CN114591402B - Moringa oleifera antioxidant peptide and preparation method and application thereof - Google Patents

Abstract

The invention discloses a moringa oleifera antioxidant peptide, and a preparation method and application thereof. The method comprises the following steps: dissolving the moringa leaf powder in water, regulating the pH value, performing ultrasonic-assisted extraction, centrifugally collecting supernatant, precipitating with ammonium sulfate, and dialyzing to obtain moringa protein; step (2) peppery wooden protein is dissolved and adjusted pH, alkaline protease is added for enzymolysis, enzyme deactivation and centrifugation are carried out, and peppery wooden protein enzymolysis liquid is obtained; step (3) ultrafiltration to obtain moringa oleifera antioxidant peptide, and then separating and purifying by gel electrophoresis; and (4) carrying out molecular weight and sequence identification on the moringa antioxidant peptide. The moringa oleifera antioxidant peptide obtained by treating moringa oleifera leaves as a raw material has high purity, strong activity and good antioxidant effect, has the effect of scavenging DPPH free radicals, superoxide free radicals and hydroxyl free radicals, has an obvious protective effect on oxidative damage of HepG2 cells, and can be widely used for producing nutritional foods, functional foods, cosmetics, antioxidant medicines and the like.

Description

Moringa oleifera antioxidant peptide and preparation method and application thereof

Technical Field

The invention relates to the field of antioxidant peptides, in particular to a moringa oleifera antioxidant peptide, and a preparation method and application thereof.

Background

Moringa oleifera (Morinfga oleifera lam.) belongs to the genus Moringa of the family Moringaceae, is a perennial deciduous arbor plant, and has about 14 varieties worldwide. Is native to India and is a tree species with wide growth and extremely high value in moringa oleifera family. The root of the tree is spicy, so the tree is named as "moringa oleifera", and the trunk of the tree is named as "drumstick tree". Moringa oleifera is rich in nutrients such as proteins, vitamins, mineral elements and active enzymes, and is called as "miracle tree", "medical treasures" and the like because of its good cure ability for various diseases. The moringa oleifera has strong adaptability to soil conditions and rainfall, and can grow rapidly and be widely planted in tropical and subtropical areas of Asia and Africa. The method is mainly used for planting the moringa oleifera in the areas of Hainan, sichuan, guangdong, guangxi, yunnan and the like in China, wherein the area of moringa oleifera planted in the Yunnan accounts for more than half of the area of moringa oleifera planted in the whole country.

The whole moringa oleifera plant can be used, and is a tropical plant with rich nutrition and unique economic value. The protein content of moringa oleifera has large difference in different positions. 100g of fresh moringa leaves contain 6.7g of protein, which is more than twice that of spinach (2.8 g/100 g); the protein content in the dry moringa oleifera leaf powder is highest, and 100g of the dry moringa oleifera leaf powder contains 27.1g of protein, which is 9 times of the protein content in the yoghourt; the tender pod of Moringa oleifera contains the least protein, and 100g of the pod contains 2.5g of protein. The leaves are the organ with the highest protein content in the moringa oleifera, and new resource foods are obtained, so that the research on the high-quality utilization of the protein in the moringa oleifera leaves has important significance. In the moringa oleifera leaf protein, gluten accounts for 80%, prolamin accounts for 14%, albumin accounts for 3.5%, globulin accounts for 1%, and the moringa oleifera leaf protein is a high-quality plant protein which can be compared with soybean protein. Compared with other vegetable proteins such as peanut, soybean, cotton seed and the like, the moringa oleifera leaf protein has higher biological potency and nutritive value, and is a high-quality protein with great development potential.

The human liver cancer cell (HepG 2) has the characteristics of easy culture, strong representativeness and the like, so that an oxidative damage model of a chemical reagent on the HepG2 cell is often selected to evaluate the protective effect of natural antioxidants and phytochemicals on the liver, the oxidative damage model is built by the HepG2 cell, the influence of polypeptide Lys-Asp-His-Cys-His (KDCH) on the intracellular antioxidant and apoptosis activity is researched, the oxidative damage model is built by the HepG2 cell, and the capability of a corn 1kDa protein peptide component (CPfS) for scavenging intracellular Reactive Oxygen Species (ROS) and the antioxidant enzyme regulating capability are researched. At present, the antioxidant research of moringa leaves in various countries is mainly focused on water extracts, and literature research shows that the moringa leaves extract has a protective effect on oxidative stress and liver and kidney injury caused by methotrexate (mtx) treatment.

However, the water extract of moringa oleifera leaves has complex components and cannot clearly show effective components, and has certain defects in the aspects of deep research and product development. Therefore, the invention starts from the moringa leaves as raw materials, extracts and separates the moringa leaf antioxidant polypeptide, researches the oxidation injury effect of the moringa leaf antioxidant polypeptide on HepG2 cells, and clarifies the oxidation action components of the moringa leaf antioxidant and the action mechanism of the antioxidant polypeptide. Provides a theoretical basis for producing functional foods and health-care foods, increases the utilization rate of moringa oleifera, has great economic value and social benefit for the comprehensive utilization of moringa oleifera biological resources, and effectively promotes the economic development of moringa oleifera industry and China.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides moringa oleifera antioxidant peptide, and a preparation method and application thereof. According to the invention, moringa oleifera leaf powder is used as a raw material to extract moringa oleifera protein, polypeptides with antioxidant activity are prepared by an enzymolysis technology, the antioxidant activity of different peptide fragments is measured by an ultrafiltration technology, target peptide fragments with higher antioxidant activity are screened, sequence identification is carried out on the target peptide fragments by a mass spectrum technology, and the corresponding peptide fragments are obtained by separation and synthesis, so that the specific target peptide fragments have higher antioxidant activity in vitro and have obvious protective effect on oxidative damage of HepG2 cells.

In order to solve the technical problems, the technical scheme of the invention is as follows: a preparation method of moringa oleifera antioxidant peptide comprises the following steps:

and (2) extracting moringa oleifera protein: dissolving Moringa oleifera leaf powder in water, adjusting pH, extracting with ultrasonic wave, centrifuging to collect supernatant, precipitating with ammonium sulfate, centrifuging, washing the precipitate with water, dialyzing with dialysis bag to obtain Moringa oleifera protein, and freeze drying;

and (2) enzymolysis: dissolving moringa oleifera protein, regulating pH, adding alkaline protease for enzymolysis, inactivating enzyme after the reaction is finished, cooling, centrifuging, and collecting supernatant to obtain moringa oleifera protein enzymolysis liquid;

And (3) separating: dialyzing the moringa oleifera protein enzymolysis liquid, regulating the pH to be neutral, pressurizing, sequentially passing through ultrafiltration membranes with the molecular weight cut-off of 10K Da, 5K Da, 3K Da and 1K Da to obtain moringa oleifera antioxidant peptide components less than 1K Da, purifying the moringa oleifera antioxidant peptide components less than 1K Da by using a centrifugal ultrafiltration tube, freeze-drying, and further separating and purifying by using Tricine SDS-PAGE gel electrophoresis;

and (4) identification: and (3) carrying out molecular weight and sequence identification on the moringa oleifera antioxidant peptide with the molecular weight less than 1K Da by utilizing LC-MS.

Further, the detailed operation of step (1) is as follows: the ratio of the moringa powder to the feed liquid is 1:30-40 are dissolved in water and stirred uniformly, the pH is regulated to 8-10, after ultrasonic treatment is carried out for 50-70min at the temperature of 50-60 ℃, the supernatant is taken and added with ammonium sulfate, the saturation degree of the ammonium sulfate is controlled to be 35-45%, the mixture is stirred uniformly and kept stand, and the supernatant is removed by centrifugation to obtain the precipitate.

Further, the enzymolysis conditions of the step (2) are as follows: the mass concentration of the moringa oleifera protein is 3-4%, the pH value is 8.5-9.5, the addition amount of alkaline protease is 3000-5000U/g, the temperature is 40-55 ℃, and the enzymolysis time is 4-5h.

Further, the Tricine SDS-PAGE gel in the step 3 adopts a structure of 3 layers of discontinuous gel and consists of separating gel, interlayer gel and concentrated gel;

spotting and electrophoresis: placing the electrophoresis tank into an ice water bath, adding an anode buffer solution into an outer tank, adding a cathode buffer solution into an inner tank, carrying out pre-electrophoresis for 8-12min at 25-35V, adding a sample subjected to Tricine special sample loading buffer treatment into a sample application hole, carrying out electrophoresis at 25-35V for 1-1.5 hours, and stopping electrophoresis after 80-120V electrophoresis until bromophenol blue reaches the bottom of the gel, and carrying out subsequent coomassie brilliant blue staining;

Dyeing and decoloring: adopting a coomassie brilliant blue method for dyeing, putting colloid into a fixing solution for fixing for 20-40min, pouring the fixing solution, rinsing for 2-3 times by using ultrapure water, adding the coomassie brilliant blue dyeing solution for dyeing overnight, adding a decolorizing solution for multiple times after dyeing is finished, changing eluent until electrophoresis strips are clear, and eluting gel background completely.

