KR20100012980A - Antioxidant composition comprising enzymatic hydrolysates of venison - Google Patents

Abstract

PURPOSE: A composition containing venison hydrolysate having anti-oxidation effects is provided to protect DNA and effectively suppress or treat aging and various diseases. CONSTITUTION: An anti-oxidation composition contains venison hydrolysate, anti-oxidative peptide, or their mixture as an active ingredient. The anti-oxidation peptide has an amino acid sequence of sequence number 1 or 2. The anti-oxidation composition is used in the form of medicinal and pharmaceutical product, supplementary health food, cosmetic product, food or food additive. The venison hydrolysate is obtained by treating protease in pH 1.0-10.0 buffer solution, at 25-70°C for 6 to 10 hours. The buffer is phosphate buffer or glycine-HCl buffer. The anti-oxidation peptide is purified through 4 steps comprising ion exchange liquid chromatography using anion resin, high performance chromatography having C18 column, high performance chromatography having C18 for two times.

Description

Antioxidant composition comprising enzymatic hydrolyzate {Antioxidant composition comprising enzymatic hydrolysates of venison}

The present invention relates to a composition containing an extract of venison hydrolase having an antioxidant effect and a method for preparing the same, and more particularly, a method for producing venison proteolytic enzyme extract through proteolytic reaction of venison. And to compositions containing them.

Oxygen is the most abundant element on earth (53.8%), accounting for 21% of dry air, and aerobic organisms acquire energy through breathing with this rich oxygen as an electron acceptor. However, the ground state triplet oxygen, an oxygen but stable molecular state that is absolutely necessary to maintain life, has various physical, chemical, and environmental factors such as enzyme system, reduction metabolism, chemicals, pollutants, and photochemical reactions. Reactivity such as superoxide radical (O ₂ ·), hydroxyl radical (HO ·), hydrogen peroxide (H ₂ O ₂ ), singlet oxygen (HO ·) The conversion to this very large active oxygen has a double sided effect that causes fatal oxygen toxicity to the living body. In other words, these free radicals have non-selective and irreversible destruction effects on cell components such as lipids, proteins, sugars, DNA, etc., as well as aging, cancer, stroke, Parkinson's disease, cerebrovascular heart disease, ischemia, arteriosclerosis, It is known to cause various diseases such as skin diseases, digestive diseases, inflammation, rheumatism and autoimmune diseases (Halliwell B. 1991. Drug antioxidant effects. Drugs 42: 596-605). In addition, various peroxides in the body, including lipid peroxides resulting from lipid peroxidation by these free radicals, also cause various functional disorders due to oxidative destruction of cells, thereby causing aging and disease.

On the other hand, even in normal cells, some free radicals and other free radicals and peroxides are generated during metabolism. However, in vivo, SOD (superoxide dismutase), peroxidase, and catalase as a defense against them are produced. Along with antioxidant enzymes such as (catalase), antioxidants such as vitamin E, vitamin C, glutathione, ubiquinone (ubiquinone, coemzyme Q10), and uric acid exist to protect themselves. However, oxidative stress is caused when abnormality is caused in such a bioprotective device or the generation of active oxygen exceeds the capacity of the bioprotective system by various physical and chemical factors. Accordingly, antioxidants such as compounds capable of scavenging such free radicals or inhibitors of peroxide production are expected as agents for inhibiting or treating aging and various diseases caused by these oxides.

The study of antioxidants began in earnest when McCord and Fridovich discovered SOD, an enzyme that eliminates superoxide radicals, in 1969. Recently, active corals and peroxides are directly responsible for various diseases and aging. With this knowledge, antioxidant research is being turned into antioxidant research as an anti-aging and disease treatment.

Conventional antioxidants include synthetic antioxidants such as tert-butylhydroxytoluene (BHT) and tert-butylhydroxyanisol (BHA), tocopherol (α-tocopherol), vitamin C (vitamin C), tannins, carotenoids, flavonoids, and the like. There are some natural antioxidants and enzyme-based superoxide dismutases (SODs), and other types of antioxidants are being developed (Kitahara K., Matsumoto Y., Ueda HA and Ueoka R. 1992. Chem. Pharm. Bull. 40: 2208-2209; Hatano T. 1995. Natural Medicines 49: 357-363).

However, these antioxidants are limited to use due to various problems such as toxicity, low activity and limit of use.

Accordingly, the present inventors have made diligent efforts to develop antioxidants derived from natural products without toxicity and side effects, confirming the excellent antioxidant effect of venison hydrolase extract according to the present invention and completed the present invention.

After all, the main object of the present invention is to provide a venison proteolytic enzyme extract prepared by proteolytic enzyme reaction by treating the venison enzyme.

It is still another object of the present invention to provide a natural antioxidant composition that is stable and effective in the suppression or treatment of aging and various diseases caused by the oxides produced by active oxygen containing the venison protease extract.

In order to achieve the above object, the present invention provides a deer protein hydrolase extract and an antioxidant peptide having an amino acid sequence isolated and purified from the extract and represented by SEQ ID NO: 1 or SEQ ID NO: 2.

The present invention also provides an antioxidant composition containing the extract or / and the antioxidant peptide as an active ingredient.

In the present invention, the venison protease extract is characterized in that the venom is prepared by treating the proteolytic enzyme in a buffer of pH 1.0 ~ 10.0, the enzyme is papain (Papain), pepsin (pepsin) It may include one or more selected from the group consisting of Trypsin, Alpha-chymotrypsin, Alcalase, and Neutrase.

Deer protein hydrolase extract according to the present invention, the antioxidant peptide isolated from the extract, and the composition comprising the extract exhibits excellent antioxidant and DNA protective effects on neurons, and is produced by free radicals. It is effective in suppressing or treating aging caused by oxides and various diseases.

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

Hereinafter, the present invention will be described in more detail.

The present invention provides venison proteinase extract and antioxidant peptide isolated and purified from the extract.

Specifically, the present invention provides venison proteinase extract and antioxidant peptide having an amino acid sequence isolated and purified from the extract and represented by SEQ ID NO: 1 or SEQ ID NO: 2.

In the present invention, the venison proteolytic enzyme extract is characterized in that the venom is prepared by treating proteolytic enzymes in a buffer of pH 1.0 ~ 10.0, the proteolytic enzymes are papain (papain), pepsin (pepsin), trypsin (Trypsin), alpha-chymotrypsin (α-Chymotrypsin), alcalase (Alcalase) and may include one or more selected from the group consisting of Neutrase (Neutrase).

