Degreased evening primrose seed protein hydrolysate, preparation method thereof and application of degreased evening primrose seed protein hydrolysate in prevention and treatment of diabetic retinopathy

Abstract

The invention discloses degreased evening primrose seed protein hydrolysate, a preparation method thereof and application of the degreased evening primrose seed protein hydrolysate in prevention and treatment of diabetic retinopathy, and belongs to the technical field of biological medicine. The degreased evening primrose seed protein hydrolysate is obtained by hydrolyzing degreased evening primrose seed protein with compound protease, and the degreased evening primrose seed protein is prepared by alkali extraction and acid precipitation of degreased evening primrose seeds. An extraction method of the degreased evening primrose seed protein is optimized, an enzyme most suitable for hydrolyzing the degreased evening primrose seed protein and an optimal hydrolysis condition are found through experiments, and the experiments prove that the degreased evening primrose seed protein hydrolysate prepared by the invention has a remarkable intervention effect on high-glucose-induced zebra fish retinal vascular injury, and can be used for preparing a medicine for treating hyperglycemia-induced zebra fish retinal vascular injury. The invention provides a new data reference and action mechanism for prevention and treatment of diabetic retinopathy.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a defatted evening primrose seed protein hydrolyzate, a preparation method thereof, and application thereof in preventing and treating diabetic retinopathy. Background Art

[0002] Evening primrose is a plant of the Willow family with a variety of biological characteristics. Most of the evening primrose seeds are used to extract oil, which is rich in unsaturated fatty acid γ-linolenic acid. The by-products after evening primrose seed oil extraction are rich in protein, most of which are used as ordinary fertilizers or directly discarded, and are not fully and effectively utilized, which not only causes serious waste of resources but also causes environmental pollution. From this perspective, how to maximize the added value of by-products after evening primrose seed oil extraction has become an important research direction. Studies have shown that evening primrose seeds have high nutritional value, and the protein content isolated from evening primrose seeds is as high as 75%. The protein has a complete range of amino acids and is rich in glutamic acid, aspartic acid and arginine amino acids. Many studies have found that enzymatic protein hydrolysates have antioxidant, anti-inflammatory and anti-diabetic biological activities. However, there are many studies on evening primrose seed oil in the prior art, but there are few studies on evening primrose seed protein hydrolysates.

[0003] DR (diabetic retinopathy) is the most common microvascular complication, often accompanied by symptoms such as retinal vascular damage, oxidative stress, and neovascularization. It is also the main cause of visual blindness, which seriously affects the health of diabetic patients and reduces their quality of life. Although the occurrence of diabetic complications can be delayed by controlling blood sugar, it cannot prevent the occurrence of diabetic complications. Therefore, improving diabetic retinal vascular damage may be an important strategy for treating DR. At present, many biological activities of evening primrose seeds have been reported, and it has been found that the chemical components of evening primrose, especially evening primrose seed oil, are beneficial in the treatment of chronic inflammation. And studies have reported that enzymatic protein hydrolysates generally have antioxidant, anti-inflammatory, and anti-diabetic biological activities. However, there are few studies on defatted evening primrose seed protein hydrolysates.

[0004] Alkaline extraction and acid precipitation is a traditional protein extraction method that uses the change in protein solubility under different pH conditions to achieve separation and purification. However, there is no report on the alkaline extraction and acid precipitation technology for obtaining defatted evening primrose seed protein. Different proteases will have different effects on the hydrolysis of the same protein, and will also lead to different degrees of hydrolysis, thereby producing peptides of different molecular weights, resulting in different physical and chemical properties and functional properties of protein hydrolysates. At present, there is no literature reporting the best conditions for the hydrolysis of defatted evening primrose seed protein.

[0005] The present invention uses zebrafish as an experimental animal model to explore the intervention effect of defatted evening primrose seed protein hydrolysate on diabetic retinal vascular damage, and characterizes the physicochemical properties and toxicity of DEPPr (defatted evening primrose seed protein complex protease hydrolysate). The intervention mechanism of DEPPr on high sugar-induced zebrafish retinal vascular damage is preliminarily explored, providing new data reference for the application of DEPPr in the prevention and treatment of diabetic retinal vascular damage. Summary of the invention

[0006] The purpose of the present invention is to provide a defatted evening primrose seed protein hydrolyzate and a preparation method thereof and an application thereof in preventing and treating diabetic retinopathy, so as to solve the problems existing in the above-mentioned prior art. The present invention optimizes the preparation method of defatted evening primrose seed protein hydrolyzate, and the protein hydrolyzate has a significant intervention effect on high sugar-induced zebrafish retinal vascular damage, providing new data reference and action mechanism for the prevention and treatment of DR.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a defatted evening primrose seed protein hydrolysate, which is obtained by hydrolyzing the defatted evening primrose seed protein using a composite protease;

[0009] The defatted evening primrose seed protein is prepared from defatted evening primrose seeds through alkali extraction and acid precipitation.

[0010] The present invention also provides a method for preparing the defatted evening primrose seed protein hydrolysate, comprising the following steps:

[0011] Mixing defatted evening primrose seed powder with water, adjusting the solution pH to 11.5, and subjecting the mixture to ultrasonic treatment and first centrifugation to obtain an alkaline extract of defatted evening primrose seeds;

[0012] The pH of the defatted evening primrose seed alkali extract is adjusted to 3.0, allowed to stand, centrifuged for a second time, and the precipitate is collected to obtain defatted evening primrose seed protein;

[0013] The defatted evening primrose seed protein is hydrolyzed with a composite protease to obtain the defatted evening primrose seed protein hydrolysate;

[0014] The CAS number of the composite protease is: 9014-01-1.

