Antioxidant peptide as well as preparation method and application thereof

Abstract

The invention provides an antioxidant peptide as well as a preparation method and application thereof, and belongs to the technical field of biology. The 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. Two antioxidative peptides are identified from the gastrodia elata protein hydrolysate through enzymolysis, ultrafiltration, gel chromatography purification and mass spectrometry technologies in combination with ABTS and DPPH free radical scavenging activity, the molecular weights of the two antioxidative peptides are 937.08 Da and 1153.50 Da respectively, and the two antioxidative peptides have high antioxidative activity, heat stability and in-vitro safety and can be used for preparing antioxidative peptides for the gastrodia elata protein hydrolysate. The cell viability decline and ROS level rise caused by oxidative stress injury of cells can be effectively relieved, and the neuroprotective effect can be achieved by activating an Nrf2 pathway. The antioxidant peptide is suitable for daily chemical products, skin care and beauty cosmetics, health foods and medicines with antioxidant efficacy requirements.

Description

Technical Field

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

[0002] Oxidative stress caused by the accumulation of reactive oxygen species (ROS) is one of the pathological factors of multiple incurable neurodegenerative diseases (NDs) and cerebral ischemia-reperfusion injury (CIRI). These aging-related diseases are characterized by the progressive degeneration of neurons and dysfunction of glial cells, leading to severe morbidity and mortality in patients, and placing a heavy burden on the aging population. Such diseases caused by oxidative damage can be prevented by the use of antioxidant peptides in food. Therefore, the search for novel food-derived antioxidant peptides that can alleviate oxidative stress has important practical application value.

[0003] At present, new food-derived antioxidant peptides generally have defects such as poor thermal stability or unstable effects, which limit the application of new food-derived antioxidant peptides as functional active ingredients and drug product development. Summary of the invention

[0004] In view of the defects in the prior art, the object 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 objective of the present invention is achieved through the following technical solutions: The present invention provides an antioxidant peptide, 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.

[0006] The present invention provides a method for preparing the antioxidant peptide described in the above technical solution, comprising: Mixing the crushed Gastrodia elata tubers with water to dissolve them, and collecting the supernatant; After the protein in the supernatant is precipitated, the obtained protein is dialyzed to obtain crude Gastrodia elata protein; The crude protein of Gastrodia elata is enzymatically hydrolyzed by using papain to obtain the crude protein hydrolyzate of Gastrodia elata; After ultrafiltration of crude protein hydrolysate from Gastrodia elata, fragments smaller than 3 kDa were separated and identified to obtain antioxidant peptides.

[0007] Preferably, during enzymatic hydrolysis, the mass volume ratio of papain to the Gastrodia crude protein solution is (0.1-0.5) g:100 mL; and the mass concentration of Gastrodia crude protein in the Gastrodia crude protein solution is 5%-10%.

[0008] Preferably, the method for precipitating the protein in the supernatant comprises: mixing the supernatant with saturated ammonium sulfate to precipitate the protein.

[0009] 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.

[0010] 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.

[0011] Preferably, the disease includes ischemic stroke and / or neurodegenerative disease.

[0012] Preferably, the drug has any one or more of the following effects (1) to (5): (1) Alleviate the decrease in cell viability caused by oxidative stress; (2) Alleviate the increase of ROS in cells induced by oxidative stress; (3) Improve DPPH free radical scavenging activity; (4) Improve the ABTS free radical scavenging activity; (5) Increased the expression level of Nrf2 protein.

[0013] The present invention provides an antioxidant product, comprising the antioxidant peptide described in the above technical solution or the antioxidant peptide prepared by the preparation method described in the above technical solution.

[0014] Preferably, the antioxidant product comprises any one or more of medicines, foods and daily chemical products.

[0015] Beneficial effects of the present invention: The present invention provides an antioxidant peptide, including: 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. The present invention identifies two novel antioxidant peptides from fresh Gastrodia elata protein hydrolysate by enzymatic hydrolysis, ultrafiltration, gel chromatography purification and mass spectrometry technology, combined with ABTS and DPPH free radical scavenging activity, namely PR-9 as shown in SEQ ID NO.2 and CT-10 as 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 activity, thermal stability and in vitro safety. In addition, the antioxidant peptides PR-9 and CT-10 can also effectively alleviate the decrease in cell viability and increase in ROS levels caused by oxidative stress damage to cells, indicating their potential neuroprotective effects. Further, immunoblotting analysis revealed that the antioxidant peptides PR-9 and CT-10 can exert neuroprotective effects by activating the Nrf2 pathway. In summary, the antioxidant peptide provided by the present invention has antioxidant activity and good thermal stability, can protect nerves, and effectively alleviate damage caused by oxidative stress, etc. Furthermore, the antioxidant peptide is suitable for daily chemical products, skin care and cosmetics, health foods and medicines that require antioxidant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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 labor.

