Cottonseed Antioxidant Peptide E5R and Its Application

Abstract

The present invention relates to the field of antioxidant peptides, and in particular to cottonseed antioxidant peptide E5R and its application. The present invention separates a cottonseed antioxidant peptide with high antioxidant activity from cottonseed protein, and determines its sequence to be ETEDACR. The antioxidant effect of the cottonseed antioxidant peptide is stronger than that of reduced glutathione at the same concentration; the cottonseed antioxidant peptide of the present invention can alleviate cellular oxidative stress. As a natural antioxidant, it is used in food, medicine, cosmetics and feed additives.

Description

Technical Field

[0001] The present invention relates to the field of antioxidant peptides, and in particular to cottonseed antioxidant peptide E5R and application thereof. Background Art

[0002] By supplementing with exogenous dietary antioxidants, the level of free radicals in the body can be significantly reduced and its antioxidant capacity can be enhanced, which is beneficial for preventing diseases and maintaining health.

[0003] Compared with synthetic antioxidants, natural antioxidants are widely available, well absorbed and highly safe. Antioxidant peptides are small peptides composed of multiple amino acid residues that have been successfully extracted from various animal and plant proteins, but their extraction methods are usually complicated, time-consuming and costly.

[0004] Cottonseed protein is a defatted concentrated protein produced from cottonseed meal through low-temperature oil pressing and defatting extraction techniques. Its crude protein content is between 40% and 70%. It is an important protein resource and also a rich library of antioxidant peptides. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a cottonseed antioxidant peptide with higher antioxidant activity.

[0006] Another object of the present invention is to provide the application of the above cottonseed antioxidant peptide.

[0007] The amino acid sequence of the cottonseed antioxidant peptide according to the present invention is ETEDACR (SEQ ID No: 1).

[0008] The present invention provides application of the cottonseed antioxidant peptide in preparing antioxidant medicines.

[0009] The present invention also provides application of the cottonseed antioxidant peptide as a feed additive.

[0010] Beneficial effects of the present invention:

[0011] The present invention separates cottonseed antioxidant peptides with high antioxidant activity from cottonseed protein. The antioxidant effect of the cottonseed antioxidant peptides is stronger than that of reduced glutathione at the same concentration; the cottonseed antioxidant peptides of the present invention can relieve cellular oxidative stress. As a natural antioxidant, they are used in food, medicine, cosmetics and feed additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 A technical roadmap for the preparation of the cottonseed antioxidant peptide of the present invention;

[0013] Figure 2 The effect of the cottonseed antioxidant peptide of the present invention on the viability of IPEC-J2 cells under H2O2 stimulation is shown;

[0014] Figure 3 The effect of the cottonseed antioxidant peptide of the present invention on ROS in IPEC-J2 cells stimulated by H2O2 is shown;

[0015] Figure 4 The effect of the cottonseed antioxidant peptide of the present invention on CAT enzyme activity in IPEC-J2 cells under H2O2 stimulation is shown;

[0016] Figure 5 The effect of the cottonseed antioxidant peptide of the present invention on the SOD enzyme activity in IPEC-J2 cells under H2O2 stimulation is shown. DETAILED DESCRIPTION

[0017] In the following examples, the cottonseed protein is preferably 65% ​​cottonseed protein, and there is no particular limitation on the specific source of the cottonseed protein. The technical scheme of the present invention is described in detail below in conjunction with the examples. In the following examples, unless otherwise specified, all are conventional methods.

[0018] Example 1 Preparation and identification of cottonseed antioxidant peptides

