CN114657162A - Antioxidant pentapeptide with vascular endothelial cell protection function and application thereof - Google Patents
Antioxidant pentapeptide with vascular endothelial cell protection function and application thereof Download PDFInfo
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Abstract
The invention discloses an antioxidant pentapeptide with a vascular endothelial cell protection function and application thereof. The antioxidant pentapeptide contains 5 amino acid residues, the molecular weight is 679.76g/mol, the theoretical isoelectric point is 11.05, and the amino acid sequence is as follows: leucine-alanine-histidine-phenylalanine-arginine. The pentapeptide is derived from an edible yeast enzymolysis product and can be prepared by solid phase synthesis. The polypeptide of the invention has good antioxidant activity and is resistant to H2O2The induced vascular endothelial cell oxidative damage has obvious protective action, can be used for developing and preparing antioxidant functional food, and has good application prospect.
Description
Technical Field
The invention belongs to the field of functional foods and biomedicines, and particularly relates to an antioxidant pentapeptide with a vascular endothelial cell protection function and application thereof.
Background
Reactive Oxygen Species (ROS) are widely present in cells and tissues of living organisms, and have important physiological actions such as signal transduction and maintenance of internal environment stability. However, excessive ROS accumulation can cause the organism to generate oxidative stress, cause oxidative damage of intracellular bioactive macromolecules, cause vascular endothelial cell damage, cause endothelial dysfunction, and cause chronic diseases such as diabetes, hypertension, cardiovascular diseases and the like. In addition, oxidative stress is one of the major causes of aging.
Antioxidants can mitigate the hazards associated with oxidative stress. Food is an important source of antioxidants, and the World Health Organization (WHO) has long recognized the important health effects of antioxidant and has claimed to increase antioxidants in the diet worldwide. Compared with common antioxidants such as BHT, BHA and the like, the antioxidant polypeptide has the advantages of small molecular weight, simple structure, strong activity, easy absorption, no toxicity and the like.
Therefore, the research and the acquisition of the novel antioxidant peptide without toxic and side effects from food have wide development prospect and urgent need.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the antioxidant pentapeptide with the vascular endothelial cell protection function. The pentapeptide is derived from a natural product, is obtained by identifying and screening an edible yeast enzymolysis product through LC-MS/MS, and has good antioxidant activity through research.
The invention also aims to provide application of the antioxidant pentapeptide with the vascular endothelial cell protection function.
The purpose of the invention is realized by the following technical scheme:
an antioxidant pentapeptide with vascular endothelial cell protection function has amino acid sequence of leucine-alanine-histidine-phenylalanine-arginine (Leu-Ala-His-Phe-Arg).
The antioxidant pentapeptide can be prepared by the conventional technical means in the field, such as solid phase synthesis.
The antioxidant pentapeptide with the vascular endothelial cell protection function is applied to the preparation of an antioxidant.
The antioxidant pentapeptide with the vascular endothelial cell protection function is applied to preparing antioxidant functional food.
The antioxidant pentapeptide with the vascular endothelial cell protection function is applied to the preparation of vascular endothelial cell protection medicines.
An antioxidant contains the antioxidant pentapeptide with the function of protecting vascular endothelial cells.
An antioxidant functional food contains the antioxidant pentapeptide with the function of protecting vascular endothelial cells.
A vascular endothelial cell protection medicine contains the antioxidant pentapeptide with the vascular endothelial cell protection function.
Compared with the prior art, the invention has the following advantages and effects:
1. the pentapeptide has good antioxidant activity and can improve the capability of resisting oxidative damage of vascular endothelial cells.
2. The pentapeptide is a small molecular peptide, the theoretical isoelectric point is 11.05, the molecular weight is 642.75g/mol, the structure is easy to regulate and control, and the pentapeptide is easy to synthesize and modify so as to obtain better activity and has obvious application potential.
