CN114657162B - 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 protecting function and application thereof. The antioxidant pentapeptide comprises 5 amino acid residues, the molecular weight is 679.76g/mol, the theoretical isoelectric point is 11.05, and the amino acid sequence is: leucine-alanine-histidine-phenylalanine-arginine. The pentapeptide of the invention is derived from edible yeast enzymolysis products and can be prepared by solid phase synthesis. The polypeptide of the invention has good antioxidant activity to H 2 O 2 The induced vascular endothelial cell oxidative damage has remarkable protective effect, can be used for developing and preparing antioxidant functional foods, and has good application prospect.
Description
Technical Field
The invention belongs to the field of functional foods and biological medicines, and particularly relates to an antioxidant pentapeptide with a vascular endothelial cell protecting function and application thereof.
Background
Reactive Oxygen Species (ROS) are widely present in cells and tissues of living organisms, and have important physiological roles such as signal transduction and maintenance of stable internal environment. However, excessive ROS accumulation can cause oxidative stress in the body, cause oxidative damage to biologically active macromolecules in the cells, cause vascular endothelial cell damage, cause endothelial dysfunction, and cause chronic diseases such as diabetes, hypertension, cardiovascular diseases, etc. In addition, oxidative stress is also one of the important causes of aging.
Antioxidants can alleviate the hazards associated with oxidative stress. Food is an important source of antioxidants, and the World Health Organization (WHO) has long recognized an important role in health for antioxidants, and has been maintaining a worldwide proposition to increase dietary antioxidants. 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 new antioxidant peptide with no toxic or side effect is researched and obtained from food, and has wide development prospect and urgent need.
Disclosure of Invention
The primary aim of the invention is to overcome the defects and shortcomings of the prior art and provide an antioxidant pentapeptide with a vascular endothelial cell protecting function. The pentapeptide is derived from natural products, is obtained by identifying and screening edible yeast enzymolysis products through LC-MS/MS, and has good antioxidant activity.
The invention also aims at providing the application of the antioxidant pentapeptide with the vascular endothelial cell protecting function.
The aim of the invention is achieved by the following technical scheme:
an antioxidant pentapeptide with vascular endothelial cell protecting function has amino acid sequence leucine-alanine-histidine-phenylalanine-arginine (Leu-Ala-His-Phe-Arg).
The antioxidant pentapeptides may be prepared by means conventional in the art, for example, by solid phase synthesis.
The application of the antioxidant pentapeptide with vascular endothelial cell protecting function in preparing antioxidant.
The application of the antioxidant pentapeptide with the vascular endothelial cell protecting function in preparing antioxidant functional food.
The application of the antioxidant pentapeptide with the vascular endothelial cell protecting function in preparing vascular endothelial cell protecting medicines.
An antioxidant comprising said antioxidant pentapeptide having vascular endothelial cell protecting function.
An antioxidant functional food contains the antioxidant pentapeptide with vascular endothelial cell protecting function.
A vascular endothelial cell protecting medicine contains the antioxidant pentapeptide with vascular endothelial cell protecting 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 capacity of vascular endothelial cells for resisting oxidative damage.
2. The pentapeptide is a small molecular peptide, has a theoretical isoelectric point of 11.05, a molecular weight of 642.75g/mol, is easy to regulate and control the structure, is easy to synthesize and modify, can obtain better activity, and has obvious application potential.
3. The pentapeptides of the present invention screen enzymatic products from edible yeasts, endogenous proteins derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae (strain ATCC 204508/S288 c)). Transglyoxylase 1 (protein accession number: P23254). Edible yeasts are listed in the FDA release of "generally recognized as safe" (GRAS) food ingredients for high safety.
Drawings
FIG. 1 is a solid phase synthesis pentapeptide LAHFR chromatogram.
FIG. 2 is a mass spectrum of the solid phase synthesis of pentapeptide LAHFR.
FIG. 3 is a graph showing the analysis of DPPH scavenging activity of pentapeptide LAHFR at various concentrations.
FIG. 4 is a graph of analysis of the results of the activity of pentapeptide LAHFR on scavenging superoxide anions at various concentrations.
FIG. 5 is a graph showing the analysis of the effect of pentapeptide LAHFR on human umbilical vein endothelial cell proliferation; wherein, x and x represent the sample group was very significant in difference (p < 0.01) and significant in difference (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, x and x represent the sample group was very significant in difference (p < 0.01) and significant in difference (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, x and x represent the sample group was very significant in difference (p < 0.01) and significant in difference (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 embodiments of the present invention are not limited thereto.
