CN112111535A

CN112111535A - Preparation method and application of antioxidant grape seed polyphenol nano material

Info

Publication number: CN112111535A
Application number: CN202011013680.2A
Authority: CN
Inventors: 李乙文; 王天佑; 陈展; 顾志鹏
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-09-24
Filing date: 2020-09-24
Publication date: 2020-12-22
Anticipated expiration: 2040-09-24
Also published as: CN112111535B

Abstract

The invention discloses a preparation method and application of an antioxidant grape seed polyphenol nano material, wherein the preparation method comprises the following steps: (1) mixing grape seed polyphenol solution with horseradish peroxidase aqueous solution, and preheating to a temperature stable at 60-70 ℃; wherein the solvent of the grape seed polyphenol solution is a mixed liquid phase of deionized water and ethanol; (2) adding the hydrogen peroxide solution into the preheated mixed solution by using an injector, and keeping stirring for 4-8 hours in the air atmosphere to obtain a brown-yellow turbid reaction stock solution; (3) cooling the reaction stock solution to room temperature under the state of keeping stirring, and sequentially centrifuging, washing and freeze-drying the reaction stock solution to obtain the grape seed polyphenol nano material with the particle size of 100-400 nm. The invention has stronger controllability in the synthesis process, effectively controls the catalytic activity of the horseradish peroxidase, and can simply and quickly adjust the particle size of the grape seed polyphenol nano material to obtain the nano material with uniform distribution and good appearance.

Description

Preparation method and application of antioxidant grape seed polyphenol nano material

Technical Field

The invention relates to the technical field of biomedicine, in particular to a preparation method and application of an antioxidant polyglucose seed polyphenol nano material.

Background

The skin covers the surface of the human body, is the largest organ of the human body, is also an important organ in physiology and anatomy, is directly contacted with the external environment, has various functions of excretion, protection, external stimulation sensing, body temperature regulation and the like, and is vital to the self protection of the human body. When the skin is damaged and injured to a certain degree, due to the change of microenvironment and the invasion of inflammation, several problems such as bleeding, infection, scars and the like can occur, which cause serious injury to human body, even life danger in serious cases, so that the rapid, effective and safe skin repair is very important. Dressing materials for covering wounds are available, and can create a suitable microenvironment to promote cell proliferation and migration, inhibit inflammation, and promote wound healing. But also has the problems that the dressing material has limited inflammation eliminating effect, has certain toxic and side effects, needs multi-step synthesis and purification in the preparation method, has high preparation cost, is not suitable for large-scale application and the like, so that the provision of the natural, simple, efficient and safe biocompatible material for skin repair is vital.

Natural polyphenol widely exists in nature, has quite unique chemical structure and excellent physical and chemical properties, and is widely researched and applied whether being used alone or being used as a basic element of a construction material. The herbal medicine used in ancient China for wound repair contains a plurality of compounds with polyphenol structures, and the material based on natural polyphenol has good biocompatibility, excellent oxidation resistance and active oxygen radical removing capacity, can effectively inhibit the development of inflammation and promote the proliferation and migration of cells, thereby effectively accelerating the repair process of skin and optimizing the repair effect of skin.

The grape seeds are byproducts of red wine processing, have wide sources and low price, contain a large amount of natural polyphenol, have very strong oxidation resistance and have very friendly action with organisms. Under certain conditions, grape seed polyphenol can be subjected to oxidative polymerization to form a macromolecular material, so that the excellent performance of the grape seed polyphenol is maintained, the self irritation of small molecular substances is reduced, and the safety and biocompatibility in application are improved. However, no mature method is available at present, and the grape seed polyphenol can be stably and controllably made into macromolecules under the condition of not introducing a large amount of external additives, so that the further application of the grape seed polyphenol is limited.

