addition, the oyster protein hydrolyzate of the present invention and the functional peptide separated therefrom may be usefully used in medicines, health supplements, cosmetics, foods, food additives, etc. for the prevention or treatment of aging and various diseases in addition to anticancer agents, It is not limited by.

In addition, when the oyster protein hydrolyzate of the present invention and the functional peptide separated therefrom are used as a medicine, it may further include appropriate carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions, It can be prepared in a pharmaceutically acceptable formulation, including the pharmaceutical composition made in this scope. In addition, the pharmaceutical use of the pharmaceutical composition as an anticancer agent is also included in the scope of the present invention.

The carrier or excipient includes water, dextrin, calcium carbonate, lactose, propylene glycol, liquid, paraffin, physiological saline, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals, which may be used one or more, but are not limited thereto. Both carriers and excipients can be used. In addition, when the anticancer agent is formulated, conventional fillers, extenders, binders, disintegrants, surfactants, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, or preservatives may be further included.

In addition, when the oyster protein hydrolyzate of the present invention and the functional peptide separated therefrom are used as cosmetics, wrinkle improvement and whitening functional cosmetics, softening lotion, nourishing cosmetics, eye cream, nourishing cream, massage cream, cleansing cream, essence, etc. In the cosmetic composition of each formulation, it can be prepared in the formulation of the formulation, including the protein hydrolyzate and functional peptide, other ingredients as wrinkles and whitening raw materials or conventional cosmetic raw materials according to the formulation or purpose of use of cosmetics, etc. It can be selected and combined without difficulty.

According to the present invention as described above, the oyster protein hydrolyzate, the anticancer peptide isolated from the hydrolyzate, and the composition comprising the hydrolyzate have anticancer activity and thus can be used as functional medicines and health functional foods.

In addition, the oyster protein hydrolyzate of the present invention can be used safely without toxicity and side effects as a substance derived from natural products.

1 is a schematic diagram showing a manufacturing process of oyster protein hydrolyzate.
Figure 2 shows the anticancer effect of oyster protein hydrolysates (1, flavozyme; 2, neutrases; 3, protamax; 4, alcalase; 5, papain; 6, alpha-chymotrypsin; 7, Trypsin; 8, pepsin).
Figure 3 shows the anticancer activity of the oyster protein hydrolyzate (flavozyme) by molecular weight (> 30,000 Da; 10,000 ~ 30,000 Da; 5,000 ~ 10,000 Da; 5,000 Da <).
Figure 4 shows the separation of the protein corresponding to the molecular weight of less than 5,000 Da of the oyster protein hydrolyzate (flavozyme) by protein liquid chromatography equipped with an anion exchange column.
Figure 5 shows the result of measuring the anticancer activity by lyophilizing the separation of the protein corresponding to the molecular weight of less than 5,000 Da of the oyster protein hydrolyzate (flavozyme) with an anion exchange column.
Figure 6 is a protein of less than 5,000 Da molecular weight of the oyster protein hydrolyzate (flavozyme) separated by protein liquid chromatography equipped with an anion exchange column to demonstrate the efficacy of the high performance chromatography equipped with a GPC column It shows what was separated using.
Figure 7 is a protein of less than 5,000 Da molecular weight of the oyster protein hydrolyzate (flavozyme) is separated by protein liquid chromatography equipped with an anion exchange column to prove the high-performance chromatography equipped with a GPC column It shows the result of measuring anticancer activity by freeze-drying the separated using.
Figure 8 is a protein of less than 5,000 Da molecular weight of the oyster protein hydrolyzate (flavozyme) is separated by protein liquid chromatography equipped with an anion exchange column using high performance chromatography equipped with a GPC column This proven fraction is shown by high performance chromatography equipped with a preparative column.
9 is a protein of less than 5,000 Da molecular weight of the oyster protein hydrolyzate (flavozyme) is separated by protein liquid chromatography equipped with an anion exchange column and separated by high performance chromatography equipped with a GPC column The resultant fractions were separated by high performance chromatography equipped with a preparative column and lyophilized to show anticancer activity.
Figure 10 is a protein of less than 5,000 Da molecular weight of the oyster protein hydrolyzate (flavozyme) of the present invention is separated by protein liquid chromatography equipped with an anion exchange column and separated using high performance chromatography equipped with a GPC column After that, the fractions proved efficacy were separated by high performance chromatography equipped with a preparative column, and the fractions demonstrated efficacy were separated by high performance chromatography equipped with an analytical column.
11 is a protein of less than 5,000 Da molecular weight of the oyster protein hydrolyzate (flavozyme) of the present invention is separated by protein liquid chromatography equipped with an anion exchange column and separated by high performance chromatography equipped with a GPC column After separating the fractions proved efficacy by high performance chromatography equipped with a preparative column and then the fractions demonstrated efficacy separated by high performance chromatography equipped with an analytical column.
Figure 12 is a protein of less than 5,000 Da molecular weight of the oyster protein hydrolyzate (flavozyme) of the present invention is separated by protein liquid chromatography equipped with an anion exchange column and separated by high performance chromatography equipped with a GPC column The fractions from which the potency was proved were separated by high performance chromatography equipped with a preparative column. The fractions were then separated by high performance chromatography equipped with the analytical column. It shows the separation by high performance chromatography.
Figure 13 is a protein of less than 5,000 Da molecular weight of the oyster protein hydrolyzate (flavozyme) of the present invention is separated by protein liquid chromatography equipped with an anion exchange column and separated using high performance chromatography equipped with a GPC column The fractions from which the potency was proved were separated by high performance chromatography equipped with a preparative column. The fractions were then separated by high performance chromatography equipped with the analytical column. It shows the anticancer activity of the isolate by high performance chromatography.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  1. Protein in Oysters Hydrolyzate  Produce

