An antioxidant peptide, its preparation method and application

Abstract

The present invention provides an antioxidant peptide, a preparation method and an application thereof, belonging to the field of biotechnology. The present invention provides an antioxidant peptide with an amino acid sequence as shown in SEQ ID NO.2 and / or an antioxidant peptide with an amino acid sequence as shown in SEQ ID NO.4. Through enzymatic hydrolysis, ultrafiltration, gel chromatography purification and mass spectrometry technology, and combined with ABTS and DPPH free radical scavenging activities, the present invention identifies 2 antioxidant peptides from gastrodia protein hydrolysate, with molecular weights of 937.08 Da and 1153.50 Da respectively. They have high antioxidant activity, thermal stability and in vitro safety, can effectively alleviate the decrease in cell viability and the increase in ROS level caused by oxidative stress damage of cells, and can play a neuroprotective role by activating the Nrf2 pathway. The antioxidant peptide is applicable to daily chemical products, skin care and beauty products, health food and drugs that require antioxidant efficacy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an antioxidant peptide, a preparation method thereof, and an application thereof. Background Art

[0002] Oxidative stress caused by the accumulation of reactive oxygen species (ROS) is one of the pathological factors of various incurable neurodegenerative diseases (NDs) and cerebral ischemia-reperfusion injury (CIRI). These age-related diseases are characterized by the gradual degeneration of neurons and the dysfunction of glial cells, resulting in severe morbidity and mortality of patients and imposing a heavy burden on the elderly population. The use of antioxidant peptides in food can prevent such diseases caused by oxidative damage. Therefore, it is of great practical application value to search for new food-derived antioxidant peptides that can alleviate oxidative stress.

[0003] Currently, there are common defects in new food-derived antioxidant peptides, such as poor thermal stability or unstable effects, which limit the application of new food-derived antioxidant peptides in the development of functional active ingredients and pharmaceutical products. Summary of the Invention

[0004] Aiming at the defects in the prior art, the purpose of the present invention is to provide an antioxidant peptide with good thermal stability and high antioxidant capacity, which can effectively alleviate the damage caused by oxidative stress.

[0005] The purpose of the present invention is achieved by the following technical solutions:

[0006] The present invention provides an antioxidant peptide, including: the antioxidant peptide with the amino acid sequence shown in SEQ ID NO.2 and / or the antioxidant peptide with the amino acid sequence shown in SEQ ID NO.4.

[0007] The present invention provides a preparation method of the antioxidant peptide described in the above technical solution, including:

[0008] Mix the crushed gastrodia tuber with water and dissolve it, and collect the supernatant;

[0009] After precipitating the protein in the supernatant, dialyze the obtained protein to obtain crude gastrodia protein;

[0010] Hydrolyze the crude gastrodia protein with papain to obtain a crude gastrodia protein hydrolysate;

[0011] After ultrafiltrating the crude gastrodia protein hydrolysate, separate and identify the fragments smaller than <3 kDa to obtain the antioxidant peptide.

[0012] Preferably, when performing enzymatic hydrolysis, the mass-volume ratio of papain to the crude Gastrodia elata protein solution is (0.1~0.5) g:100 mL; the mass concentration of the crude Gastrodia elata protein in the crude Gastrodia elata protein solution is 5%~10%.

[0013] Preferably, the method for precipitating proteins in the supernatant includes: mixing the supernatant with saturated ammonium sulfate to precipitate the proteins.

[0014] The present invention provides the use of the antioxidant peptide described in the above technical solution or the antioxidant peptide prepared by the preparation method described in the above technical solution in the preparation of products with antioxidant and / or neuroprotective effects.

[0015] The present invention provides the use of the antioxidant peptide described in the above technical solution or the antioxidant peptide prepared by the preparation method described in the above technical solution in the preparation of products for preventing and / or treating diseases caused by oxidative stress.

[0016] Preferably, the diseases include ischemic stroke and / or neurodegenerative diseases.

[0017] Preferably, the drug has any one or more of the following effects (1)~(5):

[0018] (1) Alleviating the decrease in cell viability caused by oxidative stress;

[0019] (2) Alleviating the increase in ROS in cells induced by oxidative stress;

[0020] (3) Improving the DPPH free radical scavenging activity;

[0021] (4) Improving the ABTS free radical scavenging activity;

[0022] (5) Increasing the expression level of Nrf2 protein.

[0023] The present invention provides an antioxidant product, including the antioxidant peptide described in the above technical solution or the antioxidant peptide prepared by the preparation method described in the above technical solution.

[0024] Preferably, the antioxidant product includes any one or more of pharmaceuticals, foods, and daily chemical products.

[0025] The beneficial effects of the present invention:

[0026] The present invention provides an antioxidant peptide, including: the antioxidant peptide with the amino acid sequence shown in SEQ ID NO.2 and / or the antioxidant peptide with the amino acid sequence shown in SEQ ID NO.4. Through enzymatic hydrolysis, ultrafiltration, gel chromatography purification and mass spectrometry techniques, and combined with ABTS and DPPH free radical scavenging activities, the present invention identifies 2 novel antioxidant peptides from fresh Gastrodia elata protein hydrolysates, namely PR-9 shown in SEQ ID NO.2 and CT-10 shown in SEQ ID NO.4. The molecular weights of PR-9 and CT-10 are 937.08 Da and 1153.50 Da respectively, and they have high antioxidant activities, thermal stabilities and in vitro safety. In addition, the antioxidant peptides PR-9 and CT-10 can also effectively alleviate the decrease in cell viability and the increase in ROS level caused by oxidative stress damage of cells, indicating their potential neuroprotective effects. Further, immunoblot analysis reveals that the antioxidant peptides PR-9 and CT-10 can play a neuroprotective role by activating the Nrf2 pathway. In summary, the antioxidant peptide provided by the present invention has antioxidant activity, good thermal stability, can protect nerves, and can effectively alleviate the damage caused by oxidative stress. Further, the antioxidant peptide is applicable to daily chemical products, skin care and beauty products, health foods and drugs that require antioxidant efficacy. Brief Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0028] Figure 1 It is a diagram for the preparation and identification of Gastrodia elata antioxidant peptide; wherein A is a fresh Gastrodia elata tuber; B is a diagram of the results of hydrolyzing Gastrodia elata protein with different concentrations of papain for SDS-PAGE analysis; C is a detection diagram of the <10 kDa fragment purified by a Sephacryl S-200 chromatographic column; D is a diagram of the results of the effect of the purified <3 kD fragment on the ABTS free radical scavenging activity, with ascorbic acid as the positive control; E is a diagram of the results of the effect of the purified <3 kD fragment on the DPPH free radical scavenging activity, with ascorbic acid as the positive control; F is a test diagram of the effect of the purified <3 kD fragment on the DPPH free radical scavenging activity; G is a diagram of identifying 78 polypeptides from the F2 high antioxidant activity fragment (<3 kDa) by LC-MS / MS; H is that the scores of 39 potential active peptides determined by the Peptide Ranker database are >0.5;

[0029] Figure 2It is the MS / MS spectrum of the F2 fragment in the gastrodia protein hydrolysate;

[0030] Figure 3 It is the liquid chromatography chart of peptide PR-9;

[0031] Figure 4 It is the liquid chromatography chart of peptide CT-10;

[0032] Figure 5 It is the mass spectrum of peptide PR-9;

[0033] Figure 6 It is the structural formula chart of peptide PR-9;

[0034] Figure 7 It is the mass spectrum of peptide CT-10;

[0035] Figure 8 It is the structural formula chart of peptide CT-10;

[0036] Figure 9 It is the chart of the antioxidant activity characteristics of chemically synthesized gastrodia peptides. Among them, A is the result chart of the effect of 7 synthetic peptides on the scavenging activity of DPPH free radicals; B is the result chart of the effect of 7 synthetic peptides on the scavenging activity of ABTS free radicals; C is the detection result of the thermal stability of antioxidant peptides PR-9 and CT-10; D is the result chart of the hemolytic activity test of antioxidant peptide PR-9; E is the result chart of the hemolytic activity test of antioxidant peptide CT-10; F is the statistical result of the hemolysis rate of antioxidant peptides PR-9 and CT-10;

[0037] Figure 10Figure for the neuroprotective effect and mechanism of gastrodia antioxidant peptides; A shows the effect of different concentrations of H2O2 on the viability of PC12 cells; B shows the effect of PR-9 on cell viability in H2O2-stimulated PC12 cells; C shows the effect of CT-10 on cell viability in H2O2-stimulated PC12 cells; The left graph in D shows the effect of H2O2 stimulation on ROS in PC12 cells after treatment with PR-9 detected by flow cytometry; The right graph in D shows the effect of H2O2 stimulation on ROS in PC12 cells after treatment with CT-10 detected by flow cytometry; E shows the effect of peptide PR-9 on the viability of H2O2-stimulated PC12 cells; F shows the effect of peptide CT-10 on the viability of H2O2-stimulated PC12 cells; G shows the expression level of Nrf2 protein in H2O2-stimulated PC12 cells after treatment with PR-9 and CT-10. The + in G indicates the addition of the substance on the left, and - indicates the non-addition of the substance on the left; H shows the quantitative result of the expression level of Nrf2 protein in H2O2-stimulated PC12 cells after treatment with PR-9; I shows the quantitative result of the expression level of Nrf2 protein in H2O2-stimulated PC12 cells after treatment with CT-10; Data are expressed as mean ± SEM. Compared with the control group, *P<0.05, **P<0.001, ***P<0.0001; Compared with the H2O2 group, #P<0.05, ##P<0.001, P<0.0001. Detailed implementation mode

[0038] The present invention provides an antioxidant peptide, including: an antioxidant peptide with an amino acid sequence shown in SEQ ID NO.2 and / or an antioxidant peptide with an amino acid sequence shown in SEQ ID NO.4.

[0039] For the convenience of description in the present invention, the antioxidant peptide with an amino acid sequence shown in SEQ ID NO.2 can be called antioxidant peptide PR-9, or directly called PR-9; the antioxidant peptide with an amino acid sequence shown in SEQ ID NO.4 can be called antioxidant peptide CT-10, or directly called CT-10.

[0040] The antioxidant peptides PR-9 and CT-10 provided by the present invention are two novel antioxidant peptides identified from fresh gastrodia protein hydrolysates; the molecular weight of PR-9 is 937.08 Da; the molecular weight of CT-10 is 1153.50 Da.

[0041] The antioxidant peptides PR-9 and CT-10 provided by the present invention have high antioxidant activity and can be used to prepare antioxidant products. The results of the examples of the present invention show that the antioxidant peptides PR-9 and CT-10 exhibit high antioxidant activity, have high DPPH free radical scavenging rate and ABTS free radical scavenging rate, which is beneficial to improving the DPPH free radical scavenging activity and ABTS free radical scavenging activity of the body.

[0042] The antioxidant peptides PR-9 and CT-10 provided by the present invention have good thermal stability. The results of the examples of the present invention show that the antioxidant peptides PR-9 and CT-10 can maintain high stability in the temperature range of 25-90 °C and maintain stable antioxidant ability.

[0043] The antioxidant peptides PR-9 and CT-10 provided by the present invention have in vitro safety. The results of the examples of the present invention show that the antioxidant peptides PR-9 and CT-10 do not have hemolytic activity and have high safety.