At present, a plurality of natural polyphenol large-molecule means exist to convert small molecules into large-molecule materials with stronger practicability, such as oxidation (including alkaline condition autooxidation, enzymatic oxidation, oxidant oxidation and the like), copolymerization with other functional materials, grafting with the existing polymers, chelation with metal ions, precise organic synthesis to prepare new materials and the like. The horseradish peroxidase is a natural, efficient, universal, green and safe biological catalytic enzyme, and can be widely used for catalyzing polymerization of natural polyphenol. The catalyst is usually used together with hydrogen peroxide in a catalysis manner, so that the decomposition of the hydrogen peroxide is initiated to promote the oxidation of polyphenol molecules, and the polymerization and assembly of the oxidized polyphenol molecules are further promoted to form the macromolecular material based on natural polyphenol. Many materials have been constructed based on this approach, but there are still significant problems with the controllability of this process. The horseradish peroxidase has extremely high activity, and can accelerate the decomposition of hydrogen peroxide and initiate the oxidative polymerization of polyphenol at an extremely high speed under common conditions, so that the cross-linking degree of the commonly formed polyphenol material is high, and the polyphenol material is difficult to control and regulate effectively.

Disclosure of Invention

In summary, the closest prior art of the present invention is generally deficient: the natural polyphenol micromolecules have strong antioxidant capacity and obvious effect of removing free radicals, but have some problems, such as low stability, easy invalidation, strong irritation, easy adverse reaction, permeability and potential toxicity, so that the natural polyphenol micromolecules are often required to be subjected to macro-polymerization. The traditional macromolecular technology often has the problems of complex operation, reduced effect after compounding, potential toxicity caused by introducing foreign substances and the like, particularly the enzymatic oxidation method has the problems of high reaction activity, poor controllability, difficulty in constructing uniform and stable polyphenol nano materials and the like, and the formed macromolecular material also has the defects of low repair speed, low inflammation clearance, poor repair effect, higher application cost and the like when being applied to skin repair.

Aiming at the defects, the invention provides a preparation method and application of an antioxidant grape seed polyphenol nano material.

The invention solves the problem that the enzymatic polyphenol polymerization process is too fast and uncontrollable, and can flexibly regulate and control the size of the obtained nano material within the range of 100-400 nm; the antioxidant capacity of the obtained grape seed polyphenol nano material has intentional expression in vitro and on a cell level, and can be applied to skin repair to accelerate the repair speed and the repair effect.

The technical scheme of the invention is as follows:

a method for preparing an antioxidant grape seed polyphenol nano material comprises the following steps:

(1) mixing grape seed polyphenol solution with horseradish peroxidase aqueous solution, and preheating to a temperature stable at 60-70 ℃; the solvent of the grape seed polyphenol solution is a mixed liquid phase of deionized water and ethanol, wherein the mass percent of the deionized water is 85%, and the mass percent of the ethanol is 15%;

(2) adding the hydrogen peroxide solution into the preheated mixed solution by using an injector, and keeping stirring for 4-8 hours in the air atmosphere to obtain a brown-yellow turbid reaction stock solution;

(3) cooling the reaction stock solution to room temperature under the state of keeping stirring, and sequentially centrifuging, washing and freeze-drying the reaction stock solution to obtain the polyglucose seed polyphenol nano-material with the particle size of 100-400 nm;

wherein the weight ratio of the grape seed polyphenol to the horseradish peroxidase to the hydrogen peroxide is 6: 0.1-0.8: 1-8.

Preferably, the weight ratio of the grape seed polyphenol to the horseradish peroxidase to the hydrogen peroxide is 6: 0.102-0.514: 1.312-6.56.

In order to ensure that the grape seed polyphenol solution and the horseradish peroxidase aqueous solution are fully dissolved and dispersed, before the step (1), the grape seed polyphenol solution and the horseradish peroxidase aqueous solution are respectively subjected to ultrasonic treatment for 2-3 minutes.

The poly grape seed polyphenol nano material prepared by the invention has excellent antioxidant effect on both in vitro and biological cell layers, and can be applied to antioxidants.

In addition, the grape seed polyphenol nano material prepared by the invention can effectively accelerate wound healing and reduce inflammation, and can be applied to skin repair wound dressing, thereby realizing skin wound repair more efficiently and safely.