Oyster samples used in this experiment were purchased from Yeosu fish market, and stored at -20 ° C to prepare hydrolyzate. The finely crushed lyophilized oysters were dissolved at a ratio of 0.1 M buffer (buffer, pH 2.0-8.0) and oyster: buffer = 1: 50 (w / w), then preheated to 30-60 ° C. with stirring at 150 rpm for 30 minutes. Then, the proteolytic enzyme: oyster = 1: 50 (w / w) in the ratio of proteolytic enzymes, flavozyme, neutrases, papain, pepsin, trypsin, alpha-chymotrypsin, alkalase and prota Max was added and stirred at 180 rpm for 8 hours to hydrolyze at 30 to 60 ° C, and then heated at 100 ° C for 10 minutes to terminate the hydrolysis reaction.

After completion of the reaction, the solution was filtered using Whatman filter No. 41 (Whatman filter No. 41), concentrated under a reduced pressure evaporator, and lyophilized at −80 ° C. to prepare an oyster protein hydrolyzate. The composition and reaction conditions of the enzyme used at this time are shown in Table 1 below.

However, PB in the above means phosphate buffer (Phosphate Buffer), GHB means glycine-HCl buffer (Glycine-HCl Buffer).

Experimental Example  1. Oyster Protein Hydrolyzate  Anticancer effect measure

Prostate cancer cell PC3 (ATCC, CRL-1435) was measured by staining with propidium iodide (PI) to determine whether oyster protein hydrolyzate could inhibit cancer. Iodine propidium is a fluorescence pigment frequently used to measure apoptosis, which is an automatic cell death. Because it binds to DNA, apoptosis and cell cycle can be inferred from the amount of DNA stained with PI.

That is, 24 hours after inoculating an appropriate amount of cancer cells in a 12-well plate, eight oyster protein hydrolysates (1, flavozyme; 2, neutrases; 3, protamax; 4, alkalase; 5, Papain; 6, alpha-chymotrypsin; 7, trypsin; 8, pepsin) was treated with 0.5 mg / ml and then reacted in a CO ₂ incubator at 37 ° C. for 24 hours. When the reaction was completed, harvested and centrifuged for 3 minutes at 4 ℃, 13,000rpm, the supernatant was discarded, 300μl of phosphate buffer was added and mixed well, 1ml of cold ethanol containing tween 20 0.5% was added and fixed at 4 ℃. After 24 hours, the supernatant was discarded after centrifugation at 4 ° C. and 13,000 rpm for 3 minutes, 1 ml of phosphate buffer was added thereto, mixed well and centrifuged again at 4 ° C. and 13,000 rpm for 3 minutes, and the supernatant was discarded. Add 300 µl of iodine propidium solution (final concentration 50 µg / ml) containing RNase 10 µg / ml, mix well, and stain for 30 minutes at 37 ° C for flow cytometry (FACScan, BD Science, USA). Death and cell cycle were measured.