Further, the step (4) is analyzed and identified to obtain 21 amino acid sequences, the molecular weight of the polypeptide is 722.4329 Da-1791.7952 Da, the peptide amino acid content is rich, and leucine, isoleucine, aspartic acid, glutamic acid, lysine and arginine are rich. Because ultrafiltration by adopting an ultrafiltration membrane has certain error, the molecular weight of the obtained polypeptide segment is not less than 1kDa, and the molecular weight of the polypeptide obtained by ultrafiltration by the ultrafiltration membrane of 1kDa is between 700 and 1800.

In a second aspect, the invention provides a moringa oleifera antioxidant peptide prepared by the preparation method, wherein the moringa oleifera antioxidant peptide comprises the following sequences:

preferably, the antioxidant peptide comprises one or more of the following sequences: SEQ ID No.7, SEQ ID No.9, SEQ ID No.10, SEQ ID No.12 and SEQ ID No.21.

As a further preference, the antioxidant peptide is SEQ ID No.7.

The third aspect of the invention also provides application of the moringa oleifera antioxidant peptide, which has a protective effect on HepG2 cell oxidative damage.

Further, the moringa oleifera antioxidant peptide can be used for preparing foods, cosmetics or antioxidant medicines based on the antioxidant effect of the moringa oleifera antioxidant peptide.

The invention is characterized in that: 1. and (5) researching the extraction process and physical characteristics of moringa oleifera leaf proteins. The method adopts an alkali-soluble ammonium sulfate precipitation method to extract protein, and based on factors such as temperature, pH, feed-liquid ratio, extraction time and the like, the optimal extraction conditions are as follows: the temperature is 55 ℃, the pH value is 9, the liquid-to-material ratio is 1:40, the extraction time is 60min, the protein extraction rate under the condition is highest and can reach 68.34%, and the emulsion has good emulsifying property, emulsifying stability, water holding property and oil absorption property. The obtained Moringa oleifera leaf protein has optimal sedimentation effect at pH value of about 4.0, and has highest emulsifying power and emulsion stability at pH value of 10.0 of 19.15m ² G, 144.87%; at 50 ℃, the water holding capacity and the oil absorption are respectively 1.23g/g and 1.45g/g.

2. And (5) researching the preparation process of the moringa leaf antioxidant peptide. Taking DPPH free radical clearance and polypeptide content as investigation indexes, selecting trypsin, alkaline protease, neutral protease and papain to carry out enzymolysis on peppery leaf protein under the most suitable condition, and screening out the alkaline protease as the most suitable hydrolase. And a Box-Behnken Design experiment is adopted to determine that the optimal enzymolysis condition is that the enzymolysis time is 5 hours, the enzyme adding amount is 3000U/g, the enzymolysis temperature is 50 ℃, the content of the prepared polypeptide can reach 23.3mg/g, and the DPPH free radical clearance of an enzymolysis product is 73.64%.

3. And (3) separating, purifying and identifying the structure of the moringa oleifera leaf antioxidant peptide. The antioxidant activity is used as an evaluation index, the polypeptide is segmented according to the molecular weight by ultrafiltration, and the oxidation capability sequence of different molecular weights is 1kDa & gt 3K Da & gt 5K Da & gt 10K Da, namely the smaller the molecular weight is, the better the antioxidant effect is. The small molecular peptide is separated and purified by Tricine-SDS-PAGE, and the LC-MS/MS identification analysis is carried out on the active peptide segment to obtain 21 amino acid sequences, wherein the molecular weight of the polypeptide is 722.4329 Da-1791.7952 Da, the amino acid content of the peptide segment is rich, and the peptide segment is rich in amino acids with antioxidant activity such as leucine, isoleucine, aspartic acid, glutamic acid, lysine and arginine, wherein hydrophobic amino acids are arranged at the middle C end or N end of the peptide chain of the peptide segment LALPVYN, GHVALVFVN, FHEEDDAKLF, LDEGKWQHVK, LHIAALVFQ, and the peptide segment contains aromatic rings, imidazole groups and sulfur-containing groups, so that the antioxidant activity of the peptide segment is enhanced.

4. Protection effect of moringa oleifera leaf antioxidant peptide on oxidative damage of HepG2 cells. Culturing HepG-2 cells, establishing an oxidative damage model, and exploring cytotoxicity and cell oxidative damage protection effect of moringa oleifera leaf antioxidant peptide. The study shows that the optimal conditions for inducing oxidative damage of HepG2 cells are as follows: at 500 mu M H ₂ O ₂ The survival rate of the model is 64.03% after 4 hours of action under the condition. After the cells are treated for 24 hours by using moringa oleifera leaf antioxidant peptides (50, 100, 200 mug/mL) with different concentrations, the cell survival rate, the content of GSH, CAT, MDA, SOD in the cells and the apoptosis rate are detected by a flow technology. The cell survival rate of the components treated by the drug is improved to 80.12-85.11%, the GSH content 33.06U/mg Pro, CAT content 7.39U/mg Pro, MDA content 11.16U/mg Pro and ROS content 103.4% in the cells of the damaged group, compared with the lost group, the drug group (50, 100, 200 mu g/mL can obviously improve the activity of GSH (42.72, 50.93, 58.72U/mg Pro) and CAT (13.15, 17.15, 17.50U/mg Pro) in the cells, the drug group can also effectively reduce the MDA content (3.49, 5.54, 6.97nmol/mg Pro) in the cells, and can also reduce the activity of H ₂ O ₂ Resulting in an increase in intracellular ROS (71.45%, 62.91%, 57.91%). H compared with normal ₂ O ₂ Can change cell morphology and damage groupThe apoptosis rate is 17.1%, and the apoptosis rate is reduced to 12.93%, 4.76% and 4.23% after being treated by the moringa oleifera leaf antioxidant peptide.

Compared with the prior art, the invention has the following beneficial effects: the moringa oleifera antioxidant peptide is prepared by treating moringa oleifera leaves serving as a raw material, is mostly small peptide with the concentration of less than 1000Da, has strong activity and good antioxidant effect, has the effect of scavenging DPPH free radicals, superoxide free radicals and hydroxyl free radicals, has an obvious protective effect on oxidative damage of HepG2 cells, and can be widely used for producing nutritional foods, functional foods, cosmetics, antioxidant medicines and the like.

Drawings

FIG. 1 is a graph showing the effect of feed liquid comparison on moringa protein extraction;

FIG. 2 is a graph showing the effect of temperature on moringa protein extraction;

FIG. 3 effect of ultrasound time on moringa protein extraction;

FIG. 4 pH effect on moringa protein extraction;

FIG. 5 effect of ammonium sulfate saturation on moringa protein extraction;

FIG. 6 different protease hydrolysate polypeptide content and DPPH radical scavenging rate;

FIG. 7 effect of enzymolysis time on polypeptide content and DPPH radical scavenging rate;

FIG. 8 effect of enzymatic hydrolysis temperature on polypeptide content and DPPH radical scavenging rate;

FIG. 9 effect of enzyme addition on polypeptide content and DPPH radical scavenging rate;

FIG. 10 effect of substrate concentration on polypeptide content and DPPH radical scavenging rate;

FIG. 11 DPPH radical scavenging Activity of Moringa protein peptides;

FIG. 12 hydroxyl radical scavenging activity of moringa oleifera antioxidant peptides;

FIG. 13 superoxide anion radical scavenging activity of moringa oleifera antioxidant peptides;

FIG. 14 ABTS radical scavenging activity of moringa antioxidant peptides;

FIG. 15 LC-MS/MS mass spectrum of each peptide fragment;

FIG. 16 antioxidant capacity assay for each peptide fragment, crude peptide 1kDa ultrafiltration peptide;

FIG. 17 effect of LALPVYN pretreatment on H2O2 mediated oxidative damage HepG2 cell viability. Survival of (a) HepG2 cells treated with LAL at different concentrations, (B) survival of HepG2 cells treated with H2O2 at different concentrations, (C) survival of HepG2 cells treated with lalvyn, VC and H2O2 simultaneously. Data are expressed as mean ± SEM of three independent experiments (n=3). Effect of LALPVYN pretreatment on H2O2 mediated oxidative damage HepG2 cell viability. Survival of (a) HepG2 cells treated with LAL at different concentrations, (B) survival of HepG2 cells treated with H2O2 at different concentrations, (C) survival of HepG2 cells treated with lalvyn, VC and H2O2 simultaneously. Data are expressed as mean ± SEM of three independent experiments (n=3);

FIG. 18 effect of LALPVYN pretreatment on SOD, CAT, GSH-P and MDA of H2O2 mediated oxidative damage to HepG2 cells. (A) the effect of LALPVYN on CAT, (B) the effect of LALPVYN on GSH-Px, (C) the effect of LALPVYN on SOD, and (D) the effect of LALPVYN on MDA. Data are expressed as mean ± SEM of three independent experiments (n=3);

FIG. 19 effect of LALPVYN pretreatment on H2O2 mediated oxidative damage HepG2 cell ROS. Pictures (a-F) were photographed by a laser scanning confocal microscope (G) and the average fluorescence intensity was analyzed by ImageJ. Data are expressed as mean ± SEM of three independent experiments (n=3);

FIG. 20 effect of LALPVYN pretreatment on apoptosis of H2O2 mediated oxidative damage HepG2 cells. (A-F) analysis of cell cycle by flow cytometry (G) analysis of apoptosis rate by flowjo software. Data are expressed as mean ± SEM of three independent experiments (n=3).

