Application of eel peptide in preparing antioxidant products

Abstract

The present invention provides an application of an eel peptide in the preparation of an antioxidant product, belonging to the technical field of antioxidant active peptides. The present invention proposes for the first time that 12 eel peptides with amino acid sequences such as SEQ ID NO.1-SEQ ID NO.12 have antioxidant biological activity, and the antioxidant properties are stronger than the standard antioxidant 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), and its ABTS free radical scavenging ability and oxygen free radical absorption capacity (ORAC) are 1.17-3.28 times and 2.03-5.67 times that of Trolox, respectively. The eel antioxidant peptide provided by the present invention is an effective functional antioxidant, which can be used as a natural antioxidant in the food and pharmaceutical industries.

Description

Technical Field

[0001] The invention belongs to the technical field of antioxidant active peptides, and in particular relates to an application of an eel peptide in the preparation of an antioxidant product. Background Art

[0002] In organisms, excessive production of free radicals can lead to oxidative stress, which damages the DNA, proteins and lipids of cells. This complex process is closely related to the occurrence and development of many diseases. Therefore, maintaining the balance of free radicals is essential for health and overall balance. At the same time, oxidation caused by free radicals will also have a direct impact on food quality, changing the nutrition, flavor and taste of food. Antioxidants can provide electrons for free radicals, thereby reducing oxidative stress in organisms and food systems. Compared with chemically synthesized drugs, the adverse effects of natural antioxidants are minimal or non-existent. Therefore, natural antioxidants have great potential and broad prospects in the fields of medicine and food.

[0003] As a class of natural antioxidants, antioxidant peptides have good water solubility, high stability, easy absorption and no immunoreactivity, and have been widely studied in recent years. Through enzymatic hydrolysis, the nutritional components of the raw materials can be retained, while the solubility and functionality of the protein can be improved. In order to identify the antioxidant peptides in the enzymatic hydrolysis process, a series of separation, purification and identification experiments focusing on in vitro antioxidant activity are usually used. However, this method is time-consuming and labor-intensive, and active peptides are easily overlooked. As a high-protein, low-fat fish, the eel has a rich variety of protein amino acids and high nutritional value, but there is currently no research on the antioxidant peptides of the eel and how to obtain the antioxidant peptides of the eel quickly, efficiently and accurately. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an application of eel peptide in the preparation of antioxidant products. Another object of the present invention is to provide a method for preparing eel antioxidant peptide quickly, efficiently and accurately.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an application of an eel peptide in the preparation of an antioxidant product. The eel peptide comprises at least one of the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.12.

[0007] Preferably, the antioxidant product includes antioxidant medicines, food antioxidants or cosmetics.

[0008] The invention also provides a method for preparing an eel peptide with an antioxidant effect, comprising the following steps: enzymolyzing eels with Alcalase alkaline protease to obtain an eel hydrolysate; separating and purifying the eel hydrolysate with gel column chromatography, collecting eluates according to peak types, and selecting eluates with the best antioxidant activity for UPLC-Q-TOF-MS / MS identification; predicting the biological activity score of the identified polypeptide with a PeptideRanker database, predicting the potential allergenicity, potential toxicity and physicochemical properties of the identified polypeptide with AllerTOP and ToxinPred databases, searching the BIOPEP and Uniprot databases for the predicted antioxidant activity of the identified polypeptide, and selecting an eel peptide with a predicted value higher than 0.5, no allergenicity, no toxicity, a ratio of antioxidant amino acids Y and W greater than 30%, and a biological activity score higher than 0.6; the eel peptide comprises an amino acid sequence such as at least one of SEQ ID NO.1-SEQ ID NO.12.

[0009] Preferably, the temperature of the enzymolysis is 50-60°C, the pH of the enzymolysis is 7.5-8.5, and the time of the enzymolysis is 2.5-5h.

[0010] Preferably, the amount of Alcalase alkaline protease used is 3-5g Alcalase alkaline protease per 100g rice field eel.

[0011] Preferably, the gel column is a Sephadex G-25 gel chromatography column.

[0012] Preferably, the chromatographic column model used for UPLC-Q-TOF-MS / MS identification is ACQUITYUPLC HSS T3, and the column temperature is 40-50°C.

[0013] Preferably, the mobile phase A for UPLC-Q-TOF-MS / MS identification is an aqueous solution containing 0.1% formic acid, the mobile phase B is an 80% acetonitrile solution, the flow rate is set to 0.4 mL / min, and the gradient elution program is: 0-2.0 min, 92.0% A; 2-45 min, 92.0-72.0% A; 45.0-55.0 min, 72.0-60.0% A; 55.0-56.0 min, 60.0-5.0% A; 56.0-66.0 min, 5.0% A.

[0014] The present invention also provides an antioxidant product, wherein the active ingredient of the antioxidant product comprises at least one of the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.12.

[0015] Beneficial effects of the present invention:

[0016] The present invention proposes for the first time that 12 yellow eel peptides with amino acid sequences as shown in SEQ ID NO.1-SEQ ID NO.12 have antioxidant biological activity, and their antioxidant properties are stronger than the standard antioxidant 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), and their ABTS free radical scavenging ability and oxygen free radical absorption capacity (ORAC) are 1.17-3.28 times and 2.03-5.67 times that of Trolox, respectively. The yellow eel antioxidant peptide provided by the present invention is an effective functional antioxidant, which can be used as a natural antioxidant in the food and pharmaceutical industries.