[0017] Figure 1 Figure 1 is the preparation and identification of antioxidant peptides from Gastrodia elata; A is fresh tuber of Gastrodia elata; B is the result of SDS-PAGE analysis of hydrolysis of Gastrodia elata protein with different concentrations of papain; C is the detection of fragments <10 kDa purified by Sephacryl S-200 column; D is the result of the effect of purified <3 kD fragments on ABTS free radical scavenging activity, with ascorbic acid as a positive control; E is the result of the effect of purified <3 kD fragments on DPPH free radical scavenging activity, with ascorbic acid as a positive control; F is the result of the test of purified <3 kD fragments on DPPH free radical scavenging activity; G is 78 peptides identified by LC-MS / MS from F2 high antioxidant activity fragments (<3 kDa); H is the score of 39 potential active peptides determined by Peptide Ranker database >0.5; Figure 2 This is the MS / MS spectrum of the F2 fragment in the Gastrodia elata protein hydrolysate; Figure 3 is the liquid chromatogram of peptide PR-9; Figure 4 is the liquid chromatogram of peptide CT-10; Figure 5 is the mass spectrum of peptide PR-9; Figure 6 is the structural formula diagram of peptide PR-9; Figure 7 is the mass spectrum of peptide CT-10; Figure 8 is the structural formula diagram of peptide CT-10; Fig. 9 Figure 1 is a graph showing the antioxidant activity characteristics of chemically synthesized Gastrodia elata peptides, wherein A is a graph showing the effects of seven synthetic peptides on DPPH free radical scavenging activity; B is a graph showing the effects of seven synthetic peptides on ABTS free radical scavenging activity; C is a graph showing the thermal stability test results of antioxidant peptides PR-9 and CT-10; D is a graph showing the results of the hemolytic activity test of antioxidant peptide PR-9; E is a graph showing the results of the hemolytic activity test of antioxidant peptide CT-10; and F is a statistical result of the hemolytic rate of antioxidant peptides PR-9 and CT-10. Fig.10 Figure 2 is the neuroprotective effect and mechanism of Gastrodia elata antioxidant peptides; A is the H 2 O 2 Effects of PR-9 on PC12 cell activity; B is the effect of PR-9 on PC12 cell activity; 2 O 2 Effect of CT-10 on cell viability in PC12 cells stimulated by H 2 O 2 The left image in D shows the effect of PR-9 treatment on cell viability in PC12 cells. 2 O 2 The effect of stimulation on ROS in PC12 cells; the right figure in D is the detection of H after CT-10 treatment by flow cytometry 2 O 2 Effects of stimulation on ROS in PC12 cells; E is the effect of peptide PR-9 on H 2 O 2 Effect of peptide CT-10 on the viability of PC12 cells stimulated by H 2 O 2 Effects of stimulation on PC12 cell viability; G is H after PR-9 and CT-10 treatment 2 O 2 Nrf2 protein expression level in stimulated PC12 cells. + in G indicates that the substance on the left was added, and - indicates that the substance on the left was not added. H is H after PR-9 treatment 2 O 2Quantitative results of Nrf2 protein expression level in PC12 cells stimulated by CT-10; I is the expression level of Nrf2 protein in PC12 cells after CT-10 treatment. 2 O 2 Quantitative results of Nrf2 protein expression levels in stimulated PC12 cells; data are presented as mean ± SEM. Compared with the control group, *P < 0.05, **P < 0.001, ***P < 0.0001; compared with H 2 O 2 Compared with the other groups, #P<0.05, ##P<0.001, ###P<0.0001. DETAILED DESCRIPTION

[0018] The present invention provides an antioxidant peptide, 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.

[0019] For the convenience of description in the present invention, the antioxidant peptide with the amino acid sequence shown in SEQ ID NO.2 can be referred to as antioxidant peptide PR-9, or simply referred to as PR-9; the antioxidant peptide with the amino acid sequence shown in SEQ ID NO.4 can be referred to as antioxidant peptide CT-10, or simply referred to as CT-10.

[0020] The antioxidant peptides PR-9 and CT-10 provided by the present invention are two novel antioxidant peptides identified from fresh gastrodia elata protein hydrolysate; the molecular weight of the PR-9 is 937.08 Da; and the molecular weight of the CT-10 is 1153.50 Da.

[0021] 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 present invention shows through the results of the examples that the antioxidant peptides PR-9 and CT-10 exhibit high antioxidant activity, high DPPH free radical scavenging rate and ABTS free radical scavenging rate, which are beneficial to improving the body's DPPH free radical scavenging activity and ABTS free radical scavenging activity.

[0022] The antioxidant peptides PR-9 and CT-10 provided by the present invention have good thermal stability. The present invention shows through the results of the examples 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 capacity.

[0023] The antioxidant peptides PR-9 and CT-10 provided by the present invention have in vitro safety. The present invention shows through the results of the examples that the antioxidant peptides PR-9 and CT-10 have no hemolytic activity and have high safety.