The present invention also provides a composition containing the deer protein hydrolase extract or / and an antioxidant peptide isolated and purified from the extract.

Deer protein hydrolase extract of the present invention is a deer meat using a buffer solution, preferably one or more solvents selected from phosphate buffer (PB), glycine-HCl buffer (Glycine-HCl Buffer, GHP) After dissolving, the hydrolysis reaction may proceed while adding and stirring an enzyme or enzyme mixture thereto, followed by heating in hot water to terminate the reaction, which may be prepared by concentration and drying.

Specifically, the venison is dissolved in phosphate buffer or glycine-HCl buffer at pH 1.0-10.0, preferably pH 2.0-8.0, and after adding proteolytic enzyme thereto, 25-70 ° C., preferably 30 The hydrolysis reaction proceeds sufficiently while stirring at a temperature of ˜60 ° C. for 6 to 10 hours, preferably 8 hours, and then the reaction is terminated by heating at 80 to 120 ° C., preferably 90 to 100 ° C. for 10 minutes. After that, the reaction solution was concentrated with a reduced pressure evaporator and lyophilized to obtain venison proteinase extract.

The proteolytic enzyme is selected from the group consisting of papain, pepsin, trypsin, alpha-chymotrypsin, alcalase and neutrase. Include more than one species.

In addition, the antioxidant peptide isolated and purified from the venison proteinase enzyme extract of the present invention is ion-exchange liquid chromatography using anion resin (Diethylaminoethyl-Sephacel), high performance chromatography equipped with a preparative C ₁₈ column Using high performance chromatography with chromatography and two analytical C ₁₈ columns, peptides with strong antioxidant activity can be obtained through a series of four steps of purification.

The antioxidant peptide according to the present invention obtained as described above has an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

Deer protein hydrolase extract obtained from the above method and the antioxidant peptide isolated and purified from the extract were found to have an excellent antioxidant effect through various confirmation experiments such as hydroxyl radical scavenging ability, and also to free radicals. It was confirmed that the damaged DNA also had a protective effect.

Therefore, the composition containing the deer protein hydrolase extract of the present invention is a pharmaceutical, health supplements, cosmetics, food additives, etc. for the suppression or treatment of aging and various diseases caused by the oxides produced by the active oxygen It may be usefully used, but is not limited thereto.

When the composition of the present invention is used as a medicament, it may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions, and may be prepared in pharmaceutically acceptable formulations according to conventional methods. .

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 silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals, which may be used, but are not limited to one or more. Both carriers and excipients can be used. In addition, when formulating the antioxidant composition, it may further include conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives.

When the composition of the present invention is used as a cosmetic, it can be prepared in the form of wrinkle improvement and whitening functional cosmetics, flexible cosmetics, nourishing cosmetics, eye cream, nutrition cream, massage cream, cleansing cream, essence, etc. In the composition, components other than wrinkle improvement and whitening raw materials or general cosmetic raw materials including silver lake hydrolase extract may be appropriately selected by those skilled in the art without difficulty according to the formulation or purpose of use of cosmetics.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example Preparation of Venison Proteinase Extracts

Finely pulverized venison was dissolved in 0.1 M buffer (pH, pH 2.0-8.0) at a ratio of venison: buffer = 1: 50 (w / w), then preheated to 30-60 ° C. with stirring at 150 rpm for 30 minutes. Next, the proteolytic enzyme: venison = 1: 50 ratio (w / w) of the proteinase, papain (Papain), pepsin (Pepsin), trypsin (alpha)-chymotrypsin (α) -Chymotrypsin, Alcalase, Neutrase and Trypsin were added and stirred at 180 rpm for 8 hours to hydrolyze at 30-60 ° C, followed by heating at 100 ° C for 10 minutes The decomposition reaction was terminated.

The reaction solution was concentrated with a reduced pressure evaporator, and lyophilized at -80 ° C to prepare venison protein hydrolyzed extract. The composition and reaction conditions of the enzyme used at this time are shown in Table 1 below.

enzyme pH Temperature (℃) Buffer Papain 6.0 37.0 0.1M PB Pepsin 2.0 37.0 0.1M GHP Trypsin 8.0 37.0 0.1M PB α-Chymotrypsin 8.0 37.0 0.1M PB Alcalase 8.0 50.0 0.1M PB Neutrase 8.0 50.0 0.1M PB

However, PB is a phosphate buffer (Phosphate buffer), GHB is a glycine-HCl buffer (Glycine-HCl buffer).

Experimental Example 1: Measurement of hydroxyl radical scavenging ability I

In this experiment, in order to determine the hydroxyl radical scavenging ability of venison hydrolase extract, hydroxyl using an electron spin resonance instrument (ESR spectrometer, JEX-PX 2000-300 model, JEOL, Japan) Radical scavenging activity was measured.

The hydroxyl radical scavenging ability generates hydroxyl radicals based on the Fenton reaction in which iron (Fe) ions and hydrogen peroxide react to form an amount of DMPO (5,5-dimethyl-1-pyrroline N-oxide) -OH. It measured and measured the hydroxyl radical scavenging rate.

Specifically, 0.2 ml of 0.2 M DMPO, 0.2 ml of 10 mM FeSO _{4, and} 0.2 ml of 10 mM H ₂ O ₂ were sequentially added to 0.2 ml of deer protein hydrolase extract according to the concentration of the above Example, and then for 10 seconds. After strongly vortexing, the reaction mixture was transferred to a capillary tube and the amount of hydroxyl radicals generated was measured on an ESR spectrometer [ESR measurement conditions-central field: 3475G, modulation frequency: 100 Hz, modulation amplitude: 2G, microwave power: 1 Hz]. , gain: 6.3 × 10 ⁵ , Temperature: 298 K].

The results are shown in Figure 3, it was confirmed that the venison proteinase extract of the present invention to remove the hydroxyl radical in a concentration-dependent manner. Papain (Papain) of the venison protein hydrolyzed extract of the present invention had the highest hydroxyl radical scavenging activity, IC ₅₀ value was 0.832 mg / ㎖.

Experimental Example 2. Determination of optimal protein hydrolysis conditions

In this experiment example, pH, temperature, substrate concentration, substrate concentration compared to enzyme, and hydrolysis degree by time to determine the optimal conditions for mass hydrolysis of venison with papain among proteolytic enzymes. ) Was measured.