[0017] The present invention also proposes for the first time that the nitrogen-hydrogen bond in the indole ring of tryptophan is the key site of the antioxidant activity of the above 12 eel peptides, and establishes an AAPH-induced human red blood cell oxidative damage model. The results show that the above 12 peptides have a protective effect on the oxidative damage of red blood cells, and exert their antioxidant capacity by increasing the intracellular GSH content and reducing the activity of SOD, CAT and GSH-Px enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The antioxidant activity, protein recovery rate and protein hydrolysis degree of different enzymatic hydrolysates are shown in Figure 1, where A is the antioxidant activity result, and B is the protein recovery rate and protein hydrolysis degree result. Note: The letters on the bar indicate whether there is a significant difference. Different letters mean there is a difference. One indicator uses lowercase letters, and the other indicator uses uppercase letters.

[0019] Figure 2 The chromatograms of the Alcalase hydrolysate of the eel after separation by Sephadex G-25 and the antioxidant activity results of each component, wherein (A) is the chromatogram, F1 (tubes 12-28), F2 (tubes 29-46), F3 (tubes 47-60), F4 (tubes 61-73), F5 (tubes 74-93), (B) is the antioxidant activity results, wherein the significant differences in ABTS activity of different groups are represented by lowercase letters; the significant differences in ORAC activity of different groups are represented by uppercase letters, (p < 0.05), the Alcalase group in the figure refers to the hydrolysate before separation;

[0020] Figure 3 The statistical analysis results of the eel peptides, where (A) is the distribution of peptide numbers (outer ring) and the distribution of amino acid properties of the constituent amino acids (inner ring), (B) is the composition and distribution of amino acids, and the three groups represent: the amino acid composition of the peptide nitrogen end, the amino acid composition in the middle, and the amino acid composition of the peptide carbon end, and (C) is the amino acid distribution of peptides of different lengths;

[0021] Figure 4The results of active zone analysis of twelve peptides based on HOMO and Mulliken charge distribution and bond length;

[0022] Figure 5 The molecular docking results of the twelve peptides and MPO;

[0023] Figure 6 The results are as follows: the protective effects of twelve peptides on AAPH-induced erythrocyte hemolysis (scanning electron microscope micrographs of erythrocytes);

[0024] Figure 7 This is a possible intracellular antioxidant mechanism by which the peptides of the present invention alleviate AAPH-induced oxidative damage. DETAILED DESCRIPTION

[0025] The present invention provides an application of an eel peptide in the preparation of an antioxidant product. The eel peptide comprises at least one of the following peptide segments: WGWP (SEQ ID NO.1), DHPWH (SEQ ID NO.2), LLGPW (SEQ ID NO.3), VLYPW (SEQ ID NO.4), VYGPW (SEQ ID NO.5), WGGPL (SEQ ID NO.6), WPDAR (SEQ ID NO.7), WPYVT (SEQ ID NO.8), LLVYPW (SEQ ID NO.9), WDGTGR (SEQ ID NO.10), PSWVPPA (SEQ ID NO.11) and WGDLSPK (SEQ ID NO.12).

[0026] In the present invention, the antioxidant product preferably includes antioxidant drugs, food antioxidants or cosmetics.

[0027] The invention also provides a method for preparing an eel peptide with an antioxidant effect, comprising the following steps: enzymolyzing eels with Alcalase alkaline protease to obtain an eel hydrolysate; separating and purifying the eel hydrolysate with gel column chromatography, collecting eluates according to peak types, and selecting eluates with the best antioxidant activity for UPLC-Q-TOF-MS / MS identification; predicting the biological activity score of the identified polypeptide with a PeptideRanker database, predicting the potential allergenicity, potential toxicity and physicochemical properties of the identified polypeptide with AllerTOP and ToxinPred databases, searching the BIOPEP and Uniprot databases for the predicted antioxidant activity of the identified polypeptide, and selecting an eel peptide with a predicted value higher than 0.5, no allergenicity, no toxicity, a ratio of antioxidant amino acids Y and W greater than 30%, and a biological activity score higher than 0.6; the eel peptide comprises an amino acid sequence such as at least one of SEQ ID NO.1-SEQ ID NO.12.

[0028] The present invention uses computer simulation screening to evaluate the potential biological activity, physicochemical properties, toxicological properties, digestive stability and bioavailability of peptides by using a series of databases, and then screens the target peptides. The method provided by the present invention is efficient and accurate, and can more effectively discover and utilize antioxidant peptides. The present invention infers the antioxidant active sites of the above 12 peptides through quantum chemical analysis, and verifies the accuracy of the active sites by methylating the active sites. At the same time, the antioxidant mechanism of these 12 peptides is elucidated by molecular docking. It is crucial to understand the correlation between antioxidant capacity and the structural, physical and chemical properties of antioxidants. By deeply understanding these associations, new antioxidants can be more effectively designed and developed, and their activity and bioavailability in vivo can be optimized. In addition, the present invention establishes an AAPH-induced human red blood cell oxidative damage model, and the study finds that the 12 peptides have a protective effect on the oxidative damage of red blood cells, and the mechanism by which they exert a protective effect is explored. The results of the present invention are of great significance to the development of eel peptide resources and the potential application of antioxidant peptides in functional foods.

[0029] In the present invention, the temperature of the enzymolysis is preferably 50-60°C, more preferably 55°C, the pH of the enzymolysis is preferably 7.5-8.5, more preferably 8.0, the time of the enzymolysis is preferably 2.5-5h, more preferably 3h; the amount of the Alcalase alkaline protease is preferably 3-5g of Alcalase alkaline protease per 100g of the eel, more preferably 4g of Alcalase alkaline protease per 100g of the eel. In the present invention, preferably the eel is first made into eel minced meat, and then enzymolyzed with Alcalase alkaline protease. The present invention has no special limitation on the specific method of making the eel into eel minced meat.