Detailed Description

Example 1

A preparation method of moringa oleifera antioxidant peptide comprises the following steps:

and (2) extracting moringa oleifera protein: dissolving Moringa oleifera leaf powder in water, adjusting pH, extracting with ultrasonic wave, centrifuging to collect supernatant, precipitating with ammonium sulfate, centrifuging to wash precipitate, dialyzing with dialysis bag to obtain Moringa oleifera protein, and lyophilizing;

And (2) enzymolysis: dissolving Moringa oleifera leaf protein, regulating pH, adding alkaline protease for enzymolysis, inactivating enzyme after the reaction, cooling, centrifuging, and collecting supernatant to obtain Moringa oleifera protein enzymolysis solution;

and (3) separating: dialyzing the moringa oleifera protein enzymolysis liquid, regulating pH to neutrality, pressurizing, sequentially passing through ultrafiltration membranes with cut-off molecular weights of 10K Da, 5K Da, 3K Da and 1K Da to obtain moringa oleifera antioxidant peptide components less than 1K Da, purifying the moringa oleifera antioxidant peptide components less than 1K Da by using a centrifugal ultrafiltration tube, centrifugally ultrafiltering at 3000-4000rpm for 5-8min, freeze-drying, and further separating and purifying by using Tricine SDS-PAGE gel electrophoresis;

and (4) identification: and (3) carrying out molecular weight and sequence identification on the moringa oleifera antioxidant peptide less than 1kDa by using an LC-MS.

Moringa protein extraction experimental condition exploration

Taking the moringa protein extraction rate as an investigation index, researching the influence of the feed liquid ratio (1:20, 1:25, 1:30, 1:35, 1:40), the temperature (35, 45, 55, 65, 75 ℃) and the ultrasonic time (20, 30, 40, 50, 60 min), the pH (7, 8, 9, 10, 11) and the ammonium sulfate saturation (20%, 30%, 40%, 50%, 60%) on the moringa protein extraction rate.

Protein extraction rate: measuring protein content by Coomassie Brilliant blue method, taking bovine serum albumin as standard protein at 595nm wavelength, and drawing standard curve with regression equation of Y=1.4458X+0.0004, R ² =0.9997 (where: X is bovine serum protein concentration (mg/mL), Y is absorbance). And determining the optimal technological parameter condition according to the protein extraction rate in the solution, and preparing a standard curve.

In the formula: c is the mass concentration of protein, mg/mL; v extract volume, mL; n dilution of the extract; she Fenzhi g of M moringa oleifera.

(1) Influence of feed liquid ratio on moringa protein extraction rate

Weighing 100g of moringa oleifera powder in a beaker according to a feed liquid ratio of 1: 15. 1: 20. 1: 25. 1: 30. 1:35. 1:40, stirring uniformly, regulating pH to 9, performing ultrasonic treatment at 55deg.C for 60min, centrifuging for 20min, collecting supernatant, adding 40% ammonium sulfate, stirring uniformly, standing for 2 hr, centrifuging for 20min, removing supernatant, collecting precipitate, measuring protein content, and determining extraction rate of water-soluble protein, wherein the result is shown in figure 1.

As shown in fig. 1, as the feed-to-liquid ratio increases, the protein extraction rate gradually increases, and the feed-to-liquid ratio is 1: the protein extraction rate reaches the highest at 40. The reason is that when the concentration of the substrate is too high when the feed liquid is smaller, the viscosity of the solution can be increased, the moringa powder cannot be fully dissolved in water, the movement speed of protein molecules in the water is reduced, the dissolution capacity is reduced, and a large amount of moringa protein is relatively insoluble in the water; the feed liquid ratio exceeds 1: the protein extraction rate is not obviously improved after 35, the water consumption is increased due to the high feed liquid ratio, under the premise of ensuring the protein extraction rate, the conditions of saving cost, subsequent concentration cost and the like are considered, and the optimal extraction feed liquid ratio is comprehensively considered to be 1:35.

(2) Influence of temperature on extraction yield of moringa protein

Accurately weighing moringa oleifera powder according to a feed liquid ratio of 1: adding ultrapure water into the system, stirring uniformly, regulating the pH to 9, carrying out ultrasonic treatment at the temperature of 35, 45, 55, 65 and 75 ℃ for 60 minutes, keeping other parameters and conditions unchanged, adding water into the system, adding ammonium sulfate into the system to a specified volume, and controlling the saturation degree of the ammonium sulfate, wherein the result is shown in figure 2.

As shown in figure 2, the temperature is between 35 and 55 ℃, the extraction rate of moringa oleifera protein is obviously improved, the highest protein extraction rate is 57.26 mg/g at 55 ℃, and the protein extraction rate is rapidly reduced at the temperature exceeding 55 ℃, because the temperature is too high to cause the damage of protein space structure and protein denaturation, and the precipitation is generated to cause the reduction of protein extraction rate. The protein extraction rate increases rapidly at 35-55 ℃, because the spatial conformation of the protein changes along with the temperature rise in a low temperature range, which is favorable for the movement of protein molecules and water molecules, and the temperature rise in a certain range is favorable for the enhancement of the solubility of the protein, thereby improving the protein extraction rate.

(3) Influence of ultrasonic time on moringa protein extraction rate

Mixing at a feed-liquid ratio of 1:35, adjusting pH to 9, and performing ultrasonic treatment at 55deg.C for 20, 30, 40, 50, 60min under the same conditions, and measuring the extraction rate of water-soluble protein.

As shown in fig. 3, the influence of ultrasonic time on the extraction rate of moringa oleifera protein is more remarkable, the protein extraction rate is increased along with the increase of ultrasonic time, and the highest extraction rate is achieved in 70 min. The ultrasonic time is short, the cavitation and mechanical shearing force of the ultrasonic wave can not be effectively exerted, and along with the increase of the ultrasonic time, the cavitation effect and mechanical effect of the ultrasonic wave are enhanced, so that the rupture of the plant cell tissue structure is promoted, and the release of protein is increased. However, as the ultrasonic time is increased, the release of the protein is not effectively promoted, but energy waste and mechanical loss are caused, the time cost is increased, and the protein is subjected to certain physical hydrolysis due to overlong time, so that the extraction rate is reduced. Therefore, the ultrasonic time of moringa oleifera protein was determined to be 60min under comprehensive consideration.

(4) Influence of pH on Moringa protein extraction yield

Accurately weighing moringa oleifera powder according to a feed liquid ratio of 1:35, the temperature is 55 ℃, the pH values are 7, 8, 9, 10 and 11 after ultrasonic treatment for 60min, other parameters and conditions are unchanged, and the extraction rate of the water-soluble protein is determined, and the result is shown in figure 4.

As shown in the result 4, the extraction rate of the moringa protein increases along with the increase of the pH value, and when the pH value is within the range of 6-9, the extraction rate of the moringa protein increases remarkably, and when the pH value exceeds 9, the extraction rate of the moringa protein tends to be gentle. The reason may loosen some structures in the moringa leaves under alkaline environment, the hydrogen bond is destroyed, and some polar groups are dissociated, so that the dissolution of moringa proteins is facilitated. It is also possible that the isoelectric point of moringa oleifera leaf protein is between 4.0 and 4.5, and the further the protein colloid is from the isoelectric point, the better the stability of the protein is, and the higher the protein content in the leaching solution is along with the rising of the pH value. However, proteins undergo a change in physicochemical properties under strong alkaline conditions, resulting in precipitation of the protein. The pH of the moringa protein extraction was therefore determined to be 9. (5) Influence of ammonium sulfate saturation on moringa protein extraction yield

Accurately weighing moringa oleifera powder, uniformly stirring according to a feed-liquid ratio of 1:35, carrying out ultrasonic treatment at 55 ℃ for 60min, and determining the extraction rate of water-soluble protein with pH value of 9 and other parameters and conditions unchanged, wherein the result is shown in figure 5.