Specifically, in order to determine the optimal hydrolysis pH of venison protein, 2 g of venison protein was dispersed in 100 ml of 0.1 M phosphate buffer while changing the pH from 3 to 8, and then made into 2% substrate solution, followed by 37 ° C. The reaction was carried out at 8 hours, and the amount of added enzyme was 50: 1 (w / w).

When the reaction is completed, take 2 ml of the reaction mixture, add 20% Trichloro acetic acid (TCA) in the same amount, and centrifuge at 2370 × g for 5 minutes, and take a certain amount of the supernatant to obtain the Lowry method (Lowry, Rosebrough, Farr). and Randall, J. Biol. Chem. , 193, 265-275), the amount of 10% TCA soluble nitrogen was measured, and the degree of hydrolysis was calculated from the following equation.

In addition, in order to determine the optimal temperature, 2g of venison protein was dispersed in 100 ml of 0.1 M phosphate buffer solution at the optimum pH determined in the above experiment while changing the temperature to 37, 50, and 60 ° C, and then reacted for 8 hours. In this case, the amount of enzyme added was such that the substrate-to-enzyme ratio was 50: 1 (w / w). When the reaction is completed, 2 ml of the reaction mixture is added, and the same amount of 20% TCA is added thereto, followed by centrifugation at 2370 × g for 5 minutes, and a constant amount of the supernatant is taken to measure the amount of 10% TCA soluble nitrogen according to the Lowry method. , The degree of hydrolysis was calculated according to the above formula.

In addition, in order to determine the optimal concentration for the substrate, while varying the substrate concentration from 0.2% to 0.5% 2% substrate protein by dispersing in 100 ml of 0.1 M phosphate buffer solution at the optimum pH, optimum temperature determined in the above experiment 2% substrate solution The reaction was carried out for 8 hours, and the amount of enzyme added was such that the substrate-to-enzyme ratio was 50: 1 (w / w). When the reaction is completed, 2 ml of the reaction mixture is added, and the same amount of 20% TCA is added thereto, followed by centrifugation at 2370 × g for 5 minutes, and a constant amount of the supernatant is taken to measure the amount of 10% TCA soluble nitrogen according to the Lowry method. , The degree of hydrolysis was calculated according to the above formula.

In addition, in order to determine the optimum enzyme ratio for the substrate, 2g of the protein protein hydrolyzate was dispersed in 100 ml of 0.1 M phosphate buffer at the optimal pH and temperature determined in the above experiment to make a 2% substrate solution and then the substrate to enzyme The reaction was carried out for 8 hours while changing the ratio from 5: 1 to 500: 1 (w / w). When the reaction is completed, 2 ml of the reaction mixture is added, and the same amount of 20% TCA is added thereto, followed by centrifugation at 2370 × g for 5 minutes, and a constant amount of the supernatant is taken to measure the amount of 10% TCA soluble nitrogen according to the Lowry method. , The degree of hydrolysis was calculated according to the above formula.

In addition, in order to determine the time to show the optimum degree of hydrolysis for the substrate, 2g of venison protein was dispersed in 100ml of 0.1 M phosphate buffer at the optimum pH and temperature determined in the above experiment, and the reaction time of enzyme was 1, 2, Changed to 4, 8, 12, 18 and 24 hours. At this time, the amount of enzyme added was such that the substrate-to-enzyme ratio was 100: 1 (w / w). When the reaction is completed, 2 ml of the reaction mixture is added, and the same amount of 20% TCA is added thereto, followed by centrifugation at 2370 × g for 5 minutes, and a constant amount of the supernatant is taken to measure the amount of 10% TCA soluble nitrogen according to the Lowry method. , The degree of hydrolysis was calculated according to the above formula.

As can be seen in Figures 3 to 7 showing the results, substrate mass concentration of 2%, and substrate: enzyme ratio (substrate / enzyme, S / E) in a 37 ℃ buffer of pH 7 when the venison is mass hydrolyzed with papain After adding the substrate and the enzyme at 100, it was confirmed that the maximum yield was obtained during the hydrolysis for 8 hours.

Experimental Example 3: Measurement of hydroxyl radical scavenging ability II

This experimental example hydrolyzes venison with papain in proteolytic enzymes, and separates the hydrolyzed extracts by molecular weight of 30,000 Da, 10,000 Da, 5,000 Da using Molecular Weight Cut Off (MWCO). Four fractions were obtained after passing through.

That is, the 30,000 Da film did not pass (I), the 30,000 Da film passed but the 10,000 Da film did not pass (II), the 10,000 Da film passed but the 5,000 Da film did not pass (III), and the 5,000 Da film passed The hydroxyl radical scavenging ability of each (IV) was determined in the same manner as in Experimental Example 1.

As shown in FIG. 8, fraction III had the highest hydroxyl radical scavenging activity, but the IC ₅₀ value was 0.782 mg / ml.

Experimental Example 4: Measurement of hydroxyl radical scavenging ability III

This experimental example is the first process of separating and purifying antioxidant peptides from papain hydrolyzed extract, and performing ion-exchange liquid chromatography using anion resin (Diethylaminoethyl-Sephacel). Got.

As shown in FIG. 9 showing the result, fraction VI had the highest hydroxyl radical scavenging ability, with an IC ₅₀ value of 0.390 mg / ml.

Experimental Example 5: Measurement of hydroxyl radical scavenging ability IV

In this experimental example, a second process of separating and purifying an antioxidant peptide from venison papain hydrolyzed extract, fraction VI obtained in Experimental Example 4 was isolated and purified using high performance chromatography equipped with a preparative 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, chromatography was performed using a preparative C ₁₈ column, and finally five fractions were obtained.

As shown in FIG. 10 showing the result, the hydroxyl radical scavenging ability of fraction III was the highest, with an IC ₅₀ value of 0.231 mg / ml.

Experimental Example 6: Measurement of hydroxyl radical scavenging ability V

This experimental example is a third process of separating and purifying an antioxidant peptide from venison papain hydrolyzed extract. The fraction Ⅲ obtained in Experimental Example 5 was isolated and purified using high performance chromatography equipped with an analytical C ₁₈ column. It was.

Finally, eight fractions were obtained. As shown in FIG. 11, the hydroxyl radical scavenging ability of fraction V was the highest, with an IC ₅₀ value of 0.062 mg / ml.