[0024] The antioxidant peptides PR-9 and CT-10 provided by the present invention have neuroprotective effects and can be used to prepare products that are beneficial to neuroprotective effects. The present invention shows through the results of the examples that the antioxidant peptides PR-9 and CT-10 can effectively alleviate the decrease in cell viability and the increase in ROS levels caused by oxidative stress damage to cells, and can exert neuroprotective effects by activating the Nrf2 pathway.

[0025] In the present invention, the antioxidant peptide can be directly prepared by conventional peptide synthesis methods, or can be prepared by the preparation method provided by the present invention.

[0026] The present invention provides a method for preparing the antioxidant peptide described in the above technical solution, comprising: Mixing the crushed Gastrodia elata tubers with water to dissolve them, and collecting the supernatant; After the protein in the supernatant is precipitated, the obtained protein is dialyzed to obtain crude Gastrodia elata protein; The crude protein of Gastrodia elata is enzymatically hydrolyzed by using papain to obtain the crude protein hydrolyzate of Gastrodia elata; After ultrafiltration of crude protein hydrolysate from Gastrodia elata, fragments smaller than 3 kDa were separated and identified to obtain antioxidant peptides.

[0027] The present invention mixes the crushed Gastrodia tuber with water and then dissolves it, and collects the supernatant. As an optional embodiment of the present invention, the Gastrodia tuber is a fresh Gastrodia tuber. After obtaining the fresh Gastrodia tuber, the present invention preferably washes the Gastrodia tuber and then crushes it to obtain the crushed Gastrodia tuber. The present invention has no special restrictions on the method of crushing, 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 Gastrodia tuber, the present invention mixes the crushed Gastrodia tuber with water to obtain a Gastrodia water mixture. As an optional embodiment of the present invention, the mass volume ratio of the crushed Gastrodia tuber and water is 500g:5L. After obtaining the Gastrodia water mixture, the present invention stirs and dissolves the Gastrodia water mixture for 2h. The present invention has no special restrictions on the rotation speed of the stirring, and any conventional stirring method in the art can be used. After the stirring and dissolving is completed, the present invention collects the supernatant. The present invention has no special restrictions on the method of 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 centrifugal speed is 4000 rpm, and the centrifugal time is 30 minutes.

[0028] After obtaining the supernatant, the present invention precipitates the protein in the supernatant, and then dialyzes the obtained protein to obtain crude Gastrodia protein. The present invention does not specifically limit 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 and saturated ammonium sulfate to precipitate the protein. The present invention preferably mixes the supernatant with a saturated ammonium sulfate solution with a volume fraction of 70% of the supernatant liquid to precipitate the protein; the precipitation temperature is 4°C; and the precipitation time is 12h. After the precipitation is completed, the present invention dialyzes the obtained protein. Before the dialysis is performed in the present invention, the protein is preferably redissolved with water. As an optional embodiment of the present invention, the molecular weight cutoff of the dialysis bag for dialysis is 3500 Da; the dialysis is performed in distilled water; and the dialysis time is 24h. After the dialysis is completed, the present invention obtains a crude Gastrodia protein extract. After obtaining the crude Gastrodia protein extract, the present invention freeze-dries the crude Gastrodia protein extract to obtain crude Gastrodia protein. The present invention does not specifically limit the freeze-drying method, and any conventional freeze-drying method in the art can be used. The crude protein of Gastrodia elata can be stored at -80°C for later use.

[0029] After obtaining the crude protein of Gastrodia elata, the present invention uses papain to enzymolyze the crude protein of Gastrodia elata to obtain a crude protein hydrolyzate of Gastrodia elata. Before enzymolyzing the crude protein of Gastrodia elata, the crude protein of Gastrodia elata is preferably dissolved to obtain a crude protein solution of Gastrodia elata. As an optional embodiment of the present invention, the mass concentration of Gastrodia elata crude protein in the crude protein solution of Gastrodia elata can be 5% to 10%, or 5%, 6%, 7%, 8%, 9% or 10%. After obtaining the crude protein solution of Gastrodia elata, the present invention mixes the crude protein solution of Gastrodia elata with papain for enzymolysis. As an optional embodiment of the present invention, when the papain is mixed with the crude protein solution of Gastrodia elata, the mass volume ratio of papain to the crude protein solution of Gastrodia elata is (0.1 to 0.5) g:100 mL, which 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 optional embodiment of the present invention, the temperature of the enzymolysis is 50-60°C, which can be 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60°C; the time of the enzymolysis can be 4h. After the enzymolysis is completed, the present invention preferably inactivates the enzyme in the enzymolysis system to obtain an enzymolysis product; the method of the inactivation treatment can be heating; the heating temperature can be 95-100°C, or 95, 96, 97, 98, 99 or 100°C; the heating time can be 10-15min, or 10, 11, 12, 13, 14 or 15min. After obtaining the enzymolysis product, the present invention preferably removes the insoluble substances in the enzymolysis product by centrifugation, collects the supernatant, and obtains the crude protein hydrolyzate of Gastrodia elata.