As shown in fig. 5, the saturation of ammonium sulfate has a significant effect on the extraction rate of moringa oleifera protein, the protein extraction rate increases with the increase of the saturation of sulfuric acid, and the increase of the protein extraction rate is significant in the range of 20% -40% of the saturation of ammonium sulfate, because after the protein molecules adsorb acid ions with low concentration, the charged layer repels the protein molecules from each other, and the interaction between the protein molecules and water molecules is enhanced, so that the solubility of the extract is increased, the saturation of the ammonium sulfate solution is too high, the solution is supersaturated, the protein structure is denatured, and other impurities such as saccharides are precipitated, which affects the experimental result. The greater the molecular weight of the protein, the lower the saturation of ammonium sulfate required for more charge precipitation, and typically a 50% saturation solution of ammonium sulfate will precipitate most of the protein. The ammonium sulfate saturation was determined to be 40% for consideration of the results of subsequent experiments and cost issues.

Orthogonal experimental design of moringa protein extraction process

Based on a single-factor experiment, the temperature, the feed-liquid ratio, the pH and three factors are selected as variables to perform three-factor and three-level experiment design by utilizing an orthogonal experiment design, so that the moringa protein extraction process parameters are optimized. Response surface analysis factors and levels are shown in table 1.

TABLE 1 orthogonal test factors and horizontal design factors

Orthogonal test results and analysis

On the basis of a single factor experiment, the temperature, the feed-liquid ratio and the pH value are inspected. And 3, designing and testing orthogonal experiments to determine the optimal extraction process of the moringa protein.

The orthogonal experiment result shows that the primary and secondary sequences affecting the extraction rate of moringa oleifera protein are pH value > feed-liquid ratio > temperature. The optimal extraction process combination of the moringa protein is A ₃ B ₃ C ₂ The optimal technological parameters are that the temperature is 55 ℃, the feed-liquid ratio is 1:35, the pH value is 9, and the saturation degree of ammonium sulfate is 40%. Preparation and process optimization of moringa oleifera leaf antioxidant peptide

Determination of polypeptide content: a standard curve was prepared with the peptide concentration on the abscissa X (mg/mL) and the OD value on the ordinate Y. Standard curve regression equation y= 17.925X-0.0069, r ² =0.9995 (where: X is polypeptide concentration (mg/mL), Y is absorbance).

(1) Screening of proteases

The method is characterized in that four proteases of trypsin, papain, alkaline protease and neutral protease are selected as pre-screening enzymes, enzymolysis is carried out under the optimal enzymolysis conditions respectively at a substrate concentration of 3% for 3 hours, and polypeptide content and DPPH free radical clearance of enzymolysis products are used as investigation indexes.

The enzymolysis effect of the moringa protein by four different proteases is shown in figure 6 by taking DPPH free radical clearance and polypeptide content as indexes, and the alkaline protease enzymolysis product has the highest polypeptide content of 8.19mg/g and DPPH free radical clearance of 72.64 percent, which is obviously superior to other proteases. The alkaline protease has strong hydrolytic capacity and more enzyme cutting sites, and more small molecule peptides are generated.

(2) Single factor experiment

The polypeptide content and DPPH free radical clearance rate after peppery wooden proteolytic hydrolysis are used as indexes. And (3) examining the enzymolysis time (1, 2, 3, 4 and 5 h), the enzymolysis temperature (30, 40, 50, 60 and 70 ℃), the enzyme addition amount (1000, 2000, 3000, 4000 and 5000U/g), the influence of the substrate concentration (1%, 2%, 3%, 4% and 5%) on the peppery wooden protein polypeptide content and the DPPH free radical clearance rate, and determining the optimal factor level for preparing the optimal peppery wooden antioxidant peptide.

(1) Effect of enzymatic time on polypeptide content and DPPH radical scavenging Rate

The influence of different enzymolysis time on protein hydrolysis is studied under the conditions of 60 ℃ of test conditions, 3% of substrate concentration, 9% of pH value and 4000U/g of enzyme addition. As shown in FIG. 7, the polypeptide content and DPPH free radical scavenging rate tended to be stable after rising first with the prolongation of the enzymolysis time, possibly after the substrate is basically reacted, or the enzymolysis time is too long, and the antioxidant polypeptide obtained by enzymolysis can be further hydrolyzed into amino acid, so that 4 h is selected as the optimal enzymolysis time.

(2) Effect of enzymatic hydrolysis temperature on polypeptide content and DPPH radical scavenging Rate

And (3) researching the influence of different enzymolysis temperatures on protein hydrolysis under the conditions of 3% of substrate concentration, 9% of pH value, 4000U/g of enzyme addition amount and 4 hours of enzymolysis time. As shown in FIG. 8, the peppery wood polypeptide content and DPPH free radical clearance rate are increased firstly and then decreased along with the temperature rise, and the polypeptide content and DPPH free radical clearance rate are the highest at 50 ℃; this may be related to the optimum temperature of alkaline protease, and too high a temperature may affect the spatial structure of the enzyme, reducing the enzyme activity.

(3) Effect of enzyme addition on polypeptide content and DPPH radical scavenger

And under the test conditions, the substrate concentration is 3%, the pH value is 9, the enzymolysis time is 4 hours, the enzymolysis temperature is 50 ℃, and the influence of different enzyme addition amounts on protein hydrolysis is studied. As shown in FIG. 9, the polypeptide content increased and then tended to be gentle with increasing enzyme addition, the polypeptide content reached a maximum of 0.85mg/0.1g at 5000U/g enzyme addition, DPPH radical scavenging rate increased and then decreased, and DPPH radical scavenging rate was 66.93% at 4000U/g enzyme addition. Because the concentration of the enzyme and the substrate reaches a saturated state, the enzyme enzymolysis speed is not increased by continuously adding the enzyme.

(4) Effect of substrate concentration on polypeptide content and DPPH radical scavenging Rate

And (3) researching the influence of different substrate concentrations on the hydrolysis of the protein under the conditions of test pH value 9, enzymolysis time 4h, enzymolysis temperature 50 ℃ and enzyme addition amount 4000U/g. As shown in FIG. 10, increasing the substrate concentration, increasing the polypeptide content followed by decreasing, the polypeptide content reached a maximum of 2.26mg/0.1g at a substrate concentration of 4% and the DPPH radical scavenging rate reached a maximum of 76.89% at a substrate concentration of 3%, followed by a decrease in the tendency. At lower substrate concentrations, the interaction of the enzyme with the protein molecules is not favored, too high a concentration can affect proteolysis and clearance can be inhibited.

Response surface experimental design and results

Based on a single factor experiment, a Box-Behnken experimental design principle is adopted, 3 factors of enzymolysis temperature, pH value and enzyme addition amount are used as variables, 3-factor 3-level experimental design is carried out, and the technological parameters of the moringa oleifera antioxidant polypeptide are optimized. The factor levels are shown in Table 2.

TABLE 2 response surface factor level Table

From the single factor test results, the enzymolysis time, the pH value and the enzyme addition amount have obvious influence on the moringa polypeptide content and the DPPH clearance, the polypeptide content and the DPPH clearance are taken as response values, and the experimental design (3-factor and 3-level) optimization is carried out by adopting Box-Behnken center combination.

The regression model obtains the optimal DPPH free radical clearance of the moringa oleifera leaf enzymatic hydrolysate by a response surface method, wherein the enzymatic hydrolysis time is 4.13 hours, the pH value is 8.99, the enzyme addition amount is 4026.90U/g, the polypeptide content is 2.33mg/g, and the DPPH free radical clearance is 78.61 percent. The optimal conditions are adjusted to have the enzymolysis time of 4 hours, the pH value of 9 and the enzyme addition amount of 4000U/g according to the experimental operability, and the experimental polypeptide content is 2.33mg/0.1g and the DPPH free radical clearance rate is 79.22 percent and is similar to the theoretical value.