[0019] like Figure 1 As shown, cottonseed protein is mixed with water to prepare a suspension, the solid-liquid ratio of the suspension is 1:5, and the suspension is mixed at 60°C for 1 hour. The amount of the protease is preferably 4000U / g cottonseed protein, the enzymatic hydrolysis temperature is 53°C, the time is preferably 4h, and the pH is preferably 6.84. Enzyme inactivation treatment is required, and the enzyme inactivation condition is preferably heated at 100°C for 20min. After the enzyme inactivation is completed, the supernatant is taken as the cottonseed protein hydrolysate. The supernatant is vacuum freeze-dried to obtain a cottonseed protein hydrolysate. The cottonseed protein hydrolysate is ultrafiltered to collect peptides with a molecular weight of less than 3KDa and freeze-dried. Subsequently, the cottonseed antioxidant peptide is re-dissolved in Tris-HCl buffer with a pH of 9.0, and the negatively charged cottonseed peptide is collected by anion exchange chromatography. The above cottonseed peptide is further separated by a gel chromatography column and a reversed-phase high-performance liquid chromatography column, and finally, after mass spectrometry sequencing, the cottonseed antioxidant peptide sequence is obtained as ETEDACR.

[0020] Example 2 Determination of DPPH free scavenging ability of cottonseed antioxidant peptides

[0021] The cottonseed peptide obtained in Example 1 was synthesized, and its DPPH scavenging ability was determined. At the same time, the reduced glutathione with the same concentration was used as a control group for the test. The specific method is as follows: 0.2 mL of peptide aqueous solution with different concentrations was taken, 0.2 mL of 1 mM DPPH anhydrous ethanol solution was added to a 2 mL centrifuge tube, mixed evenly, and reacted in the dark for 30 minutes. The absorbance (A) was measured at 517 nm. i); Take 0.2mL of peptide aqueous solution in a test tube, add 0.2mL of anhydrous ethanol, and measure its absorbance at 517nm (A j ); Take 0.2mL 1mM DPPH anhydrous ethanol solution and 0.2mL water as a reference, and measure its absorbance (A0) at 517nm. The scavenging rate of the sample on DPPH free radicals is calculated according to the following formula: DPPH free radical scavenging rate = (1-(A i -A i ) / A0)×100. Where: A0 is the absorbance of 0.2mL 1mM DPPH anhydrous ethanol solution and 0.2mL anhydrous ethanol at 517nm; A i A is the absorbance value at 517nm of the reaction between 0.2mL 0.1mmol / L DPPH anhydrous ethanol solution and 0.2mL sample solution; j It is the absorbance value of 0.2mL anhydrous ethanol and 0.2mL sample solution at 517nm.

[0022] The results showed that the EC of the peptide ETEDACR for scavenging DPPH free radicals 50 The value is 0.27 mg / mL, and the EC value of reduced glutathione GSH for scavenging DPPH free radicals is 50 The value is 0.42mg / mL.

[0023] Example 3 Determination of free radical scavenging ability of cottonseed antioxidant peptide ABTS

[0024] The cottonseed peptide in Example 1 was synthesized, and its scavenging ability for ABTS free radicals was determined. At the same time, the reduced glutathione of the same concentration was used as a control group for the test. The specific method is as follows: accurately prepare 7.0mM ABTS solution and 2.45mM potassium persulfate solution, mix well, and stand at room temperature and dark for 12h to obtain ABTS mother solution. The ABTS mother solution was diluted with deionized water so that its absorbance at 734nm was 0.70±0.02, and it was balanced at 30℃ for 30min to obtain ABTS working solution. 0.1mL of small peptide I solution of different concentrations and 0.9mL of ABTS working solution were added to the test tube, mixed well, placed at room temperature and dark for 5min, and the absorbance was measured at 734nm. The reaction mixture with the same volume of deionized water instead of small peptide I solution was used as the control, and 3 repetitions were set to calculate the average value. The scavenging rate of small peptide I for ABTS free radicals was calculated according to the following formula.

[0025] ABTS free radical scavenging rate (%) = (1-As / Ab) × 100. Where As is the absorbance of the solution after adding the sample; Ab is the absorbance of the solution after adding the deionized water solution. Where As is the absorbance of the solution after adding the sample; Ab is the absorbance of the solution after adding the deionized water solution.

[0026] The results showed that the EC of the small peptide ETEDACR for scavenging ABTS free radicals 50 The value is 0.035 mg / mL, and the EC value of reduced glutathione for scavenging ABTS free radicals is 50 The value is 0.0088mg / mL.