The activity of horseradish peroxidase is controlled by adjusting the temperature atmosphere of reaction, so that the oxidative polymerization process of grape seed polyphenol is in an adjustable and controllable state; meanwhile, the oxidation process of the grape seed polyphenol is further controlled by taking the change of the polarity of the solvent and the control of the adding rate of hydrogen peroxide as auxiliary means, so that the grape seed polyphenol nanoparticles with good adjustability are obtained.

Compared with the prior art, the invention has the following characteristics and advantages:

(1) the main raw material grape seed polyphenol is naturally extracted from red wine processing byproducts, and the method has mature extraction technology, low price, safety and high efficiency; the horseradish peroxidase is naturally extracted from horseradish, so that the horseradish peroxidase is green, environment-friendly, safe and efficient; the whole reaction system does not introduce compounds with high toxicity and irritation, and the whole reaction process is green, efficient and environment-friendly, and has great potential in the application of the biological layer.

(2) The controllability of the synthesis process is strong, the catalytic activity of horseradish peroxidase is effectively controlled, the particle size of the grape seed polyphenol nano material can be simply and rapidly adjusted, the nano materials with different sizes, uniform distribution and good appearance are obtained, and the method is suitable for application in different scenes.

Experiments show that the nano-materials with different sizes can be obtained by adjusting the weight ratio of the grape seed polyphenol, the horseradish peroxidase and the hydrogen peroxide by adopting the method. When the weight ratio of the grape seed polyphenol to the horseradish peroxidase to the hydrogen peroxide is 6: 0.102: at 1.312, the particle size of the nano material is 124-156 nm; when the weight ratio of the grape seed polyphenol to the horseradish peroxidase to the hydrogen peroxide is 6: 0.172: at 2.178, the particle size of the nano material is 214-246 nm; when the weight ratio of the grape seed polyphenol to the horseradish peroxidase to the hydrogen peroxide is 6: 0.446: 5.664, the particle size of the nano material is 285-315 nm; when the weight ratio of the grape seed polyphenol to the horseradish peroxidase to the hydrogen peroxide is 6: 0.514: at 6.56, the particle size of the nano material is 327-373 nm.

(3) The prepared grape seed polyphenol nano material has very good oxidation resistance, has good oxidation resistance effect in-vitro detection and cell level detection, and can be applied to antioxidants, skin repair wound dressings and the like.

Drawings

FIG. 1 is a scanning electron microscope image of a sample obtained in example 1;

FIG. 2 is a scanning electron microscope image of the sample obtained in example 2;

FIG. 3 is a scanning electron microscope image of the sample obtained in example 3;

FIG. 4 is a scanning electron microscope image of the sample obtained in example 4;

FIG. 5 is an electron paramagnetic resonance spectrum of a sample obtained in example 1;

FIG. 6 is a DPPH radical scavenging curve for samples obtained in examples 1-4;

FIG. 7 is an ABTS radical scavenging curve for samples obtained in examples 1-4;

FIG. 8 shows the results of the cell antioxidant assay of the sample obtained in example 1;

FIG. 9 is a graph showing the fluorescence intensity of active oxygen of the sample obtained in example 1;

FIG. 10 is a graph of wound area at 0, 5, 10, 15 days after wound exposure for rat skin wounds for samples obtained in example 1;

FIG. 11 is a scanning electron microscope image of the polyglucoside prepared in comparative example 1;

FIG. 12 is a scanning electron microscope image of the polyglucoside prepared in comparative example 2;

FIG. 13 is a scanning electron microscope image of the polyglucoside prepared in comparative example 3.