Example 1
The pentapeptide with the sequence of leucine-alanine-histidine-phenylalanine-arginine (Leu-Ala-His-Phe-Arg) is prepared by adopting Fmoc solid-phase synthesis method. According to the amino acid residue composition of pentapeptide, taking various amino acids (Fmoc-Leu, fmoc-Ala, fmoc-His, fmoc-Phe and Fmoc-Arg) with fluorenylmethoxycarbonyl (Fmoc-) protecting groups at the amino terminal as raw materials, and connecting carboxyl groups of the Fmoc-Arg with a high polymer resin (Wang resin) through covalent bonds; adding Dimethylformamide (DMF) containing 20% (v/v) of piperidine, and reacting for 0.5h to remove amino protecting group Fmoc-; adding excessive Fmoc-Phe, taking hydroxybenzotriazole as a condensing agent, and reacting for 2 hours at 30 ℃ to enable carboxyl of the Fmoc-Phe to be condensed with active amino of Arg on resin; repeating deprotection and condensation reaction, sequentially connecting other amino acids, then cracking the pentapeptide from the resin, separating and purifying by a C18 column, and freeze-drying to obtain the pentapeptide. The purity of the small molecule polypeptide synthesized by the method is 98.79 percent according to the analysis of a liquid chromatogram (figure 1). The liquid chromatography-mass spectrometry (figure 2) shows that the synthesized polypeptide is Leu-Ala-His-Phe-Arg.
Example 2
DPPH was formulated in ethanol (0.1 mmol/L). The small molecule peptides of the invention are respectively prepared into 0.5-7.0 mg/mL concentration solutions by deionized water. Taking 0.15mL of pentapeptide solution, adding 0.15mL of DPPH, standing at 25 ℃ in a dark place for 1h, and measuring the absorbance value (Am) at 517 nm; taking 0.15mL of a sample, adding 0.15mL of absolute ethyl alcohol, standing for 1h at 25 ℃ in a dark place, and subtracting the absorbance value Ax of the sample from Am as a background; deionized water was used as a control group and absorbance was labeled An.
DPPH radical Scavenging (SA) was calculated according to equation (1).
As can be seen from FIG. 3, the pentapeptides of the present invention have an EC for DPPH scavenging 50 The value is 2.50mg/mL, and has certain antioxidant activity.
Example 3
The superoxide anion scavenging activity of the pentapeptides was determined by the pyrogallol method. 13. Mu.L of sample or deionized water (blank) and 174. Mu.L of Tris-HCl buffer (0.5 mol/L, pH 8.2) were added to the microplate, and the mixture was incubated at 25℃for 10 minutes in an microplate reader. 13 μl of a pyrogallol solution (3 mmol/L,10mmol/L HCl) was added, absorbance at 321nm was measured every 30s, and measurement was continued for 150s, to calculate the pyrogallol oxidation rate V, and the superoxide anion scavenger activity was calculated according to formula (2). Wherein Vc: rate of control group; vs: sample group rate.
As can be seen from FIG. 4, the pentapeptides of the present invention have a strong scavenging activity on superoxide anions, and the EC thereof 50 The value was 2.18mg/mL.
Example 4
Culture of human umbilical vein endothelial cell line EA.hy 926 cells in cell culture broth from 90% DMEM broth (NaHCO 3 1.5g/L, pH 7.0), 10% south America blood source fetal bovine serum and 1% penicillin/streptomycin diabody. Taking cells, inoculating into T25 cell culture flask at 1.0e5 cells/mL density, placing at 37deg.C, and 5% CO 2 Culturing in an incubator, changing culture solution every 2 days, and passaging every 4 days. 3-5 generations of cells were taken, cultured to logarithmic phase, digested with pancreatin, added to 6-well plates at a density of 1.0e5 cells/mL, 1mL per well, and 4 multiplex wells per group. When the cells are cultured to be more than 80% of the full hole bottom, the original culture solution is sucked and abandoned, and 1mL of serum-free culture solution with low concentration (389 mu M) or high concentration (778 mu M) of pentapeptide is respectively used for culturing and pretreating the cells for 24 hours, and the cell culture solution is sucked and abandoned. The antioxidant Glutathione (GSH) (concentration 1.63. Mu.M) was used as a positive control. 200 mu L H was then added to each group 2 O 2 Serum-free culture medium (250. Mu.M) induced oxidative damage to cells, and after further culturing for 24h, a set of samples were taken and assayed for cell viability by MTT method. Another set of samples measured intracellular MDA and SOD indicators.
The MTT method is specifically operated as follows: at 24h and 48h, 20. Mu.L MTT (5 mg/mL) was added to each well, incubated for 4h, the wells were blotted, 150. Mu.L DMSO was added and gently shaken for 10min, and absorbance at 570nm was measured using an microplate reader. The culture medium was pipetted with an equal amount of MTT after 4h and 150. Mu.L of DMSO was added as a zeroing well. Cell viability was calculated according to equation (3).