[0044] The antioxidant peptides PR-9 and CT-10 provided by the present invention have neuroprotective effects and can be used to prepare products beneficial to neuroprotection. The results of the examples of the present invention show that the antioxidant peptides PR-9 and CT-10 can effectively alleviate the decrease in cell viability and the increase in ROS level caused by oxidative stress damage of cells, and can play a neuroprotective role by activating the Nrf2 pathway.

[0045] In the present invention, the antioxidant peptide can be directly prepared by a conventional polypeptide synthesis method or can be prepared by the preparation method provided by the present invention.

[0046] The present invention provides a preparation method of the antioxidant peptide according to the above technical solution, including:

[0047] Mix the crushed gastrodia tuber with water for dissolution, and collect the supernatant;

[0048] After precipitating the protein in the supernatant, dialyze the obtained protein to obtain crude gastrodia protein;

[0049] Enzymatically hydrolyze the crude gastrodia protein with papain to obtain a crude gastrodia protein hydrolysate;

[0050] After ultrafiltration of the crude gastrodia protein hydrolysate, separate and identify the fragments smaller than <3 kDa to obtain the antioxidant peptide.

[0051] The tuber of Gastrodia elata Blume is crushed and then mixed with water for dissolution, and the supernatant is collected. As an optional embodiment of the present invention, the tuber of Gastrodia elata Blume is a fresh tuber of Gastrodia elata Blume. After obtaining the fresh tuber of Gastrodia elata Blume, the present invention preferably washes the tuber of Gastrodia elata Blume and then crushes it to obtain the crushed tuber of Gastrodia elata Blume. The present invention has no special limitation on the crushing method, and any conventional crushing method in the art can be used. As an optional embodiment of the present invention, the crushing method is grinding. After obtaining the crushed tuber of Gastrodia elata Blume, the present invention mixes the crushed tuber of Gastrodia elata Blume with water to obtain a Gastrodia elata Blume - water mixture. As an optional embodiment of the present invention, the mass - volume ratio of the crushed tuber of Gastrodia elata Blume to water is 500 g:5 L. After obtaining the Gastrodia elata Blume - water mixture, the present invention stirs and dissolves the mixture for 2 h. The present invention has no special limitation on the stirring speed, and any conventional stirring method in the art can be used. After the stirring and dissolution is completed, the present invention collects the supernatant. The present invention has no special limitation on the method for collecting the supernatant, and any conventional collection method in the art can be used. As an optional embodiment of the present invention, the collection method is centrifugation, the centrifugation speed is 4000 rpm, and the centrifugation time is 30 min.

[0052] After obtaining the supernatant, the present invention precipitates the protein in the supernatant and then dialyzes the obtained protein to obtain the crude Gastrodia elata Blume protein. The present invention has no special limitation on the precipitation method, and any conventional precipitation method in the art can be used. As an optional embodiment of the present invention, the precipitation method is: mixing the supernatant with saturated ammonium sulfate for protein precipitation. The present invention preferably mixes the supernatant with a saturated ammonium sulfate solution with a volume fraction of 70% of the supernatant for protein precipitation; the precipitation temperature is 4°C; the precipitation time is 12 h. After the precipitation is completed, the present invention dialyzes the obtained protein. Before dialysis, the present invention preferably redissolves the protein with water. As an optional embodiment of the present invention, the cut - off molecular weight of the dialysis bag used for dialysis is 3500 Da; the dialysis is carried out in distilled water; the dialysis time is 24 h. After dialysis is completed, the present invention obtains a crude Gastrodia elata Blume protein extract. After obtaining the crude Gastrodia elata Blume protein extract, the present invention lyophilizes the crude Gastrodia elata Blume protein extract to obtain the crude Gastrodia elata Blume protein. The present invention has no special limitation on the lyophilization method, and any conventional lyophilization method in the art can be used. The crude Gastrodia elata Blume protein can be stored at - 80°C for future use.

[0053] After obtaining the crude Gastrodia elata protein, the present invention enzymatically hydrolyzes the crude Gastrodia elata protein with papain to obtain a hydrolyzate of the crude Gastrodia elata protein. Before enzymatically hydrolyzing the crude Gastrodia elata protein, the present invention preferably dissolves the crude Gastrodia elata protein to obtain a crude Gastrodia elata protein solution. As an alternative embodiment of the present invention, the mass concentration of the crude Gastrodia elata protein in the crude Gastrodia elata protein solution can be 5% - 10%, or can be 5%, 6%, 7%, 8%, 9% or 10%. After obtaining the crude Gastrodia elata protein solution, the present invention mixes the crude Gastrodia elata protein solution with papain for enzymatic hydrolysis. As an alternative embodiment of the present invention, when papain is mixed with the crude Gastrodia elata protein solution, the mass - volume ratio of papain to the crude Gastrodia elata protein solution is (0.1 - 0.5) g:100 mL, and can be 0.1 g:100 mL, 0.2 g:100 mL, 0.3 g:100 mL, 0.4 g:100 mL or 0.5:100 mL. As an alternative embodiment of the present invention, the temperature of the enzymatic hydrolysis is 50 - 60 °C, and can be 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 °C; the time of the enzymatic hydrolysis can be 4 h. After the enzymatic hydrolysis is completed, the present invention preferably inactivates the enzyme in the enzymatic hydrolysis system to obtain an enzymatic hydrolysis product; the method of inactivation treatment can be heating; the temperature of heating can be 95 - 100 °C, or can be 95, 96, 97, 98, 99 or 100 °C; the time of heating can be 10 - 15 min, or can be 10, 11, 12, 13, 14 or 15 min. After obtaining the enzymatic hydrolysis product, the present invention preferably removes insoluble substances in the enzymatic hydrolysis product by centrifugation, collects the supernatant, and obtains a hydrolyzate of the crude Gastrodia elata protein.