[0030] After obtaining the crude protein hydrolysate of Gastrodia elata, the present invention ultrafilters the crude protein hydrolysate of Gastrodia elata, separates and identifies the fragments less than <3 kDa, and obtains antioxidant peptides. As an optional embodiment of the present invention, the ultrafiltration includes a first ultrafiltration and a second ultrafiltration; the molecular weight cutoff of the ultrafiltration membrane during the first ultrafiltration is 10 kDa; after the first ultrafiltration is completed, a protein hydrolysate of <10 kDa is obtained. After obtaining the protein hydrolysate of <10 kDa, the present invention preferably separates the protein hydrolysate of <10 kDa by gel chromatography to obtain different protein hydrolysis fragments. After obtaining different protein hydrolysis fragments, the present invention performs a second ultrafiltration on the protein hydrolysis fragments respectively; the molecular weight cutoff of the ultrafiltration membrane during the second ultrafiltration is 3 kDa; after the second ultrafiltration is completed, a fragment less than <3 kDa is obtained. After obtaining the <3 kDa fragment, the present invention preferably performs LC-MS / MS analysis on the <3 kDa fragment, and obtains antioxidant peptides by separation and identification.

[0031] The present invention can obtain a variety of peptide fragments through the above method. 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 predicts the toxicity of the peptide fragments using the "ToxIBTL" application in the BIOPEP-UWM database; further, through the ABTS and DPPH free radical scavenging activities, the Gastrodia elata antioxidant peptide with an amino acid sequence as shown in SEQ ID NO.2 and the Gastrodia elata antioxidant peptide with an amino acid sequence as shown in SEQ ID NO.4 are screened.

[0032] 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 medicines, foods and daily chemical products.

[0033] 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 medicines, foods and daily chemical products. As an optional embodiment of the present invention, the disease includes ischemic stroke and / or neurodegenerative diseases. As an optional embodiment of the present invention, the drug has any one or more of the following (1) to (5): (1) alleviating the decrease in cell viability caused by oxidative stress; (2) inhibiting the generation of ROS in cells induced by oxidative stress; (3) improving DPPH free radical scavenging activity; (4) improving ABTS free radical scavenging activity; (5) improving the expression level of Nrf2 protein.

[0034] The present invention provides an antioxidant product, comprising the antioxidant peptide described in the above technical solution. As an optional embodiment of the present invention, the antioxidant product comprises any one or more of medicines, foods and daily chemicals.

[0035] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] 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).

[0037] The experimental materials and reagents used in the following examples are as follows: Fresh tubers of Gastrodia elata were collected from Zhaotong City, Yunnan Province, China. Ammonium sulfate was purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd., and 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). MTS solution was from Promega Biotechnology Co., Ltd. (Beijing, China). DPPH, ABTS, and reactive oxygen species (ROS) kits were purchased from Solebo.

[0038] The instruments used in the embodiments include: a freeze dryer (Beijing Boyikang Experimental Instrument Co., Ltd.), an LC-3000 high performance liquid chromatograph (Shanghai Chuding Analytical Instrument Co., Ltd.), a centrifuge (Xiangyi Company), a dialysis bag (Solebo Company), an electrophoresis apparatus (Beijing Liuyi Instrument Factory), a cell culture incubator (BINDER Company), a clean bench (Suzhou Antai Air Technology Co., Ltd.), a 96-well plate (Wuxi Nice Biotechnology Co., Ltd.), a 6-well plate (Wuxi Nice Biotechnology Co., Ltd.), an LED inverted microscope (Leica), an electric heating blast constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd.), a low temperature refrigerator (-80°C) (Haier Special Equipment Co., Ltd.), and a flow cytometer (BD Company).

[0039] In the embodiments, each experiment was repeated at least 3 times, and all data were expressed as mean ± standard deviation (mean ± SEM). Differences among multiple groups were analyzed by one-way ANOVA. Data were analyzed using PraphPad Prism 9.4 software. P value was less than 0.05, which was statistically significant.

[0040] Example 1 Preparation and identification of Gastrodia elata antioxidant peptides 1. Preparation of Gastrodia elata protein hydrolysate (1) 500 g fresh Gastrodia elata tubers (such as Figure 1 A) was washed and crushed to obtain a homogenate, which was mixed with 5L of water and stirred for 2 h, then centrifuged at 4000 rpm for 30 min. The supernatant was collected, and 70% saturated ammonium sulfate was added to precipitate the protein at 4°C for 12 h, then centrifuged at 4000 rpm for 30 min, the precipitate was collected and re-dissolved with water, and dialyzed in distilled water with a 3500 Da dialysis bag for 24 h and then freeze-dried to obtain Gastrodia elata crude protein, which was stored in a -80°C refrigerator.