Experimental Example 7: Measurement of hydroxyl radical scavenging ability VI

This experimental example is a fourth process of separating and purifying an antioxidant peptide from venison papain hydrolyzed extract. The fraction V obtained in Experimental Example 6 was once again analyzed using high performance chromatography equipped with an analytical C ₁₈ column. Further separation, purification and finally two fractions were obtained.

As shown in FIG. 12 showing the results, IC ₅₀ values of hydroxyl radical scavenging ability of each fraction were 0.044 and 0.069 mg / ml, respectively.

Experimental Example 8: Confirmation of Antioxidant Peptide Amino Acid Sequence

In this Experimental Example, to confirm the amino acid sequence of the antioxidant peptide obtained in Experimental Example 7, the amino acid sequence from the N-termina according to Edman degradation method (Edman, P., Acta Chemica Scandinavica , 4 , 283-293, 1950) It was.

As a result, the amino acid sequence of the antioxidant peptides I and II (APVP-I and II) was Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly and Asp-Leu-Ser-Asp-Gly- Glu-Gln-Gly-Val-Leu, and its molecular weights were found to be 9,853 Da and 11,213 Da, respectively (see FIG. 13 and Sequence Listing).

Experimental Example 9: Determination of Free Radical Scavenging Activity

This Experimental Example is to confirm the free radical scavenging ability of the two antioxidant peptides obtained in Experimental Example 7 in addition to the hydroxyl radical DPPH (2,2-diphenyl-1-picrylhydrazyl), superoxide (Superoxide), peroxy radical ( Peroxyl radical) scavenging ability was measured. DPPH is dark purple and is itself a nitrogen-centered radical, which exists in a stabilized state by delocalization of radical electrons. In the present invention, an electron spin resonance device (ESR spectrometer, JEX-PX 2000-300 model, JEOL , Japan) was used to measure DPPH radical scavenging activity.

Specifically, 60 μl of 60 μM DPPH dissolved in methanol and 60 μl of antioxidant peptides prepared by concentration in ultrapure distilled water were mixed, then strongly vortexed for 10 seconds, allowed to stand at room temperature for 2 minutes, and then transferred to a capillary tube. ESR measurement conditions-central field: 3475G, modulation frequency: 100 Hz, modulation amplitude: 2G, microwave power: 5 Hz, gain: 6.3 × 10 ⁵ , Temperature: 298 K].

In addition, the amount of alkyl radicals produced by AAPH (2,2'-azobis (2-amidinopropane) dihydrochloride) was measured by the amount of reactants with 4-POBN (4-pyridyl-1-oxide-N-tert-butylnitrone). .

That is, 10 μl of PBS (phosphate buffered saline, phosphate buffered salt solution), 10 μl of concentration-specific antioxidant peptide, 10 μl of 40 mM AAPH, 10 μl of 40 mM POBN are added sequentially, and then vortexed strongly for 10 seconds. The mixture was reacted in a 37 ° C. water bath for 30 minutes, and then transferred to a capillary tube to measure the amount of alkyl radicals generated by an ESR spectrometer. [ESR measurement conditions-central field: 3475G, modulation frequency: 100 Hz, modulation amplitude] : 2G, microwave power: 10 ㎽, gain: 6.3 × 10 ⁵ , Temperature: 298 K].

In addition, the superoxide radicals produced when the UV light was exposed on the riboflavin / EDTA solution were dissolved in 10 μl 0.8 mM riboflavin, 10 μl 1.6 mM Ethylene diamine tetraacetic acid (EDTA) and ultrapure distilled water prepared by concentration. 10 μl of peptide was mixed and vigorously vortexed for 10 seconds, exposed to 365 nm UV for 1 minute, and transferred to a capillary tube and measured on an ESR spectrometer [ESR measurement conditions-central field: 3475G, modulation frequency: 100 Hz, modulation] amplitude: 2G, microwave power: 10 Hz, gain: 6.3 × 10 ⁵ , Temperature: 298 K].

As a result, as shown at 14, IC ₅₀ values of the antioxidative peptide Ⅰ (APVP-Ⅰ) IC ₅₀ values were 0.077, 0.217, was 0.085 ㎎ / ㎖ antioxidant peptide Ⅱ (APVP-Ⅱ) of respectively 0.107, 0.597 , 0.163 mg / ml.

Experimental Example 10 Confirmation of Cytotoxicity of Antioxidant Peptide I

In this Experimental Example, MTT method (Mosmann, Mosmann, To determine the neuronal cytotoxicity of the antioxidant peptide I (APVP-I, sequence listing) having the amino acid sequence of SEQ ID NO: 1 of the two antioxidant peptides obtained in Experimental Example 7 T cell viability was measured according to the Journal of Immunology Method , 65 , 55-63, 1983).

The MTT method is a non-aqueous MTT formazan (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide) having a violet color of MTT tetrazolium, a yellow water-soluble substrate by dehydrogenase action. As a test that uses the ability of mitochondria to reduce, the more the number of living cells, the more water-insoluble MTT formazan production, which makes it easy to measure the growth of cells.

Figure 15 shows the growth of neurons when the antioxidant peptide I of the present invention treated by concentration, it could be confirmed that the antioxidant peptide I of the present invention is not toxic to neurons at all.

Experimental Example 11: Effect of Antioxidant Peptide I on Neuronal Catalase Activity

In this Experimental Example, the effect of the antioxidant peptide I obtained in Experimental Example 7 on the catalase activity in neurons was measured using Sigma's Catalase assay kit.

Specifically, 1.25, 2.5, 5.0, and 10 μl of the sample were added to the Eppendorf tube (Eppendorf-tube) to make 75 μl total with the phosphate buffer solution, and then 25 μl of the substrate solution for colorimetric analysis was added. Tubes were inverted and mixed, and then allowed to stand at room temperature for 5 minutes to add 900 μl of stop solution. Take 10 µl of this solution, transfer to a new Eppendorf tube, add 1 ml of color solution, invert, mix, and leave at room temperature for 15 minutes, and measure the absorbance (OD value) at 520 nm (Sunrise-Basic, Tecan, Austria). The enzyme activity was measured according to the following formula.

Catalase is an enzyme that catalyzes the reaction of hydrogen peroxide to produce water and oxygen. Peroxides, such as hydrogen peroxide, can adversely affect cell membranes and interfere with substances that play an important role in the body, such as enzymes, so the presence of hydrogen peroxide in the body has to be destroyed quickly. Catalase plays this role.

