KR20120079977A - Anticancer composition comprising enzymatic hydrolysates of ruditapes philippinarum - Google Patents

Abstract

PURPOSE: An anti-cancer composition containing Ruditapes philippinarum hydrolysate is provided to be safely used in pharmaceutical and health products without toxicity and side effect. CONSTITUTION: An anti-cancer composition contains peptides isolated from Ruditapes philippinarum hydrolysate as an active ingredient. A method for preparing the hydrolysate comprises: a step of dissolving freeze-dried Ruditapes philippinarum in a buffer solution; a step of adding hydrolysate 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 solution.

Description

Anticancer composition comprising clam hydrolyzate as an active ingredient {Anticancer composition comprising enzymatic hydrolysates of Ruditapes philippinarum}

The present invention relates to an anticancer agent composition containing a clam hydrolyzate as an active ingredient, and more particularly, to a method for producing an anticancer composition and a clam protein hydrolyzate comprising a peptide isolated from the clam protein hydrolyzate and a pharmaceutically acceptable carrier. And a method for separating and purifying a peptide from the hydrolyzate and a primary structural sequence of a peptide derived from a clam protein 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.

Thus, the present inventors have made intensive efforts to develop anti-cancer agents derived from natural products without toxicity and side effects, and confirmed the excellent anti-cancer effect of the clam 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 drug composition containing a peptide isolated from the clam protein hydrolyzate as an active ingredient, and effective without toxicity and side effects.

Still another object of the present invention is to provide a method for preparing a clam 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 clam 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 agent composition containing a peptide isolated from the clam protein hydrolyzate as an active ingredient.

The clam protein hydrolyzate is pulverized lyophilized clam was dissolved in a buffer, the protein hydrolase was 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 clam protein hydrolyzate is (1) separating the clam protein hydrolyzate by molecular weight by the TFF (Tangetial Flow Filtration) system to measure 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 was separated using an open column chromatography using an anion exchange column (DEAE-Sephacel), and then preparative C ₁₈ column (GROM-SIL 120 C ₁₈ , 20 X 250 mm) equipped with high performance chromatography, two analytical C ₁₈ column (GROM-SIL 120 C ₁₈ , 4.0 X 250 mm) equipped with high performance chromatography equipped with a strong anti-cancer activity .

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 clam 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, the clam protein hydrolyzate of the present invention and the functional peptide isolated therefrom 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 clam protein hydrolyzate of the present invention and the functional peptide separated therefrom are used as a cosmetic, wrinkle improvement and whitening functional cosmetics, softening longevity, nourishing cosmetics, eye cream, nutrition 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 clam 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 clam 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 the clam protein hydrolyzate.
Figure 2 shows the anticancer effect of clam protein hydrolyzate (1, flavozyme; 2, neutrases; 3, protamax; 4, alcalase; 5, alpha-chymotrypsin; 6, papain; 7, Trypsin; 8, pepsin).
Figure 3 shows the anticancer activity by molecular weight of the clam protein hydrolyzate (alpha-chymotrypsin) (> 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 10,000 ~ 30,000 Da of the clam protein hydrolyzate (alpha-chymotrypsin) by open column chromatography using 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 10,000 ~ 30,000 Da of the clam protein hydrolyzate (alpha-chymotrypsin) using an anion exchange column. .
Figure 6 is a high-performance column equipped with a preparative column to separate the protein corresponding to the molecular weight 10,000 ~ 30,000 Da of clam protein hydrolyzate (alpha-chymotrypsin) by open column chromatography using an anion exchange column It shows the separation using chromatography.
7 is a high-performance column equipped with a preparative column to separate the protein corresponding to the molecular weight 10,000 ~ 30,000 Da of the clam protein hydrolyzate (alpha-chymotrypsin) by open column chromatography using an anion exchange column It shows the result of measuring anticancer activity by lyophilization of the isolate using chromatography.
8 is a protein corresponding to a molecular weight of 10,000 ~ 30,000 Da of the clam protein hydrolyzate (alpha-chymotrypsin) is separated by an open column chromatography using an anion exchange column and separated by high performance chromatography equipped with a preparative column. After that, the fractions showing efficacy were separated by high performance chromatography equipped with an analytical column.
9 is a protein corresponding to a molecular weight of 10,000 ~ 30,000 Da of the clam protein hydrolyzate (alpha-chymotrypsin) is separated by an open column chromatography using an anion exchange column and separated by high performance chromatography equipped with a preparative column. After separating the fractions proved efficacy by high performance chromatography equipped with an analytical column shows the results of the anti-cancer activity by lyophilization.
10 is a high-performance chromatography equipped with a column for preparative column separation of the protein corresponding to the molecular weight 10,000 ~ 30,000 Da of the clam protein hydrolyzate (alpha-chymotrypsin) of the present invention using an anion exchange column After separation using the fractions proved to be effective by high performance chromatography equipped with an analytical column, the fractions were once again separated by high performance chromatography equipped with an analytical column.
11 is a high-performance chromatography equipped with a preparative column and separating the protein corresponding to the molecular weight 10,000 ~ 30,000 Da of the clam protein hydrolyzate (alpha-chymotrypsin) of the present invention using an anion exchange column After separation by using a high performance chromatography equipped with an analytical column, the fractions proved to be effective shows the anticancer activity of the separation of high performance chromatography equipped with an analytical column.