Detailed Description

In the invention, horseradish peroxidase and hydrogen peroxide are cooperatively applied to the oxidative polymerization process of grape seed polyphenol, and the horseradish peroxidase has extremely high catalytic efficiency at normal temperature, can quickly decompose hydrogen peroxide and promote the polymerization of polyphenol molecules, and converts natural polyphenol micromolecules into polyphenol macromolecular materials. However, in the actual experimental process, it can be found that the polymerization process cannot be effectively controlled due to the excessively high enzymatic efficiency and the excessively fast reaction rate, so that the nanoscale material with uniform and regular size and morphology cannot be obtained. It is therefore essential to find a method for effectively controlling the enzymatic polyphenol polymerisation process. The invention successfully obtains the poly grape seed polyphenol nano material with uniform, good and stable appearance by controlling the polymerization process of the grape seed polyphenol by regulating the activity of the horseradish peroxidase for the first time, and further verifies the excellent oxidation resistance and the ability of accelerating wound healing.

The conditions for realizing the effective control of the polymerization process of the invention are as follows: the reaction temperature is 60-70 ℃, and the most preferable reaction temperature is 65 ℃; the dispersed phase of the grape seed polyphenol solution is a mixed liquid phase of deionized water and ethanol, wherein the mass percent of the deionized water is 85%, and the mass percent of the ethanol is 15%; the reaction atmosphere is air, the reaction time is 4-8 hours, and the most preferable reaction time is 6 hours; stirring was maintained during the reaction. And (3) obtaining a brown yellow and turbid reaction stock solution after the reaction is finished, cooling the reaction stock solution to room temperature under the condition of keeping stirring, and then sequentially carrying out centrifugation, washing and freeze-drying treatment to obtain brown yellow powder, namely the grape seed polyphenol nano material. The obtained material can be observed to be nano particles with uniform distribution and good appearance by a desk type scanning electron microscope.

The invention will be further described with reference to the following examples, but the invention is not limited to these specific examples, and the invention has many applications besides these.

In examples 1 to 4 of the present invention, the polyphenol component in the grape seed polyphenol powder is not less than 70% and purchased from Nanjing Dow Biotech limited; the activity of the horseradish peroxidase is more than 300U/mg, and the horseradish peroxidase is purchased from Shanghai Aladdin Biotechnology GmbH; the concentration of the hydrogen peroxide is 30 percent, and the hydrogen peroxide is purchased from Chengdu Jinshan chemical industry Co.

The amounts of grape seed polyphenol, horseradish peroxidase, hydrogen peroxide and other raw materials used in examples 1-4 are shown in table 1, and the obtained polyglucose seed polyphenol nanoparticles are denoted as PGS-i, i being 1-4.

The specific preparation steps of examples 1-4 are as follows:

(1) reaction preparation:

weighing grape seed polyphenol powder according to the table 1, dissolving the grape seed polyphenol powder in a mixed liquid phase of deionized water and ethanol to obtain a grape seed polyphenol solution with the mass concentration of 3mg/ml, and treating the solution for about 5 minutes by adopting ultrasonic to ensure that the system is uniformly dispersed; dissolving a corresponding amount of horseradish peroxidase in deionized water to obtain a horseradish peroxidase solution with the mass concentration of 10mg/ml, adding a grape seed polyphenol solution, fully mixing the two solutions by stirring, and then placing the mixture in an oil bath for preheating until the temperature is stabilized at 65 ℃.

(2) The reaction process is as follows:

diluting hydrogen peroxide with deionized water to obtain a hydrogen peroxide solution with the mass concentration of 0.3%, slowly adding the diluted hydrogen peroxide solution into the preheated solution by using a 10ml syringe, observing that after the hydrogen peroxide solution is added, the reaction solution is quickly gradually changed into a dark brown turbid solution from an initial wine red transparent solution, the reaction solution is gradually changed into brown yellow with the time being prolonged, and keeping stirring for reacting for 4-8 hours to obtain a uniform and stable brown yellow turbid reaction raw solution.

(3) And (3) post-reaction treatment:

and cooling the reaction stock solution to room temperature under the condition of keeping stirring, centrifuging the reaction stock solution, setting the rotating speed of a centrifuge to 12000-16000r/min, setting the centrifuging time to 5-10 minutes to obtain a brown yellow solid, and washing the brown yellow solid with deionized water for three times to obtain the polyglucose seed polyphenol nano-material sample PGS-i (i is 1-4).