Figure 16 shows the catalase activity of the antioxidant peptide I of the present invention, the antioxidant peptide I treatment group according to the present invention, the antioxidant peptide I increased the activity compared to the untreated group (control) increased the activity of catalase Sikkim was confirmed.

Experimental Example 12: Effect of Antioxidant Peptide I on Glutathione Peroxidase Activity in Neurons

In this experimental example, the effect of antioxidant peptide I of the present invention on glutathione peroxidase activity in neurons was measured using the Sigma glutathione peroxidase assay kit. Glutathion is the first crystalline polypeptide extracted from living organisms and is widely distributed in nature and plays an important role in redox reactions in almost all living organisms such as animals and enzymes. Glutathione peroxidase decomposes and neutralizes hydrogen peroxide through the glutathione molecule as a medium.

Specifically, 890-930 μl of buffer, 50 μl of NADPH solution, 10 μl of 30 mM t-Bu-OOH and 10-50 μl of sample were added to the Eppendorf tube, and the respective tubes were inverted and mixed for 10 seconds at 340 nm. OD total 6 times in intervals The value was measured (Sunrise-Basic, Tecan, Austria) and the enzyme activity was calculated as in the following formula.

However, the above 6.22 is the ε ^mM value of NADPH.

Figure 17 shows the glutathione peroxidase activity of the antioxidant peptide I of the present invention, the antioxidant peptide I treated group according to the present invention, the antioxidant peptide I is considered to be higher than the control group (control) glutathione peroxidase It has been shown to increase the activity of.

Experimental Example 13: Effect of Antioxidant Peptide I on Glutathione-S-Transferase Activity in Neurons

In this experimental example, the effect of the antioxidant peptide I of the present invention on glutathione-s-transferase activity in neurons was measured using the Sigma glutathione-s-transferase assay kit. Glutathione reacts slowly because the group of globulars is poorly bound to the electron-affinity substrate in the region of sushitain. At this time, glutathione-S-transferase serves as a catalyst to bind glutathione and electron-affinity substrate to neutralize hydrogen peroxide.

Specifically, 1,000 μl of substrate solution and 2 μl of each sample were added to the Eppendorf tube (however, 2 μl of GST control was added for the enzyme calibration group), followed by inversion and mixing, followed by a total of 9 ODs at 30 seconds intervals at 340 nm. The value was measured (Sunrise-Basic, Tecan, Austria) and calculated as follows.

Figure 18 shows the glutathione-S-transferase activity of the antioxidant peptide I of the present invention, antioxidant peptide I according to the present invention, the antioxidant peptide I treatment group is considered to have increased activity compared to the control group (control) It has been shown to increase the activity of glutathione-S-transferase.

Experimental Example  14: antioxidant Peptide  Ⅰ Within nerve cells  Effect on Nitric Oxide Production

In this experimental example, the effect of antioxidant peptide I of the present invention on nitric oxide production in neurons was measured.

Nitric oxide is produced when L-arginine produces citrulline in the body, and it is a free radical that promotes the oxidation of our body in excess production.

Specifically, 50 μl of sulfanilamide was added to 50 μl of standard solution (NaNO ₂ ) and antioxidant peptide I solution, which was then allowed to stand at room temperature for 10 minutes, and then 50 μl of N- (1-Naphthyl) ethylenediamine dihydrochloride was added thereto. The OD value was measured at 550 nm after being left to stand (Sunrise-Basic, Tecan, Austria).

Figure 19 shows the amount of nitric oxide produced by the antioxidant peptide I of the present invention, the antioxidant peptide I according to the present invention the antioxidant peptide I treated group in view of the reduced production compared to the control group (control) It has been confirmed to inhibit.

Experimental Example 15 Effect of Antioxidant Peptide I on the Production of Reactive Oxygen Species in Neurons

In this experimental example, the effect of antioxidant peptide I of the present invention on the generation of reactive oxygen species (ROS) in neurons was measured. Free radical species (ROS) is a highly oxidative oxygen species that is produced by various metabolic processes as oxygen enters the body during the respiration process and attacks biological tissues and damages cells. Promote

Specifically, after inoculating 2.1 x 10 ⁵ neurons in a 6-well plate and 24 hours later treated with 1 mM H ₂ O ₂ or 1 mM H ₂ O ₂ + antioxidant peptide I, 37 ℃, CO ₂ incubator 24 The reaction was carried out for a time. After 24 hours, 5 μM of 2 ', 7'-dichlorofluoroscein diacetate (DCF-DA) was added to each medium for 30 minutes, followed by harvesting and measuring the amount of reactive oxygen species produced by flow cytometry (FACScan, BD Science, USA). do.

Figure 20 shows the amount of reactive oxygen species produced by the antioxidant peptide I of the present invention, the antioxidant peptide I treated group according to the present invention when the antioxidant peptide I treated amount is reduced compared to the control group (control) It was confirmed to suppress the formation of.

Experimental Example 16: Effect of Antioxidant Peptide I on Lipid Peroxide Production in Neurons

In this experimental example, the effect of the antioxidant peptide I of the present invention on lipid peroxide production in neurons was measured. Lipid peroxides produced in vivo are the causes of diseases such as liver disease, thrombosis, heart disease, aging, and cancer. Malondialdehyde, a lipid peroxide, meets thiobarbituric acid and turns into a pink substance. This can be used to measure the level of lipid peroxide production in cells.

Specifically, after inoculating 2.1 x 10 ⁵ neurons in a 6-well plate and 24 hours later treated with 1 mM H ₂ O ₂ or 1 mM H ₂ O ₂ + antioxidant peptide I, 37 ℃, CO ₂ incubator 24 The reaction was carried out for a time. When the reaction is complete, harvest and transfer 225 μl to a new effendorf tube, grind three times for 7 seconds in a tissue grinder (VCX 750, Sonics & Materials Inc., USA) with the same amount of 20% trichloroacetic acid (TCA), then 1,800 Centrifuge at 10 g for 10 minutes, and further centrifuge at 6,940 g for 15 minutes to transfer 1 ml of the supernatant to a 15 ml cap tube, to which 2 ml of thiobarbituric acid (TBA) solution was added. After immersion in cool, the OD value was measured at 532 nm (Sunrise-Basic, Tecan, Austria).