Separation and purification of moringa antioxidant peptide

Ultrafiltration separation of moringa oleifera antioxidant peptide

After the moringa oleifera protein enzymolysis liquid is dialyzed, the pH value is regulated to 7, and the moringa oleifera protein enzymolysis liquid is sequentially subjected to ultrafiltration membranes with the molecular weight cut-off of 10kDa, 5kDa, 3kDa and 1kDa under the pressure of 0.3MPa to obtain 4 components with different molecular weights, wherein the color of each component sample is gradually deepened from small to large according to the molecular weight, and the color of the sample with the minimum molecular weight less than 1kDa is shallowest and light yellow, so that the ultra-filtration treatment can play a better decoloring role on moringa oleifera antioxidant peptide. In vitro antioxidant Activity study on the 4 fractions obtained

(1) DPPH radical scavenging Activity assay

Since DPPH free baseband has an odd electron ion, it is stable in ethanol solution and has a maximum absorption peak at 517 nm. When DPPH reacts with the proton donor polymer, the DPPH radical number decreases and the absorption peak at 517nm also decreases. As shown in FIG. 11, the moringa antioxidant peptides have certain clearance ability to DPPH, are enhanced with the increase of concentration, have dose dependency, and have clearance rates of 53.17%, 45.34%, 38.12% and 29.81% of different molecular weights respectively when the concentration is 1 mg/mL.

(2) Scavenging ability of hydroxyl radical

OH is the most active radical among oxygen radicals and can damage almost all macromolecular substances easily, such as proteins, enzymes, carbohydrates, lipids (peroxidation) and nucleic acids (mutation) to accelerate aging and the occurrence of certain diseases in the body. As shown in FIG. 12, the clearance rates of 3kDa, 5kDa and 10kDa on OH are relatively close, the clearance rates of the 3kDa, 5kDa and 10kDa are 36.58%, 33.94% and 30.76% respectively at the concentration of 1mg/mL, and the small molecule 1K Da has better clearance activity than the other three molecular weights, and the clearance activity is 44.28% at the concentration of 1 mg/mL.

(3) Superoxide anion radical scavenging ability

The pyrogallol can undergo autoxidation reaction under alkaline conditions, the generated intermediate reacts with superoxide anion free radicals with stable concentration, and the intermediate reacts with superoxide anion free radicals to obtain a colored intermediate product which is absorbed at 325nm, and the accumulation of absorbance values shows correlation with the quality of the intermediate product. As shown in FIG. 13, the moringa oleifera antioxidant peptide has a certain scavenging capacity for superoxide anions, the small molecule 1kDa has a good scavenging activity, the scavenging capacity is 44.45% at the concentration of 1mg/mL, the scavenging activity of the moringa oleifera protein peptide superoxide anions with the molecular weight of 10kDa is weak, and the scavenging capacity is 24.1% at the concentration of 1 mg/mL.

(4) ABTS radical scavenging ability

ABTS is a radical initiator that is oxidized by potassium persulfate to form a stable blue-green radical (abts+), which is prone to acquire electrons or protons from ABTS, has a strong absorption at 734nm, and fades if the added test sample has a scavenging ability for abts+. As shown in fig. 14, GSH has a remarkable scavenging effect on ABTS radicals. The moringa antioxidant peptides with different molecular weights have the activity of scavenging the ABTS free radicals, and the activity of scavenging the ABTS free radicals is increased with the increase of the concentration. The antioxidant peptide with the molecular weight of 1kDa has the strongest scavenging ability at different concentrations, and the maximum ABTS scavenging activity is 47.61% at 1mg/mL, and the ABTS scavenging activities of 3K Da, 5 kDa and 10K Da are 45.70%, 42.13% and 20.81% respectively. The antioxidant peptide with small molecular weight has strong cleaning activity.

The antioxidant peptides of 1kDa and 3kDa have stronger antioxidant activity, so that the antioxidant peptides of 1kDa and 3kDa are mainly separated and identified.

(2) Relative molecular weight distribution of moringa antioxidant peptide

As can be seen from Table 3, the antioxidant peptides after ultrafiltration mainly consist of small molecular peptides, the relative molecular mass is mainly concentrated at 600-1000Da, the relative molecular weight in solid powder after ultrafiltration of 1K Da and 3K Da is below 1000Da and respectively accounts for 84.15% and 79.61%, 309 and 494 peptide fragment numbers are obtained by searching database NCBI Quan Ku by a computer, which means that the composition of the peptide fragments after ultrafiltration is complex and further separation and purification are needed.

TABLE 3 relative molecular mass distribution of Moringa oleifera antioxidant peptides

Tricine-SDS-PAGE separation and purification

The Tricine SDS-PAGE gel adopts a structure of 3 layers of discontinuous gel, and consists of separating gel, interlayer gel and concentrated gel, wherein each layer of gel is polymerized by acrylamide and methylene bisacrylamide mixed solution which are composed of different molecules. (Table 4)

TABLE 4 concentrated gel, separator gel and interlayer gel compositions

Spotting and electrophoresis: placing the electrophoresis tank into 4 ℃ or ice water bath, adding anode buffer solution into the outer tank, adding cathode buffer solution into the inner tank, pre-electrophoresis for 10min at 30V, adding sample (subjected to Tricine special sample loading buffer treatment) into sample application holes, electrophoresis at 30V for 1 h, and stopping electrophoresis until bromophenol blue reaches the bottom of the gel after electrophoresis at 100V, and performing subsequent coomassie brilliant blue staining.

Dyeing and decoloring: staining was performed using coomassie brilliant blue. Placing the colloid into a fixing solution (ethanol: glacial acetic acid: water=5:1:4), fixing for 30min, pouring the fixing solution, rinsing with ultrapure water for 2-3 times, adding coomassie brilliant blue staining solution (1.6% phosphoric acid, 8% ammonium sulfate, 0.02% CBB, G250 and 20% ethanol), staining overnight, adding a decolorizing solution for multiple times after the staining is finished, and changing the eluent until electrophoresis bands are clear, wherein gel background elution is stopped.

LC-MS/MS analysis of purified protein peptides

Sequence identification is carried out on the electrophoresis adhesive tape, and data acquisition software is adopted: thermo xcalibur4.0 (Thermo, USA); reverse phase column information: c18 column (75. Mu. Mx 25cm, thermo, USA); chromatographic instrument: EASY-nLC 1200; mass spectrometry instrument: q-exact (Thermo, USA); chromatographic separation time: 90min A:2%ACN with 0.1%formic acid; b:80%ACN with 0.1%formic acid; flow rate: 300nL/min, EASY-nLC gradient: 0-1min 5% B, 1-41min 23% B, 41-51min 29% B, 51-57min 38% B, 57-58min 48% B, 58-59min 100% B, 59-90min stop; MS scanning range (m/z) 350-1300, collecting mode DDA; top 20 (the 20 most strongly signalled parent ions were selected for secondary fragmentation); first-order mass spectrum resolution 70000, fragmentation mode HCD; second resolution 17500, dynamic exclusion time 18s.

LC-MS/MS identification results

(1) Moringa oleifera antioxidant peptide amino acid composition

TABLE 5 amino acid composition of Moringa oleifera antioxidant peptides of different molecular weights

The antioxidant capacity of a polypeptide is closely related to the kind, number and arrangement of amino acids constituting the peptide. Many amino acids and derivatives thereof have the ability to scavenge free radicals, such as Lys, his, tyr, met, pro, arg, glu, cys and the like. Table 5 shows that the moringa antioxidant peptides 1K Da and 3K Da are rich in solid powder and amino acids in the adhesive tape, and the content of glutamic acid, lysine and arginine in the adhesive tape after being separated and purified by Tricine-SDS-PAGE electrophoresis is higher than that in the solid powder, and most of the amino acids are related to antioxidant activity, wherein glutamine has a great contribution in protecting the integrity of cell membranes, maintaining the activity of cells and reducing oxidative damage of cells.

(2) Molecular weight and sequence identification of target adhesive tape

The chemical structure is a key factor affecting the antioxidant activity of polypeptide, and the antioxidant peptide is generally composed of 2-20 amino acids, and researches show that the composition, molecular weight and arrangement sequence of polypeptide amino acids have great correlation with the antioxidant activity. The protein peptide containing P, G, A, V, L in the peptide sequence has potential antioxidant activity. The specific branched structure of amino acid residues in polypeptides is considered as a direct scavenger of free radicals, such as phenylalanine (Phe) containing a benzene ring, tryptophan (Trp) containing an indole group, histidine (His) containing an imidazole ring, tyrosin (Tyr) containing a phenolic hydroxyl group, and cysteine (Cys) and methionine (Met) containing sulfur. The N end or the C end of the peptide sequence contains hydrophobic amino acid (V, L, I, A, F), the third amino acid at the C end of the peptide chain is hydrophobic amino acid residue, and the peptide chain has low electrostatic repulsion, steric hindrance and hydrogen bonding effect (W, Y, F, M, L, I) and has an effect on the antioxidation activity; the peptide chain has acidic amino acid residues (D, E) which can enhance the antioxidant activity.