As a result, it was confirmed that cancer cell killing effect and cell proliferation inhibitory effect were shown in prostate cancer, and flavozyme hydrolyzate showed the highest cell killing effect (see FIG. 2).

Experimental Example 2. Oyster-derived enzyme From hydrolyzate  Anticancer Peptide Material Isolation and Purification I

In order to isolate and purify the anticancer peptide of hydrolyzate hydrolyzed by oyster flavozyme, less than 5,000 Da, 5,000 ~ 10,000 Da, 10,000 ~ 30,000 Da, 30,000 Da using TFF system to separate and purify protein according to membrane size The anticancer activity was measured by dividing into four fractions by molecular weight. As a result, it was confirmed that the highest activity in the size of less than 5,000 Da of the four fractions (see Figure 3).

Experimental Example 3. Oyster-derived enzyme From hydrolyzate  Anticancer Peptide Material Separation and Purification II

This experimental example is a second process of separating and purifying an anticancer peptide from oyster flabozyme hydrolyzate. The fraction of less than 5,000 Da, which is the fraction showing the best anticancer activity in the TFF method, is a protein liquid equipped with an anion exchange column. It was separated using chromatography.

Protein Liquid Chromatography is a device that specializes in the purification of proteins by applying high-performance chromatography technology. It is a fully automatic separation device using a tube (separation tube) uniformly containing a packing material suitable for ion exchange or size exclusion. In this experiment, protein liquid chromatography was performed by mounting a column suitable for anion exchange, and finally, three fractions were obtained (see FIG. 4). As a result of measuring the anticancer activity by lyophilizing each fraction, as shown in FIG. 5, the fraction showed the highest activity in the C fraction.

Experimental Example 4. Oyster-derived enzyme From hydrolyzate  Anticancer Peptide Material Separation and Purification III

This experimental example is a third process of separating and purifying an anticancer peptide from oyster flabozyme hydrolyzate. The fraction C, which shows the best anticancer activity in the above Experimental Example 3 using protein liquid chromatography, was used as the GPC column. Separation and purification was carried out using the equipped high performance chromatography.

High performance chromatography is a method in which a gas sample or a gasified liquid or a solid sample is developed by a carrier gas in a uniformly packed tube (separation tube), but separated by passing through a gaseous state without decomposition. In this experiment, chromatographic analysis was performed using a GPC column, which was easily separated by size, and finally four fractions were obtained (see FIG. 6).

As a result of measuring the anticancer activity of each fraction by lyophilization, as shown in FIG. 7, the C-II fraction showed the highest activity.

Experimental Example 5: Oyster-derived enzyme From hydrolyzate  Anticancer Peptide Material Separation and Purification IV

This Experimental Example is a fourth step of separating and purifying the anticancer peptide from the oyster flabozyme hydrolyzate. The fraction C-II obtained in Experimental Example 4 is separated by high performance chromatography equipped with a preparative column. Purified.

Finally, six fractions were obtained (FIG. 8), and each fraction was lyophilized to measure anticancer activity. As shown in FIG. 9, C-II-ii fractions showed the highest activity.

Experimental Example 6: Oyster-derived enzyme From hydrolyzate  Anticancer Peptide Material Separation and Purification V

This Experimental Example is a fifth step of separating and purifying an anticancer peptide from oyster flabozyme hydrolyzate. The fraction C-II-ii obtained in Experimental Example 5 was analyzed using high performance chromatography equipped with an analytical column. Isolate and purify.

