1. An antioxidant polypeptide derived from tachypleus tridentatus antimicrobial peptide, wherein the antioxidant polypeptide comprises an amino acid sequence as shown in the following:

Arg-Gly-Pro-Pro-Lys-Cys-Ala-Thr-Tyr-Gly-Gln-Lys-Cys-Arg-Thr-Trp。

2. the antioxidant polypeptide derived from tachypleus tridentatus antimicrobial peptide of claim 1, wherein the primary structure of the complete sequence of the antioxidant polypeptide is as follows:

Arg-Gly-Pro-Pro-Lys-Cys-Ala-Thr-Tyr-Gly-Gln-Lys-Cys-Arg-Thr-Trp。

3. the antioxidant polypeptide derived from tachypleus tridentatus antimicrobial peptide of claim 2, wherein the antioxidant polypeptide has a molecular weight of 1852.14Da, an isoelectric point of 9.85, a net charge of 4, and a destabilization factor of 1.41.

4. The method for preparing the antioxidant polypeptide derived from horseshoe crab antimicrobial peptide as claimed in any one of claims 1 to 3, wherein the antioxidant polypeptide derived from horseshoe crab antimicrobial peptide is obtained by chemical synthesis by Fmoc polypeptide solid phase synthesis method according to the amino acid sequence of the antioxidant polypeptide.

5. The method of claim 4, wherein the antioxidant polypeptide derived from horseshoe crab antimicrobial peptide is obtained by purifying the polypeptide by reverse phase high performance liquid chromatography after chemically synthesizing the polypeptide by Fmoc polypeptide solid phase synthesis.

6. Use of the anti-oxidant polypeptide derived from horseshoe crab antimicrobial peptide according to any one of claims 1 to 3 in the preparation of anti-oxidant drugs, health products, food additives and anti-oxidant cosmetics.

7. An antioxidant agent comprising the antioxidant polypeptide derived from horseshoe crab antimicrobial peptide according to any one of claims 1 to 3.

8. A health product comprising the antioxidant polypeptide derived from horseshoe crab antimicrobial peptide according to any one of claims 1 to 3.

9. A food additive comprising the antioxidant polypeptide derived from horseshoe crab antimicrobial peptide according to any one of claims 1 to 3.

10. An antioxidant cosmetic comprising the antioxidant polypeptide derived from the antimicrobial peptide of Tachypleus tridentatus according to any one of claims 1 to 3.

Background

The bioactive peptide is a molecular polymer between amino acid and protein, and has physiological regulation effect on the life activity of living organisms. Bioactive peptides have great development prospects in the industries of medicines, foods and cosmetics, wherein polypeptides with antioxidant activity are also called antioxidant peptides.

The occurrence of various diseases in human, such as cancer, aging, rheumatoid arthritis, arteriosclerosis and other chronic diseases, is related to the oxidative damage of the body. Under normal physiological environment, the body can generate a small amount of active oxygen free radicals, and when the body is stressed by the environment, a large amount of oxygen free radicals can be generated, so that the body is oxidized and damaged, and chronic diseases and organ aging are further caused. By properly ingesting substances with antioxidant activity, the human body can reduce the level of free radicals in the body, prevent oxidative stress, prevent lipid peroxidation and help the body to resist diseases. In addition, because the photoaging effect of skin is caused by the accumulation of a large amount of oxygen radicals generated by skin cells induced by ultraviolet rays, the antioxidant added into a plurality of skin care products can not only remove excessive free radicals in vivo, but also has obvious protective effect on mitochondrial damage caused by ultraviolet rays and lipid peroxidation induced by the free radicals, and also has the effects of reducing freckles, delaying aging, regulating immunity and protecting skin.