TABLE 6 identification of moringa oleifera antioxidant peptide amino acid sequence

It is generally believed that the 20 amino acids comprising the protein all react with free radicals, but the difference in activity is large, the moringa polypeptide is subjected to ultrafiltration for electrophoretic separation, molecular identification is performed on molecular adhesive tapes smaller than 1K Da, and the difference in antioxidant capacity is evaluated. As shown in Table 6, the moringa oleifera antioxidant peptide has 21 amino acid sequences, the molecular weight is 722.4329 Da-1791.7952 Da, and the moringa oleifera antioxidant peptide consists of 6-16 amino acids; more than 50% of amino acids (P, G, A, V, L) in most peptide fragments have potential antioxidant activity, and the peptide chain contains aromatic rings, imidazole groups and sulfur-containing groups, so that the antioxidant activity of the peptide fragments can be enhanced. Hydrophobic amino acids exist at the C end or N end of peptide chains of peptide segments 7, 9, 10, 12, 14, 19 and 21, and the aromatic ring, the imidazole group and the sulfur-containing group are contained in the peptide chains, so that the antioxidant activity of the polypeptide is influenced to a certain extent.

Research shows that the antioxidant capacity of antioxidant peptide has close relation with the molecular weight of polypeptide, specific amino acid, amino acid variety, sequence, configuration, etc. The presence of acidic amino acids in the peptide chain, amino acid residues with proton donor properties, hydrophobic amino acids present in the side chain or C, N end, aromatic amino acids, and the like are important features of the existence of primary structures of antioxidant peptides. The smaller the molecular weight of the polypeptide, the easier the polypeptide can penetrate the biological membrane to reach the action part to act; the compound contains hydrophobic amino acid, has positive charge, can be combined with oxygen or inhibit the release of hydrogen in lipid, delays lipid peroxidation chain reaction, plays an antioxidant role, and is rich in proline which is easy to oxidize to generate antioxidant activity; the peptide chain containing aromatic ring, imidazole group and sulfur-containing group can quench free radical directly; the third amino acid at the C end of the peptide chain is a hydrophobic amino acid residue, has low electrostatic repulsion, steric hindrance and hydrogen bond effect, has an effect on antioxidation activity, has acidic amino acid residues in the peptide chain and can enhance the antioxidation activity, and five polypeptides in the figure 15 are determined to be the small molecule peptides with the strongest antioxidation potential through comprehensive analysis.

Peptide fragment separation: treating an electrophoresis adhesive tape to obtain a protein solution, separating by adopting RP-HPLC reversed phase high performance liquid chromatography, wherein a chromatographic column is a C18 column, a mobile phase is a first mixed solution and a second mixed solution, performing linear elution, wherein the first mixed solution is formed by mixing 99.8% of water and 0.2% of trifluoroacetic acid, and the second mixed solution is formed by mixing 99.8% of acetonitrile and 0.2% of trifluoroacetic acid; the conditions for linear elution were: the elution time is 60min, the elution is started from the second mixed solution accounting for 0% to 75% of the mobile phase in volume ratio, the flow rate is 0.8mL/min, the detection wavelength is 280nm, 5 polypeptides are obtained through separation and purification and are identified, and the peptide 1 is LALPVYN; peptide 2 was FHEEDDAKLF; peptide 3 was LDEGKWQHVK; peptide 4 was LHIAALVFQ; peptide 5 was GHVALVFVN.

Example 2 determination of antioxidant Capacity of amino acid sequence

The antioxidant capacity of the separated different polypeptides is studied by taking 4 antioxidant indexes such as DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity measurement, superoxide anion free radical scavenging activity, ABTS free radical scavenging capacity measurement and the like as references.

(1) Determination of DPPH radical scavenging ability

4mg of DPPH was weighed and dissolved in 100mL of absolute ethyl alcohol to prepare an ethanol solution of 0.1mmol/L DPPH, and the solution was stored in a brown bottle in a sealed manner and placed in a refrigerator at 4 ℃. Samples were taken, 0.5mL of the solution to be measured and 0.5mL of a 0.1mmol/L ethanol solution were reacted in a 2mL centrifuge tube for 50min in the absence of light, and the absorbance was measured at a wavelength of 517nm by centrifugation (10 min 4000 r/min).

Wherein: a is that _{Empty space} The blank tube is replaced by absolute ethyl alcohol with the concentration of 0.1mmol/L DPPH; a is that _{For a pair of} 0.5mL of absolute ethanol and 0.5mL of a 0.1mmol/L DPPH ethanol solution; a is that _Sample 0.5mL of the sample solution to be tested and 0.5mL of an ethanol solution of 0.1mmol/L DPPH.

(2) Determination of the hydroxy radical scavenging Capacity

With reference to the prior art, 8.8mmol/L H is taken ₂ O ₂ ，0.5mL 9mmol/L Fe ²⁺ 0.5mL 9mmol/L salicylic acid-ethanol solution 0.5 mL) were added with 100 μL of different concentrations of Moringa oleifera proteolytic products, and distilled water was used as blank group A ₀ Measurement of absorbance A at 510nm at each concentration ₁ The hydroxyl radical scavenging capacity formula is as follows:

wherein: a is that ₀ Light absorption value of sample group; a is that ₁ Blank light absorption values

(3) Superoxide anion radical scavenging activity

Referring to the prior art, 2.25mL of Tris-HCL buffer (0.1 mol/L) with pH 8.2 was taken, L mL of distilled water and 1mL of sample solution with different concentrations were added, and then 0.25mL of 10mmol/L of pyrogallol was added. After mixing well, the reaction was stopped by adding 1 drop of concentrated HCl at 3min in a water bath at 25℃and absorbance was measured at 325 nm. Glutathione at different concentrations was used as a control. The clearance calculation formula is as follows:

wherein: a is that ₀ To replace the blank absorbance value of the sample solution with distilled water; a is that ₁ Absorbance for the sample solution; a is that ₂ The absorption value of the solution without adding the pyrogallol is obtained.

(4) ABTS radical scavenging ability assay

The ABTS free radical scavenging activity is properly modified by referring to the existing method, 2.45mmol/L potassium persulfate is easy to be mixed with 7mmol/L ABTS mother liquor according to the proportion of 1:1, the mixture is subjected to light-proof reaction for 12-16h, 5mM PBS with pH of 7.4 is used for dilution, the absorbance value of the mixture is 0.7-0.8 by measuring at 734nm, samples to be tested with different concentrations and diluted ABTS free radical working solution are mixed according to the proportion of 1:1, the mixture is subjected to light-proof reaction for 10min at room temperature, distilled water is used as blank, glutathione with different concentrations is used as positive control, and the calculation formula is as follows:

wherein A is _S Absorbance values for the sample set; a is that ₀ Absorbance values for the blank group.

Because the content of the five polypeptides separated and purified is small, in order to verify the antioxidant activity and cell injury repairing effect in the later period, a large amount of five polypeptides are obtained by adopting a synthesis method for further research on the antioxidant activity.

As can be seen from FIG. 16, the antioxidant activity of the five amino acid sequences after synthesis was determined, and the antioxidant activity of the synthesized LALPVYN, FHEEDDAKLF, LDEGKWQHVK, LHIAALVFQ, GHVALVFVN amino acid sequence was found to be stronger, wherein the antioxidant activity of LALPVYN was higher than that of the other amino acid sequences and was close to that of VC.

Example 3 protection of LALPVYN against oxidative damage to HepG2 cells

MTT cell viability assay method

HepG2 cells were all at 37℃and 5% CO ₂ Culturing in a humidified environment. To all media 10% foetal calf serum and 1% penicillin-streptomycin mixture was added. HepG2 cells in the logarithmic growth phase were seeded in 96-well plates at 1X 10 per well ⁴ Concentration of individual cells. After 24h incubation, 200. Mu.L of medium was added to the control group. The experimental groups were treated with different concentrations of LALPVYN (50, 100, 250, 500 and 1000 and 2000. Mu.g/mL). The blank group was added with LALPVYN without HepG2 cells. After 24h incubation, 100. Mu.L of 0.25mg/mL MTT was added to each well. After another 4 hours incubation, the medium was replaced with DMSO and the OD was measured with a microplate reader at a wavelength of 490 nm. The viability was calculated as HepG2 cell viability (%) = (odexperimental group-odblanc group)/(odcontrol group-odblanc group) ×100%

LALPVYN vs H ₂ O ₂ Effect of mediated oxidative damage HepG2 cell viability experiments

After 24h culture of HepG2 cells, 200. Mu.l of the suspension was adsorbed and inoculated in 96-well plates at a density of 1X 10 ⁴ Holes, grouped into control, protective and oxidative damage groups. The preparation of the protective group comprises the following steps: respectively add 200 mu.LVc (60. Mu.g/mL), LALPVYN (50, 100, 200. Mu.g/mL) DMEM solution were added to each well after incubation for 24h ₂ O ₂ (0.5 mmol/L) for 4h, the oxidative damage group was prepared as follows: 200 mu LDMEM culture solution was added, and after incubation for 24 hours, 200 mu L H was added to each well ₂ O ₂ (0.5 mmol/L) for 4h. 200 mu L of culture medium is added into the control group for culturing for 28 hours; the blank was incubated for 28h with LALPVYN without addition of HepG2 cells. MTT assay measures cell viability.