Table 1 raw material formulations corresponding to the examples and characterization results of the samples obtained

Characterization test tests of the samples obtained in the examples are as follows:

(1) characterization test

And (3) carrying out desktop scanning electron microscope test on the sample PGS-i (i ═ 1-4), and observing the microscopic morphology of the sample. The specific operation method comprises the following steps: preparing a 2mg/ml sample solution, spin-coating the sample solution on the surface of a smooth mica sheet, drying the sample solution, spraying gold, and observing the sample solution, wherein the obtained electron microscope images are respectively shown in figures 1-4. The obtained samples are nano-scale particles, the appearance is good, the distribution is uniform, and the particle size is sequentially increased along with the increase of the using amount of the horseradish peroxidase and the hydrogen peroxide. The particle size of the particles in the obtained electron microscope image is subjected to mathematical statistics, and the result is shown in table 1 and is consistent with the visual observation rule. In addition, the Zeta potential characterization test is carried out on the obtained sample, and the obtained potential values are about-30 mV, which shows that the surface of the obtained nano material is negative and can be stably dispersed in a water phase.

(2) Detection of antioxidant capacity

And (3) detecting the antioxidant capacity of the sample PGS-i (i is 1-4) by adopting an electron paramagnetic resonance technology test means. The electron paramagnetic resonance technology is a very common means for researching the oxidation resistance of materials, and can detect free radicals contained in the materials. The sample PGS-1 was tested using electron paramagnetic resonance testing on a Bruker EPR EMX _ Plus instrument operating at X-band (9.85GHz) to obtain a paramagnetic resonance spectrum (0.1mW,100kHz) of the sample as shown in FIG. 5. As can be seen from the figure, the spectrum has a signal peak at about 3517G, which is similar to the spectrum of many melanin materials having antioxidant ability, especially excellent antioxidant ability, and it is proved that the sample PGS-1 contains a large amount of hydroxyl radicals and has the basis of being an antioxidant material, and therefore, the radical scavenging ability thereof will be further explored below.

The ethanol phase free radical scavenging ability of the sample PGS-i (i ═ 1-4) was assessed using the 2, 2-diphenyl-1-picrylhydrazine (DPPH) method. The specific operation steps are as follows: a0.1 mM ethanol solution of DPPH and a 1mg/ml ethanol phase solution of sample PGS-i (i ═ 1-4) were prepared. The removal rate was determined by mixing 300. mu.L of DPPH solution with 2600. mu.L of ethanol, adding 100. mu.L of sample solution, and mixing. The removal effect was evaluated by absorbance at 517nm to evaluate the ethanol phase antioxidant capacity of the samples, and the change in absorbance was measured at different time points within 30 minutes. The obtained DPPH radical scavenging curve is shown in fig. 6, and it can be found from the graph that the scavenging effect of the sample PGS-i (i ═ 1-4) has a certain dependence on the sample size, and the sample with smaller size has stronger antioxidant activity.

Further, the aqueous-phase radical scavenging ability of the sample PGS-i (i ═ 1-4) was evaluated using the 2,2' -diaza bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) method. The specific operation steps are as follows: preparing a 7mM ABTS aqueous solution and a 2.45mM potassium persulfate aqueous solution respectively; mixing the ABTS aqueous solution with the potassium persulfate aqueous solution in a ratio of 1: 2, left overnight at room temperature and left to stand in the dark to give the final ABTS detection reagent. A 1mg/ml aqueous phase solution of sample PGS-i (i ═ 1-4) was prepared, 100 μ L of labes reagent was added to 2800 μ L of deionized water and mixed well, and then 100 μ L of sample solution was added and mixed well to start the determination of the clearance rate. The removal effect was evaluated by using the absorbance at 734nm to evaluate the antioxidant capacity of the aqueous phase of the sample, and the change in absorbance was measured at different time points within 30 minutes. The obtained ABTS free radical scavenging curve is shown in fig. 7, and it can be found from the graph that the scavenging effect of the sample PGS-i (i ═ 1-4) has a certain dependence on the sample size, and the sample with smaller size has stronger antioxidant activity.