[0015] Preferably, the solid-liquid ratio of the defatted evening primrose seed powder to water is 1 g:20 mL; the ultrasonic treatment is performed at 250 W for 60 min; and the first centrifugation is performed at 4° C. and 3000 r / min for 20 min.

[0016] Preferably, the standing condition is 4° C. for 2 hours; and the second centrifugation condition is 4° C. and 3000 r / min for 30 minutes.

[0017] Preferably, the enzyme activity of the composite protease is 3000U / g;

[0018] Preferably, the hydrolysis conditions are 50° C., pH 7, and shaking at 200 r / min for 3 h.

[0019] The present invention also provides application of the defatted evening primrose seed protein hydrolyzate in preparing medicine for preventing and treating diabetic retinopathy.

[0020] The present invention also provides a medicine for preventing and treating diabetic retinopathy, comprising the defatted evening primrose seed protein hydrolyzate.

[0021] The concentration of the defatted evening primrose seed protein hydrolyzate is 10 μg / mL-50 μg / mL.

[0022] Optionally, it further comprises pharmaceutically acceptable excipients.

[0023] Optionally, the dosage form of the drug includes a liquid dosage form.

[0024] The present invention discloses the following technical effects:

[0025] The invention optimizes the defatted evening primrose seed protein extraction method: the most suitable alkali extraction and acid precipitation technical conditions for the defatted evening primrose seed protein are determined through experiments, thereby improving the extraction efficiency and quality of the protein.

[0026] Through experiments, the most suitable enzyme (compound protease) for hydrolyzing defatted evening primrose seed protein and the optimal hydrolysis conditions (temperature 50℃, pH7, enzyme activity 3000U / g, constant temperature oscillation time 3 hours) were found. The hydrolyzate obtained under these conditions has the best antioxidant activity. Compared with the original DEP (defatted evening primrose seed protein), DEPPr (compound protease hydrolyzate) has a lower molecular weight, average particle size and polydispersity index, which indicates that the compound protease hydrolysis improves the solubility and stability of DEP, making it more suitable for use as a food or drug ingredient. Through Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD) and ultraviolet spectroscopy analysis, DEPPr shows a different secondary and tertiary structure from DEP, indicating that the compound protease hydrolysis changes the protein structure of DEP and may enhance its biological activity. The surface hydrophobicity of DEPPr is significantly higher than that of DEP, which may help to improve its absorption and utilization in the body, thereby enhancing its biological activity.

[0027] By converting the byproducts of evening primrose seed oil extraction into protein-rich defatted evening primrose seed protein hydrolysate, the utilization efficiency of evening primrose seeds is improved, and resource waste and environmental pollution are reduced.

[0028] Experiments have confirmed that DEPPr prepared by using DEP has a significant intervention effect on high-sugar-induced retinal vascular damage in zebrafish. Specifically, compared with the high-sugar exposure group, the diameter of zebrafish retinal blood vessels in the DEPPr-treated group was significantly reduced from 5.89μm to 4.27μm; through non-targeted metabolomics technology, it was found that DEPPr may alleviate high-sugar-induced retinal vascular damage in zebrafish by regulating amino acid metabolism, providing new data references and mechanisms of action for the prevention and treatment of DR, showing potential therapeutic effects on DR. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 Response surface analysis diagram; (a): pH and liquid-to-solid ratio interaction surface diagram; (b): pH and ultrasonic power interaction surface diagram; (c): pH and time interaction surface diagram; (d): ultrasonic power and liquid-to-solid ratio interaction surface diagram; (e): liquid-to-solid ratio and time interaction surface diagram; (f): ultrasonic power and time interaction surface diagram;

[0031] Figure 2 The SDS-PAGE result diagram of DEP and various protease hydrolysates; wherein M is a marker, DEP is defatted evening primrose seed protein, 1-6 are trypsin hydrolysate, neutral protease hydrolysate, composite protease hydrolysate, papain hydrolysate, flavor protease hydrolysate, and alkaline protease hydrolysate, respectively;

[0032] Figure 3 The protective effect of defatted evening primrose seed protein hydrolysate on retinal vascular damage in zebrafish; (a): fluorescence image of retinal vascular morphology in each group; (b): statistical image of retinal vascular diameter in each group;

[0033] Figure 4 Statistical diagrams of particle size (a), PDI (b) and Zeta potential (c) of DEP and DEPPr;

[0034] Figure 5 is the Fourier infrared spectra of DEP and DEPPr;

[0035] Figure 6 is the CD chromatogram of DEP and DEPPr;

[0036] Figure 7is the UV spectra of DEP and DEPPr;

[0037] Figure 8 is the surface hydrophobic fluorescence value of DEP and DEPPr;

[0038] Fig. 9 is the intrinsic fluorescence spectra of DEP and DEPPr;

[0039] Fig.10 Effects of different concentrations of DEPPr on the growth and development of zebrafish embryos; (a): hatching rate; (b): survival rate; (c): morphological changes; BT indicates tail bending and PE indicates pericardial edema;

[0040] Fig.11 The intervention effect of DEPPr on retinal vascular damage in zebrafish; (a): fluorescence image of retinal vascular morphology in each group; (b): statistical image of retinal vascular diameter in each group;

[0041] Fig.12 The effect of DEPPr intervention on the morphology of zebrafish retina induced by high glucose; (a): HE staining sections of zebrafish retina in each group (400×); (b): Quantitative images of zebrafish retinal layers in each group; GCL represents ganglion cell layer, IPL represents inner plexiform layer, INL represents inner nuclear layer, ONL represents outer nuclear layer, and RPE represents pigment epithelium;