[0030] In the present invention, when the eel hydrolysate is separated and purified, a Sephadex G-25 gel chromatography column is preferably used, eluted with deionized water, the absorbance is detected at wavelengths of 214nm and 254nm, the eluate is collected according to the peak shape, and the component with the best antioxidant activity is selected for subsequent UPLC-Q-TOF-MS / MS mass spectrometry identification. The chromatographic column model used for the UPLC-Q-TOF-MS / MS identification is preferably ACQUITYUPLC HSS T3, and the column temperature is preferably 40-50°C, more preferably 45°C. The mobile phase A for UPLC-Q-TOF-MS / MS identification is preferably an aqueous solution containing 0.1% formic acid, and the mobile phase B is preferably an acetonitrile solution of 80%. The flow rate is set to 0.4 mL / min, and the gradient elution program is: 0-2.0 min, 92.0% A; 2-45 min, 92.0-72.0% A; 45.0-55.0 min, 72.0-60.0% A; 55.0-56.0 min, 60.0-5.0% A; 56.0-66.0 min, 5.0% A. In the above elution program, when the mobile phase A is 92%, it indicates that the mobile phase B is 8%, and so on. Electrospray ionization (ESI) is used as the ionization mode, and the scan type is set to MSE. The mass spectrometer scanning range is preferably 50-2000 m / z, and the wavelength range is preferably 201-799 nm. The present invention does not specifically limit the specific operation methods of each database used in the screening of antioxidant peptides, and the general operation methods of the database can be used.

[0031] The present invention also provides an antioxidant product, wherein the active ingredient of the antioxidant product comprises at least one of the amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 12. In the present invention, the antioxidant product is preferably an antioxidant drug, a food antioxidant or an antioxidant cosmetic.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] All data in the following examples are mean values ​​± standard deviations of three to seven independent experiments. The data were analyzed using PRISM 6 statistical software for one-way analysis of variance (ANOVA), and Dunnett's multiple comparison method was used for significant difference analysis.

[0036] The sources of the materials used in the following examples are as follows: Ricefield eel was obtained from Zhuangxing Base of Shanghai Academy of Agricultural Sciences (Shanghai, China). Human red blood cells (1%) were purchased from Cenxi Hongquan Biotechnology Co., Ltd. Composite protease, papain, neutral protease, Alcalase and flavor protease were purchased from Beijing Dahong Lihui Biotechnology Center. 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) and 2,2'-azo[2-methylpropionamide] dihydrochloride (AAPH) were purchased from Beijing Yishan Huitong Technology Co., Ltd. 2,2-Azo-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) and fluorescein sodium were purchased from Beijing Zhongcheng Jinnian Technology Co., Ltd. Sephadex G-25 was purchased from Shanghai Yuanye Biotechnology Co., Ltd. The synthesized peptides and active site methylated peptides were purchased from Nanjing Jie Peptide Biotechnology Co., Ltd., including AVLW (SEQ ID NO.14), VPWP (SEQ ID NO.15), VWPS (SEQ ID NO.16), WGWP (SEQ ID NO.1), DHPWH (SEQ ID NO.2), LLGPW (SEQ ID NO.3), VLYPW (SEQ ID NO.4), VYGPW (SEQ ID NO.5), WGGPL (SEQ ID NO.6), WPDAR (SEQ ID NO.7), WPYVT (SEQ ID NO.8), LLVYPW (SEQ ID NO.9), WDGTGR (SEQ ID NO.10), PSWVPPA (SEQ ID NO.11), WGDLSPK (SEQ ID NO.12) and HWDGSLPR (SEQ ID NO.13), with a purity of more than 95%.

[0037] BCA total protein quantitative detection kit, catalase (CAT) detection kit, plant malondialdehyde (MDA) detection kit, lactate dehydrogenase (LDH) detection kit, glutathione peroxidase (GSH-Px) detection kit, superoxide dismutase (SOD) enzyme activity detection kit, reduced glutathione (GSH) detection kit and oxidized glutathione (GSSG) detection kit were purchased from Nanjing Jiancheng Haihao Biotechnology Co., Ltd. (Nanjing, China). All other reagents used in the following examples are of analytical grade and are sourced from China.

[0038] Example 1

[0039] First, 60g of minced eel meat was dispersed in 200mL of water, and then the pH was adjusted to 8.0. Subsequently, the corresponding Alcalase alkaline protease was added at a ratio of 4% of the mass of the eel, and the enzymatic reaction was carried out at a pH of 8.0 and a temperature of 55°C for 3 hours. To terminate the enzymatic reaction, the enzymatic solution was placed at 100°C to inactivate the enzyme for 10 minutes and then cooled to room temperature. Next, the enzymatic solution was separated by centrifugation, and the supernatant was collected and freeze-dried for later use.

[0040] Comparative Example 1

[0041] The difference from Example 1 is that the enzymes used are replaced by neutral protease (pH 7.0 and 50°C, Neutrase), flavor protease (pH 7.0 and 55°C, Flavorzyme), papain (pH 7.0 and 55°C, Papain) and composite protease (pH 7.0 and 55°C, Protease M), respectively. The enzymolysis conditions of the corresponding enzymes are shown in brackets, and the rest are the same as Example 1.

[0042] Example 2

[0043] Comparison of the antioxidant activity, protein recovery rate and protein hydrolysis degree of different enzymatic hydrolysates of Example 1 and Comparative Example 1, the specific test method is as follows:

[0044] (1) Determination of degree of hydrolysis (DH)

[0045] OPA reagent (200 mL) was prepared as follows: 160.0 mg OPA, 7.62 g borax were added to 4.0 mL ethanol, 200.0 mg SDS and 176.0 mg DTT were added to 150 mL deionized water. The mixture was then mixed with OPA reagent and diluted to a final volume of 200 mL with deionized water. 20 μL of sample solution was thoroughly mixed with 150 μL OPA reagent. After an accurate reaction time of 2 min, the absorbance at 340 nm (A sample Deionized water was used as blank control (A blank ), and a standard reference group (A standard ). The serine NH2 equivalent (SerNH2) and DH of the sample were calculated as follows:

[0046]

[0047]

[0048] Among them C sample (g / L) represents the protein concentration of the sample, α, β, h totThe values ​​of are 1.0, 0.4 and 8.6 mmol / g respectively. In addition, the molar concentration of 0.1 mg / mL serine is 0.9516 mM.