At present, research on antioxidant peptides is increasing at home and abroad, and research on preparation, separation and purification of the antioxidant peptides to obtain detection methods and properties of the antioxidant peptides is increasing day by day, and patent document CN 107226841B provides an antioxidant ultrashort peptide and application thereof, wherein the antioxidant ultrashort peptide contains 5-6 amino acids; patent document CN110256530A provides an antioxidant peptide and application thereof, wherein the molecular weight is 700-800 Da; patent document CN 108373496a discloses an antioxidant polypeptide GTMGAVGPRG and a preparation method thereof. The technology for synthesizing the polypeptide by using the chemical solid-phase synthesis method is more and more mature, and the method is a quick and effective method for synthesizing the antioxidant short peptide in the future. Although the method can successfully obtain the antioxidant peptide, the antioxidant performance of the product has further improved space.

Therefore, the development of an antioxidant peptide with excellent antioxidant performance is of great practical significance.

Disclosure of Invention

The invention aims to provide an antioxidant peptide with excellent antioxidant performance, in particular to an antioxidant polypeptide derived from tachypleus tridentatus antibacterial peptide, and preparation and application thereof.

Limulus is an ancient marine organism belonging to the families of arthropoda, acropoda, caudate and limulus, has been alive for 4 billion years on the earth, called marine "activating stone" and has more than 50 bioactive substances in blood. According to the invention, the 7 th-22 th amino acid residue at the N tail end of the antibacterial peptide Tachystatin C found in the blood of the horseshoe crab is intercepted according to the amino acid sequence DYDWSLRGPPKCATYGQKCRTWSPPNCCWNLRCKAFRCRPR, and the intercepted small molecule short peptide RW-16 with the amino acid sequence RGPPKCATYGQKCRTW is found, so that the antibacterial peptide Tachystatin C has extremely strong antioxidant activity and extremely high stability. The full length of the primary sequence has 16 amino acid residues, the molecular weight is small, the structure is simple, no protein modification is caused, the preparation method is a chemical synthesis method, the process is simple and convenient, the yield is high, and the purity is high.

In order to achieve the purpose, the invention provides the following technical scheme:

an antioxidant polypeptide derived from tachypleus tridentatus antimicrobial peptide, the antioxidant polypeptide comprising an amino acid sequence (i.e., SEQ ID NO:1) as shown below:

Arg-Gly-Pro-Pro-Lys-Cys-Ala-Thr-Tyr-Gly-Gln-Lys-Cys-Arg-Thr-Trp is RGPPKCATYGQKCRTW.

The antioxidant polypeptide has strong antioxidant activity, strong free radical scavenging capacity, small molecular weight, simple structure and easy synthesis, can be used as antioxidant medicaments, food additives and antioxidant cosmetics, and has high popularization value.

As a preferred technical scheme:

the antioxidant polypeptide derived from tachypleus tridentatus antibacterial peptide is named as RW-16 because the antioxidant polypeptide contains 16 amino acid residues, and the primary structure of the whole sequence of the antioxidant polypeptide is as follows:

Arg-Gly-Pro-Pro-Lys-Cys-Ala-Thr-Tyr-Gly-Gln-Lys-Cys-Arg-Thr-Trp。

RW-16 is rich in three essential amino acids such as lysine (Lys), threonine (Thr) and tryptophan (Trp), and contains cysteine (Cys), and has strong antioxidant activity and high stability, with a concentration of 0.2mg/mL, a clearance rate for DPPH free radicals of 95.58% and a clearance rate for superoxide free radicals of 52.97%; the clearance rate of DPPH free radical is 95.85% when the concentration is 2mg/mL, and the clearance rate of superoxide radical is 95.07%.

The antioxidant polypeptide derived from tachypleus tridentatus antibacterial peptide has the molecular weight of 1852.14Da, the isoelectric point of 9.85, the net charge of 4 and the instability coefficient of 1.41 (extremely stable). The antioxidant polypeptide has the advantages of small molecular weight, easy absorption and utilization by organisms, alkalescence in aqueous solution, positive charge, capability of neutralizing gastric acid by theoretically eating (needing clinical verification subsequently), contribution to gastrointestinal absorption, higher stability when the instability coefficient is less than 40 and lower stability when the instability coefficient is more than 40, and the instability coefficient of the antioxidant polypeptide is 1.41, which indicates that the antioxidant polypeptide is extremely stable in the environment and is not easy to inactivate.