[0041] Dissolve the crude protein of Gastrodia elata in deionized water and adjust the mass concentration to 5% to obtain the crude protein solution of Gastrodia elata. Mix papain with the crude protein solution of Gastrodia elata, and add papain to the crude protein solution of Gastrodia elata at mass concentrations of 0.1%, 0.3% and 0.5% (m / v), respectively. After hydrolysis at 55°C for 4 h, heat at 95°C for 10 min to inactivate the enzyme activity, then centrifuge at 4000 xg for 30 min at 4°C to remove insoluble substances, collect the supernatant, perform SDS-PAGE analysis, and then freeze-dry for further purification. The SDS-PAGE results are shown in Figure 2. Figure 1 B. From the SDS-PAGE electrophoresis, it can be seen that the enzymatic hydrolysis effect is most significant when the mass ratio of enzyme to substrate is 0.5%, so it is selected as the enzyme concentration for preparing Gastrodia elata protein hydrolysate.

[0042] The papain and the crude protein solution of Gastrodia elata were mixed and hydrolyzed at a mass concentration of 0.5%, and freeze-dried to obtain crude protein hydrolyzate of Gastrodia elata for subsequent processes. The crude protein hydrolyzate of Gastrodia elata was ultrafiltered through a 10 kDa membrane, and the fragments less than 10 kDa were separated into 5 fragments (F1, F2, F3, F4 and F5) by gel chromatography (such as Figure 1 The purified 5 fragments were then ultrafiltered through a 3 kDa membrane, and the fractions smaller than 3 kDa were collected and recorded as F1, F2, F3, F4, and F5, which were freeze-dried and stored at -20°C.

[0043] (2) The obtained fractions F1, F2, F3, F4 and F5 with a size less than 3 kDa were collected and the ABTS free radical scavenging activity of F1, F2, F3, F4 and F5 was tested respectively. At the same time, the ABTS free radical scavenging activity of vitamin C was tested as a positive control group. The mass concentration of vitamin C was 300 μg / mL, and a PBS negative control group was set up.

[0044] ABTS free radical scavenging activity detection method: The T-AOC assay kit (ABTS) was used to detect the ABTS free radical scavenging activity. According to the requirements of the kit operation manual, the ABTS working solution was prepared in the dark at room temperature for 16 h. Then 20 μL of 5 mg / mL sample (purified fragment) was mixed with 180 μL of ABTS working solution and reacted in the dark at room temperature for 5 minutes. The ABTS free radical scavenging rate was detected at a wavelength of 734 nm using a microplate reader. The test results are shown in Tables 1 and Figure 1 The D in the figure and the Vc in the figure represent vitamin C.

[0045] Table 1 Effect of purified <3 kD fragments on ABTS free radical scavenging activity

[0046] After the F1 fragment was ultrafiltered with a 3kDa membrane, the amount of fragments less than 3kDa was too small. Therefore, only F2, F3, F4 and F5 less than 3 kDa were subsequently tested for DPPH free radical scavenging ability, and the DPPH free radical scavenging activity of vitamin C was tested as a positive control group, with a mass concentration of vitamin C of 500μg / mL, and a PBS negative control group was set up.

[0047] DPPH free radical scavenging activity assay: 30 μL of 5 mg / mL sample (purified fragment) was mixed with 120 μL of DPPH solution and incubated at 37°C in the dark for 60 min. The DPPH free radical scavenging activity was measured at a wavelength of 515 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). The test results are shown in Table 2 and Figure 1 E and Figure 1 As shown in F, NC in F is the test result of PBS negative control group; PC is the test result of vitamin C positive control group.

[0048] Table 2 Effect of purified <3 kD fragments on DPPH free radical scavenging activity

[0049] As shown in Table 1~2, Figure 1 As shown in Figures D and E, the F2 fragment less than 3 kDa showed significant antioxidant activity, with high ABTS and DPPH free radical scavenging activities. Figure 1 The dashed arrow in C showed significant antioxidant activity. Therefore, LC-MS / MS analysis of the <3 kDa fragment in F2 was performed to determine the amino acid sequence of the peptide.

[0050] (3) LC-MS / MS analysis of the <3 kDa fragment in F2 was performed. The results are as follows Figure 1 G in the figure and Vc in the figure represent vitamin C, the same below.

[0051] The TIC spectrum of the fragment <3 kDa in F2 is as follows Figure 2 shown.

[0052] A total of 78 peptide chains were identified from the F2 fraction less than 3 kDa. Among the identified peptides, there were 3, 55, and 20 peptides with molecular weights between 2000-3000 Da, 1000-2000 Da, and <1000 Da, respectively ( Figure 1 The molecular weights of the identified peptides were mainly less than 2000 Da.

[0053] 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 peptides. Among the 20 peptides with a molecular weight of less than 1000 Da, 3 peptides had a score greater than 0.5. Among the 55 peptides with a molecular weight between 1000 and 2000 Da, 30 peptides had a score greater than 0.5. However, in the peptide group with a molecular weight greater than 2000 Da, no peptides with a score greater than 0.5 were found ( Figure 1 This suggests that small molecular weight peptides may have more potential functional activities.