Analysis of antioxidant Capacity in HepG2 cells

HepG2 cells were grown at 3X 10 ⁵ Each dish was inoculated with 4mL in 6 60mm cell culture dishes. After various treatments (group and 2.5.2), the total superoxide dismutase (SOD), catalase (CAT) activity and the content of Malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in the cell lysate were determined according to the method in the kit instructions.

ROS was assayed using a reactive oxygen species assay kit, and HepG2 cells were 2X 10 ⁵ The cells were treated according to the experimental grouping method described above by inoculating the cells/wells into laser confocal dishes. Pictures were taken with a laser confocal microscope and ImageJ was subjected to fluorescence intensity analysis.

Apoptosis detection

At 3X 10 ⁵ HepG2 cell apoptosis was detected in 6-well plates seeded with individual/well HepG2 cells using flow cytometry (Ex=488 nm, FL1Em=525+ -20 nm, FL2Em=585+ -21 nm) and FlowJo software according to the Annexin V-FITC kit instructions.

Statistical analysis

All tests were performed in three separate experiments and the mean ± standard deviation of the data is reported. Differences between the different treatment groups were analyzed for significance (P < 0.05) using SPSS19.0 (IBM corp., armonk, NY, USA) for one-way analysis of variance (ANOVA).

H ₂ O ₂ Is a well-known hepatotoxic chemical substance with long half-life, can be directly converted into hydroxyl free radicals and oxygen free radicals, and is an important cause of oxidative stress injury in vivo. To demonstrate the antioxidant effect of LALPVYNActivity by H ₂ O ₂ And (5) establishing an oxidation damage model and evaluating the antioxidant activity of the model.

The antioxidant effect of LALPVYN is shown in figure 17, and the effect of LALPVYN on the survival rate of HepG2 cells is studied first (A), and the result shows that the peppery wood protein peptide has no growth promoting effect on HepG2 cells, and the survival rate of HepG2 cells tends to be reduced along with the increase of the concentration. Compared with the control group, the cell viability is (97.95+/-1.18-93.03+/-0.56%) in the range of 50-500 mug/mL, and the cell viability is nontoxic to cells (> 90%). When LALPVYN concentration reached 1000. Mu.g/mL, the concentration was significantly reduced compared with the blank group, and the toxicity effect on cells was generated, and 50, 100 and 200. Mu.g/mL were selected for experiments under comprehensive consideration. Next, an oxidation damage model (B) is established, and the result shows that H with different concentrations is used ₂ O ₂ Induced cells for 4h all had damaging effects on cells and cell viability decreased with increasing concentration. 0.5mmol/L H ₂ O ₂ Inducing the cells for 4 hours, wherein the cell survival rate is (54.09 +/-1.58)%, and the concentration is close to the concentration of the semi-inhibited cells, and determining the concentration for establishing a cell oxidative damage model. Finally, the antioxidant effect (C) of LALPVYN is studied, and the result shows that the cell survival rate after LALPVYN pretreatment is between (80.12+/-1.47 and 85.11 +/-3.24)%, and is close to V _C The pretreated cell viability indicates that the moringa oleifera antioxidant peptide pretreatment can effectively reduce the cell oxidative damage effect.

Effect of LALPVYN pretreatment on HepG2 cells SOD, CAT, GSH-Px and MDA

The antioxidant capacity of intracellular antioxidants can be divided into two aspects, direct and indirect. The direct oxidation resistance of antioxidants is achieved by providing hydrogen atoms or electrons to remove intracellular ROS. The indirect antioxidant properties of antioxidants are those that are resistant to oxidative damage by mediating the expression of antioxidant enzymes and antioxidant genes. Previous studies have demonstrated that the intracellular antioxidant enzyme system is an important defense system against oxidative stress damage, in order to elucidate the role of LALPVYN in H ₂ O ₂ The mediated protection mechanism of oxidative stress injury of HepG2 cells is studied, and the influence of LALPVYN on the CAT and GSH-Px, MDA, SOD contents of the HepG2 cells is shown in figure 18.

Changes in antioxidant enzyme activity levels can be considered an important biomarker for cellular responses to oxidative stress. Superoxide dismutase (SOD) catalyzes the conversion of highly active superoxide anions from oxidative stress to hydrogen peroxide (H) ₂ O ₂ ) And is further decomposed by hydrogen peroxide (CAT) into water and oxygen. As can be seen from FIG. 18A, the CAT enzyme activity in normal cells is higher ((27.15 + -2.29) U/mg pro), the enzyme activity in the injured group is lowest (7.38+ -0.63U/mg pro), and the CAT enzyme activity pre-protected by LALPVYN is improved to different degrees compared with the injured group, namely (13.15+ -2.29), (17.15+ -2.13) and (17.50+ -1.75) U/mgpro. As can be seen from FIG. 18B, the SOD activity in normal cells was (11.16.+ -. 0.32) U/mg Pro, the lowest enzyme activity in the injured group ((6.83.+ -. 0.35) U/mg Pro), and the SOD activity after LALPVYN pretreatment was (8.33.+ -. 0.053) U/mg Pro, (8.74.+ -. 0.17) U/mg Pro, and (9.04.+ -. 0.19) U/mg Pro were significantly improved.

Peroxidase (GSH-Px) is an important peroxide decomposing enzyme widely existing in the body, and can catalyze GSH to GSSG, reduce toxic peroxide to nontoxic hydroxy compound, and promote H ₂ O ₂ The decomposition of the cell membrane, protecting the structure and function of the cell membrane from interference and damage by the peroxide. Compared with the normal group (67.72 +/-1.36) Umg/Pro), the GSH-Px content in the damaged group is obviously reduced to (33.06 +/-1.46) Umg/Pro, and the GSH-Px enzyme activities of the drug group pre-protected by LALPVYN are increased to different degrees compared with the damaged group, and the GSH-Px content after the LALPVYN treatment is (42.72+/-1.36, 50.93 +/-1.25 and 58.72 +/-1.36 Umg/Pro) respectively. The GSH-Px content of the drug group is obviously increased compared with that of the injury group. Indicating that LALPVYN can improve the cause H ₂ O ₂ Content of GSH-Px in resulting oxidatively damaged HepG2 cells.

Malondialdehyde is a tricarbon compound, an indicator of lipid peroxidation, and is a component of the decomposition of peroxidized polyunsaturated fatty acids. ROS produced by cells during metabolism can trigger lipid peroxidation to form MDA with polyunsaturated fatty acids associated with phospholipids, enzymes and membrane receptors of biological membranes. MDA is an important indicator of oxidative damage to cells, and the higher the MDA content, the more severe the oxidative damage to cells. The MDA level of the cells in the normal group is lower (2.49+/-0.25) nmol/mg pro, and the MDA content of the injured group is obviously increased to (11.16+/-0.088) nmol/mg pro compared with that of the normal control group. Compared with the model group, the MDA content of the drug group (50, 100, 200 mug/mL) is obviously reduced, the MDA content is (3.49+/-0.28), (5.54+/-0.11) and (6.97+/-0.52) nmol/mg pro (P < 0.001) respectively, and the drug group have obvious dose effect relation. The moringa antioxidant peptide can effectively inhibit lipid peroxidation, reduce malondialdehyde, and has a certain protective effect on cell oxidative damage due to the fact that hydrophobic amino acid is contained in LALPVYN as a factor.

The LALPVYN can not only remove free radicals in cells, but also enhance endogenous antioxidant defense system including antioxidant enzyme defense system and glutathione system, thereby reducing H ₂ O ₂ MDA and free radicals generated by induced HepG2 cells achieve the effect of protecting cells. Study of watermelon seed antioxidant peptide pair H with Chao et al ₂ O ₂ In cytoprotective effect of induced oxidative stress, treatment with antioxidant peptide significantly weakens H ₂ O ₂ The results of the decrease in the induced antioxidant enzyme activity were consistent. The antioxidant effect is superior to that of duck embryo peptide studied by YIng et al, and the effect of reducing the antioxidant enzyme activity can be remarkably relieved by a small dosage of LALPVYN.