[0042] Fig.13 TIC overlap diagram of quality control samples; (a): positive ion mode; (b): negative ion mode;

[0043] Fig.14 The PCA score graphs of each group of samples; (a): positive ion mode; (b): negative ion mode;

[0044] Fig.15 PCA score diagrams of Glu group and DEEPr group; (a): positive ion mode; (b): negative ion mode;

[0045] Fig.16 OPLS-DA analysis and permutation test diagrams of the Glu group and the DEPPr group; (a): positive ion mode; (b): negative ion mode;

[0046] Fig.17 Volcano plot of differential metabolites between Glu group and DEPPr group; (a): positive ion mode; (b): negative ion mode;

[0047] Fig.18 KEGG enrichment bubble diagram of differential metabolites between Glu group and DEPPr group; (a): positive ion mode; (b): negative ion mode. DETAILED DESCRIPTION

[0048] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0049] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0050] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0051] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0052] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0053] Currently, there are few studies on defatted evening primrose seed protein. The present invention has found the most suitable alkali extraction and acid precipitation technical conditions for defatted evening primrose seed protein through experiments: the extraction method of defatted evening primrose seed protein by alkali extraction and acid precipitation is optimized, the optimal extraction conditions and the optimal precipitation isoelectric point are determined, and the extraction efficiency of defatted evening primrose seed protein is improved.

[0054] Preparation of defatted evening primrose seed powder: A certain amount of evening primrose seeds are placed in an oven for drying. After drying, the evening primrose seeds are defatted: the dried evening primrose seeds are mixed with n-hexane in a ratio of 1:3 (g:mL), placed on a shaker for stirring for 3h, taken out and allowed to stand for 30min, and then the upper yellow liquid is gently poured out. Next, the evening primrose seeds are mixed with n-hexane again in a ratio of 1:2, stirred on a shaker for 2h, allowed to stand for 30min, and the upper yellow liquid is poured out again. Repeat this operation until the upper liquid becomes colorless. Afterwards, the defatted evening primrose seeds are placed back in the oven to accelerate the volatilization of n-hexane and complete the drying process. After drying, the evening primrose seeds are crushed and sieved (80 mesh) using a grinder to obtain defatted evening primrose seed powder.

[0055] The trypsin, neutral protease, composite protease, papain, flavor protease and alkaline protease used in the present invention were purchased from Beijing Solebow Technology Co., Ltd., and the CAS numbers are: 9002-07-7, 9068-59-1, 9014-01-1, 9001-73-4, 9001-92-7, 9014-01-1, respectively.

[0056] Example 1 Optimization of the extraction conditions of defatted evening primrose seed protein

[0057] The optimal extraction conditions of defatted evening primrose seed protein were obtained based on the results of single factor experiment and response surface methodology (RSM).

[0058] 1. Optimize the alkali extraction and acid precipitation method

[0059] 1.1 Determine the optimal alkaline extraction conditions

[0060] 1.1.1 Single Factor Experiment

[0061] First, the present invention conducted a single factor test to evaluate the effect of each independent variable (such as pH, solid-liquid ratio, ultrasonic time, ultrasonic power and temperature) on the extraction rate of defatted evening primrose seed protein. At this stage, each variable was adjusted separately, while the other variables remained unchanged to observe the effect of each variable on the extraction rate.

[0062] 1.1.2 Response surface experimental design

[0063] On the basis of the single factor test, the present invention selects factors (pH, solid-liquid ratio, ultrasonic time and ultrasonic power) that have a significant impact on the extraction rate, and uses the response surface method (RSM) for further optimization. The response surface method is a statistical technique for evaluating the impact of multiple variables and their interactions on the response (protein extraction rate in this embodiment). In this way, the optimal combination of variables can be determined.

[0064] Box-Behnken design (BBD): In response surface experiments, the Box-Behnken design is used, which is a commonly used RSM design to find the optimal levels of multiple variables. BBD can evaluate the interaction of variables while reducing the number of experiments required.

[0065] The response surface diagram of the interaction between pH (A), solid-liquid ratio (B), ultrasonic power (C) and ultrasonic time (D) on the extraction rate of defatted evening primrose seeds is shown in Figure 2. Figure 1 As shown. The steeper the surface, the more significant the influence of the factor. As can be seen from the figure, the response surface of pH (A) and liquid-to-liquid ratio (B) is the steepest, indicating that the interaction between pH and liquid-to-liquid ratio has the most significant effect on the extraction rate of defatted evening primrose seeds. Similarly, the order of the significant degree of the interaction between the four factors on the extraction rate of defatted evening primrose seeds is: AB>AC>AD>CD>BD>BC. The optimal extraction conditions were determined to be: pH 11.94, liquid-to-liquid ratio of 1:20.95, extraction time 55.89min, and ultrasonic power 250W.

[0066] Data analysis: The present invention analyzes the response surface test data by using statistical software such as Design-Expert software, and establishes a regression model to predict the protein extraction rate. By analyzing this model, the optimal levels of pH, material-liquid ratio, ultrasonic time and ultrasonic power are determined.

[0067] Regression Model:

[0068] Y=6.36+0.0667A-0.1293B-0.1899C+0.1182D+0.1140AB-0.0589AC+0.1556AD-0.3028BC+0.2317BD+0.3426CD-0.4166A 2 -0.5125B 2 -0.2902C 2 -0.2118D 2 (-1≤A≤1, -1≤B≤1, -1≤C≤1, -1≤D≤1).