[0054] Next, the toxicity of the seven 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 seven candidate peptides, all identified peptide sequences were searched in the SciFinder and BIOPEP databases to determine their novelty. After searching these peptides in these two databases, it was found that these peptides were not found; therefore, the seven peptides identified from Gastrodia elata were novel and chemically synthesized (Table 3).

[0055] Table 3 7 candidate peptides

[0056] Example 2 Chemical synthesis of Gastrodia elata antioxidant peptides and their antioxidant activity characteristics 1. Evaluation of Antioxidant Activity of Chemically Synthesized Peptides The antioxidant activity of the 7 synthetic peptides in Table 3 was tested using the antioxidant activity evaluation method in Example 1. The DPPH and ABTS free 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. The DPPH and ABTS free radical scavenging activities of vitamin C were also tested as a positive control, and the mass concentration of vitamin C was 400 μg / mL.

[0057] The antioxidant activity evaluation results of chemically synthesized peptides are shown in Tables 4~5 and Fig. 9 As shown in A~B.

[0058] Table 4 Effects of different concentrations of 7 synthetic peptides on DPPH free radical scavenging activity

[0059] Table 5 Effects of different concentrations of seven synthetic peptides on ABTS free radical scavenging activity As shown in Tables 4~5 and Fig. 9 As shown in Figures A to B, compared with other synthetic peptides, synthetic peptides PR-9 and CT-10 showed higher DPPH and ABTS free radical scavenging activities, and the antioxidant activity was concentration-dependent. Therefore, the two peptides PR-9 and CT-10 were selected to further study their antioxidant properties. Among them, the liquid chromatogram of peptide PR-9 is shown in Figure 2. Figure 3 The liquid chromatography of peptide CT-10 is shown in Figure 4 The mass spectrum of peptide PR-9 is shown in Figure 5 As shown, the structural formula is Figure 6 The mass spectrum of peptide CT-10 is shown in Figure 7 As shown, the structural formula is Figure 8 shown.

[0060] 2. Thermal stability of antioxidant peptides PR-9 and CT-10 The antioxidant peptides PR-9 and CT-10 were prepared with PBS solvent to an initial concentration of 2 mg / mL, treated at 25°C, 50°C, 70°C, and 90°C for 30 min, 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, reacted at 37°C for 1 h, and the absorbance of each well was read at 515 nm using a microplate reader. The DPPH free radical scavenging ability was measured at a wavelength of 515 nm using a microplate reader to evaluate the thermal stability of the peptides.

[0061] The thermal stability test results of antioxidant peptides PR-9 and CT-10 are shown in Tables 6 and Fig. 9 As shown in C.

[0062] Table 6 Thermal stability test results of antioxidant peptides PR-9 and CT-10

[0063] As shown in Table 6 and Fig. 9 As shown in C, the DPPH free radical scavenging activity of peptides PR-9 and CT-10 changed little with increasing temperature, while the antioxidant activity did not change much. The above results indicate that peptides PR-9 and CT-10 have high thermal stability in vitro.

[0064] 3. Hemolytic activity of antioxidant peptides PR-9 and CT-10 A 5% (adjusted with PBS) rabbit erythrocyte suspension was mixed with an equal volume of antioxidant peptides (final concentration of 400 μg / mL) and incubated at 37°C for 1 h. An equal volume of PBS was used instead of antioxidant peptides to mix with a 5% rabbit erythrocyte suspension as a negative control; an equal volume of 0.5% Triton X-100 was used instead of antioxidant peptides to mix with a 5% rabbit erythrocyte suspension as a positive control. At the same time, PBS without rabbit erythrocyte suspension was used as a blank group.

[0065] After incubation, the supernatant was collected after centrifugation 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: Hemolysis rate (%) = [(Asample-A0) / (A1-A0)] × 100; Asample is the OD415 nm of the peptide solution, A0 is the OD415 nm of the blank group PBS, and A1 is the OD415 nm of 0.5% Triton X-100.

[0066] The results of the hemolytic activity test of the antioxidant peptides PR-9 and CT-10 are shown in Tables 7 and Fig. 9 As shown in D~F.

[0067] Table 7 Hemolytic activity test results of antioxidant peptides PR-9 and CT-10

[0068] Note: NC is the hemolysis rate of negative control; PC is the hemolysis rate of positive control.

[0069] As shown in Table 7 and Fig. 9As shown in D to F, compared with the PBS-treated group, peptides PR-9 and CT-10 had no hemolytic activity, which is consistent with the predicted toxicity of peptides PR-9 and CT-10 determined using the above database. The above results show that peptides PR-9 and CT-10 have high safety in vitro.

[0070] Example 3 Neuroprotective effect and mechanism of Gastrodia elata antioxidant peptides 1.H 2 O 2 Stimulated PC12 cell viability assay PC-12 cells were cultured in serum-containing medium (RPMI1640 medium containing fetal bovine serum) at a rate of 1×10 4 The cells were seeded in a 96-well plate at a density of 100 μM and cultured for more than 8 h. 2 O 2 The cells were stimulated for 2 h by adding the above-mentioned different concentrations of H 2 O 2 The cells were stimulated and cultured for 2 h at 37°C, 5% CO 2 The corresponding culture stimulation process was carried out in the incubator. Then 100 μL of MTS working solution was added to each well. After incubation at 37℃ for 2 h, the absorbance of each well was monitored at a wavelength of 490 nm.