[0049] (2) Determination of protein recovery rate

[0050] The protein content of the enzymatic hydrolysate was determined by the Kjeldahl method in GB 5009.5-2016, and the recovery volume of the enzymatic hydrolysate was determined at the same time. The protein recovery rate was calculated according to the following formula.

[0051]

[0052] (3) Antioxidant activity assay

[0053] 1) Determination of ABTS free radical scavenging ability

[0054] ABTS (7 mM) solution and potassium persulfate (2.45 mM) were mixed in equal volumes and incubated in the dark at room temperature for 12-16 h to prepare ABTS· + Before use, ABTS· + The stock solution was appropriately diluted to make its absorbance at 734 nm 0.700 ± 0.020. 150 μL of the diluted ABTS· + The stock solution was added to 50 μL of the sample, incubated at 30°C for 30 min, and the absorbance at 734 nm was measured. PBS and Trolox were used as blank control and positive control, respectively. The calculation of ABTS free radical scavenging activity is as follows:

[0055]

[0056] Among them, A sample and A control The results are expressed as Trolox equivalents (μmol TE / μmol) of sample.

[0057] 2) Determination of oxygen radical absorbance capacity (ORAC)

[0058] Mix 20 μL of sample and 120 μL of sodium fluorescein (117 nM) and place at 37 ° C in the dark for 15 minutes. After adding 60 μL of AAPH (40 mM), the fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm, respectively, and the measurement was performed every 1 minute for a total of 100 minutes. PBS and Trolox were used as blank controls and positive controls, respectively. Both FL and AAPH solutions were prepared with 75 mM PBS (pH 7.4). The fluorescence intensity calculation formula of ORAC is as follows:

[0059]

[0060] Net AUC=AUC sample -AUC blank

[0061] where f i is the fluorescence intensity at the nth measurement point, f0 is the fluorescence intensity at the initial time, and AUC represents the area under the fluorescence decay curve. The ORAC value can be calculated based on the regression equation between the net fluorescence decay area and the antioxidant concentration, and the result is expressed as Trolox equivalent (μmolTE / μmol sample).

[0062] The results are as follows Figure 1 Compared with the other four enzymatic hydrolysates, the DH and protein recovery rates and antioxidant activity of the Alcalase enzymatic hydrolysate were significantly higher (p<0.05).

[0063] Example 3

[0064] (1) After obtaining the enzymatic hydrolysate according to the method of Example 1, the eel enzymatic hydrolysate was separated and purified using a Sephadex G-25 gel chromatography column (3.7×27.8 cm), eluted with deionized water, and the absorbance was detected at wavelengths of 214 nm and 254 nm. The eluate was collected according to the peak shape. The results are as follows: Figure 2 As shown in (A), the collected eluate was subjected to antioxidant activity assay according to the method of (3) antioxidant activity assay in Example 2. The results are shown in Figure 2 (B) The results showed that the antioxidant activity of F3 was significantly higher.

[0065] (2) UPLC-Q-TOF-MS / MS (TripleTOP5600+LC / MS system, ABSCIEX, USA) was used to identify the sequence of the active peptide in F3. The chromatographic column model was ACQUITYUPLC HST3, and the column temperature was maintained at 45°C. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an 80% acetonitrile solution. The flow rate was set to 0.4 mL / min. The gradient elution program was as follows: 0-2.0 min, 92.0% A; 2-45 min, 92.0-72.0% A; 45.0-55.0 min, 72.0-60.0% A; 55.0-56.0 min, 60.0-5.0% A; 56.0-66.0 min, 5.0% A. Electrospray ionization (ESI) was used as the ionization mode, and the scan type was set to MSE. The mass spectrometer scan range was 50-2000 m / z and the wavelength range was 201-799 nm. The acquired data were analyzed using the Mascot database. Using a filter setting with a threshold of ≥85%, 245 peptides were identified from F3 by UPLC-Q-TOF-MS / MS. The length of the peptides analyzed by the Mascot database and the nature and distribution of the constituent amino acids are shown in Figure 3 .

[0066] The results showed that these peptides were mainly composed of pentapeptides (18%), hexapeptides (23%), heptapeptides (21%) and octapeptides (15%). Figure 3 (A)-external ring). Among them, hydrophobic amino acids account for the highest proportion, accounting for 53%, followed by polar uncharged amino acids, accounting for 22%. Basic amino acids account for 18%, and acidic amino acids account for 7% ( Figure 3 (A) - inner ring). From Figure 3 (B) It can be seen that the constituent amino acids of these peptides include 20 different amino acids. Among them, L, H, P, T and V are the top five amino acids with relatively high frequencies, accounting for 12.01%, 9.95%, 9.42%, 8.36% and 8.12%, respectively. Wen et al. (2020) emphasized that hydrophobic amino acids (such as L, P and V) can enhance antioxidant activity, especially when these hydrophobic amino acids are located at the N-terminus and C-terminus of the peptide chain. The proportion of hydrophobic amino acids at the N-terminus of these peptides is 33.88%, and the proportion of hydrophobic amino acids at the C-terminus is 44.08%. The results show that eel peptides have significant antioxidant potential. Seqlogo was used to analyze the distribution of amino acids in peptides of different lengths. The larger the amino acid letter, the higher its occurrence rate. From Figure 3 (C) It can be seen that in peptides of different lengths, hydrophobic amino acids are still the most important amino acids, followed by polar uncharged amino acids. At the same time, as the length of the peptide increases, the types of amino acids decrease, and the proportion of acidic and basic amino acids increases, especially in nonapeptides.