Figure 21 shows the amount of lipid peroxide produced by the antioxidant peptide I of the present invention, antioxidant peptide I according to the present invention is the production of lipid peroxide in view of the reduced amount of the antioxidant peptide I treatment compared to the control (control) It was confirmed to suppress.

Experimental Example 17 Measurement of Inhibitory Activity of Neuronal Cell Death Due to Oxidative Stress of Antioxidant Peptide I

In this experimental example, it was determined whether the antioxidant peptide I of the present invention can inhibit neuronal damage caused by oxidative stress. Iodide (propidium iodide, PI) is a fluorescent dye often used to measure apoptosis, which is an automatic cell death, and binds to DNA, which can infer cell death and cell cycle from the amount of DNA stained with PI.

Specifically, after inoculating 2.1 x 10 ⁵ neurons in a 6-well plate and 24 hours later treated with 1 mM H ₂ O ₂ or 1 mM H ₂ O ₂ + antioxidant peptide I, 37 ℃, CO ₂ incubator 24 The reaction was carried out for a time. After the reaction was completed, harvested and centrifuged at 4 ° C and 13,000 rpm for 3 minutes, discard the supernatant, add 300 µl of phosphate buffer solution, mix well, add 1 ml of cold ethanol containing tween 20 0.5%, and fix it at 4 ° C. It was. After 24 hours, the supernatant was discarded after centrifugation at 4 ° C. and 13,000 rpm for 3 minutes, 1 ml of phosphate buffer solution was added thereto, mixed well, followed by centrifugation at 4 ° C. and 13,000 rpm for 3 minutes, and the supernatant was discarded. Next, 300 μl of an iodine propidium solution containing a RNase 10 μg / ml (final concentration 50 μg / ml) was added and mixed well, followed by staining for 30 minutes at 37 ° C. (FACScan, BD Science, USA) Apoptosis and cell cycle were measured at.

Figure 22 shows the degree of neuronal cell damage caused by the oxidative stress of the antioxidant peptide I of the present invention, antioxidant peptide I treatment group in view of the reduced number of apoptosis compared to the control group (control) antioxidant peptide I according to the present invention Has been shown to inhibit neuronal cell death.

Experimental Example 18 Measurement of Neuronal Cell Death Inhibition Due to Oxidative Stress of Antioxidant Peptide I

In this experimental example, it was determined whether the antioxidant peptide I of the present invention can inhibit neuronal damage caused by oxidative stress. Annexin-V is also a fluorescence dye mainly used to measure apoptosis, which is autoapoptotic, and stains phosphatidylserine when phosphatidylserine in the mitochondria is released out of the mitochondria when apoptosis occurs. Simultaneous staining with PI can be used to infer the apoptosis period (early, late).

Specifically, after inoculating 2.1 x 10 ⁵ neurons in a 6-well plate and 24 hours later treated with 1 mM H ₂ O ₂ or 1 mM H ₂ O ₂ + antioxidant peptide I, 37 ℃, CO ₂ incubator 24 The reaction was carried out for a time. After the reaction was completed, harvested and centrifuged at 4 ° C. and 13,000 rpm for 3 minutes, discard the supernatant, add 1 ml of cold phosphate buffer solution, mix well, and centrifuge for 3 minutes at 4 ° C. and 13,000 rpm. Discard it and mix well by adding 1 ml of binding buffer to it, and transfer 100 µl into a 5 ml tube for flow cytometry. 5 μl of Annexin-V and iodine propidium were added thereto, followed by 15 minutes of reaction at room temperature and in the dark, followed by 400 μl of binding buffer, and then apoptosis was measured on a flow cytometer (FACScan, BD Science, USA).

Figure 23 shows the degree of neuronal cell damage due to the oxidative stress of the antioxidant peptide I of the present invention, antioxidant peptide I treatment group antioxidant peptide I according to the present invention when the number of apoptosis is reduced compared to the control (control) Has been shown to inhibit neuronal cell death.

Experimental Example 19 Measurement of Changes in Expression of Neuronal Cell Death-related Genes Due to Oxidative Stress of Antioxidant Peptide I

In this experimental example, gene changes related to neuronal cell death caused by oxidative stress of antioxidant peptide I were measured by PCR. PCR (polymerase chain reaction) is a technique for amplifying a large amount of a specific region of DNA or RNA in vitro. In this experiment, mRNA before protein expression was amplified and the amount of change was measured.

Specifically, after diluting the DNA extract of neurons, calculate the amount of reagents required per reaction, mix them all, put them in 48 μl of PCR tubes, and PCR reaction in the tube using 5 μl of one tube as the DNA template. (Takara PCR Thermal Cycler MP, Takara, Japan). The PCR reaction was initially denatured at 94 ° C. for 2 minutes, then denatured at 94 ° C. for 2 seconds, stable at 58 ° C. for 30 seconds, and repeated 35 cycles of polymerization at 72 ° C. for 35 minutes, and finally, polymerization at 72 ° C. for 2 minutes. The finished product was stained with Ethidium bromoblue (ETBR), and then electrophoresed in 1.2% agarose gel to observe its expression under ultraviolet light.

At this time, the forward primer of the used iNOS is 5'-AAATCCAGATAAGTGACA-3 ', the reverse primer is 5'-TGAACGTCCAGGTTTAGA-3', the forward primer of Bax is 5'-ATGGCTGGGGAGA CACCTGAG-3 ', the reverse primer is 5'-CTAGCAAAGTAGAAAAGGGCAAC- 3 ', the forward primer of Bcl-2 is 5'-CACCCCTGGCATCT TCTCCTT-3', the reverse primer is 5'-CACAATCCTCCCCCAGTTCACC-3 ', the forward primer of GAPDH is 5'-CCC CTTCATTGACCTCAACT-3', and the reverse primer is 5 '. '-TTGTCATGGATGTCCTTGGC-3'.

Figure 24 shows the neuronal cell death gene changes due to the oxidative stress of the antioxidant peptide I of the present invention by PCR, the antioxidant peptide I treatment group inhibits apoptosis, iNOS, Bax and the like apoptosis induction compared to the control group (control) When the gene Bcl-2 was reduced, the antioxidant peptide I caused by stress according to the present invention was found to inhibit neuronal cell death.