[0071] The FlexStatonTM3 multi-mode microplate reader was used to measure the absorbance at 490 nm, and the cell viability was calculated according to the following formula. Cell viability (%) = (OD sample - OD blank) / (OD control group - OD blank) × 100, where the OD value is the absorbance of each well monitored at 490 nm. The OD blank is the absorbance value of the treatment group with only culture medium added but no cells; the OD control group is the treatment group with cells added but no H added. 2 O 2 The absorbance test results of the negative control group of the solution; OD samples are different concentrations of H 2 O 2 Absorbance test results of the stimulation group. In the above experiment, each treatment group was subjected to 4 parallel tests. The OD blank was 0.049 at a wavelength of 490 nm.

[0072] The results are shown in Tables 8~9 and Fig.10 As shown in A.

[0073] Table 8 OD values ​​of each treatment group at 490 nm wavelength

[0074] Table 9 Different concentrations of H 2 O 2 Effects on PC12 cell viability

[0075] From Tables 8-9, we can see that 400 μM H 2 O 2 The solution can induce a 20% decrease in PC12 cell viability, which is a suitable oxidative stress model. 2 O 2 Under solution treatment, more than half of the cells were dead and were not suitable for preparing oxidative stress models. 2 O 2 The solution had little effect on cell viability and was not sufficient to induce the oxidative stress model. Therefore, 400 μM H 2 O 2 The oxidative stress model was prepared by using the solution and the corresponding experiments were carried out.

[0076] 2. Using serum-containing culture medium (RPMI1640 culture medium containing fetal bovine serum) as the culture medium, PC-12 cells were cultured at a rate of 1×10 4 The cells were seeded at a density of 100 μg / mL in a 96-well plate and cultured for more than 8 h. The cells were pretreated with different concentrations of PR-9 and CT-10 (0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) for 24 h, that is, different concentrations of PR-9 were added to RPMI1640 culture medium or different concentrations of CT-10 were added to RPMI1640 culture medium for 24 h. After the treatment, 400 μM H 2 O 2 Stimulate cells for 2 h at 37°C, 5% CO 2 The corresponding culture stimulation process was carried out in the incubator. At the same time, a PBS control group was set up, that is, PBS was used instead of PR-9 or CT-10, and no H 2 O 2 Stimulation. Then add 100µL of MTS working solution to each well. After incubation at 37℃ for 2 h, monitor the absorbance of each well at 490 nm.

[0077] The FlexStatonTM3 multi-mode microplate reader was used to measure the absorbance at 490 nm, and the cell viability was calculated according to the following formula. Cell viability (%) = (OD sample - OD blank) / (OD control group - OD blank) × 100, where the OD value is the absorbance of each well monitored at 490 nm. The OD blank is the absorbance value of the treatment group with only culture medium added but no cells; the OD control group is the treatment group with cells added but no peptide and H added.2 O 2 The absorbance value detection result of the negative control group is shown in Figure 1; the absorbance value detection result of the OD sample is shown in Figure 2 for each experimental group. In the above experiments, each treatment group was subjected to 3 parallel experiments, the same below.

[0078] The results are shown in Tables 10~13 and Fig.10 As shown in B~C.

[0079] Table 10 OD values ​​of each treatment group at 490 nm wavelength Table 11 Effect of peptide PR-9 on H 2 O 2 Effects of stimulation on PC12 cell viability

[0080] Table 12 OD values ​​of each treatment group at 490 nm wavelength

[0081] Table 13 Effect of peptide CT-10 on H 2 O 2 Effects of stimulation on PC12 cell viability

[0082] From Tables 10~13 and Fig.10 From B~C in the figure, we can see that 2 O 2 Compared with the treatment groups, peptides PR-9 and CT-10 increased cell viability in a dose-dependent manner, especially 200 μg / mL PR-9 and CT-10 treatment could increase cell viability by about 17% and 19%, respectively.

[0083] 3. ROS detection: PC-12 cells were cultured in serum-containing medium (RPMI1640 medium containing fetal bovine serum) at a rate of 5×10 5 The cells were inoculated into 6-well plates at a density of 100 μg / mL and cultured for more than 8 h. 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, i.e., 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 treatment, the cells were treated with 400 μM H 2 O 2 Stimulation for 2 h at 37°C, 5% CO 2The corresponding culture stimulation process was carried out in the incubator. Then, the cell supernatant was removed and washed twice with PBS. Subsequently, the cells were incubated with 10 μM DCFH-DA (saolibio, Shanghai, China) at 37°C in the dark for 20 min, and then the cells were collected and washed with PBS. Finally, the fluorescence intensity of DCFH-DA was measured by flow cytometry at 480 nm (excitation) and 530 nm (emission). ROS content was compared with PR-9 and CT-10 and H 2 O 2 The mean fluorescence intensity of the treatment groups is compared.