[0069] Where: A represents extraction pH, B represents solid-liquid ratio (g: mL), C represents ultrasonic power (W), and D represents extraction time (min). The optimal levels of pH, solid-liquid ratio, ultrasonic time and ultrasonic power are pH 11.5, solid-liquid ratio 1:20, ultrasonic power 250W, and time 60min.

[0070] 80g of defatted evening primrose seed powder was mixed in distilled water at a solid-liquid ratio of 1:20 (m / v), and the pH of the defatted evening primrose seed powder solution was adjusted to 11.5 using 2.5mol / L NaOH. After ultrasonic treatment (250W, 60min), the solution was cooled to room temperature at room temperature. The cooled solution was centrifuged at 4°C for 20min (3000r / min), and the supernatant was taken to obtain the alkaline extract of defatted evening primrose seeds.

[0071] 1.2 Determine the optimal acid precipitation conditions

[0072] In order to determine the optimal acid precipitation isoelectric point, 5 50mL centrifuge tubes were taken, 5mL of defatted evening primrose seed alkali extract was added to each tube, and the pH was adjusted to 2.5, 3.0, 3.5, 4.0, and 4.5 respectively using 0.1mol / L HCl, and then centrifuged at 3000r / min for 30min at 4℃. The supernatant was transferred to a 10mL volumetric flask, and the Coomassie Brilliant Blue G-250 method was used. With bovine serum albumin as the standard curve, the isoelectric point was determined using an enzyme marker to determine the optimal acid precipitation isoelectric point of defatted evening primrose seed alkali extract. By measuring the amount of protein precipitation at different pH values, the pH value with the most protein precipitation was determined, and this pH value was the optimal isoelectric point. The optimal isoelectric point was determined to be pH=3.0.

[0073] The pH of the defatted evening primrose seed alkali extract was adjusted to 3.0 (isoelectric point), and after standing at 4°C for 2 hours, the extract was centrifuged at 3000 r / min for 30 minutes, the supernatant was discarded, and the precipitate was collected to obtain the defatted evening primrose seed protein.

[0074] 2. Optimize protease and hydrolysis conditions

[0075] 2.1 Protease

[0076] The present invention extracts defatted evening primrose seed protein (yield 46.7%), and then uses trypsin, neutral protease, composite protease, papain, flavor protease and alkaline protease to hydrolyze defatted evening primrose seed protein under optimal conditions, and prepares trypsin hydrolysate (DEPTr), neutral protease hydrolysate (DEPNe), composite protease hydrolysate (DEPPr), papain hydrolysate (DEPPa), flavor protease hydrolysate (DEPFl), and alkaline protease hydrolysate (DEPAl). In order to characterize the changes in molecular weight after defatted evening primrose seed protein is enzymatically degreased by different proteases, an SDS-PAGE experiment is performed. The results are as follows: Figure 2As shown, lanes 1-6 are DEPTr, DEPNe, DEPPr, DEPPa, DEPFl and DEPAl, respectively. The intensity of the four protein bands in DEPTr and DEPAl (lanes 1 and 6) weakens or partially disappears. The DEPTr band is generally concentrated in the molecular weight of 70-25kDa, and the DEPAl band is generally concentrated in the molecular weight of 70-15kDa, with a small amount at 10kDa. The four protein bands of DEPNe, DEPPr, DEPPa and DEPFl (lanes 2, 3, 4, 5) almost disappear, without obvious bands, and the weak bands appear at about 35KDa (lane 5), 25kDa (lanes 2, 3, 4), and 15KDa (lanes 2, 3, 4, 5) at the same time. There are certain differences in the degree of hydrolysis of the six different protease hydrolysates. The hydrolysis degree of DEPTr and DEPAl is poor, while the hydrolysis degree of DEPNe, DEPPr, DEPPa and DEPFl is good, especially DEPPr, DEPPa and DEPFl.

[0077] The different hydrolysis degrees of the six protease hydrolysates may cause differences in the physicochemical and functional properties of the protease hydrolysates. In order to evaluate the activity of the six protease hydrolysates, the changes in the retinal vascular diameter of zebrafish embryos exposed to a high glucose environment under the intervention of the six protease hydrolysates were compared using the retinal vascular diameter of zebrafish embryos as an indicator. Figure 3 As shown in the figure, in the Con group, the diameter of the retinal blood vessels of zebrafish embryos was 4.59 μm. However, when zebrafish embryos were exposed to 130 mM glucose, the diameter of their retinal blood vessels increased significantly to 5.89 μm. After intervention with DEP, DEPTr, DEPNe, DEPPr, DEPPa, DEPFl and DEPAl, the blood vessel diameters decreased to 5.80 μm, 4.68 μm, 4.93 μm, 4.27 μm, 4.76 μm, 5.14 μm and 5.48 μm, respectively. There was no significant effect on the diameter of the retinal blood vessels after intervention with DEP. The six protease hydrolysates generally had a certain intervention effect. Although the intervention effects of DEPFl and DEPAl were compared with the high glucose group, they were not significant. DEPTr, DEPNe, DEPPr and DEPPa showed significant effects in the intervention effect, among which the intervention effect of DEPPr was the most significant. The results showed that there were certain differences in the functional properties of different protease hydrolysates, among which DEPPr had the best activity. Therefore, the most suitable protease for hydrolysis of defatted evening primrose seeds was screened out as the composite protease.