[0067] (3) Screening of antioxidant peptides: PeptideRanker database was used to predict the bioactivity score of peptides; AllerTOP and ToxinPred databases were used to predict potential allergenicity, potential toxicity and physicochemical properties; BIOPEP and Uniprot databases were searched to predict the antioxidant activity of peptides. Non-toxic, non-allergenic, antioxidant amino acid Y and W ratio greater than 30% and bioactivity score higher than 0.6 were screened out. The results are shown in Tables 1 and 2.

[0068] PeptideRanker is a predictive bioactivity server that can be used to predict and screen antioxidant peptides. If the predicted value is higher than the threshold of 0.5, the peptide is considered active. From 245 peptides, 36 peptides with scores greater than 0.5, non-sensitizing and non-toxic were screened. Aromatic amino acids (especially Y and W) and hydrophobic amino acids can enhance the efficacy of antioxidant peptides (see Table 1).

[0069] Table 1 In silico screening results of peptides (including predicted physicochemical properties and activity scores)

[0070]

[0071]

[0072] According to the high ratio of aromatic amino acids to hydrophobic amino acids, 16 peptides (AVLW, VPWP, VWPS, WGWP, DHPWH, LLGPW, VLYPW, VYGPW, WGGPL, WPDAR, WPYVT, LLVYPW, WDGTGR, PSWVPPA, WGDLSPK and HWDGSLPR) were selected from the 36 peptides for synthesis, and the antioxidant activity was determined according to the method of (3) antioxidant activity determination in Example 2. The results are shown in Table 2. The synthetic peptides have higher scores, usually composed of 4-8 amino acids, with steric hindrance values ​​between 0.32 and 0.64, amphipathicity of 0 or 0.58, and hydrophobicity between -0.39 and 0.42. In the ABTS free radical scavenging test, except for VWPS, the antioxidant activities of the other fifteen peptides were stronger than the positive control; at the same time, in the ORAC test, the TE values ​​of the sixteen peptides were all greater than 1.0 μmol TE / μmol, indicating that their activities exceeded the positive control Trolox (Table 2). The ABTS free radical absorption capacity of the peptides VLYPW, VYGPW, WPYVT and LLVYPW containing both Y and W was significantly higher than that of other peptides (Table 2). The ABTS free radical scavenging capacity of these four peptides reached 2.68±0.11μmol TE / μmol to 3.28±0.13μmol TE / μmol. The ORAC values ​​of VLYPW, VYGPW and WPYVT were also significantly higher, with WPYVT reaching 5.67±0.12μmol TE / μmol, and the ORAC values ​​of VLYPW and VYGPW being 5.00±0.00μmol TE / μmol and 5.49±0.14μmol TE / μmol, respectively. In addition, the activity of WGWP containing two W residues was also higher, with ABTS free radical absorbance capacity and ORAC values ​​of 2.20 ± 0.06 μmol TE / μmol and 4.35 ± 0.20 μmol TE / μmol, respectively.

[0073] Table 2 Physicochemical properties, antioxidant activity and quantum computing energy of the sixteen peptides. Data with different letters in the same test indicate significant differences (p<0.05)

[0074]

[0075] Example 4

[0076] Density functional theory (DFT) calculations

[0077] The initial conformations of the 12 antioxidant peptides obtained in Example 3 (WGWP, DHPWH, LLGPW, VLYPW, VYGPW, WGGPL, WPDAR, WPYVT, LLVYPW, WDGTGR, PSWVPPA and WGDLSPK) were constructed using Chem 3D software. Subsequently, the structures were optimized by Gaussian density functional theory (DFT) calculations at the M062X / 6-31G (d) level. The absence of imaginary frequencies was confirmed by frequency calculations to ensure conformational stability. After obtaining the stable conformations of the antioxidant peptides, the molecular frontier orbitals (HOMO and E HOMO ) and structural parameters such as atomic Mulliken charge distribution and bond length. All calculations were performed using the Gaussian 09 software package. The results are shown in Figure 4 As shown in Table 3 and Table 4.

[0078] Table 3 Atomic charge distribution and bond lengths in the active regions of twelve peptides

[0079]

[0080] Table 4 Mulliken charge distribution and bond lengths of predicted active sites in 12 peptides

[0081]

[0082]

[0083] In order to further study the relationship between the structure and properties of antioxidant peptides, the structure of the peptides was calculated using quantum chemical methods. The ability of a molecule to donate electrons is closely related to the effectiveness of its antioxidant activity. According to the frontier molecular orbital theory, E HOMO The higher the value, the stronger the ability of the molecule to donate electrons. In addition, HOMO, as a frontier orbital, plays a key role in reactions with nucleophiles (especially hydrogen-withdrawing and electron-withdrawing free radicals). Figure 4The HOMO distribution of twelve synthetic peptides is presented, with a focus on the indole molecules on W in the peptides. These findings suggest that these particular sites are most likely to lose electrons when the peptides interact with free radicals. In addition, previous studies have shown that antioxidants primarily scavenge free radicals at specific sites within the HOMO orbital region, which are typically associated with relatively negatively charged atoms and longer bond lengths. These features enhance the ability of antioxidants to donate hydrogen atoms during the scavenging process. Table 3 details the Mulliken atomic charge distribution and bond lengths within the HOMO orbital region of these 12 peptides. These peptides exhibit similar charge distribution patterns, with the negative charge primarily located on the nitrogen atom of the tryptophan indole ring. It is speculated that the active sites of WGWP, DHPWH, LLGPW, VLYPW, VYGPW, WGGPL, WPDAR, WPYVT, LLVYPW, WDGTGR, PSWVPPA and WGDLSPK may be located at the positions 9N-48H, 37N-78H, 39N-83H, 46N-94H, 42N-82H, 9N-46H, 9N-54H, 9N-56H, 55N-113H, 10N-58H, 26N-75H and 10N-65H on the indole ring of tryptophan, respectively (Table 4). These sites are considered to be hydrogen donors for scavenging free radicals, which contribute to their antioxidant activity. These findings suggest that the nitrogen-hydrogen bond atoms on the indole ring of tryptophan play a key role in antioxidant activity, regardless of whether tryptophan is located at the N-terminus, C-terminus or in the middle. Quantum chemical calculations once again confirmed the key role of tryptophan.