[0054] After obtaining the hydrolyzate of the crude Gastrodia elata protein, the present invention ultrafilters the hydrolyzate of the crude Gastrodia elata protein, separates and identifies fragments smaller than <3 kDa, and obtains antioxidant peptides. As an alternative embodiment of the present invention, the ultrafiltration includes a first ultrafiltration and a second ultrafiltration; when performing the first ultrafiltration, the cut - off molecular weight of the ultrafiltration membrane is 10 kDa; after the first ultrafiltration is completed, a protein hydrolyzate with a molecular weight less than <10 kDa is obtained. After obtaining the protein hydrolyzate with a molecular weight less than <10 kDa, the present invention preferably separates the protein hydrolyzate with a molecular weight less than <10 kDa by gel chromatography to obtain different protein hydrolysis fragments. After obtaining different protein hydrolysis fragments, the present invention separately ultrafilters the protein hydrolysis fragments; when performing the second ultrafiltration, the cut - off molecular weight of the ultrafiltration membrane is 3 kDa; after the second ultrafiltration is completed, fragments smaller than <3 kDa are obtained. After obtaining fragments smaller than <3 kDa, the present invention preferably performs LC - MS / MS analysis on the fragments smaller than <3 kDa, and through separation and identification, obtains antioxidant peptides.

[0055] Through the above method, the present invention can obtain a variety of peptide fragments. The present invention preferably screens small molecular weight polypeptides with more potential functional activities through the Peptide Ranker database. For the screened small molecular weight polypeptides, the present invention uses the "ToxIBTL" application program in the BIOPEP-UWM database to predict the toxicity of the peptide segments; further through the ABTS and DPPH free radical scavenging activities, the Gastrodia antioxidant peptides with the amino acid sequence shown in SEQ ID NO.2 and the Gastrodia antioxidant peptides with the amino acid sequence shown in SEQ ID NO.4 are screened out.

[0056] The present invention provides the use of the antioxidant peptide described in the above technical solution or the antioxidant peptide prepared by the preparation method described in the above technical solution in the preparation of antioxidant products. As an optional embodiment of the present invention, the product includes any one or more of drugs, foods, and daily chemical products.

[0057] The present invention provides the use of the antioxidant peptide described in the above technical solution or the antioxidant peptide prepared by the preparation method described in the above technical solution in the preparation of products for preventing and / or treating diseases caused by oxidative stress. As an optional embodiment of the present invention, the product includes any one or more of drugs, foods, and daily chemical products. As an optional embodiment of the present invention, the diseases include ischemic stroke and / or neurodegenerative diseases. As an optional embodiment of the present invention, the drug has any one or more of the following effects (1) to (5): (1) alleviating the decrease in cell viability caused by oxidative stress; (2) inhibiting the production of ROS in cells induced by oxidative stress; (3) increasing the DPPH free radical scavenging activity; (4) increasing the ABTS free radical scavenging activity; (5) increasing the expression level of Nrf2 protein.

[0058] The present invention provides an antioxidant product, including the antioxidant peptide described in the above technical solution. As an optional embodiment of the present invention, the antioxidant product includes any one or more of drugs, foods, and daily chemical products.

[0059] In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the drawings and embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0060] The cell line used in the following examples is: rat adrenal pheochromocytoma cell line (PC-12); the cell line was purchased from Procell Life Science Co., Ltd. (Wuhan, China).

[0061] The experimental materials and reagents used in the following examples are as follows: Fresh Gastrodia elata Blume tubers were collected from Zhaotong City, Yunnan Province, China. Ammonium sulfate was purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd. Sephacryl S-200 was purchased from GE Healthcare Co. (Uppsala, Sweden). Fetal bovine serum (FBS), RPMI1640 medium, phosphate buffered saline (PBS, pH 7.2), penicillin and streptomycin were purchased from Biological Industries (Beit Haemek, Israel). The MTS solution was from Promega Biotechnology Co., Ltd. (Beijing, China). DPPH, ABTS, and reactive oxygen species (ROS) kits were purchased from Solarbio.

[0062] The instruments used in the examples include: freeze dryer (Beijing Boyikang Experimental Instrument Co., Ltd.), LC-3000 high performance liquid chromatograph (Shanghai Chuding Analytical Instrument Co., Ltd.), centrifuge (Xiangyi Co.), dialysis bag (Solarbio Co.), electrophoresis apparatus (Beijing Liuyi Instrument Factory), cell culture incubator (BINDER Co.), laminar flow hood (Suzhou Antai Air Technology Co., Ltd.), 96-well plate (Wuxi Naisi Biotechnology Co., Ltd.), 6-well plate (Wuxi Naisi Biotechnology Co., Ltd.), LED inverted microscope (Leica), electrothermal blast thermostatic incubator (Shanghai Yiheng Scientific Instrument Co., Ltd.), low temperature refrigerator (-80 °C) (Haier Special Equipment Co., Ltd.), flow cytometer (BD Co.).

[0063] In the examples, each experiment was performed at least 3 times, and all data were expressed as mean ± standard error of the mean (mean ± SEM). One-way analysis of variance (one-way ANOVA) was used to analyze the differences among multiple groups. Data analysis was performed using PraphPad Prism 9.4 software. A P value less than 0.05 was considered statistically significant.

