CN108794574B

CN108794574B - Antioxidant active peptide and application thereof

Info

Publication number: CN108794574B
Application number: CN201810574097.5A
Authority: CN
Inventors: 盛剑勇; 杨晓宇; 曾坚; 刘卫; 杨祥良; 万江陵
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2018-06-06
Filing date: 2018-06-06
Publication date: 2021-03-19
Anticipated expiration: 2038-06-06
Also published as: CN108794574A

Abstract

The invention provides an antioxidant active peptide and application thereof. The amino acid sequence of the active peptide comprises one or more of the sequences shown in SEQ ID NO. 1-12. The active peptides provided by the invention are all derived from walnut pulp hydrolysate and can also be obtained by solid phase synthesis respectively. The active peptide provided by the invention has high antioxidant activity and strong nerve cell proliferation promoting capability, particularly can obviously promote the proliferation of SH-SY5Y cells with oxidative damage, and has high oral administration absorption efficiency.

Description

Antioxidant active peptide and application thereof

Technical Field

The invention relates to the technical field of food-derived bioactive peptides, and more particularly relates to an antioxidant bioactive peptide and application thereof.

Background

Some short peptides can provide nutrients necessary for human growth and development and regulate human functions, and these bioactive polypeptides are called active peptides. The active peptide is an important effective component of medicines or functional foods, and compared with protein, the polypeptide has smaller molecular weight, better stability in gastrointestinal tract and more favorable absorption. Different biological peptides have different structures and physiological functions, and the active peptides mainly comprise the following peptides: binding peptides, phosphopeptides, glutathione, antimicrobial peptides, neuropeptides, immunopeptides, flavor peptides, hormone peptides, nutritional peptides, antioxidant peptides, and the like. The natural antioxidant peptide is a peptide with antioxidant activity existing in animals and plants, has the effects of scavenging free radicals, chelating metal ions, quenching singlet oxygen, decomposing peroxide, inhibiting lipid peroxidation and the like, is easy to be absorbed by human bodies, has no toxic or side effect, and has good nutrition and processing characteristics, thereby having huge development prospects in human foods and medicines.

Research shows that many polypeptides from different sources have antioxidant capacity, and polypeptides obtained by hydrolyzing collagen, casein, bovine serum albumin, soybean protein, corn protein and the like have certain antioxidant capacity. However, the oral absorption of active peptides is generally poor, and the bioavailability is low, which is not favorable for the exertion of the in vivo efficacy of active peptides. Therefore, it is necessary to screen out polypeptides with high biological activity and oral absorption efficiency, which provides a basis for the intensive research of active peptides.

Disclosure of Invention

The first purpose of the invention is to provide an antioxidant active peptide, wherein the amino acid sequence of the active peptide comprises one or more of the following sequences:

EQEEEESTGRMK, as shown in SEQ ID NO. 1;

AGNDGFEYVTLK, as shown in SEQ ID NO. 2;

LAGNPHQQQQN, as shown in SEQ ID NO. 3;

HNLDTQTESDV, as shown in SEQ ID NO. 4;

QQRQQQGL as shown in SEQ ID No. 5;

AELQVVDHLGQTV, as shown in SEQ ID NO. 6;

VEGNLQVLRPR, as shown in SEQ ID NO. 7;

WSREEQELEDR, as shown in SEQ ID NO. 8;

ADAFNVDTETA, as shown in SEQ ID NO. 9;

LQVVGQDGQNVFDGELR, as shown in SEQ ID NO. 10;

QLQVLRPR, shown in SEQ ID NO. 11;

LGLLPSFSNAPR, as shown in SEQ ID NO. 12.

The invention also provides application of the active peptide in preparing antioxidant drugs.

The invention also provides a medicament containing the active peptide.

The invention also provides application of the active peptide in preparing medicines for promoting nerve cell proliferation.

The invention also provides application of the active peptide in preparing a medicine for preventing and/or treating memory deterioration.