(3) Cell antioxidation experiment

In the embodiment, NIH mouse embryo fibroblast 3T3 cells are taken as cell strains, and the MTT colorimetric method is adopted to verify the cytotoxicity of the obtained grape seed polyphenol nano material. Wherein the cells are cultured by adding 10% Fetal Bovine Serum (FBS) to DMEM medium and incubating, and the culturing is performed in a humid atmosphere containing 5% CO2 at 37 deg.C. Cultured NIH 3T3 cells are incubated in a 96-well plate for 24h at the density of 1000 cells per well, PGS-1 samples with different concentrations (the concentrations are respectively 50 mug/ml, 80 mug/ml and 110 mug/ml) are treated for 24h and 48h, and then the corresponding cell survival rate is detected by adopting an MTT colorimetric method, wherein the detection result is shown in figure 8. It can be seen from the figure that after the poly grape seed polyphenol nano material is treated, the cell activity is higher under the three adopted concentrations, and the cell activity is sequentially increased along with the increase of the applied concentration of the material, and similar statistical results are obtained at two time nodes of 24h and 48 h.

Further, the cultured NIH 3T3 cells were seeded in a 12-well plate at 10 ten thousand per well, and incubated in the plate for 24 hours. After incubation, the culture medium is removed, 500 mu L of PGS-1 which is prepared from DMEM culture medium and contains the polyglucose seed polyphenol nanoparticle material with different concentrations is added, and 500 mu L of full-component culture solution and 100 mu L of diluted hydrogen peroxide (the concentration is 100 mu mol/L) are further supplemented. Then removing the culture solution, adding 200 μ L of pancreatin-digested cells and adding 200 μ L of culture medium to terminate the pancreatin digestion; centrifuging to remove supernatant, adding 1ml of PBS solution for resuspension, and adding 250 μ L of prepared probe; finally, the cell sample was resuspended in PBS after centrifugation and subjected to quantitative analysis using a flow cytometer for 24h and 48h, respectively, to obtain the results shown in fig. 9. Compared with the blank and the control, the fluorescence intensity of the active oxygen in the experimental group is increased, wherein the intensity of the control group without the applied material is the highest, the intensity is reduced when the materials with different concentrations are applied, and the higher the concentration is, the more the reduction is, the applied poly grape seed polyphenol nano material has certain inhibition on the active oxygen, has certain antioxidant capacity, and can be obtained in 24h and 48 h.

(4) Application of poly grape seed polyphenol nano material in skin surface wound repair

The antioxidant poly grape seed polyphenol nano material synthesized by the invention has good antioxidant effect on both chemical level and cell level, and has good biocompatibility, so that the specific application of the antioxidant poly grape seed polyphenol nano material on animal level can be further explored.

The sample PGS-1 is used in an animal model for repairing the skin injury of a rat, and the experiment is approved by the medical ethics committee of the oral hospital, western, Sichuan university (approval number is WCHSIRB-D-2017-. The animals selected were healthy female rats from large adult animals, which weigh approximately 200 g. The specific operation is as follows: the rat's back hair was first shaved and disinfected, two full-thickness circular skin wounds 15 mm in diameter were incised on the rat's back, 1mg/ml of PGS-1 nanomaterial solution was applied to the wound surface, and the wound was covered with a transparent dressing for biological evaluation. During this period, the rat wounds were debrided and the material was applied on

days

0, 3, 5, 8, 10, 13, and 15, respectively, the wound change was recorded by photographing, and the wound areas of the rat wounds on

days

0, 5, 10, and 15 were counted and analyzed, respectively, and the results are shown in fig. 10. From the figure, it can be observed that the wounds of the control group and the group applied with the PGS-1 material are gradually reduced within 15 days, but the wound repair speed of the rats applied with the PGS-1 material is obviously quicker, the wounds are basically recovered at 15 days after the wounds, and the control group still has obvious wounds, so that the obtained polyglucoside nano material has an obvious accelerating process for repairing the skin wounds and has a good effect on quickly repairing the wounded skin.