3. The pentapeptide sieve of the invention is selected from enzymolysis products of edible yeast, endogenous protein from Saccharomyces cerevisiae (strain ATCC 204508/S288 c): transhydroxyacetase 1 (protein accession No.: P23254). Edible yeast is listed as "generally recognized as safe" (GRAS) food ingredient released by FDA, and has high safety.
Drawings
FIG. 1 is a chromatogram of solid phase synthesis of pentapeptide LAHFR.
FIG. 2 is a mass spectrum of solid phase synthesis of pentapeptide LAHFR.
FIG. 3 is a graph of the results of analysis of DPPH scavenging activity by pentapeptide LAHFR at various concentrations.
FIG. 4 is a graph of the results of analysis of various concentrations of pentapeptide LAHFR on superoxide anion scavenging activity.
FIG. 5 is a graph showing the analysis of the effect of pentapeptide LAHFR on human umbilical vein endothelial cell proliferation; wherein, and indicate that the sample group differed very significantly (p <0.01) and significantly (p <0.05), respectively, compared to the oxidative damage group.
FIG. 6 is a graph showing the analysis of the effect of pentapeptide LAHFR on MDA content in human umbilical vein endothelial cells; wherein, and indicate that the sample group differed very significantly (p <0.01) and significantly (p <0.05), respectively, compared to the oxidative damage group.
FIG. 7 is a graph showing the analysis of the effect of pentapeptide LAHFR on SOD activity in human umbilical vein endothelial cells; wherein, and indicate that the sample group differed very significantly (p <0.01) and significantly (p <0.05), respectively, compared to the oxidative damage group.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
The pentapeptide with the sequence of leucine-alanine-histidine-phenylalanine-arginine (Leu-Ala-His-Phe-Arg) is artificially synthesized and prepared by adopting an Fmoc solid phase synthesis method. According to the composition of amino acid residues of pentapeptide, various amino acids (Fmoc-Leu, Fmoc-Ala, Fmoc-His, Fmoc-Phe and Fmoc-Arg) with Fmoc-protecting groups at amino terminals are used as raw materials, and carboxyl of the Fmoc-Arg is connected with high molecular resin (Wang resin) by covalent bonds; adding Dimethylformamide (DMF) containing 20% (v/v) piperidine, and reacting for 0.5h to remove the amino protecting group Fmoc-; adding excessive Fmoc-Phe, taking hydroxybenzotriazole as a condensing agent, and reacting for 2h at 30 ℃ to condense carboxyl of the Fmoc-Phe with active amino of Arg on the resin; repeating the deprotection and condensation reaction, sequentially connecting the rest other amino acids, cracking the pentapeptide from the resin, separating and purifying by a C18 column, and freeze-drying to obtain the pentapeptide. The liquid chromatogram (figure 1) analysis shows that the purity of the small molecule polypeptide synthesized by the method is 98.79%. The liquid chromatography-mass spectrometry (FIG. 2) shows that the synthesized polypeptide is Leu-Ala-His-Phe-Arg.
Example 2
DPPH was prepared in ethanol (0.1 mmol/L). The small molecular peptides are respectively prepared into solutions with the concentration of 0.5-7.0 mg/mL by deionized water. Taking 0.15mL of pentapeptide solution, adding 0.15mL of DPPH, standing for 1h at 25 ℃ in a dark place, and measuring the light absorption value (Am) at 517 nm; adding 0.15mL of absolute ethyl alcohol into 0.15mL of a sample, standing for 1h at 25 ℃ in a dark place, and deducting a light absorption value Ax of the sample from Am as a background; deionized water was used as a control and absorbance was labeled An.
DPPH radical clearance (SA) was calculated according to equation (1).
As can be seen from FIG. 3, the EC for DPPH clearance of the pentapeptide of the present invention50The value is 2.50mg/mL, and the antioxidant activity is certain.