[0084] The results are shown in Tables 14~15 and Fig.10 D~F in.

[0085] Table 14 Flow cytometry detection of H 2 O 2 ROS content in stimulated PC12 cells

[0086] Table 15 Flow cytometry detection of H 2 O 2 ROS content in stimulated PC12 cells

[0087] As shown in Tables 14~15 and Fig.10 As shown in D~F, in H 2 O 2 In the PC12 cell injury model induced by H2O2, peptides PR-9 and CT-10 could significantly inhibit the generation of ROS (p<0.001). These results indicate that peptides PR-9 and CT-10 can effectively inhibit H2O2 2 O 2 induced the production of ROS in PC12 cells, thereby providing antioxidant protection to the cells.

[0088] 4. Immunoblotting Analysis Methods: PC-12 cells were treated as described in step 3 of Example 3. After drug treatment, cells were rinsed twice with cold PBS and lysed on ice for 30 min with RIPA lysis buffer containing 1 mM PMSF. Samples were separated by 10% SDS-PAGE, transferred to PVDF (0.45 mm pore size) membranes, blocked with 5% skim milk powder, and incubated with the following primary antibodies: Nrf2 (1:1000, #4621, CST) anti-GAPDH (1:5000, ab181602, Abcam). Then, they were incubated with the following horseradish peroxidase-labeled secondary antibodies, goat anti-rabbit IgG H&L (1:5000, ab6721, Abcam). Finally, immunoblot bands were visualized (Trans-Blot Turbo System, BIO-RAD) using chemiluminescence (ECL) detection reagents (Tiangen, Beijing, China), and signal intensity was analyzed using Image J software. Among them, PBS is the result of PBS control group, that is, PBS was used instead of PR-9 or CT-10, and H 2 O 2 Stimulate.

[0089] The results are shown in Tables 16~17 and Fig.10 In G~I.

[0090] Table 16 PR-9 vs H 2 O 2 Effect of stimulation on Nrf2 protein expression level in PC12 cells

[0091] Table 17 CT-10 to H 2 O 2 Effect of stimulation on Nrf2 protein expression level in PC12 cells

[0092] For H 2 O 2 Western blot analysis was performed to analyze the protein expression of Nrf2 in the PC12 cell injury model induced by β-lactamase. Fig.10 As shown in G~I, peptides PR-9 and CT-10 significantly increased the expression level of Nrf2 protein. These results indicate that peptides PR-9 and CT-10 activate the Nrf2 pathway to regulate H 2 O 2 The induced PC12 cell injury has a neuroprotective effect.

[0093] In summary, the present invention identified two novel antioxidant peptides from fresh Gastrodia elata protein hydrolysate through enzymatic hydrolysis, ultrafiltration, gel chromatography purification and mass spectrometry technology, combined with ABTS and DPPH free radical scavenging activity. They have high antioxidant activity, thermal stability and in vitro safety, can effectively alleviate the decrease in cell viability and increase in ROS levels caused by oxidative stress damage, and can exert a neuroprotective effect by activating the Nrf2 pathway.

[0094] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

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

4.

2. The method for preparing the antioxidant peptide according to claim 1, characterized in that: include: Mixing the crushed Gastrodia elata tubers with water to dissolve them, and collecting the supernatant; After the protein in the supernatant is precipitated, the obtained protein is dialyzed to obtain crude Gastrodia elata protein; The crude protein of Gastrodia elata is enzymatically hydrolyzed by using papain to obtain the crude protein hydrolyzate of Gastrodia elata; After ultrafiltration of crude protein hydrolysate from Gastrodia elata, fragments smaller than 3 kDa were separated and identified to obtain antioxidant peptides.

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

4. The preparation method according to claim 2, characterized in that: The method for precipitating protein in the supernatant comprises: mixing the supernatant with saturated ammonium sulfate to precipitate the protein.

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 to 4 in the preparation of products with antioxidant and / or neuroprotective effects.

6. 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 to 4 in the preparation of products for preventing and / or treating diseases caused by oxidative stress.

7. The use according to claim 6, characterized in that: The diseases include ischemic stroke and / or neurodegenerative diseases.

8. The use according to claim 5 or 6, characterized in that: The drug is used for any one or more of the following (1) to (5): (1) Alleviate the decrease in cell viability caused by oxidative stress; (2) Alleviate the increase of ROS in cells induced by oxidative stress; (3) Improve DPPH free radical scavenging activity; (4) Improve the ABTS free radical scavenging activity; (5) Increased the expression level of Nrf2 protein.

9. An antioxidant product, characterized in that: The invention comprises the antioxidant peptide according to claim 1 or the antioxidant peptide prepared by the preparation method according to any one of claims 2 to 4.

10. The antioxidant product according to claim 9, characterized in that: The antioxidant products include any one or more of medicines, foods and daily chemical products.

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