[0064] Example 1 Preparation and Identification of Gastrodia elata Blume Antioxidant Peptides

[0065] 1. Preparation of Gastrodia elata Blume Protein Hydrolysate

[0066] (1) 500 g of fresh Gastrodia elata Blume tubers (such as Figure 1 A therein) were washed and crushed to obtain a homogenate. The homogenate was mixed with 5 L of water and stirred for 2 h to dissolve, and then centrifuged at 4000 rpm for 30 min. The supernatant was collected, 70% saturated ammonium sulfate was added to precipitate proteins at 4 °C for 12 h, and then centrifuged at 4000 rpm for 30 min. The precipitate was collected and redissolved in water, dialyzed against distilled water using a 3500 Da dialysis bag for 24 h, and then freeze-dried to obtain crude Gastrodia elata Blume protein, which was stored in a -80 °C refrigerator.

[0067] The crude Gastrodia elata protein was dissolved in deionized water and adjusted to a mass concentration of 5% to obtain a crude Gastrodia elata protein solution. Papain was mixed with the crude Gastrodia elata protein solution, and papain was added to the crude Gastrodia elata protein solution at mass concentrations of 0.1%, 0.3%, and 0.5% (m / v), respectively. After hydrolysis at 55 °C for 4 h, it was heated at 95 °C for 10 min to inactivate the enzyme activity, and then centrifuged at 4000 x g at 4 °C for 30 min to remove insoluble substances. The supernatant was collected, analyzed by SDS-PAGE, and freeze-dried for further purification. The SDS-PAGE results are as Figure 1 shown in B of []. It can be seen from the SDS-PAGE electrophoresis pattern that the enzymatic hydrolysis effect is the most significant when the enzyme-to-substrate mass ratio is 0.5%. Therefore, it was selected as the enzyme concentration for preparing Gastrodia elata protein hydrolysate.

[0068] The above papain and the crude Gastrodia elata protein solution were mixed and enzymatically hydrolyzed at a mass concentration of 0.5%, freeze-dried to obtain the crude Gastrodia elata protein hydrolysate for the subsequent process. The crude Gastrodia elata protein hydrolysate was ultrafiltered through a 10 kDa membrane, and the fragments smaller than 10 kDa were further separated into 5 fragments (F1, F2, F3, F4, and F5) by gel chromatography (as Figure 1 shown in C of []). Then, the purified 5 fragments were ultrafiltered through a 3 kDa membrane, and the components smaller than 3 kDa were collected and denoted as F1, F2, F3, F4, and F5, respectively. After freeze-drying, they were stored at -20 °C.

[0069] (2) The collected components F1, F2, F3, F4, and F5 smaller than 3 kDa were respectively subjected to the corresponding detection of ABTS radical scavenging activity. At the same time, the ABTS radical scavenging activity of vitamin C was detected as a positive control group, and the mass concentration of vitamin C was 300 μg / mL. A PBS negative control group was set up.

[0070] Method for detecting ABTS radical scavenging activity: The ABTS radical scavenging activity was detected using a T-AOC analysis kit (ABTS). According to the requirements of the kit operation manual, the ABTS working solution was prepared by standing in the dark at room temperature for 16 h. Then, 20 μL of a 5 mg / mL sample (purified fragment) was mixed with 180 μL of the ABTS working solution and reacted in the dark at room temperature for 5 min. The ABTS radical scavenging rate was detected using a microplate reader at a wavelength of 734 nm. The detection results are shown in Table 1 and Figure 1 shown in D of [], where Vc in the figure represents vitamin C.

[0071] Table 1 Effects of purified <3 kD fragments on ABTS radical scavenging activity

[0072]

[0073] After ultrafiltration of the F1 fragment with a 3 kDa membrane, the amount of the fragment smaller than 3 kDa obtained was too small. Therefore, subsequently, only the DPPH radical scavenging capacities of F2, F3, F4, and F5 smaller than 3 kDa were determined respectively, and the DPPH radical scavenging activity of vitamin C was detected as a positive control group. The mass concentration of vitamin C was 500 μg / mL, and a PBS negative control group was set up.

[0074] Method for detecting DPPH radical scavenging activity: Mix 30 μL of a 5 mg / mL sample (purified fragment) with 120 μL of DPPH solution and incubate for 60 min under dark conditions at 37 °C. Use a microplate reader (Molecular Devices, Sunnyvale, CA) to measure the DPPH radical scavenging capacity at a wavelength of 515 nm. The detection results are shown in Table 2 and Figure 1 E in Figure 1 F shown in

[0075] Table 2 Effects of purified <3 kD fragments on DPPH radical scavenging activity

[0076]

[0077] As shown in Tables 1 - 2, Figure 1 D and E in Figure 1 the F2 fragment smaller than 3 kDa showed significant antioxidant activity, with relatively high ABTS and DPPH radical scavenging activities. The F2 fragment (as indicated by the arrow in the dashed box in Figure 1 C) showed significant antioxidant activity. Therefore, LC-MS / MS analysis was performed on the <3 kDa fragment in F2 to determine the amino acid sequence of the peptide.

[0078] (3) LC-MS / MS analysis was performed on the <3 kDa fragment in F2, and the results are shown in Figure 1 G in

[0079] The TIC chromatogram of the <3 kDa fragment in F2 is shown in Figure 2 shown in

[0080] A total of 78 peptide chains were identified from the fractions of F2 smaller than 3 kDa. Among the identified peptide segments, there were 3, 55, and 20 peptide segments with molecular weights between 2000 - 3000 Da, 1000 - 2000 Da, and <1000 Da respectively ( Figure 1 G in

[0081] When the score of a peptide in the Peptide Ranker database is greater than 0.5, it may have potential functional activity. Therefore, the Peptide Ranker database was used to predict the scores of antioxidant polypeptides. Among the 20 polypeptides with a molecular weight less than 1000 Da, 3 polypeptides had scores greater than 0.5. Among the 55 peptides with a molecular weight between 1000 and 2000 Da, 30 peptides had scores greater than 0.5. However, in the polypeptide group with a molecular weight greater than 2000 Da, no polypeptide with a score greater than 0.5 was found ( Figure 1 H), indicating that small-molecular-weight polypeptides may have more potential functional activities.