[0078] 2.2 Hydrolysis conditions

[0079] 2.2.1 Effect of temperature on the activity of hydrolysates

[0080] The DPPH and ABTS free radical scavenging experiments were carried out at different temperatures (40℃, 45℃, 50℃, 55℃, 60℃) to observe the effect of different temperatures on the antioxidant activity of defatted evening primrose seed protein hydrolysate. The experimental results showed that the antioxidant activity of the hydrolysate was the best when the temperature was 50℃.

[0081] 2.2.2 Effect of pH on the activity of hydrolysates

[0082] Hydrolysis was performed at different pH values ​​(5, 6, 7, 8, 9), and the antioxidant activity was then evaluated by DPPH and ABTS assays. The results showed that the antioxidant activity of the hydrolysate was highest when pH was 7.

[0083] 2.2.3 Effect of compound protease concentration on hydrolysis activity

[0084] By changing the concentration of the composite protease (80mg / mL, 100mg / mL, 120mg / mL, 140mg / mL, 160mg / mL), hydrolysis was carried out under fixed temperature and pH conditions, and the antioxidant activity was evaluated by DPPH and ABTS experiments. The experiment found that when the enzyme activity was 3000U / g, the antioxidant activity of the hydrolyzate reached the maximum.

[0085] 2.2.4 Effect of constant temperature oscillation time on hydrolysis activity

[0086] Under the same temperature, pH and enzyme concentration conditions, the constant temperature oscillation (200r / min) time (1h, 2h, 3h, 4h, 5h) was changed, and DPPH and ABTS experiments were carried out. The results showed that the antioxidant activity of the hydrolyzate was the best when the reaction time was 3 hours.

[0087] Based on the above experimental results, the optimal extraction process parameters for the composite protease hydrolyzed defatted evening primrose seed protein were determined to be: temperature 50°C, pH 7, enzyme activity 3000U / g, constant temperature shaking time 3 hours. Under these conditions, the defatted evening primrose seed protein hydrolyzate obtained had the best antioxidant activity.

[0088] Add composite protease to the protein solution to ensure that the final enzyme to substrate ratio is 3000U / g, and shake at a constant temperature (200r / min) for 3 hours at a temperature of 50°C and pH 7 to obtain defatted evening primrose seed protein composite protease hydrolysate (DEPPr).

[0089] 3. Effect of compound protease on DEP

[0090] 3.1 Particle size and dispersibility

[0091] like Figure 4As shown in (a), the average particle sizes of DEP and DEPPr are 5766.00±602.11nm and 2998.31±538.14nm, respectively. The particle size of DEPPr is significantly lower than that of DEP (p<0.05). The reason for this result may be that the composite protease hydrolysis can convert DEP into more amino acids or small molecular weight short peptides. The polydispersity index (PDI) mainly reflects the molecular weight distribution of the polymer. The more uniform the molecular weight distribution, the smaller the PDI value. The PDI value of the polymer shows an opposite trend to its dispersity. Figure 4 As shown in (b), the PDI values ​​of DEP and DEPPr are 0.515±0.05 and 0.479±0.03, respectively. The PDI of DEPPr is significantly lower than that of DEP. This indicates that the composite protease hydrolysate has good dispersibility in water. Zeta potential is an indicator for evaluating the dispersibility of a solution. The higher the Zeta potential, the more dispersed the solution. Figure 4 As shown in (c), compared with DEP (-22.24±5.61mV), the Zeta potential of DEPPr (-30.16±5.73mV) is significantly higher than that of DEP, indicating that the surface charge of DEP has been significantly increased after the treatment of composite protease hydrolysis. According to studies, the high Zeta potential of the hydrolyzate can effectively enhance the electrostatic repulsion between molecules, thereby promoting the unfolding of proteins, destroying or preventing the formation of protein aggregates, and thus improving the dispersibility of the hydrolyzate. Therefore, the high Zeta potential of DEPPr makes it have better dispersibility in water.

[0092] 3.2 Secondary structure

[0093] Figure 5 This indicates that the hydrolysis of the complex protease affects the secondary structure of DEP. -1 The absorption peaks in the range are the symmetric and asymmetric stretching vibrations of CH, which characterize the hydrophobic region of the protein and are similar to DEP (2924.18 cm -1 ) absorption peak, DEPPr (2935.61cm -1 ) red-shifted and the peak intensity decreased, indicating that the hydrophobic region of DEP was destroyed by the composite protease hydrolysis. The absorption peak of the amide I band is located at 1600-1700 cm -1 As shown in the figure, the characteristic absorption peaks of DEP and DEPPr hydrolyzates are amide I band at 1647.08 cm-1 (belonging to random coil-irregular curl), DEPPr at 1600-1700 cm -1 The peak intensity at 400 μmol / L was weaker than that of DEP, indicating that the hydrolysis of DEP by the composite protease destroyed the hydrogen bond between C=O and NH. The above results show that the hydrolysis of DEP by the composite protease can affect the hydrophobic properties of proteins by changing the secondary structure and functional groups of DEP.

[0094] 3.3CD spectrum

[0095] CD spectroscopy is a detection technique for analyzing protein secondary structure. Figure 6 As shown, the CD spectrum of DEP shows a positive peak near 190nm and a weak negative peak at 208nm, indicating that it is an α-helical structure. DEPPr shows two positive peaks at 190nm and 195nm, and a negative peak near 207nm, which is accompanied by a weak blue shift from 208nm (evening primrose seed protein) to 207nm compared with DEP, indicating a change in the α-helical structure and an increase in the β-folded structure. These results indicate that the complex protease hydrolysis changes the secondary structure of DEP.