According to the traditional enzymatic oxidation method for polymerizing polyphenol compounds, grape seed polyphenol, horseradish peroxidase and hydrogen peroxide are directly mixed and then react at room temperature, so that the reaction process is uncontrollable, and the nano material with uniform and stable size is difficult to obtain. The invention controls the reaction rate of the reaction system by means of adjusting the reaction temperature, changing the polarity of the solvent, changing the adding mode of hydrogen peroxide and the like, so that the polymerization process of the grape seeds is at a proper polymerization rate, and uniform and stable nano materials are obtained.

Comparative examples will be provided below to demonstrate the advantageous effects of the production process of the present invention.

Comparative example 1

The process conditions of the comparative example are the same as those of example 2, except that the comparative example is carried out in an air atmosphere at normal temperature, the obtained material is also nano-sized, but has larger size and random aggregation, and the scanning electron microscope image thereof is shown in FIG. 11. In addition, multiple experiments show that the reaction under the atmosphere of normal temperature air cannot obtain uniform and stable nano materials no matter how reaction conditions such as monomer molecule concentration, solvent polarity, pH value and the like are adjusted.

Comparative example 2

The process conditions of the comparative example are the same as those of example 2, the difference is only that the adding mode of the hydrogen peroxide in the comparative example is different, the hydrogen peroxide is added at one time in the comparative example, the size of the obtained material is large and micron-sized, the morphology of the material is random and uneven, and the scanning electron microscope image of the material is shown in figure 12.

Comparative example 3

The process conditions of this comparative example were the same as those of example 2 except that the mass percentage of ethanol in the polar solvent was different. In the comparative example, three solvents were selected to dissolve grape seed polyphenols, the first was 100% deionized water, the second was a mixed liquid phase of 70% deionized water and 30% ethanol, and the third was a mixed liquid phase of 50% deionized water and 50% ethanol. The scanning electron microscope images of the obtained material are respectively shown in a figure (a), a figure (b) and a figure (c) in fig. 13, and the excessive crosslinking of the material can be observed from the figures, so that the uniform and dispersed nano material is not formed.

The materials prepared in comparative examples 1-3 are all random aggregates, large in size, unstable in structure, and uneven in system, and the performance and further application of the obtained materials are limited. When such a random aggregate material is applied to a living body, problems such as clogging of blood vessels, ineffective action on a target site, and unstable action effect are likely to occur.

Those skilled in the art will appreciate that, in the embodiments of the methods of the present invention, the sequence numbers of the steps are not used to limit the sequence of the steps, and it is within the scope of the present invention for those skilled in the art to change the sequence of the steps without inventive work. The examples described herein are intended to aid the reader in understanding the practice of the invention and it is to be understood that the scope of the invention is not limited to such specific statements and examples. Those skilled in the art can make various other specific changes and combinations based on the teachings of the present invention without departing from the spirit of the invention, and these changes and combinations are within the scope of the invention.

Claims

1. A preparation method of an antioxidant grape seed polyphenol nano material is characterized by comprising the following steps:

2. The method for preparing the antioxidant polyglucose seed polyphenol nanomaterial as claimed in claim 1, wherein:

the weight ratio of the grape seed polyphenol to the horseradish peroxidase to the hydrogen peroxide is 6: 0.102-0.514: 1.312-6.56.

3. The method for preparing the antioxidant polyglucose seed polyphenol nanomaterial as claimed in claim 1, wherein:

before the step (1), the grape seed polyphenol solution and the horseradish peroxidase aqueous solution are respectively subjected to ultrasonic treatment for 2-3 minutes.

4. Use of the polyglucose seed polyphenol nanomaterial prepared by the preparation method of any one of claims 1-3 in an antioxidant.

5. Use of the polyglucose seed polyphenol nanomaterial prepared by the preparation method of any one of claims 1-3 in skin repair wound dressing.