The invention also provides a method for preparing the antioxidant polypeptide from the tachypleus tridentatus antibacterial peptide, and the antioxidant polypeptide from the tachypleus tridentatus antibacterial peptide is obtained by adopting Fmoc polypeptide solid phase synthesis method to chemically synthesize according to the amino acid sequence of the antioxidant polypeptide. The protection scope of the present invention is not limited thereto, and those skilled in the art can adopt an appropriate preparation method according to actual needs as long as the amino acid sequence of the antioxidant polypeptide is ensured to be correct.

As a preferred technical scheme:

according to the method, after the polypeptide is chemically synthesized by adopting an Fmoc polypeptide solid-phase synthesis method, the polypeptide is purified by using reverse-phase high performance liquid chromatography to obtain the antioxidant polypeptide derived from the tachypleus tridentatus antibacterial peptide.

In addition, the invention also provides application of the antioxidant polypeptide derived from the tachypleus tridentatus antimicrobial peptide in preparation of antioxidant drugs, health-care products, food additives and antioxidant cosmetics.

In addition, the invention also provides an antioxidant medicine containing the antioxidant polypeptide derived from the tachypleus tridentatus antimicrobial peptide.

A health product contains the above antioxidant polypeptide derived from Tachypleus tridentatus antibacterial peptide.

A food additive contains the above antioxidant polypeptide derived from horseshoe crab antibacterial peptide.

An antioxidant cosmetic contains the above antioxidant polypeptide derived from horseshoe crab antibacterial peptide.

Has the advantages that:

(1) the antioxidant polypeptide derived from the tachypleus tridentatus antibacterial peptide has strong antioxidant activity, strong free radical scavenging capacity, small molecular weight, simple structure and high popularization value, is easy to synthesize, and can be used as an antioxidant medicine, a food additive and an antioxidant cosmetic;

(2) the preparation method of the antioxidant polypeptide derived from the tachypleus tridentatus antimicrobial peptide has the advantages of simple process, low cost, large-scale production and wide application prospect.

Drawings

FIG. 1 is a high performance liquid chromatogram of the crude polypeptide obtained in step (5) of example 1 when purified;

FIG. 2 is a mass spectrum of the antioxidant polypeptide prepared in example 1.

Detailed Description

The present invention will be described in more detail with reference to the accompanying drawings, in which embodiments of the invention are shown and described, and it is to be understood that the embodiments described are merely illustrative of some, but not all embodiments of the invention.

Example 1

A preparation method of antioxidant polypeptide derived from tachypleus tridentatus antimicrobial peptide comprises the following steps:

(1) Fmoc-Trp (Boc) -Wang Resin (wang Resin) was put into a polypeptide synthesizer.

(2) Deprotection: the Fmoc protecting group on the resin was removed with 20% piperidine (DMF: hexahydropyridine ═ 4:1), reacted for 20 minutes, the resin was washed with DMF and subjected to ninhydrin test (Kaiser test) and the resin appeared blue to confirm that the Fmoc protecting group had been removed.

(3) Amino acid condensation: DIC, HOBT and FMOC-Thr (tBu) -OH are added into a synthesizer, condensation reaction is carried out for 40 minutes, DMF is used for washing the resin, ninhydrin test (Kaiser test) shows that the resin is colorless, and the amino acid is successfully grafted, so that the next amino acid can be jointed. Repeating the steps (2) and (3), and sequentially joining according to the polypeptide sequences shown as follows:

Arg-Gly-Pro-Pro-Lys-Cys-Ala-Thr-Tyr-Gly-Gln-Lys-Cys-Arg-Thr-Trp。

(4) after the last amino acid deprotection and indene detection are finished, residual liquid is pumped out, washed by DCM, pumped out after washing, and pumped out until the resin is loose and has no caking.

(5) Adding a cutting agent trifluoroacetic acid into the resin, cutting the polypeptide from the resin, extracting with diethyl ether, and freeze-drying to obtain a crude polypeptide product.