Finally, five fractions were obtained (FIG. 10), and each fraction was lyophilized to measure anticancer activity. As shown in FIG. 11, C-II-ii-a fraction showed the highest activity.

Experimental Example 7: Oyster-derived enzyme From hydrolyzate  Anticancer Peptide Material Separation and Purification VI

This Experimental Example is the sixth process of separating and purifying the anticancer peptide from the oyster flabozyme hydrolyzate. The fraction C-II-ii-a obtained in Experimental Example 6 was once again equipped with an analytical column. Finally, once again separated and purified by chromatography to obtain a single fraction (Fig. 12), as a result, as shown in Figure 13 the anti-cancer activity inhibition of the fraction showed a 35.2% scavenging rate.

Experimental Example 8: Confirmation of peptide amino acid sequence having anticancer activity

In this experimental example, in order to confirm the amino acid sequence of the peptide having anticancer activity obtained in Experimental Example 7, Edman degradation method (Edman, P., Acta Chemica The amino acid sequence was analyzed from N-termina according to Scandinavica , 283-293, 1950).

As a result, the amino acid sequence of the anticancer peptide was confirmed as His-Phe-Asn-Ile-Gly-Asn-Arg-Cys-Leu-Cys, respectively (see Table 2).

As described above, specific portions of the contents of the present invention have been described in detail, and for those skilled in the art, these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University Industrial Cooperation Corp. <120> Anticancer composition comprising enzymatic hydrolysates of          Crassostrea gigas <130> P10-E506 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 10 <212> PRT <213> Crassostrea gigas <400> 1 His Phe Asn Ile Gly Asn Arg Cys Leu Cys   1 5 10

Claims (10)

An anticancer composition comprising a peptide isolated from oyster protein hydrolyzate as an active ingredient.
The method of claim 1,
The oyster protein hydrolyzate
(1) dissolving the lyophilized oyster in buffer;
(2) hydrolysis by adding proteolytic enzyme to the solution;
(3) heating the hydrolyzate in hot water;
(4) filtering, concentrating and drying the heated hydrolyzate; anticancer composition, characterized in that prepared by the method comprising a.
The method of claim 2,
The buffer of step (1) is an anticancer composition, characterized in that any one of phosphate buffer (Phosphate buffer) or glycine-HCl buffer (Glycine-HCl buffer).
The method of claim 2,
The pH of the buffer of step (1) is the anticancer composition, characterized in that 1.0 ~ 10.0.
The method of claim 2,
The proteolytic enzymes are Flavorzyme, Neutrase, Papain, Pepsin, Trypsin, Alpha-Chymotrypsin, Alcalase And Protamex (Protamex) anticancer composition, characterized in that at least one member selected from the group consisting of.
The method of claim 2,
Proteolysis of the step (2) is the anticancer composition, characterized in that the enzyme treatment with a reaction time of 6 to 10 hours at a reaction temperature of 25 ~ 70 ℃.
The method of claim 1,
The peptide is
(1) separating oyster protein hydrolyzate by molecular weight by TFF (Tangetial Flow Filtration) system and measuring the physiological activity of each fraction;
(2) separating the fraction having the most physiological activity among the separated fractions by chromatography;
(3) measuring the biological activity of each of the separated fractions; And
(4) purifying the fraction having the highest physiological activity among the separated fractions; prepared by the method comprising a, characterized in that the separation and purification to a single material by repeating the steps (2) to (4) Anticancer agent composition.
8. The method of claim 7,
Step (2) is an open column chromatography using an ion bonding column (DEAEFF 16/10), high performance chromatography equipped with a GPC column (GPC-SB 802.5) for easy separation of sizes, and a preparative C ₁₈ column (GROM). High performance chromatography with SIL 120 C ₁₈ , 20 X 250 mm) and high performance chromatography with two analytical C ₁₈ columns (GROM-SIL 120 C ₁₈ , 4.0 X 250 mm) Anticancer agent composition, characterized in that carried out.
An anticancer agent comprising the composition of any one of claims 1 to 8 as an active ingredient.
Health functional food containing the composition of any one of Claims 1-8 as an active ingredient.

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