Cell viability (%) = experimental/control absorbance formula (3)
MDA and SOD measurement operations were: the pellet was kept by digestion, centrifugation and washed with sterile PBS buffer (4 ℃) and repeated twice, and the cells were repeatedly freeze-thawed with liquid nitrogen to break the cells. The total protein in the cells is measured by a total protein kit (Nanjing institute of biological engineering, product number: A045-4-2), the content of MDA in the cells is measured by a Malondialdehyde (MDA) kit (Nanjing institute of biological engineering, product number: A003-1-2), and the Nanjing institute of biological engineering, product number: a001-3-2) the activity of intracellular SOD was measured.
As shown in FIG. 5, 250. Mu.M H 2 O 2 The treatment can generate oxidative damage, obviously inhibit the proliferation of EA.hy 926 cells, and the cell survival rate of a damage model group is 47.80+/-4.62 percent. The survival rates of cells after low and high dose pentapeptide treatment were 54.98 + -6.34% and 59.15 + -2.80%, respectively. Compared with the injury model group, the pentapeptide can remarkably improve the survival rate of cells, has remarkable protective effect on oxidative injury of cells (p<0.01)。
As can be seen from FIG. 6, H compared with the control group 2 O 2 The MDA content in the cells of the treated oxidative damage model is increased from 3.09+/-0.65 mu M/mgprot (milligram protein) to 11.09+/-1.38 mu M/mgprot. Positive control GSH can significantly reduce intracellular MDA content. The MDA measurement in the cells after the low and high concentration pentapeptide treatment is 11.23+ -0.95 mu M/mgprot and 9.19+ -0.73 mu M/mgprot (p)<0.05 MDA is an intracellular oxidative stress marker, and the reduction of the content thereof indicates that the pentapeptide can protect endothelial cells from H 2 O 2 Induced oxidative damage.
As can be seen from FIG. 7, H compared with the control group 2 O 2 The SOD activity in the treated oxidative damage model cells is drastically reduced from 44.08+/-1.97U/mgprot to 23.62+/-2.23U/mgprot (p)<0.01). After the pentapeptide is treated in low concentration and high concentration, the intracellular SOD activity is respectively increased to 24.42+/-1.55U/mgprot and 30.21+/-2.49U/mgprot (p)<0.05). The pentapeptide of the invention can obviously promote the intravascular reactionActivity of antioxidant enzyme SOD in skin cells.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (8)
1. An antioxidant pentapeptide with vascular endothelial cell protecting function, which is characterized in that:
the amino acid sequence is leucine-alanine-histidine-phenylalanine-arginine.
2. The antioxidant pentapeptide with vascular endothelial cell protecting function according to claim 1, wherein:
the antioxidant pentapeptide is prepared by solid phase synthesis.
3. Use of an antioxidant pentapeptide with vascular endothelial cell protecting function according to any one of claims 1 or 2 for the preparation of an antioxidant, characterized in that: the concentration of the antioxidant pentapeptide is 778 mu mol/L.
4. Use of the antioxidant pentapeptide with vascular endothelial cell protecting function according to any one of claims 1 or 2 for preparing antioxidant functional food, characterized in that: the concentration of the antioxidant pentapeptide is 778 mu mol/L.
5. Use of the antioxidant pentapeptide with vascular endothelial cell protecting function according to any one of claims 1 or 2 for preparing vascular endothelial cell protecting medicine, characterized in that: the concentration of the antioxidant pentapeptide is 778 mu mol/L.
6. An antioxidant, characterized in that:
an antioxidant pentapeptide having a vascular endothelial cell protecting function according to any one of claims 1 or 2; the concentration of the antioxidant pentapeptide is 778 mu mol/L.
7. An antioxidant functional food, which is characterized in that:
an antioxidant pentapeptide having a vascular endothelial cell protecting function according to any one of claims 1 or 2; the concentration of the antioxidant pentapeptide is 778 mu mol/L.
8. A vascular endothelial cell protective medicament, characterized in that:
an antioxidant pentapeptide having a vascular endothelial cell protecting function according to any one of claims 1 or 2; the concentration of the antioxidant pentapeptide is 778 mu mol/L.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2343351A1 (en) * | 1998-09-10 | 2000-03-16 | Incyte Pharmaceuticals, Inc. | Human transferase proteins |
| 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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| KR20100018040A (en) * | 2007-06-06 | 2010-02-16 | 도만티스 리미티드 | Methods for selecting protease resistant polypeptides |
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Patent Citations (4)
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|---|---|---|---|---|
| CA2343351A1 (en) * | 1998-09-10 | 2000-03-16 | Incyte Pharmaceuticals, Inc. | Human transferase proteins |
| 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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| Title |
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| Fletcher,T.S. et al..RecName: Full=Transketolase 1 * |
| Short=TK 1 GENBANK ACCESSION NO. P23254.4.GENBANK.2021,第1-8页. * |
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