Wherein, the medicament can be in oral dosage form, intravenous, subcutaneous, transdermal or intramuscular administration dosage form, and is preferably oral dosage form.

The invention also provides a health product for strengthening brain and improving intelligence, which comprises the active peptide.

The active peptide can be used as a functional active factor to be added into health products or medicines for preventing and/or treating memory deterioration. The active peptides of the invention may be present as a single active ingredient or as a synergistic effect of one of the active ingredients with the other active ingredients.

Wherein, the health product is preferably in oral dosage form.

The active peptides provided by the invention are all derived from walnut pulp hydrolysate and can also be obtained by solid phase synthesis.

The active peptide provided by the invention has high antioxidant activity, strong nerve cell proliferation promoting capacity and high oral absorption efficiency.

Drawings

FIG. 1 is an HPLC profile of each of 12 active peptides provided by the present invention;

FIG. 2 is the mass spectrum of each of 12 active peptides provided by the present invention;

FIG. 3 is a graph showing the hydroxyl radical scavenging rate of 12 active peptides provided by the present invention;

FIG. 4 is a diagram of the pro-oxidative damaged nerve cell proliferation potency of 12 active peptides provided by the present invention;

fig. 5 is a graph of the oral absorption rate of 12 active peptides provided by the present invention;

in the figure, P1-P12 refer to the polypeptides of SEQ ID NO.1-12, respectively.

Detailed Description

The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional technical means well known to those skilled in the art. The reagents used in the examples are commercially available unless otherwise specified.

The walnut pulp hydrolysate DWMH used in the invention is walnut peptide prepared in the specification of example 1 in the publication number CN 105063152A.

The active peptides used in the examples were obtained by solid phase synthesis, wherein EQEEEESTGRMK, AGNDGFEYVTLK, LAGNPHQQQQN, HNLDTQTESDV, QQRQQQGL, AELQVVDHLGQTV, VEGNLQVLRPR, WSREEQELEDR, ADAFNVDTETA, LQVVGQDGQNVFDGELR, QLQVLRPR, LGLLPSFSNAPR are numbered P1-P12, and their HPLC and mass spectrograms are shown in FIGS. 1 (A) - (L) and 2 (A) - (L), respectively, and FIGS. 1 and 2 show that 12 polypeptides were successfully synthesized, respectively.

Experimental example 1

Method for measuring antioxidant capacity of 12 polypeptides by hydroxyl radical scavenging method

1mL of 1mg/mL polypeptide solution was prepared, and 2mL of 1.8mM ferrous sulfate, 1.5mL of 1.8mM salicylic acid-ethanol solution, and 1mL of 8.8mM hydrogen peroxide solution were added in this order, followed by incubation in a water bath at 37 ℃ for 30 min.

1mL of 1mg/mL walnut meal hydrolysate solution is prepared, 2mL of 1.8mM ferrous sulfate, 1.5mL of 1.8mM salicylic acid-ethanol solution and 1mL of 8.8mM hydrogen peroxide solution are sequentially added, and the mixture is incubated in a water bath at 37 ℃ for 30 min.

The absorbance A1 was measured at 510 nm. The absorbance measured by the above method using distilled water instead of the sample was used as control A0. Hydroxyl radical clearance was calculated as follows:

hydroxyl radical scavenging rate (%) - (A)₀-A₁)/A₀]×100％

The hydroxyl radical clearance rate of the 12 polypeptides is shown in fig. 3, which indicates that the 12 polypeptides have certain antioxidant capacity, and the antioxidant capacity of the 12 polypeptides is obviously superior to that of walnut pulp hydrolysate DWMH in P1, P3, P8 and P9.

Experimental example 2:

CCK-8 method for evaluating proliferation effect of 12 polypeptides on oxidative damaged nerve cells

Construction of SH-SY5Y cell model of oxidative damage: SH-SY5Y cells in logarithmic growth phase are inoculated in a 96-hole cell culture plate and cultured overnight, and 200 mu mol/L H is added₂O₂The solution was incubated with the cells for 24h, with no treatment provided for the control group. 10 microliter of CCK-8 solution is added into each well, the culture is stopped after 4 hours, and the absorbance at 450nm is detected by using a microplate reader. The cell survival rate of the model group is measured to be 73.1 percent of that of the control group, which indicates that the oxidative damage model is successfully constructed.