[0096] 3.4 UV-Visible Spectroscopy

[0097] The UV-visible spectra of DEP and DEPPr are shown in Figure 7 As shown, the spectra of the two groups are very similar. The absorption peak wavelength is near 280nm, indicating the characteristic absorption of Tyr (tyrosine) and Trp (tryptophan) near 280nm. DEP has a weak absorption near 280nm, while the peak wavelength of DEPPr is less than 280nm. The ultraviolet spectrum has a blue shift around 276nm, which indicates that after the composite protease hydrolyzes DEP, part of Tyr and Trp are embedded in the peptide structure, and the peptide may form a conjugated double bond, which increases the ultraviolet absorption near 276nm. Tyr and Trp are usually present in the hydrophobic interior of proteins, indicating that the microenvironment of the peptide becomes weak after enzymatic hydrolysis.

[0098] 3.5 Surface hydrophobicity

[0099] ANS reacts with anionic proteins and can be used as an exogenous fluorescent marker to reflect the hydrophobicity level of proteins. Figure 8 As shown in the figure, the highest fluorescence values ​​of DEP and DEPPr are 864.5 and 1360 respectively, and the fluorescence peak wavelengths are both at 390.8nm. It can be seen that the surface hydrophobicity of DEPPr is much greater than that of DEP, indicating that the hydrolysis of the composite protease is conducive to the exposure of the hydrophobic area and significantly improves the surface hydrophobicity of DEP. The fluorescence peak can reflect the hydrophobic strength of the protein and further reveal the spatial structure and functional characteristics of the protein. The change in the fluorescence peak of DEPPr indicates the damage of the DEP structure by the composite protease hydrolysis. The composite protease hydrolysis unfolds the structure of DEP, exposing a large number of internal hydrophobic groups and enhancing the surface hydrophobicity.

[0100] 3.6 Intrinsic fluorescence spectroscopy

[0101] Intrinsic fluorescence spectra can reflect changes in the tertiary structure of proteins. Fig. 9As shown in the figure, the intrinsic fluorescence spectra of DEP and its DEPPr are described. Compared with the fluorescence peak of DEP (460.2nm), the fluorescence peak of DEPPr (438.2nm) has a significant blue shift, indicating that the tertiary structure of the protein has undergone certain changes and the tryptophan (Trp) residue is in a more hydrophobic microenvironment, which is consistent with the conclusion of the surface hydrophobicity of DEPPr. Compared with DEP, the fluorescence intensity of DEPPr at the maximum fluorescence peak is also reduced. The fluorescence intensity is mainly affected by the energy transfer from tyrosine (Tyr) to tryptophan (Trp) (depending on the distance between the two residues) and the fluorescence quenching neighboring groups. The fluorescence intensity of DEPPr is lower than that of DEP, which indicates that the enzymatic hydrolysis of the complex protease may reduce the energy transfer from Tyr to Trp.

[0102] In summary, by studying the effect of compound protease hydrolysis on the physicochemical properties of DEP, it can be concluded that DEP is easily hydrolyzed by DEPPr. DEPPr has a lower molecular weight, average particle size and PDI. The compound protease cleaves the peptide bonds between hydrophobic amino acids such as Tyr and Trp and aromatic amino acids in DEP, destroys the secondary, tertiary and spatial structures of DEP, makes the structure of DEP more flexible and loose, exposes a large number of internal hydrophobic groups, and enhances the surface hydrophobicity of DEPPr. It can be concluded that compound protease hydrolysis improves the dispersibility of DEP in water.

[0103] Example 2

[0104] 1.1 Determine the reasonable concentration of DEPPr

[0105] Zebrafish embryos were taken and set up into 7 groups (control group (Con group), Glu group and different concentrations of DEPPr groups (10, 25, 50, 75, 100 μg / mL). The embryos in each group were placed in the corresponding treatment solution (see Table 1), and the morphological changes at 24h, 48h, 72h and 96h were observed, and the survival rate and hatching rate were calculated. There were 100 embryos in each group, and the experiment was repeated 3 times independently.

[0106] Table 1 Treatment solutions corresponding to each group

[0107] Group Treatment fluid Con Group Embryo culture medium Glu group 130 mM glucose 10 μg / mL DEPPr group 130 mM glucose + 10 μg / mL DEPPr 25 μg / mL DEPPr group 130 mM glucose + 25 μg / mL DEPPr 50 μg / mL DEPPr group 130 mM glucose + 50 μg / mL DEPPr 75 μg / mL DEPPr group 130 mM glucose + 75 μg / mL DEPPr 100 μg / mL DEPPr group 130 mM glucose + 100 μg / mL DEPPr

[0108] Note: Embryo culture medium is the culture medium used to culture zebrafish embryos, specifically methylene blue culture medium.

[0109] Fig.10The results showed that 10, 25, 50, 75, and 100 μg / mL of composite protease hydrolysate significantly improved the hatching rate of zebrafish embryos. Combined with the survival rate of zebrafish embryos, the toxic effect of DEPPr in the high concentration dose group (75, 100 μg / mL) was greater. Therefore, 10, 25, and 50 μg / mL of composite protease hydrolysate are reasonable concentrations that can be used to study the zebrafish retinal vascular injury model.