[0084] Verification of antioxidant active sites

[0085] In order to verify the active sites predicted by HOMO distribution, these sites were methylated to block their activity. The hydrogen atoms 9N-48H, 37N-78H, 39N-83H, 46N-94H, 42N-82H, 9N-46H, 9N-54H, 9N-56H, 55N-113H, 10N-58H, 26N-75H and 10N-65H on the active site tryptophan indole in 12 peptide sequences were methylated, and twelve new antioxidant peptides were obtained, with the sequences being W(me)GW(me)P, DHPW(me)H, LLGPW(me), VLYPW(me), VYGPW(me), W(me)GGPL, W(me)PDAR, W(me)PYVT, LLVYPW(me), W(me)DGTGR, PSW(me)VPPA and W(me)GDLSPK (Table 5). The modified peptides were evaluated for their in vitro antioxidant activity using the same method as (3) in Example 2. The results are shown in Table 5. The ABTS free radical scavenging ability of the methylated peptides decreased (compared with Table 2). In addition, after the active sites of the peptides were modified, the ORAC values ​​of VYGPW(me), VLYPW(me), W(me)PYVT and LLVYPW(me) were reduced to 5.2208±0.006μmol TE / μmol, 3.7322±0.1176μmol TE / μmol, 3.6607±0.1409μmol TE / μmol and 2.5418±0.0686μmol TE / μmol, respectively, which were 61.43%-95.01% of those before modification. The results showed that the modified active peptides almost lost the ability to scavenge ABTS free radicals; four peptides (VYGPW, VLYPW, WPYVT and LLVYPW) retained some ORAC values, and the others almost lost ORAC. This indicates that methylation of the active sites predicted by HOMO will weaken their ability to scavenge free radicals in vitro. The hydrogen atoms 9N-48H, 37N-78H, 39N-83H, 46N-94H, 42N-82H, 9N-46H, 9N-54H, 9N-56H, 55N-113H, 10N-58H, 26N-75H and 10N-65H on the active site tryptophan indole in the twelve peptide sequences made important contributions to the antioxidant capacity of antioxidant peptides.

[0086] Table 5 Antioxidant activity test results of 12 methylated (modified) peptides. Data with different letters in the same test indicate significant differences (p<0.05)

[0087]

[0088]

[0089] Example 5

[0090] Molecular docking of antioxidant peptides and MPO protein

[0091] Molecular docking analysis was used to study the interaction between the peptide and the MPO protein. The crystal structure of the MPO protein (PDB ID: 3F9P) was downloaded from the Protein Data Bank (https: / / www.rcsb.org / structure / 3F9P) (Ren et al.). Water molecules were deleted using PyMol software, and hydrogen atoms and charges were added using MGLTools. The three-dimensional structure of the antioxidant peptide was drawn using ChemDraw 20.0 software. The ligand and MPO protein were docked using Autodock Tools 1.5.6, and the complex with the highest score obtained by docking was considered the best docking result. The complex of the MPO protein and the ligand was visualized using PyMol and Discovery Studio 4.5 software. The results are shown in Figure 2. Figure 5 shown.

[0092] Based on the molecular docking results, the molecular mechanism of the antioxidant activity of the synthetic peptides was further analyzed. Figure 5 The optimal conformations and main forces of 12 peptides with MPO receptors are shown, mainly including hydrogen bonds (conventional hydrogen bonds, carbon-hydrogen bonds), electrostatic interactions (salt bridges, attractive charges, Pi-cations, Pi-anions) and hydrophobic interactions (alkyl, Pi-alkyl, Pi-Sigma, Pi-Pi T-shape, amide-Pi stacking, Pi-Pi stacking, Pi-sulfur bonds). Generally speaking, the binding energy is less than -5 kcal / mol, indicating that the binding force is very good, and when the binding energy is less than -7 kcal / mol, the binding effect is very strong. The binding energies of the synthetic peptides to the MPO protein were -11.0 kcal / mol (WGWP), -10.3 kcal / mol (DHPWH), -9.0 kcal / mol (LLGPW), -9.8 kcal / mol (VLYPW), -10.0 kcal / mol (VYGPW), -8.9 kcal / mol (WGGPL), -9.4 kcal / mol (WPDAR), -9.9 kcal / mol (WPYVT), -9.3 kcal / mol (LLVYPW), -9.2 kcal / mol (WDGTGR), -9.3 kcal / mol (PSWVPPA) and -8.9 kcal / mol (WGDLSPK), respectively, indicating that these yellow eel protein peptides can bind tightly to the MPO protein. Figure 5As can be seen from the three-dimensional diagram, the eel peptide molecule docks with the active cavity of MPO. When the peptide binds to MPO, it interacts with 27 amino acid residues, and these interactions are listed in detail in Table 6. It is worth noting that key residues including Arg405, Arg499, Arg590, Asp 260, Asp264, Gln257, Glu268, His261, His502 and Thr495 mainly interact with the peptide through hydrogen bonds and electrostatic interactions. Hydrogen bond interactions mainly occur at Arg405, Arg499, Arg590, Gln257, Glu268, His261, and Thr266, and these electrostatic interactions mainly occur between the basic amino acids (Arg405, Arg499, Arg590, His261 and His502) and acidic amino acids (Asp260 and Glu268) in MPO and the tryptophan and C-terminal carboxyl groups in the twelve peptides.