[0082] Next, the toxicity of 7 peptides was predicted using the "ToxIBTL" application in the BIOPEP-UWM database, and it was found that none of them had potential toxicity (Table 3). In addition, to verify the novelty of these 7 candidate peptides, all identified peptide sequences were searched in the SciFinder and BIOPEP databases to determine their novelty. After searching for these peptides in these two databases, no such peptides were found; therefore, the 7 peptides identified from Gastrodia elata were novel and chemically synthesized (Table 3).

[0083] Table 3 Seven candidate peptides

[0084]

[0085] Example 2 Chemical synthesis of Gastrodia elata antioxidant peptides and their antioxidant activity characteristics

[0086] 1. Evaluation of the antioxidant activity of chemically synthesized peptides

[0087] The antioxidant activities of the 7 synthetic peptides in Table 3 were detected using the antioxidant activity evaluation method in Example 1. The DPPH and ABTS radical scavenging activities of the 7 synthetic peptides at different concentrations (400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL) were evaluated respectively. At the same time, the DPPH and ABTS radical scavenging activities of vitamin C were detected as positive controls, and the mass concentration of vitamin C was 400 μg / mL.

[0088] The evaluation results of the antioxidant activities of the chemically synthesized peptides are shown in Tables 4-5 and Figure 9 A-B as follows.

[0089] Table 4 Effects of 7 synthetic peptides at different concentrations on DPPH radical scavenging activity

[0090]

[0091] Table 5 Effects of Seven Synthetic Peptides with Different Concentrations on ABTS Radical Scavenging Activity

[0092]

[0093] As shown in Tables 4 - 5 and Figure 9 A - B, compared with other synthetic peptides, synthetic peptides PR - 9 and CT - 10 showed higher DPPH and ABTS radical scavenging activities, and the antioxidant activity was concentration - dependent. Therefore, two peptides, PR - 9 and CT - 10, were selected to further study their antioxidant properties. Among them, the liquid chromatography diagram of peptide PR - 9 is as shown in Figure 3 Figure [Figure number corresponding to 0000223]. The liquid chromatography diagram of peptide CT - 10 is as shown in Figure 4 Figure [Figure number corresponding to 0000224]. The mass spectrometry diagram of peptide PR - 9 is as shown in Figure 5 Figure [Figure number corresponding to 0000225], and the structural formula is as shown in Figure 6 Figure [Figure number corresponding to 0000226]. The mass spectrometry diagram of peptide CT - 10 is as shown in Figure 7 Figure [Figure number corresponding to 0000227], and the structural formula is as shown in Figure 8 Figure [Figure number corresponding to 0000228].

[0094] 2. Thermal Stability of Antioxidant Peptides PR - 9 and CT - 10

[0095] The antioxidant peptides PR - 9 and CT - 10 were prepared into an initial concentration of 2 mg / mL with PBS solvent, and were treated at 25℃, 50℃, 70℃ and 90℃ for 30 min respectively, and then cooled to room temperature. Then, 30 μL of PR - 9 and CT - 10 peptides (final concentration of 400 μg / mL) were added to 120 μL of DPPH working solution, and reacted at 37℃ for 1 h. The absorbance values of each well were read at 515 nm with an enzyme - linked immunosorbent assay reader. The DPPH radical scavenging ability was measured at a wavelength of 515 nm using a microplate reader to evaluate the thermal stability of the polypeptide.

[0096] The detection results of the thermal stability of antioxidant peptides PR - 9 and CT - 10 are shown in Table 6 and Figure 9 Figure C.

[0097] Table 6 Detection Results of the Thermal Stability of Antioxidant Peptides PR - 9 and CT - 10

[0098]

[0099] As shown in Table 6 and Figure 9 Figure C, the DPPH radical scavenging activities of peptides PR - 9 and CT - 10 changed less with the increase of temperature. The above results indicate that peptides PR - 9 and CT - 10 have high thermal stability in vitro.

[0100] 3. Hemolytic Activity of Antioxidant Peptides PR - 9 and CT - 10

[0101] A rabbit red blood cell suspension with a concentration of 5% (adjusted with PBS) was mixed with an equal volume of antioxidant peptide (final concentration of 400 μg / mL) and incubated at 37 °C for 1 h. An equal volume of PBS was used to replace the antioxidant peptide and mixed with the 5% rabbit red blood cell suspension as a negative control; an equal volume of 0.5% Triton X-100 was used to replace the antioxidant peptide and mixed with the 5% rabbit red blood cell suspension as a positive control. At the same time, PBS without adding rabbit red blood cell suspension was used as a blank group.

[0102] After incubation, the supernatant was collected by centrifuging at 3000 rpm for 10 min at 25 °C, and the absorbance was monitored at a wavelength of 415 nm. The hemolysis rate was calculated as follows:

[0103] Hemolysis rate (%) = [(Asample - A0) / (A1 - A0)] × 100;

[0104] where Asample is the OD415 nm of the polypeptide solution, A0 is the OD415 nm of the blank group PBS, and A1 is the OD415 nm of 0.5% Triton X-100.

[0105] The hemolytic activity test results of antioxidant peptides PR-9 and CT-10 are shown in Table 7 and Figure 9 D - F in.

[0106] Table 7 Hemolytic activity test results of antioxidant peptides PR-9 and CT-10

[0107]

[0108] Note: NC is the hemolysis rate of the negative control; PC is the hemolysis rate of the positive control.

[0109] As shown in Table 7 and Figure 9 D - F in, compared with the PBS treatment group, neither polypeptide PR-9 nor CT-10 had hemolytic activity, which was consistent with the predicted toxicity of polypeptides PR-9 and CT-10 determined using the above database. The above results indicate that peptides PR-9 and CT-10 have high safety in vitro.