[0110] 1.2 Intervention effect of DEPPr on high glucose-induced retinal vascular damage in zebrafish

[0111] In order to use the optimal concentration of DEPPr to study its effect on the diameter of zebrafish retinal vessels induced by high glucose, the present invention used a fluorescent inverted microscope to take pictures of retinal blood vessels in the Con group, Glu group, and groups with different concentrations of DEPPr (10 μg / mL, 25 μg / mL, and 50 μg / mL). Fig.11 As shown in (a), the diameters of the eyeballs of different experimental groups were measured according to the scale. The results are shown in Fig.11 As shown in (b). In the Con group, the diameter of the retinal blood vessels of zebrafish embryos was 4.32 μm. However, when zebrafish embryos were exposed to 130 mM glucose, the diameter of their retinal blood vessels was significantly thickened to 5.86 μm. After intervention with DEPPr, the blood vessel diameters decreased with increasing concentrations to 5.74 μm, 4.73 μm, and 4.27 μm, respectively. There was no significant difference between the Con group and the DEPPr group at 50 μg / mL. There were significant differences between DEPPr 25 μg / mL and 50 μg / mL compared with the Glu group, especially 50 μg / mL DEPPr. The above results indicate that DEPPr can alleviate high glucose-induced vascular damage in the zebrafish retina.

[0112] 1.3 HE staining analysis

[0113] In order to study the effect of compound protease hydrolysate on high glucose-induced retinal vascular damage in zebrafish, the eyeballs of zebrafish embryos (larvae) cultured to day 6 were frozen and stained with HE, and the thickness of the retinal structures GCL (ganglion cell layer), IPL (inner plexiform layer), INL (inner nuclear layer), ONL (outer nuclear layer), and RPE (pigment epithelium) were quantified and statistically analyzed. Fig.12 As shown in (a), the retina of zebrafish in the control group had normal morphology and structure, with neatly arranged cells and orderly layers. It was further observed that the retina of zebrafish treated with the composite protease hydrolysate was slightly smaller, as shown in Figure 2. Fig.12As shown in (b), compared with the Con group, the Glu group did not significantly change the thickness of each retinal layer of zebrafish, but the retinal structure was slightly chaotic compared with the Con group; after intervention with DEPPr, there was no significant difference in the thickness of each retinal layer between the DEPPr group and the Glu group. After intervention with 10μg / mLDEPPr, there was no improvement in the thickness of GCL and ONL, and the thickness of IPL and INL decreased slightly; after intervention with 25μg / mLDEPPr, the thickness of GCL increased, and IPL, INL, ONL, and RPE tended to normal; after intervention with 50μg / mLDEPPr, the thickness of GCL, INL, and ONL decreased, and IPL and RPE tended to normal. The results show that DEPPr can improve the retinal pathological changes caused by the Glu group to a certain extent.

[0114] In summary, the activity of DEPPr was evaluated by the intervention of DEPPr on high glucose-induced zebrafish retinal vascular injury and HE staining. First, a toxic exposure experiment was conducted by establishing a high glucose-induced zebrafish vascular injury model. The results showed that 10, 25, and 50 μg / mL DEPPr can be used to study the reasonable concentrations of intervention in zebrafish retinal vascular injury. The morphological changes in the diameter of retinal vessels and the increase in the diameter of retinal vessels induced by high glucose were analyzed. DEPPr made the diameter of zebrafish retinal vessels induced by high glucose thinner; and as its concentration increased, the diameter of zebrafish retinal vessels gradually became thinner, especially 50 μg / mL had a significant change in the diameter of zebrafish retinal vessels. Then, quantitative analysis of the stained sections showed that DEPPr could improve the retinal pathological changes caused by Glu. The above results show that DEPPr can alleviate high glucose-induced vascular injury in the zebrafish retina.

[0115] 1.4 Study on the intervention mechanism of DEPPr on high glucose-induced retinal vascular injury in zebrafish based on non-targeted metabolomics

[0116] 1.4.1 UHPLC-MS / MS metabolic profile analysis

[0117] During the instrument analysis, mixed QC samples were collected and run in duplicate to examine the repeatability of the analysis process. Fig.13 As shown in the figure, the TIC overlap diagrams of the quality control samples in positive and negative ion modes are shown. The TIC overlap effect of the QC samples at different times is relatively good, indicating that the stability of the machine is relatively good. After data preprocessing (alignment and standardization), all sample data are used for multivariate statistical analysis, and QC samples are randomly run throughout the analysis process to evaluate system stability.

[0118] 1.4.2 Multidimensional statistical analysis

[0119] To observe the changes in metabolites in zebrafish after DEEPr administration, the LCMC data of the Con group, Glu group, DEEPr group, and quality control group (QC) were preprocessed and imported into the software for principal component analysis (PCA). The PCA scores were as follows: Fig.14 As shown, all QC samples were tightly clustered together in both positive and negative ion modes, indicating that the method used for this experiment was considered to be repeatable and the system was stable. The samples of the three groups were well clustered within a certain range, indicating that each group had similarities. In both positive and negative ion modes, the data of the three groups were also well separated, which meant that the metabolites of the three groups were different, and an important trend was observed. The DEEPr group was between the Glu group and the Con group, and the DEEPr was shifting closer to the Con group, showing the alleviating and improving effect of the DEEPr group, indicating that the DEEPr group may improve the retinal vascular damage induced by high glucose in zebrafish.

[0120] In order to more intuitively reflect the protective effect of DEEPr on high glucose-induced retinal vascular damage in zebrafish, the QC samples were removed and orthogonal partial least squares discriminant analysis (OPLS-DA) was further performed to distinguish the differences in metabolites, and the Glu group and the DEEPr group were selected for metabolomics analysis. Fig.15 As shown, the individual samples in each group can be clearly distinguished, and the samples within the group are relatively concentrated, indicating that there are differences in the metabolic conditions of the zebrafish larvae in the Glu group and the DEEPr group, indicating that DEEPr administration of the Glu group has a certain regulatory effect on the metabolism of zebrafish larvae.