Example 3
The superoxide anion scavenging activity of the pentapeptide was determined by the pyrogallol method. mu.L of sample or deionized water (blank control) and 174. mu.L of Tris-HCl buffer (0.5mol/L, pH 8.2) were added to the microplate, and incubated in an microplate reader at 25 ℃ for 10 min. Adding 13 μ L of pyrogallol solution (3mmol/L, 10mmol/L HCl), measuring absorbance at 321nm every 30s, measuring for 150s, and calculating oxidation rate V of pyrogallol and superoxide anion scavenging activity according to formula (2). In the formula, Vc: a control group rate; vs: sample set rate.
As can be seen from FIG. 4, the pentapeptide of the present invention has strong scavenging activity on superoxide anion, and its EC50The value was 2.18 mg/mL.
Example 4
Culturing human umbilical vein endothelial cell line EA.hy 926 cell, wherein the cell culture solution is 90% DMEM culture solution (NaHCO)31.5g/L, pH 7.0), 10% of south American blood source fetal calf serum and 1% of penicillin/streptomycin double antibody. The cells were harvested and plated at 1.0e5 cells/mL in T25 cell culture flasks at 37 ℃ with 5% CO2Culturing in an incubator, changing culture solution every 2 days, and subculturing every 4 days. 3-5 generation cells were taken, cultured to log phase, digested with pancreatin, added to 6-well plates at a density of 1.0e5 cells/mL, 1mL per well, and 4 wells per group. When the cells grow to more than 80% of the bottom of the hole, the original is discardedThe cells were pretreated for 24 hours by replacing the culture medium with 1mL of a serum-free medium containing pentapeptide at a low concentration (389. mu.M) or a high concentration (778. mu.M), respectively, and the cell culture medium was discarded. The antioxidant Glutathione (GSH) (concentration 1.63. mu.M) was used as a positive control. Then 200. mu. L H was added to each group2O2Serum-free medium (250. mu.M) induced oxidative damage to cells, and a group of samples was taken after 24h of continuous culture to determine cell viability by MTT method. Another set of samples was tested for intracellular MDA and SOD markers.
The MTT method is specifically operated as follows: at 24h and 48h, 20. mu.L MTT (5mg/mL) was added to each well, the wells were incubated for 4h, the solution was aspirated, 150. mu.L DMSO was added, the mixture was gently shaken for 10min, and the absorbance at 570nm was measured using a microplate reader. The cell-free medium was added with the same amount of MTT, and after 4 hours the medium was aspirated off, 150. mu.L of DMSO was added as a zero well. The cell viability was calculated according to formula (3).
Cell viability (%) ═ absorbance for experiment/control (3)
MDA and SOD assay operations were: the pellet was retained by digestion and centrifugation, and the cell pellet was washed twice with sterile PBS buffer (4 ℃), and the cells were repeatedly frozen and thawed with liquid nitrogen to disrupt the cells. The total protein in the cells is measured by adopting a total protein kit (Nanjing institute for bioengineering, product number: A045-4-2), the MDA content in the cells is measured by adopting an Malondialdehyde (MDA) kit (Nanjing institute for bioengineering, product number: A003-1-2), and the SOD kit Nanjing institute for bioengineering, product number: a001-3-2) determining the activity of SOD in cells.
As shown in FIG. 5, 250. mu.M of H2O2The treatment can generate oxidative damage, obviously inhibit the proliferation of EA.hy 926 cells, and the cell survival rate of the damage model group is 47.80 +/-4.62%. The survival rate of the cells after the low and high dose pentapeptide treatment was 54.98 + -6.34% and 59.15 + -2.80%, respectively. Compared with a damage model group, the pentapeptide can remarkably improve the cell survival rate and has remarkable protective effect on cell oxidative damage (p)<0.01)。
As can be seen from FIG. 6, H is compared with that of the control group2O2Treated oxidative damage model cellsThe content of inner MDA increases from 3.09 + -0.65 μ M/mgprot (milligram protein) to 11.09 + -1.38 μ M/mgprot. The positive control GSH can reduce the MDA content in the cells very obviously. The intracellular MDA values after low and high concentration pentapeptide treatment were 11.23 + -0.95. mu.M/mgprot and 9.19 + -0.73. mu.M/mgprot (p), respectively<0.05), MDA is an intracellular oxidative stress marker, and the reduction of the content indicates that the pentapeptide can protect endothelial cells against H2O2Induced oxidative damage.