[0093] Table 6 Docking sites and interaction forces between synthetic peptides and MPO receptors

[0094]

[0095]

[0096] Note: "+" indicates the number of interactions between the peptide and the receptor (green is hydrogen bond; red is electrostatic interaction; purple is hydrophobic interaction).

[0097] Example 6

[0098] Cell antioxidant assay

[0099] (1) Cell culture and model establishment

[0100] According to the method of Du et al. (2020), an AAPH-induced human red blood cell oxidative damage model was established. 1% human red blood cells were centrifuged at 8000 rpm / min for 10 min at 4°C to separate red blood cells from plasma. The red blood cell pellet was carefully collected and washed four times with PBS buffer with a pH of 7.4. The PBS buffer consisted of 137mM NaCl, 2.7mM KCl, 8.1mMNa2HPO4, and 1.5mM KH2PO4. After the washing step, a certain amount of red blood cell pellet was diluted with PBS and shaken to form a 10% red blood cell suspension.

[0101] Subsequently, 100 μL of 10% red blood cell suspension was added to each well of a 96-well culture plate. Then, 50 μL of sample solution (sample group, i.e., the 12 peptides obtained in Example 3 (WGWP, DHPWH, LLGPW, VLYPW, VYGPW, WGGPL, WPDAR, WPYVT, LLVYPW, WDGTGR, PSWVPPA, and WGDLSPK)) or PBS (referred to as the AAPH group) was added to each well. The culture plate was placed in a biochemical incubator at 37°C for 30 minutes. After pre-culture, 100 μL of 200 mM AAPH solution was added to induce oxidative damage for 3 hours. The red blood cell suspension of the control group was mixed with PBS buffer and cultured under the same conditions. Both the sample and AAPH were prepared using the above-mentioned PBS buffer. Normal human red blood cells treated with PBS without the addition of AAPH solution were used as the Control group.

[0102] (2) Scanning electron microscopy (SEM) observation of red blood cell morphology

[0103] In order to confirm the protective effect of the synthetic peptides on the morphological changes of red blood cells caused by AAPH, the morphology of red blood cells was examined by scanning electron microscopy (SEM). Specifically, the red blood cell pellet treated with the above different peptides (obtained in step (1)) was added with 2.5% glutaraldehyde and then placed in a 4°C refrigerator for fixation for 12 h. Then, the cells were centrifuged at 4000 rpm / min for 5 min at 4°C and the supernatant was discarded. The cells were rinsed three times with PBS to remove excess glutaraldehyde. A small amount of fixed red blood cell samples was applied to a thin layer on a coverslip and then gradually dehydrated with 30%, 50%, 70% and 90% ethanol and air-dried at room temperature (Lins et al., 2018). After the coverslip was attached to the sample plate, the red blood cell samples were analyzed using a gold spray instrument (ETD-2000, Beijing Aliberton Technology Development Co., Ltd.). Finally, the samples were scanned and photographed under an electron microscope (EVO 18, Carl Zeiss, Germany).

[0104] The results are as follows Figure 6 As shown, the red blood cells in the Control group exhibited a characteristic persimmon shape, characterized by a biconcave disc with a smooth surface and flat edges. In contrast, the cell membrane of the AAPH group was damaged by free radicals, resulting in an imbalance of ions inside and outside the cell membrane. This caused the cells to be severely deformed, showing many spines and a rough surface. In addition, some red blood cells were rounded and spherical, during which the fluidity of the cell membrane phospholipid bilayer was affected and approached a state of swelling and potential rupture. This change in morphology is a precursor to hemolysis. In the group treated with synthetic peptides, the red blood cell morphology was partially preserved, and most of the red blood cells maintained a normal appearance. Only a small number of red blood cells showed spines or spherical shapes.

[0105] (3) Determination of hemolysis rate and hemoglobin oxidation rate, as well as determination of BCA, MDA, LDH, GSH / GSSG, SOD, CAT and GSH-Px in human erythrocytes

[0106] Hemolysis rate determination: Transfer 200 μL of the red blood cell reaction solution obtained in step (1) to a 2 mL centrifuge tube. Then add 600 μL of PBS buffer and centrifuge at 8000 rpm / min for 10 min at 4°C. After centrifugation, carefully collect 200 μL of the supernatant and transfer it to a 96-well transparent microplate. Measure the absorbance at a wavelength of 540 nm and record this value as A PBS The same procedure was repeated using an equal amount of 10% erythrocyte suspension. To ensure complete lysis of erythrocytes, ultrapure water at 4°C was used. The absorbance value at 540 nm was recorded as A water The calculation formula of hemolysis rate is as follows: Hemolysis rate (%) = (A PBS / A water )×100% (Du et al., 2020).

[0107] Determination of hemoglobin oxidation rate: The oxidation rate of hemoglobin in human red blood cells was determined according to the method of Quds et al. (2023). Specifically, the cells obtained in step (1) were collected and lysed with ultrapure water at 4°C. The absorbance at wavelengths of 630 and 700 nm was measured and recorded as A 630 and A 700 200 μL of sample solution was mixed with 10 μL of 5% potassium ferrocyanide solution to prepare methemoglobin (MetHb), and the absorbance at wavelengths of 630 and 700 nm was recorded as A 100％metHb630 and A 100％metHb700 The formula for calculating the oxidation rate of hemoglobin is: metHb (%) = [(A 630 -A 700 ) / (A 100％metHb630 -A 100％metHb700 )]×100%.

[0108] Determination of BCA, MDA, LDH, GSH / GSSG, SOD, CAT and GSH-Px in human erythrocytes: The erythrocyte reaction solution obtained in step (1) was centrifuged at 4000 rpm / min for 10 min. The supernatant was collected and stored at -80°C for future use, and the cell pellet was rinsed three times with PBS. It was then completely lysed with 200 μL of ultrapure water at 4°C and stored at -80°C for future use. The BCA, MDA, LDH, GSH, GSSG, SOD, CAT and GSH-Px contents in the lysed cells were determined using a kit (Ma et al., 2023). The LDH enzyme activity in the supernatant was determined using a kit.