[0121] 1.4.3 Identification of different metabolites

[0122] In order to further study the differences between the metabolites of each group, an OPLSDA model was established for the Glu group and the DEPPr group. To check the reliability of the OPLS-DA model, 200 permutation validations were performed. Fig.16 The OPLS-DA model validation in the positive and negative ion modes for the Glu and DEPPr groups. The results show that the original model can well explain the differences between the two groups of samples. At the same time, as the substitution retention gradually decreases, the proportion of the substituted Y variable increases, and the R2 and Q2 of the random model gradually decrease. All the blue Q2 values ​​on the left and all the green R2 values ​​are lower than the original points on the right. The intercept of the Q2 regression line is lower than 0.05, indicating that the original model does not have an overfitting phenomenon, reflecting that the OPLS-DA model has good predictive ability and reliability. The differential metabolites can be selected according to the VIP value analysis in the subsequent sequence. Fig.17To screen out the variable importance in the projection (VIP) of the first principal component of the OPLS-DA model, the VIP value indicates the influence of the inter-group difference of the corresponding metabolite in the classification and discrimination of each group of samples in the model, and the metabolites with VIP ≥ 1 are significantly different. Based on the above, SPSS software was used to perform the t-Test hypothesis test and select p ≤ 0.05, indicating that the significant difference is statistically significant. In the positive ion mode, a total of 55 different differential metabolites were found in the Glu group and the DEPPr group, 44 of which were upregulated and 11 were downregulated; in the negative ion mode, a total of 133 different differential metabolites were found in the Glu group and the DEPPr group, 71 of which were upregulated and 62 were downregulated.

[0123] 1.4.4 Identification of metabolic pathways affected by differential metabolites

[0124] In order to gain a deeper understanding of the mechanism of DEPPr intervention on high glucose-induced zebrafish retinal vascular injury, the metabolic pathways of significantly different metabolites were analyzed. The screened metabolites were entered into the KEGG website to determine the metabolite pathways, and MetaboAnalystR was used to perform KEGG pathway enrichment. The results are shown in the KEGG enrichment network ( Fig.18), the light yellow nodes in the middle are pathway names, the node size represents the number of metabolites annotated to the pathway, the gray nodes connected to them are specific metabolites annotated to the pathway, and the connecting line colors represent different pathways. In positive ion mode, 6 metabolic pathways were found, namely glycerophospholipid metabolism, purine metabolism, folate biosynthesis, pyrimidine metabolism, cysteine ​​and methionine metabolism, and tyrosine metabolism; 8 metabolic pathways were found under negative ion mode, namely phenylalanine, tyrosine and tryptophan biosynthesis. Metabolic pathway analysis depends on potential biomarkers. The results of pathway analysis show that the intervention effect of DEPPr on high glucose-induced retinal vascular injury in zebrafish may be related to these metabolic pathways.

[0125] Therefore, the experimental results of the present invention show that DEPPr can reduce high-glucose-induced zebrafish retinal vascular damage through amino acid metabolism, slow down the pathological process of DR, and lay a foundation for promoting the utilization of DEPPr. The present invention proves that defatted evening primrose seed protein hydrolysate has a protective effect on high-glucose-induced zebrafish retinal vascular damage, providing a new theoretical basis for the prevention and treatment of DR.

[0126] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A defatted evening primrose seed protein hydrolyzate, characterized in that: It is obtained by hydrolyzing and defatting evening primrose seed protein using composite protease; The defatted evening primrose seed protein is prepared from defatted evening primrose seeds through alkali extraction and acid precipitation.

2. A method for preparing the defatted evening primrose seed protein hydrolyzate according to claim 1, characterized in that: The following steps are involved: Mixing defatted evening primrose seed powder with water, adjusting the solution pH to 11.5, and subjecting the mixture to ultrasonic treatment and first centrifugation to obtain an alkaline extract of defatted evening primrose seeds; The pH of the defatted evening primrose seed alkali extract is adjusted to 3.0, allowed to stand, centrifuged for a second time, and the precipitate is collected to obtain defatted evening primrose seed protein; The defatted evening primrose seed protein is hydrolyzed with a composite protease to obtain the defatted evening primrose seed protein hydrolysate; The CAS number of the composite protease is: 9014-01-1.

3. The preparation method according to claim 2, characterized in that: The solid-liquid ratio of the defatted evening primrose seed powder to water is 1 g:20 mL; the ultrasonic treatment condition is 250 W treatment for 60 min; the first centrifugation condition is 4° C., 3000 r / min centrifugation for 20 min.

4. The preparation method according to claim 2, characterized in that: The static condition is 4° C. for 2 h; the second centrifugation condition is 4° C., 3000 r / min for 30 min.

5. The preparation method according to claim 2, characterized in that: The enzyme activity of the composite protease is 3000U / g; The hydrolysis conditions are as follows: temperature 50°C, pH 7, and shaking at 200 r / min for 3 hours.

6. Use of the defatted evening primrose seed protein hydrolyzate according to claim 1 in the preparation of a medicament for preventing and treating diabetic retinopathy.

7. A drug for preventing and treating diabetic retinopathy, characterized in that: The invention comprises the defatted evening primrose seed protein hydrolyzate according to claim 1.

8. The drug according to claim 7, characterized in that The concentration of the defatted evening primrose seed protein hydrolyzate is 10 μg / mL-50 μg / mL.

9. The drug according to claim 7, characterized in that Also contains pharmaceutically acceptable excipients.

10. The drug according to claim 7, characterized in that The dosage form of the drug includes a liquid dosage form.

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