As can be seen from FIG. 7, H is compared with that of the control group2O2The SOD activity in the cells of the treated oxidative damage model is sharply reduced from 44.08 +/-1.97U/mgprot to 23.62 +/-2.23U/mgprot (p)<0.01). After the pentapeptide is treated at low concentration and high concentration, the SOD activity in cells is respectively promoted to 24.42 +/-1.55U/mgprot and 30.21 +/-2.49U/mgprot (p)<0.05). The pentapeptide can obviously improve the activity of antioxidant enzyme SOD in vascular endothelial cells.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. An antioxidant pentapeptide with vascular endothelial cell protection function is characterized in that:
the amino acid sequence is leucine-alanine-histidine-phenylalanine-arginine.
2. The antioxidant pentapeptide having a vascular endothelial cell protective function according to claim 1, wherein:
the pentapeptide with the antioxidant activity is prepared by solid phase synthesis.
3. Use of the antioxidant pentapeptide having a vascular endothelial cell protective function of any one of claims 1 or 2 for preparing an antioxidant.
4. Use of the antioxidant pentapeptide having a vascular endothelial cell protective function of any one of claims 1 or 2 for the preparation of an antioxidant functional food.
5. Use of the antioxidant pentapeptide having a vascular endothelial cell protective function according to any one of claims 1 or 2 for the preparation of a vascular endothelial cell protective drug.
6. An antioxidant, characterized by:
contains the antioxidant pentapeptide with the function of protecting vascular endothelial cells according to any one of claims 1 or 2.
7. An antioxidant functional food, which is characterized in that:
contains the antioxidant pentapeptide with the function of protecting vascular endothelial cells according to any one of claims 1 or 2.
8. A vascular endothelial cell protective agent, characterized in that:
contains the antioxidant pentapeptide with the function of protecting vascular endothelial cells according to any one of claims 1 or 2.
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Citations (5)
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|---|---|---|---|---|
| CA2343351A1 (en) * | 1998-09-10 | 2000-03-16 | Incyte Pharmaceuticals, Inc. | Human transferase proteins |
| US20090148437A1 (en) * | 2007-06-06 | 2009-06-11 | Cook Fiona | Polypeptides, antibody variable domains & antagonists |
| CN105131085A (en) * | 2015-09-28 | 2015-12-09 | 华南理工大学 | Pentapeptide and application thereof |
| CN105218639A (en) * | 2015-09-28 | 2016-01-06 | 华南理工大学 | A kind of seven peptides and application thereof |
| CN112292452A (en) * | 2018-02-23 | 2021-01-29 | Cj第一制糖株式会社 | Microorganism producing mycosporine-like amino acid and method for producing mycosporine-like amino acid using the same |
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|---|---|---|---|---|
| CA2343351A1 (en) * | 1998-09-10 | 2000-03-16 | Incyte Pharmaceuticals, Inc. | Human transferase proteins |
| US20090148437A1 (en) * | 2007-06-06 | 2009-06-11 | Cook Fiona | Polypeptides, antibody variable domains & antagonists |
| CN105131085A (en) * | 2015-09-28 | 2015-12-09 | 华南理工大学 | Pentapeptide and application thereof |
| CN105218639A (en) * | 2015-09-28 | 2016-01-06 | 华南理工大学 | A kind of seven peptides and application thereof |
| CN112292452A (en) * | 2018-02-23 | 2021-01-29 | Cj第一制糖株式会社 | Microorganism producing mycosporine-like amino acid and method for producing mycosporine-like amino acid using the same |
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