[0110] Example 3 Neuroprotective effect and mechanism of Gastrodia antioxidant peptide

[0111] 1. Determination of PC12 cell viability stimulated by H2O2

[0112] Using a serum-containing medium (RPMI1640 medium containing fetal bovine serum) as the medium, PC-12 cells were seeded at 1 × 10 per well 4Cells were seeded at a density into 96-well plates and cultured for more than 8 h. The cells were stimulated with different concentrations of H2O2 (0, 50, 100, 200, 400, 800 μM) for 2 h, that is, the above different concentrations of H2O2 were added to the serum-free medium (RPMI1640 medium) to stimulate the cells for 2 h, and the corresponding culture and stimulation process was carried out in a 37 °C, 5% CO2 incubator. Then 100 μL of MTS working solution was added to each well. After incubating at 37 °C for 2 h, the absorbance of each well was monitored at a wavelength of 490 nm.

[0113] The FlexStatonTM3 multimode microplate reader was used to measure the absorbance value at a wavelength of 490 nm, and the cell viability was calculated according to the following formula. Cell viability (%) = (ODsample - ODblank) / (ODcontrol - ODblank) × 100, where the OD value is the absorbance of each well monitored at a wavelength of 490 nm. Among them, ODblank is the detection result of the absorbance value of the treatment group that only added the medium without adding cells; ODcontrol is the detection result of the absorbance value of the negative control group that added cells but did not add H2O2 solution in the medium; ODsample is the detection result of the absorbance value of the H2O2 stimulation group with different concentrations. In the above experiments, 4 parallel experiments were carried out for each treatment group. Among them, ODblank was 0.049 at a wavelength of 490 nm.

[0114] The results are shown in Tables 8-9 and Figure 10 as shown in A in

[0115] Table 8 OD values of each treatment group at a wavelength of 490 nm

[0116]

[0117] Table 9 Effects of different concentrations of H2O2 on the viability of PC12 cells

[0118]

[0119] It can be seen from Tables 8-9 that the 400 μM H2O2 solution can induce a 20% decrease in the viability of PC12 cells, which is a suitable oxidative stress model. Under the treatment of the 800 μM H2O2 solution, more than half of the cells have died, which is not suitable for preparing an oxidative stress model. The 200 μM H2O2 solution has little effect on cell viability and is not sufficient to induce an oxidative stress model. Therefore, subsequently, a 400 μM H2O2 solution was used to prepare an oxidative stress model for corresponding experiments.

[0120] 2. Using the medium containing serum (RPMI1640 medium containing fetal bovine serum) as the medium, PC-12 cells were seeded at 1×10 per well 4Seed cells at a density into 96-well plates and culture for more than 8 h. Pretreat the cells with different concentrations of PR-9 and CT-10 (0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) for 24 h respectively, that is, add different concentrations of PR-9 to RPMI1640 medium or add different concentrations of CT-10 to RPMI1640 medium to pretreat the cells for 24 h. After the treatment, stimulate the cells with 400 μM H2O2 for 2 h, and carry out the corresponding culture and stimulation process in a 37 °C, 5% CO2 incubator. At the same time, set up a PBS control group, that is, use PBS instead of PR-9 or CT-10 and do not stimulate with H2O2. Then add 100 μL of MTS working solution to each well. After incubating at 37 °C for 2 h, monitor the absorbance of each well at a wavelength of 490 nm.

[0121] Use a FlexStatonTM3 multimode microplate reader to measure the absorbance value at a wavelength of 490 nm, and calculate the cell viability according to the following formula. Cell viability (%) = (ODsample - ODblank) / (ODcontrol - ODblank) × 100, where the OD value is the absorbance monitored for each well at a wavelength of 490 nm. Among them, ODblank is the detection result of the absorbance value of the treatment group that only adds medium without cells; ODcontrol is the detection result of the absorbance value of the negative control group that adds cells to the medium without adding peptides and H2O2; ODsample is the detection result of the absorbance value of each experimental group. In the above experiments, 3 parallel experiments were carried out for each treatment group, the same below.

[0122] The results are shown in Tables 10 - 13 and Figure 10 as shown in B - C in

[0123] Table 10 OD values of each treatment group at a wavelength of 490 nm

[0124]

[0125] Table 11 Effects of peptide PR-9 on the viability of H2O2-stimulated PC12 cells

[0126]

[0127] Table 12 OD values of each treatment group at a wavelength of 490 nm

[0128]

[0129] Table 13 Effects of peptide CT-10 on the viability of H2O2-stimulated PC12 cells

[0130]

[0131] From Tables 10 - 13 andFigure 10 As can be seen from B to C in [reference], compared with the H2O2 treatment group, polypeptides PR-9 and CT-10 dose-dependently increased cell viability. In particular, treatment with 200 μg / mL PR-9 and CT-10 increased cell viability by approximately 17% and 19% respectively.

[0132] 3. ROS detection Using serum-containing medium (RPMI1640 medium containing fetal bovine serum) as the culture medium, PC-12 cells were seeded into 6-well plates at a density of 5×10 5 cells per well and cultured for more than 8 h. Then, they were treated with PR-9 (0 μg / mL, 100 μg / mL, 200 μg / mL) and CT-10 (0 μg / mL, 100 μg / mL, 200 μg / mL) for 12 h, that is, different concentrations of PR-9 were added to RPMI1640 medium or different concentrations of CT-10 were added to RPMI1640 medium to pretreat the cells for 12 h. After the treatment, the cells were stimulated with 400 μM H2O2 for 2 h, and the corresponding culture and stimulation processes were carried out in a 37°C, 5% CO2 incubator. Then, the cell supernatant was taken out and the cells were rinsed twice with PBS. Subsequently, the cells were incubated with 10 μM DCFH-DA (saolibio, Shanghai, China) for 20 min at 37°C in the dark, and then the cells were collected and rinsed with PBS. Finally, the fluorescence intensity of DCFH-DA was measured by flow cytometry at wavelengths of 480 nm (excitation) and 530 nm (emission). The ROS content was expressed as the comparison of the average fluorescence intensity of PR-9 and CT-10 with the H2O2 treatment group.