Effect of LALPVYN pretreatment on HepG2 cell ROS

Studies have shown that excessive ROS oxidize proteins and lipids, which in turn lead to disruption of nuclear DNA and mitochondrial integrity and ultimately cell death. DCFH-DA is a specific fluorescent probe for intracellular reactive oxygen species, which can penetrate cell membranes and be degraded by esterases in the membranes into DCFH, which further specifically binds to intracellular ROS to generate DCF that can emit fluorescent signals. Thus, photographs can be taken by confocal laser, and the ImageJ software measures the change in fluorescence intensity, reflecting the change in ROS content in the cells.

LALPVYN vs H ₂ O ₂ The effect of mediating HepG2 cell ROS content is shown in figure 19. ROS mean fluorescence intensity (36.57.+ -. 0.44) in the injured group versus normal controlSignificant differences in group (11.26.+ -. 0.027) compared to H ₂ O ₂ Can cause elevation of ROS levels in HepG2 cells; ROS mean fluorescence intensity compared to H in drug group ₂ O ₂ The injury group is obviously reduced (32.93+/-1.1, 25.92+/-0.51, 24.99+/-0.57), wherein the ROS content of the component with the polypeptide concentration of 200 mug/mL is close to that of positive control V _C The group (23.14.+ -. 0.69) also had significant differences in the fractions with polypeptide concentrations of 50. Mu.g/mL and 100. Mu.g/mL compared to the injured group. This cytoprotective effect of LALPVYN may be due to its antioxidant capacity to scavenge intracellular ROS. In conclusion, the moringa oleifera leaf antioxidant peptide LALPVYN has an antioxidant effect by removing intracellular ROS.

Effect of LALPVYN pretreatment on HepG2 apoptosis

Apoptosis is an important component of the processes of normal cell turnover, normal development and function of the immune system, embryonic development, chemically induced cell death, and hormone-dependent atrophy. Apoptosis abnormalities are associated with many human diseases such as ischemic injury, autoimmune diseases, and cancer. Apoptosis assays are always used to evaluate the potential use of bioactive ingredients as food supplements or chemotherapeutics.

HepG2 cells were treated with different concentrations of LALPVYN (50, 100, 200. Mu.g/mL) and the effect of LALPVYN on HepG2 apoptosis was examined by flow cytometry. The apoptosis rate of the HepG2 cell of the normal group is lower than (1.43+/-0.19)%, and the apoptosis rate of the damaged group is obviously increased to (15.93+/-0.93)%; the apoptosis rate is obviously reduced by treating cells with 50-200ug/mL LALPVYN, and the apoptosis rate of 200ug/mL LALPVYN is the lowest (4.28+/-0.22)%. And the apoptosis rate of the LALPVYN treatment group is lower than that of the GSGH treatment group with the concentration of 200ug/mL, which shows that the LALPVYN has better effect of relieving the apoptosis caused by oxidative stress than that of the GSGH. The results in conclusion show that LALPVYN is opposite to H ₂ O ₂ The mediated oxidative stress injury has a protective effect, wherein the protective effect of LALPVYN of 200ug/mL is most obvious.

In summary, the present invention uses alkaline protease to hydrolyze moringa oleifera leaf protein effectively to obtain components with antioxidant activity, and the hydrolysate is separated into three components by membrane ultrafiltration. The low molecular weight fraction (< 1 kDa) has a relatively strong free radical scavenging capacity. Further separating by ultrafiltration tube, and screening to obtain 5 components (LALPVYN, FHEEDDAKLF, LDEGKWQHVK, LHIAALVFQ, GHVALVFVN) with strong antioxidant activity, wherein the LALPVYN component has molecular weight of 788.44Da and clearance rate of each free radical is superior to that of other polypeptides. Meanwhile, LALPVYN has better protection effect on the oxidative damage of the HepG2 cells induced by H2O 2. In particular, the comprehensive analysis of LALPVYN has strong protection effect on HepG2 cells in the aspects of improving CAT, GSH, SOD activity, reducing MDA content, reducing ROS content and inhibiting apoptosis, has a certain potential compared with VC, and MLPH can become a natural antioxidant in foods, and meanwhile, moringa polypeptide smaller than 1KD can also be used as a raw material of antioxidant foods.

The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.

SEQUENCE LISTING

<110> university of agriculture in Yunnan

<120> an antioxidant peptide of Moringa oleifera, and its preparation method and application

<130> 20220406

<160> 21

<170> PatentIn version 3.3

<210> 1

<211> 13

<212> PRT

<213> Moringa oleifera

<400> 1

Thr Val Leu Ile Met Glu Leu Ile Asn Asn Val Ala Lys

1 5 10

<210> 2

<211> 10

<212> PRT

<213> Moringa oleifera

<400> 2

Gln Ile Lys Thr Ile Pro Lys Lys Pro Asn

1 5 10

<210> 3

<211> 9

<212> PRT

<213> Moringa oleifera

<400> 3

Gly Ala Val Gly Ser Gly Leu Ser Lys

1 5

<210> 4

<211> 12

<212> PRT

<213> Moringa oleifera

<400> 4

Leu Ile Lys Val Leu Leu Thr Ala Val Lys Asp Phe

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Moringa oleifera

<400> 5

Lys Ala Pro Ala Tyr Ser Val

1 5

<210> 6

<211> 6

<212> PRT

<213> Moringa oleifera

<400> 6

Thr Phe Leu Lys Ser Lys

1 5

<210> 7

<211> 7

<212> PRT

<213> Moringa oleifera

<400> 7

Leu Ala Leu Pro Val Tyr Asn

1 5

<210> 8

<211> 8

<212> PRT

<213> Moringa oleifera

<400> 8

Glu Tyr Asp Leu Ser Lys Ala Gln

1 5

<210> 9

<211> 10

<212> PRT

<213> Moringa oleifera

<400> 9

Phe His Glu Glu Asp Asp Ala Lys Leu Phe

1 5 10

<210> 10

<211> 10

<212> PRT

<213> Moringa oleifera

<400> 10

Leu Asp Glu Gly Lys Trp Gln His Val Lys

1 5 10

<210> 11

<211> 17

<212> PRT

<213> Moringa oleifera

<400> 11

Leu Asp Glu Gly Lys Trp Gln His Val Lys Pro Lys Gly Leu Lys Thr

1 5 10 15

Asn

<210> 12

<211> 9

<212> PRT

<213> Moringa oleifera

<400> 12

Leu His Ile Ala Ala Leu Val Phe Gln

1 5

<210> 13

<211> 16

<212> PRT

<213> Moringa oleifera

<400> 13

Lys Ala Tyr Gly Gln Asn Leu Ser Ile Gly Tyr Asp Asp Phe Asp Ser

1 5 10 15

<210> 14

<211> 9

<212> PRT

<213> Moringa oleifera

<400> 14

Gly His Val Ala Leu Val Phe Val Asn

1 5

<210> 15

<211> 8

<212> PRT

<213> Moringa oleifera

<400> 15

Leu Leu Val Val Ser Gly Ile Asn

1 5

<210> 16

<211> 9

<212> PRT

<213> Moringa oleifera

<400> 16

Thr Val Asn Ile Ile Ser Ser Lys Arg

1 5

<210> 17

<211> 8

<212> PRT

<213> Moringa oleifera

<400> 17

Ser Lys Ser Leu Ile Ala Ile Asn

1 5

<210> 18

<211> 10

<212> PRT

<213> Moringa oleifera

<400> 18

His Thr Glu Leu Ala Leu Lys Tyr Val Asn

1 5 10

<210> 19

<211> 7

<212> PRT

<213> Moringa oleifera

<400> 19

Gly Pro Ile Ile Leu Pro Asn

1 5

<210> 20

<211> 9

<212> PRT

<213> Moringa oleifera

<400> 20

Pro Ala Ala Leu Ala Lys Met Lys Asn

1 5

<210> 21

<211> 9

<212> PRT

<213> Moringa oleifera

<400> 21

Gly His Val Ala Leu Val Phe Val Asn

1 5

Claims (3)

1. The moringa oleifera antioxidant peptide is characterized by comprising one or more of the following sequences: SEQ ID No.7, SEQ ID No.9, SEQ ID No.10, SEQ ID No.12 and SEQ ID No.21.

2. The moringa oleifera antioxidant peptide of claim 1, wherein the moringa oleifera antioxidant peptide is SEQ ID No.7.

3. The use of the moringa oleifera antioxidant peptide according to claim 1 or 2, wherein the moringa oleifera antioxidant peptide is used for preparing cosmetics or antioxidant medicines.