Experimental Example 20 Measurement of Changes in Expression of Neuronal Cell Death-related Genes Due to Oxidative Stress of Antioxidant Peptide I

In this experimental example, the neuronal cell death associated with oxidative stress of antioxidant peptide I was measured using Western blotting. Western blots are methods of finding specific proteins from a mixture of several proteins using antigen-antibody reactions.

Specifically, the protein extracted from the neuron is boiled for 3 minutes at 100 ° C to denature the protein, and the sample and size markers are flown on a 15% gel, followed by Polyvinylidene Fluoride (PVDF). Transferred to the membrane and washed three times for 5 minutes with Tris-buffered saline (TBS) containing 0.5% of Tween20. Then, shake for 1 hour in blocking solution (5% battery oil dissolved in TBS containing 5% of Tween20), dissolve 0.1% primary antibody in blocking solution, shake for 2 hours at room temperature, and TBS containing 0.5% of Tween20. (Hereinafter, TBS-T) was washed three times for 5 minutes. Finally, 0.2% secondary antibody was dissolved in a blocking solution, shaken for 1 hour at room temperature, washed three times with TBS-T for 5 minutes, and the film was immersed in the developing solution, and the image was taken with an imaging device (LAS 3000, Fujifilm, Japan). Photographed.

Figure 25 shows the neuronal cell death gene changes due to the oxidative stress of the antioxidant peptide I of the present invention by Western blot, the antioxidant peptide I treated group iNOS, Bax, cleaved apoptosis inducing gene compared to the control (control) It was confirmed that antioxidant peptide I according to the present invention inhibits neuronal cell death due to stress when caspase 3 and the like have decreased Bcl-2, an apoptosis inhibitory gene.

Experimental Example 21 Measurement of Position Change of Neuronal Cell Death Related Genes due to Oxidative Stress of Antioxidant Peptide I

In this experimental example, the change in the position of the neuronal cell death related gene, Cytochrome C, due to the oxidative stress of the antioxidant peptide I was measured by immunocytochemistry. In immunocytochemistry, a protein antibody to be measured is added and a secondary fluorescent antibody is reacted to observe the morphology and migration of cells through a fluorescence microscope. Cytochrome C is a gene that flows out of the mitochondria early in apoptosis. The position can be used to determine the presence of apoptosis.

Specifically, the harvested cells were attached to the slides and fixed with cold 4% paraformaldehyde for 20 minutes, washed three times for 5 minutes with Phosphate Buffered Saline (PBS), followed by cold 0.2% triton X-100. After shaking for 10 minutes, washed three times for 10 minutes with phosphate buffer, 1% primary antibody was added at 37 ℃ for 1 hour, washed three times in phosphate buffer solution for 5 minutes, 2% secondary antibody at room temperature 1 After the application, the cover slide was covered and measured with a confocal microscope (LSM 410, Carl Zeizz, Germany).

FIG. 26 shows changes in neuronal cell death related genes, namely, cytochrome C, due to oxidative stress of the antioxidant peptide I of the present invention through immunocytochemistry, and the antioxidant peptide I treatment group apoptosis compared to the control group. Antioxidant peptide I according to the present invention was found to suppress neuronal cell death due to stress, since the outflow of cytochrome C, which is an induction gene, was reduced.

Experimental Example 22 Measurement of Nuclear Morphology of Neurons due to Oxidative Stress of Antioxidant Peptide I

In this experimental example, the nuclear morphology of neurons due to the oxidative stress of the antioxidant peptide I was observed by staining with the fluorescent pigment Heochst 33342.

Specifically, inoculate 2.1 x 10 ⁵ neurons in a 6 well plate and 24 hours after 1 mM H ₂ O ₂ or 1 mM H ₂ O ₂ + After treatment with antioxidant peptide I, the reaction was incubated for 24 hours at 37 ℃, CO ₂ incubator, harvested and cytospin for 3 minutes at 1,300 rpm to attach the cells to the slide fixed by cold 4% paraformaldehyde for 20 minutes I was.

The fixed cells were washed three times with phosphate buffer solution (PBS) for 10 minutes, and then stained with 0.8 μg / ml Heochst 33342 for 30 minutes at 37 ° C. and observed by fluorescence microscope.

Figure 27 shows the change in the nucleus of neurons due to the oxidative stress of the antioxidant peptide I of the present invention, the antioxidant peptide I treatment group in the present invention when the nucleus cleavage is reduced compared to the control (control) According to the antioxidant peptide I was confirmed to suppress neuronal cell death caused by stress.

Experimental Example 23 Measurement of DNA Damage Protection Capacity of Antioxidant Peptide I Due to Oxidative Stress

In this experimental example, electrophoresis was carried out using plasmid DNA to determine DNA protection ability of venison protease extract from free radicals, and the DNA protection ability from impact induced by free radicals was measured. . Electrophoresis of normal plasmid DNA is divided into supercoiled DNA, linear DNA, and open circular DNA, with the highest amount of super helical DNA. When a Fenton reaction is applied to the DNA and a shock is applied to the DNA, the DNA is lost and the super helical DNA is changed into a ring-opening DNA.

Specifically, 0.5 μg of pBR 322 DNA, 3 μl of ultrapure distilled water, 3 μl of 2 mM FeSO ₄ , 2 μl of deer protein hydrolase extract, and 4 μl of 30% H ₂ O ₂ were mixed. The reaction was carried out at 37 ° C. for 1 hour. The reaction solution was subjected to 0.8% agarose gel electrophoresis stained with EDTA to observe the band.

The results are shown in Figure 28, it can be seen in the venison proteinase extract of the present invention compared to the normal plasmid DNA (lane 1), the ultra-strand DNA was completely converted to the ring opening DNA under the Fenton reaction (lane 2) ). Venison proteinase extract of the present invention showed a DNA protection effect in a concentration-dependent manner.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a schematic diagram showing a manufacturing process of venison proteinase extract of the present invention,

2 is a diagram showing the hydroxyl radical scavenging activity (a) and the hydroxyl radical scavenging activity (ESR) of the venison proteinase extract (6 species) of the present invention,

Figure 3 is a diagram showing the degree of hydrolysis by pH of the venison protein hydrolase (papaine) extract of the present invention,

Figure 4 is a diagram showing the degree of hydrolysis by temperature of venison protein hydrolase (papaine) extract of the present invention,

Figure 5 shows the degree of hydrolysis by substrate concentration of venison protein hydrolase (papain) extract of the present invention,