[0027] Example 4 Determination of the ability of cottonseed antioxidant peptides to alleviate cellular oxidative stress

[0028] H2O2 is widely used in the construction of cell models of oxidative stress injury. H2O2 simulates oxidative stress injury by generating hydroxyl radicals and inducing lipid peroxidation, leading to damage to cell membranes, proteins and DNA. It can also further aggravate cell damage and dysfunction by regulating cell signaling pathways and inducing cell apoptosis. IPEC-J2 cells are a porcine small intestinal epithelial cell line that is often used to study intestinal physiological and pathological processes.

[0029] 1. Cottonseed antioxidant peptides improve the survival rate of IPEC-J2 cells stimulated by H2O2

[0030] After culturing IPEC-J2 cells for 24 h, 1×10 4 The cells were inoculated into a 96-well plate at a density of 100 cells / well. After the cells were cultured to 70-80%, the small peptide was added for 12 hours. Except for the NC group, the other groups were treated with 100 μL of culture medium containing 200 μM H2O2 for 12 hours. After the H2O2 treatment, the culture medium was removed, the cells were washed with PBS, and the culture medium containing CCK-8 reagent was added to each well. After incubation for 4 hours, the absorbance at 450 nm was measured using an enzyme reader. The results showed that ( Figure 2 ), the cell viability of the H2O2-treated group was significantly lower than that of the control group ( P< 0.05), when antioxidant peptides were added, the cell viability of the added group was significantly increased compared with the H2O2 treatment group ( P < 0.05).

[0031] 2. Cottonseed antioxidant peptide can reduce the content of intracellular ROS under H2O2 stimulation

[0032] After culturing IPEC-J2 cells for 24 h, 1×10 4The cells were inoculated into a black 96-well plate at a density of 100 cells / well. After the cells were cultured to 70-80%, the small peptide was added for 12 hours. Except for the NC group, the other groups were treated with 100 μL of culture medium containing 200 μM H2O2 for 12 hours. After the H2O2 treatment, the culture medium was removed, the cells were washed with PBS, and the culture medium containing 10 μM DCFH-DA probe was added and incubated for 20 minutes. The cells were washed with PBS to fully remove the DCFH-DA that did not enter the cells, and the fluorescence intensity was detected using an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The results show ( Figure 3 ) Adding H2O2 can significantly stimulate cells to secrete ROS ( P <0.05), when antioxidant peptides were added, the content of intracellular ROS was significantly downregulated compared with the H2O2 treatment group ( P <0.05).

[0033] 3. Cottonseed antioxidant peptides increase the activity of cellular antioxidant enzymes under H2O2 stimulation

[0034] After culturing IPEC-J2 cells for 24 h, 1×10 5 The cells were inoculated into 6-well plates at a density of 100 cells / well. After the cells were cultured to 70-80%, the small peptide was added for 12 hours. Except for the NC group, the other groups were treated with 100 μL of culture medium containing 200 μM H2O2 for 12 hours. After the H2O2 treatment, the culture medium was removed, the cells were washed with PBS, and the antioxidant enzyme activity of the cells was measured. The results showed that under the stimulation of H2O2, the catalase (CAT) of the cells ( Figure 4 ) and superoxide dismutase (SOD) ( Figure 5 ) were significantly lower than those in the control group (NC) ( P <0.05). The CAT and SOD enzyme activities in the small peptide-added group were significantly higher than those in the H2O2 group ( P <0.05).

[0035] From the above results, it can be concluded that the antioxidant cottonseed peptides described in the present invention have a high in vitro free radical scavenging ability. At the cellular level, it is shown that cottonseed antioxidant peptides can improve the survival rate of cells under oxidative stress, reduce the production of intracellular ROS, increase the enzymatic activity of antioxidant enzymes, and effectively alleviate the oxidative stress of cells.

[0036] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cottonseed antioxidant peptide, characterized in that The amino acid sequence of the cottonseed antioxidant peptide is ETEDACR.

2. Use of the cottonseed antioxidant peptide according to claim 1 for preparing antioxidant drugs.

3. Use of the cottonseed antioxidant peptide described in claim 1 as a feed additive.

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