[0133] The results are shown in Tables 14-15 and Figure 10 D-F in [reference].

[0134] Table 14 ROS content in H2O2-stimulated PC12 cells after PR-9 treatment detected by flow cytometry

[0135]

[0136] Table 15 ROS content in H2O2-stimulated PC12 cells after CT-10 treatment detected by flow cytometry

[0137]

[0138] As shown in Tables 14-15 and Figure 10 D-F in [reference], in the H2O2-induced PC12 cell injury model, polypeptides PR-9 and CT-10 could significantly inhibit the production of ROS (p<0.001). These results indicate that polypeptides PR-9 and CT-10 can effectively inhibit the production of ROS in H2O2-induced PC12 cells, thereby providing antioxidant protection for the cells.

[0139] 4. Immunoblot analysis

[0140] Method: PC-12 cells were processed according to the method described in step 3 of Example 3. After drug treatment, the cells were rinsed twice with cold PBS and lysed on ice for 30 min with RIPA lysis buffer containing 1 mM PMSF. The samples were separated by 10% SDS-PAGE, transferred to a PVDF (0.45 mm pore size) membrane, blocked with 5% non-fat milk powder, and incubated with the following primary antibodies: Nrf2 (1:1000, #4621, CST) and anti-GAPDH (1:5000, ab181602, Abcam). Then, it was incubated with the following horseradish peroxidase-labeled secondary antibody, goat anti-rabbit IgG H&L (1:5000, ab6721, Abcam). Finally, the immunoblot bands were visualized using a chemiluminescence (ECL) detection reagent (Tiangen, Beijing, China) (Trans-Blot Turbo System, BIO-RAD), and the signal intensity was analyzed using Image J software. Among them, PBS was the result of the PBS control group, that is, PBS was used instead of PR-9 or CT-10, and at the same time, H2O2 stimulation was not performed.

[0141] The results are shown in Tables 16-17 and Figure 10 G-I in

[0142] Table 16 Effects of PR-9 on the expression level of Nrf2 protein in H2O2-stimulated PC12 cells

[0143]

[0144] Table 17 Effects of CT-10 on the expression level of Nrf2 protein in H2O2-stimulated PC12 cells

[0145]

[0146] Western blot analysis was performed on the protein expression of Nrf2 in the H2O2-induced PC12 cell injury model. As can be seen from Tables 16-17 and Figure 10 G-I in, both polypeptides PR-9 and CT-10 significantly increased the expression level of Nrf2 protein. These results indicate that polypeptides PR-9 and CT-10 have neuroprotective effects on H2O2-induced PC12 cell injury by activating the Nrf2 pathway.

[0147] In summary, through enzymatic hydrolysis, ultrafiltration, gel chromatography purification and mass spectrometry techniques, and combined with ABTS and DPPH free radical scavenging activities, two novel antioxidant peptides were identified from fresh Gastrodia elata protein hydrolysates. They have high antioxidant activity, thermal stability and in vitro safety, can effectively alleviate the decrease in cell viability and the increase in ROS level caused by oxidative stress damage in cells, and can play a neuroprotective role by activating the Nrf2 pathway.

[0148] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antioxidant peptide, characterized in that, Comprising: An antioxidant peptide with an amino acid sequence as shown in SEQ ID NO.2 and / or an antioxidant peptide with an amino acid sequence as shown in SEQ ID NO.

4.

2. The preparation method of the antioxidant peptide according to claim 1, characterized in that, Comprising: Mix the crushed Gastrodia elata tuber with water for dissolution, and collect the supernatant. After precipitating the protein in the supernatant, dialyze the obtained protein to obtain crude Gastrodia elata protein. Digest the crude Gastrodia elata protein with papain to obtain a crude Gastrodia elata protein hydrolysate. After ultrafiltration of the crude Gastrodia elata protein hydrolysate, separate and identify the fragments smaller than <3 kDa to obtain antioxidant peptides.

3. The preparation method according to claim 2, characterized in that, When performing enzymatic hydrolysis, the mass-to-volume ratio of papain to the crude Gastrodia elata protein solution is (0.1 - 0.5) g:100 mL; the mass concentration of the crude Gastrodia elata protein in the crude Gastrodia elata protein solution is 5% - 10%.

4. The preparation method according to claim 2, wherein The method for precipitating the protein in the supernatant includes: mixing the supernatant and saturated ammonium sulfate for protein precipitation.

5. Use of the antioxidant peptide according to claim 1 or the antioxidant peptide prepared by the preparation method according to any one of claims 2 - 4 in the preparation of a product for treating ischemic stroke.

6. The application according to claim 5, wherein The product is used for any one or more of the following aspects (1) - (5): (1) Alleviating the decrease in cell viability caused by oxidative stress; (2) Alleviating the increase in ROS in cells induced by oxidative stress; (3) Improving the DPPH free radical scavenging activity; (4) Improving the ABTS free radical scavenging activity; (5) Increasing the expression level of Nrf2 protein.

7. An antioxidant product, characterized in that, Comprising the antioxidant peptide according to claim 1 or the antioxidant peptide prepared by the preparation method according to any one of claims 2 - 4.

8. The antioxidant product according to claim 7, wherein The antioxidant product includes any one or more of pharmaceuticals, health foods, and daily chemical products.

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