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 of the clam Hydrolyzate  Produce

The clam samples used in this experiment were purchased from Yeosu Fish Market, and stored at -20 ° C to prepare hydrolyzate. The pulverized lyophilized clam was dissolved in 0.1M buffer (buffer, pH 2.0 ~ 8.0) and clam: buffer = 1: 50 (w / w), then preheated to 30 ~ 60 ℃ with stirring at 150rpm for 30 minutes. Then, the proteolytic enzyme: clam = 1: 50 (w / w) in the ratio of proteolytic enzyme, flavozyme, neutrase, 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 freeze-dried at -80 ° C to prepare a clam 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. Clam Protein Hydrolyzate  Anticancer effect measure

Whether the clam protein hydrolyzate can inhibit cancer was measured by staining prostate cancer cell PC3 (ATCC, CRL-1435) with iodine propidium (PI). 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, 8 kinds of clam protein hydrolysates (1, flavozyme; 2, neutrases; 3, protamax; 4, alcalase; 5, Alpha-chymotrypsin; 6, papain; 7, trypsin; 8, pepsin) was treated with 0.5 mg / ml and reacted at 37 ° C. in a CO ₂ incubator 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 alpha chymotrypsin hydrolyzate showed the highest activity (see FIG. 2).

Experimental Example 2. Enzyme derived from clam protein From hydrolyzate  Anticancer Peptide Material Isolation and Purification I

In order to isolate and purify the anticancer peptide of the hydrolyzate hydrolyzed by the clam with alpha-chymotrypsin, the TFF system is used to separate and purify proteins according to membrane size, less than 5,000 Da, 5,000 to 10,000 Da, 10,000 to 30,000 Da, Anticancer activity was measured by dividing into four fractions by molecular weight of 30,000 Da or more. As a result, it was confirmed that the highest activity in the size of 10,000 ~ 30,000 Da of the four fractions (see Figure 3).

Experimental Example 3. Enzyme derived from clam protein From hydrolyzate  Anticancer Peptide Material Separation and Purification II

This experimental example is the second process of separating and purifying the anticancer peptide from the clam alpha-chymotrypsin hydrolyzate.The fraction of 10,000-30,000 Da, the fraction showing the best anticancer activity in the TFF method, was used in the anion exchange column. Isolation using open column chromatography.

Open column chromatography involves the passage of liquid or gas to be analyzed via a long capillary tube, which is filled with fine solids coated with a liquid adsorbent, or a thin membrane of adsorbent liquid, whereby each component of the mixture It is one of the analytical chemistry techniques to separate mixtures using different passage rates by interaction. In this experiment, open column chromatography was performed using a column suitable for anion exchange, and finally 5 fractions were obtained (see FIG. 4). As a result of measuring the anticancer activity of each fraction by lyophilization, as shown in FIG. 5, it showed the highest activity in the D fraction.

Experimental Example 4. Enzyme derived from clam protein From hydrolyzate  Anticancer Peptide Material Separation and Purification III

This experimental example is a third process of separating and purifying the anticancer peptide from the clam alpha-chymotrypsin hydrolyzate. The fraction D, which is the fraction showing the best anticancer activity in the above Experimental Example 3 using open column chromatography, is used for preparative Separation and purification was performed using high performance chromatography equipped with a column.

High performance chromatography is a method in which gas components or gasified liquid or solid samples are 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, a preparative column was mounted and chromatographed. Finally, three fractions were obtained (see FIG. 6).

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

Experimental Example 5: Enzyme derived from clam protein From hydrolyzate  Anticancer Peptide Material Separation and Purification IV

This experimental example is a fourth process of separating and purifying the anticancer peptide from the clam alpha-chymotrypsin hydrolyzate, and fraction D-I obtained in Experimental Example 4 was separated by high performance chromatography equipped with an analytical column. And purified.

Finally, two fractions were obtained (FIG. 8), and each fraction was lyophilized to measure anticancer activity. As shown in FIG. 9, the highest fraction was obtained from the D-I-i fraction.

Experimental Example 6: Enzyme derived from clam protein From hydrolyzate  Anticancer Peptide Material Separation and Purification V

This experimental example is a fifth process of separating and purifying the anticancer peptide from the clam pepsin hydrolyzate. The fraction D-I-i obtained in the above Experimental Example 5 was once again subjected to high performance chromatography equipped with an analytical column. Finally, once again separated and purified to obtain a single fraction (Fig. 10), as a result as shown in Figure 11 the anti-cancer activity inhibition rate of the fraction showed a 36.8% scavenging rate.

Experimental Example 7: 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 the anticancer activity obtained in Experimental Example 6, 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 Ala-Val-Leu-Val-Asp-Lys-Gln-Cys-Pro-Asp, 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          Ruditapes philippinarum <130> P10-E507 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 10 <212> PRT <213> Tapes philippinarum <400> 1 Ala Val Leu Val Asp Lys Gln Cys Pro Asp   1 5 10

Claims (10)

An anticancer agent composition comprising a peptide isolated from the clam protein hydrolyzate as an active ingredient.
The method of claim 1,
The clam protein hydrolyzate is
(1) dissolving the lyophilized clam 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) measuring the physiological activity of each fraction after separating the clam protein hydrolysates by molecular weight by a TFF (Tangetial Flow Filtration) system;
(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 binding column (DEAE-Sephacel), high performance chromatography equipped with a preparative C ₁₈ column (GROM-SIL 120 C ₁₈ , 20 X 250 mm), two assays Anticancer composition characterized in that the purification process using high performance chromatography equipped with a C ₁₈ column (GROM-SIL 120 C ₁₈ , 4.0 X 250 mm).
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.

Images