Figure 6 is a diagram showing the degree of hydrolysis by substrate concentration of enzyme protein hydrolase (papine) extract of the present invention,

7 is a diagram showing the degree of hydrolysis of the venison protein hydrolase (papain) extract of the present invention over time,

8 is a molecular weight (I,> 30,000 Da; II, 10,000-30,000 Da; III, 5.000-10,000 Da; IV, 5,000 Da <) hydroxyl radical scavenging according to the molecular weight of the venison protease (papine) extract of the present invention Is a diagram showing an ESR signal (b) showing activity (a) and hydroxyl radical scavenging activity,

Figure 9 is a diagram showing the hydroxyl radical scavenging activity after separating the protein corresponding to the molecular weight 5,000 ~ 10,000 Da of the protein proteinase (papine) extract of the present invention by ion exchange liquid chromatography,

10 is a diagram showing the hydroxyl radical scavenging activity after separating the venison proteolytic enzyme (papaine) extract of the present invention by high-performance chromatography equipped with ion exchange liquid chromatography and preparative column;

11 is a diagram showing the hydroxyl radical scavenging activity of the venison protein hydrolase (papaine) extract of the present invention after separation using ion exchange liquid chromatography, high performance chromatography equipped with a preparative column and an analytical column. ego;

Figure 12 is isolated from the deer protein hydrolase (papine) extract of the present invention using ion exchange liquid chromatography, high performance chromatography equipped with a preparative column, analytical column and once again analytical column hydroxyl Diagram showing radical scavenging activity;

FIG. 13 shows the amino acid sequence and molecular weight of two antioxidant peptides separated using ion exchange liquid chromatography, high performance chromatography equipped with a preparative column, and an analytical column;

14 is a diagram showing the free radical scavenging ability of the antioxidant peptide of FIG. 13;

15 is a diagram showing the neuronal cytotoxicity test of the highly active peptide (antioxidant peptide I) in the purified antioxidant peptide isolated from venison proteolytic enzyme (papaine) extract of the present invention;

Figure 16 is a diagram showing the effect on the catalase activity in neurons of the antioxidant peptide I of the present invention;

17 is a diagram showing the effect on the glutathione peroxidase activity in neurons of the antioxidant peptide I of the present invention;

18 is a diagram showing the effect of the antioxidant peptide I of the present invention on glutathione-S-transferase activity in neurons;

19 is a diagram showing the effect of the antioxidant peptide I of the present invention on the production of nitric oxide in neurons;

20 is a diagram showing the effect on the production of reactive oxygen species in neurons of the antioxidant peptide I of the present invention;

21 is a diagram showing the effect of the antioxidant peptide I of the present invention on the production of lipid peroxide in neurons;

Figure 22 is a measure of the protection against neuronal loss due to oxidative stress of the antioxidant peptide I of the present invention after staining with iodide pyropidium;

Figure 23 is a measure of the protection against neuronal loss due to oxidative stress of the antioxidant peptide I of the present invention after staining with Annexin-V;

FIG. 24 is a diagram showing PCR changes in genes involved in neuronal cell death due to oxidative stress of the antioxidant peptide I of the present invention; FIG.

25 is a diagram showing the change in genes involved in neuronal cell death due to oxidative stress of the antioxidant peptide I of the present invention measured by Western blot;

FIG. 26 is a diagram showing changes in genes related to neuronal cell death, cytochrome C, due to oxidative stress of the antioxidant peptide I of the present invention measured by immunocytochemistry;

FIG. 27 is a diagram illustrating nuclear changes of neurons due to oxidative stress of the antioxidant peptide I of the present invention after staining with Hoechst 33342; FIG.

28 is a diagram measuring the DNA protection capacity of the antioxidant peptide I of the present invention against DNA loss due to oxidative stress.

<110> Konkuk University Industrial Cooperation Corp. <120> Antioxidant composition comprising enzymatic hydrolysates of          venison <130> P08-E209 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 10 <212> PRT <213> venison <400> 1 Met Gln Ile Phe Val Lys Thr Leu Thr Gly   1 5 10 <210> 2 <211> 10 <212> PRT <213> venison <400> 2 Asp Leu Ser Asp Gly Glu Gln Gly Val Leu   1 5 10 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 3 aaatccagat aagtgaca 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 4 tgaacgtcca ggtttaga 18 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 5 atggctgggg agacacctga g 21 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 6 ctagcaaagt agaaaagggc aac 23 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 7 cacccctggc atcttctcct t 21 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 8 cacaatcctc ccccagttca cc 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 9 ccccttcatt gacctcaact 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 10 ttgtcatgga tgtccttggc 20  

Claims (10)

Venison protein hydrolase extract having antioxidant effect and antioxidant peptide isolated and purified from the extract. The method of claim 1, The peptide is an antioxidant peptide, characterized in that having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. The method of claim 1, The proteolytic enzyme is 1 selected from the group consisting of papain, pepsin, trypsin, trypsin, alpha-chymotrypsin, alcalase, and nutrase. A composition characterized in that the species or more. An antioxidant composition comprising any one or a mixture of the venison proteolytic enzyme extract of claim 1 and the antioxidant peptide isolated and purified from the extract as an active ingredient. The method of claim 4, wherein The antioxidant composition is a composition, characterized in that used as medicines, health supplements, cosmetics, food, food additives. Method for producing venison protein hydrolase extract, characterized in that the venison is treated with a proteolytic enzyme at a reaction temperature of 25 to 70 ℃ and a reaction time of 6 to 10 hours in a buffer of pH 1.0 ~ 10.0. The method of claim 6, PH of the buffer is characterized in that the production method of 2.0 to 8.0. The method of claim 6, The buffer is a manufacturing method, characterized in that the phosphate buffer (Phosphate buffer) or glycine-HCl buffer (Glycine-HCl buffer). The method of claim 6, The proteolytic enzyme is 1 selected from the group consisting of papain, pepsin, trypsin, trypsin, alpha-chymotrypsin, alcalase, and nutrase. The production method characterized by the above-mentioned species. In addition to the method of claim 6, the deer protein hydrolyzate extract was subjected to ion exchange liquid chromatography using diethylaminoethyl-sephacel, high performance chromatography equipped with a preparative C ₁₈ column, and two analytical C ₁₈ columns. A method for separating and purifying peptides having strong antioxidant power through a continuous four-step purification process using the equipped high performance chromatography.

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