Evaluation of proliferation promoting ability: SH-SY5Y cells in logarithmic growth phase are inoculated in a 96-hole cell culture plate and cultured overnight, and 200 mu mol/L H is added₂O₂The solution was incubated with the cells for 24 h. Then, the cells were incubated in serum-free medium containing 300. mu.g/mL of the polypeptide for 24 hours as an experimental group; cells were incubated in serum-free medium for 24h as a blank control. mu.L of CCK-8 solution was added to each well and the incubation was terminated after 4 h. And detecting the light absorption value at 450nm by using a microplate reader. Cell viability for each group is shown in figure 4. The 12 polypeptides can obviously promote the proliferation of SH-SY5Y cells damaged by oxidation.

Experimental example 3:

oral absorption efficiency assay for 12 polypeptides

And (3) constructing a Caco-2 monolayer cell model, namely, selecting the Caco-2 cell monolayer model to research the transport of the representative peptide. Caco-2 cells with good growth status were selected for digestion at 1X 10⁵Per cm²The density of (A) was seeded on a polycarbonate membrane of a Millicell cell, the Millicell cell was inserted into a 24-well plate, and placed in an incubator at 37 ℃ with 5% CO₂And (5) culturing under an environment. The solution was changed 24h after inoculation, followed by every 2 days. After 21 days of culture, the transmembrane resistance TEER was determined. TEER values are all larger than 300 omega m²The success of Caco-2 monolayer cell model construction is demonstrated.

Caco-2 cell transport experiments: first, 15 polypeptides were fluorescently labeled with 5-FAM, and a solution of 3mg/mL was prepared as a dosing solution. Then, the top and bottom sides of the Millicell scaffold were aspirated, HBSS was added at 0.2mL and 1.3mL on the top and bottom sides, respectively, and incubated for 20min on an air bath shaker at a temperature of 37 ℃ and shaking at 80 rpm. Next, the Millicell scaffold apical side liquid was again aspirated and HBSS 0.2mL was added for 1 wash, and HBSS 1.3mL was added at the basal side. Then, 0.2mL of the dosing solution was added to the top side of the chamber and 1.3mL of blank HBSS was added to the bottom side. Incubate at 37 ℃ with shaking at 80rpm and sample 0.2mL from the bottom side at 1h, 2h and 3h, respectively, and immediately replenish the same amount of HBSS. Finally, a multi-label analyzer is selected to measure the fluorescence intensity of the sample under the conditions that the excitation wavelength is 490nm and the emission wavelength is 510nm, and the apparent permeability coefficient P is calculated_appThe value means the oral absorption efficiency. The results in FIG. 5 show that the apparent permeability coefficients P of P8, P10 and P11_appThe value is between 0.1 and 10X 10^-6cm/s, which belongs to polypeptide with medium oral absorption efficiency and is absorbed in vivo by 1-10%; and other polypeptides P_appAll values are greater than 10 × 10^-6cm/s, which belongs to polypeptide with high oral absorption efficiency.

Finally, the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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Gln Leu Gln Val Leu Arg Pro Arg

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Claims

1. An antioxidant active peptide, wherein the amino acid sequence of said active peptide is VEGNLQVLRPR.

2. Use of the active peptide of claim 1 for the preparation of an antioxidant medicament.

3. A pharmaceutical comprising the active peptide of claim 1.

4. Use of the active peptide of claim 1 for the manufacture of a medicament for promoting nerve cell proliferation.

5. The use of claim 4, wherein the medicament is in an oral dosage form.

6. A health product for strengthening brain and improving intelligence, comprising the active peptide of claim 1.

7. The nutraceutical of claim 6, wherein said nutraceutical is in an oral dosage form.