(6) The crude peptide obtained was purified by reverse phase high performance liquid chromatography (FIG. 1), passed through a reverse phase column Kromasil C18(4.6 x 250mm,5um) and treated with CH containing 0.1% trifluoroacetic acid₃CN/H₂And performing gradient elution on the O at the flow rate of 1mL/min for 80min, monitoring light absorption at 214nm, collecting a main peak, and performing freeze-drying to obtain the purified antioxidant polypeptide. Finally, the molecular weight of the peptide was determined by mass spectrometry (fig. 2).

As can be seen from FIG. 1, the purity of the purified antioxidant polypeptide was greater than 95%, and the antioxidant polypeptide RW-16 obtained from FIG. 2 had a relative molecular weight of 1852.14 Da.

Then, the antioxidant activity of the antioxidant polypeptide RW-16 prepared in the step (6) is measured, and the content of the measurement comprises the measurement of DPPH free radical scavenging capacity and superoxide radical (O)₂ ^-) The specific contents of the measurement of the clearing capacity are as follows:

1) determination of DPPH radical scavenging Capacity

The polypeptide was diluted with distilled water to give solutions of concentrations of 0.02, 0.2 and 2mg/mL, respectively. Preparing a DPPH solution with the concentration of 0.2mmol/L by using 95% ethanol, uniformly mixing 2mL of a polypeptide sample and 2mL of the DPPH solution, carrying out constant-temperature water bath at 28 ℃ for 30min in a dark condition, and measuring the absorbance at 517nm after the reaction is finished. The control group is the absorbance of the mixed solution of 2mL of absolute ethyl alcohol solution and 2mL of sample solution, the blank group is the absorbance of the mixed solution of 2mL of DPPH solution and 2mL of distilled water, and absolute ethyl alcohol is used for zero setting.

Wherein, As is the absorbance of the experimental group; ac is the absorbance of the control group; ab is absorbance of blank.

2) Superoxide radical (O)₂ ^-) Determination of the scavenging Capacity

200uL of polypeptide solutions with concentrations of 0.02, 0.2 and 2mg/mL, respectively, were mixed with 1.3mL of 50mM Tris-HCl (pH 8.8), 100. mu.L of 1.5mM pyrogallol (prepared with 10mM HCl) was added to react at room temperature for 10min, and then 50. mu.L of 8M HCl was added to terminate the reaction, absorbance A1 was measured at 420nm, 200. mu.L of distilled water was used as a blank A0 instead of the sample, and 100. mu.L of HCl was used as a control A2 instead of the pyrogallol.

The test results are shown in table 1;

TABLE 1

Polypeptide concentration (mg/mL)	DPPH clearance (%)	O2 -Clearance (%)
			0.02	24.84	50.01
0.2	95.58	52.97
			2	95.85	95.07

As can be seen from Table 1, the antioxidant polypeptide RW-16 has strong antioxidant activity, and when the sample concentration is 2mg/mL, the clearance rate of the antioxidant polypeptide RW-16 to DPPH is as high as 95.85%, and to O₂ ^-The clearance rate of the product is as high as 95.07 percent.

The following results were obtained after testing the molecular weight, isoelectric point, net charge and instability coefficient of the antioxidant polypeptide RW-16: the molecular weight is 1852.14Da, the isoelectric point is 9.85, the net charge is 4, and the instability coefficient is 1.41.

Proved by verification, the antioxidant polypeptide derived from the tachypleus tridentatus antibacterial peptide has strong antioxidant activity, strong free radical scavenging capacity, small molecular weight, simple structure and easy synthesis, can be used as an antioxidant medicine, a food additive and an antioxidant cosmetic, and has extremely high popularization value; the preparation method has simple process and low cost, can be used for large-scale production, and has wide application prospect.

Although specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these embodiments are merely illustrative and various changes or modifications may be made without departing from the principles and spirit of the invention.

Sequence listing

<110> Shanghai ocean university

<120> an antioxidant polypeptide derived from Tachypleus tridentatus antimicrobial peptide, its preparation and application

<141> 2021-04-29

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 1

Arg Gly Pro Pro Lys Cys Ala Thr Tyr Gly Gln Lys Cys Arg Thr Trp

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