[0109] The results are shown in Table 7. The hemolysis rate and hemoglobin oxidation rate of the Control group were significantly lower, only 2.52±0.11% and 5.85±0.03%, respectively. In contrast, the hemolysis rate (39.95±1.08%) and hemoglobin oxidation rate (36.09±0.23%) of erythrocytes in the AAPH group were significantly higher. Compared with the AAPH injury group, the hemolysis rate and hemoglobin oxidation rate of all peptide-treated groups were significantly decreased, indicating that the peptides can effectively protect erythrocytes from oxidative damage (p<0.05). Among the various peptides, the hemolysis rate (15.11±0.26%) and hemoglobin oxidation rate (17.06±0.16%) of erythrocytes treated with VLYPW were both lower, showing a good protective effect against oxidative hemolysis. In addition, WGWP, VYGPW, LLVYPW, and WGDLSPK also achieved good results in inhibiting hemolysis rate and hemoglobin oxidation. The ABTS free radical absorbance capacity of the different antioxidant peptide treatment groups was negatively correlated with the hemolysis rate and MetHb level; at the same time, the ORAC value was negatively correlated with the MetHb level. This indicates that the eel antioxidant peptides may reduce erythrocyte hemolysis and hemoglobin oxidation through antioxidant action.

[0110] Cell membrane integrity can be assessed by measuring the production of MDA and the release of LDH. The effects of the eel antioxidant peptides on erythrocyte MDA production and LDH release are shown in Table 7. The MDA production of the control group was 6.03±0.24nmol / mg (protein), while the MDA production of the AAPH group increased significantly to 13.93±0.20nmol / mg (protein), and the LDH release reached 305.45±5.03% of the control group. The increase in MDA and LDH content indicated that the AAPH group was significantly damaged compared with the control group. Compared with the AAPH group, the MDA and LDH content of all peptide-treated groups were significantly reduced (p<0.05). The peptide WGDLSPK had the strongest protective effect, with an MDA production of 7.34±0.38nmol / mg protein, followed by the peptide VLYPW, with an MDA production of 7.84±0.44nmol / mg protein.

[0111] GSH is usually the first line of defense against oxidative stress. As shown in Table 7, the GSH / GSSG ratio of the control group was relatively high, at 10.66±0.17%, indicating that erythrocytes naturally maintain a high GSH content under normal physiological conditions. In contrast, the GSH / GSSG ratio of the AAPH group dropped sharply to 0.53±0.02%. However, in all peptide-treated groups, the GSH / GSSG ratio was significantly higher than that of the AAPH group (p<0.05), indicating that these peptides effectively alleviated cellular oxidative damage by increasing the GSH / GSSG ratio. It is worth noting that the WPDAR-treated group had the highest GSH / GSSG ratio of 1.60±0.06%, which was significantly higher than that of the other peptide-treated groups; followed by the WGWP and DHPWH-treated groups, with GSH / GSSG ratios of 1.31±0.06%. Erythrocytes also contain a rich enzyme antioxidant system (SOD, CAT, GSH-Px, etc.), which can protect erythrocytes from oxidative damage (Liu et al., 2017). As shown in Table 7, the intracellular SOD, CAT and GSH-Px activities in the AAPH-treated group increased significantly (p<0.05), which were 3.62 times, 4.30 times and 1.99 times that of the control group, respectively. Compared with the AAPH-treated group, the enzyme activities in the peptide-treated group decreased to varying degrees. The activities of SOD (2.43±0.09U / mg protein), CAT (37.98±0.93U / mg protein) and GSH-Px (132.85±5.07U / mg protein) in the VLYPW-treated group were all low. In addition, LLGPW, WPYVT and LLVYPW also showed a decrease in the antioxidant enzyme activity of the lower peptide-treated group, which may be due to the strong ability of synthetic peptides to scavenge free radicals, thereby effectively alleviating the demand for antioxidant enzymes during oxidative stress. The above results show that synthetic peptides have a protective effect on the cellular redox system (GSH / GSSG, SOD, CAT, GSH-Px) in AAPH-induced erythrocyte oxidative stress.

[0112] Table 7 The protective effects and mechanisms of the twelve peptides on AAPH-induced erythrocyte hemolysis

[0113]

[0114]

[0115] Example 7

[0116] Study on the mechanism of antioxidant peptides inhibiting AAPH-induced erythrocyte oxidative damage

[0117] Based on the changes in the indicators of MDA, LDH, MetHb, GSH / GSSG, SOD, CAT and GSH-Px in human red blood cells obtained in Example 6, it is speculated that the antioxidant mechanism of corn antioxidant peptides in inhibiting AAPH-induced oxidative damage in human red blood cells is as follows. Figure 7 shown.

[0118] AAPH is a free radical initiator that stimulates the production of a large amount of ROS, including O2 - and H2O2. Excess ROS reacts with unsaturated fatty acids on the cell membrane to produce a large amount of MDA. MDA cross-links with phospholipids and proteins on the cell membrane, causing changes in the morphology of red blood cells. The destruction of the phospholipid bilayer causes cell membrane damage, leading to the release of LDH and hemoglobin into the extracellular space, ultimately leading to hemolysis. GSH plays a key role as a non-enzymatic antioxidant in red blood cells and is the first line of defense against oxidative stress. Under the action of GSH-Px, GSH is oxidized to GSSG. Excess GSSG can be reduced to GSH with the help of NADPH and glutathione reductase (GR). Reducing the conversion rate of GSH to GSSG can effectively inhibit lipid peroxidation. Red blood cells also have an enzymatic antioxidant defense system, including SOD, CAT, and GSH-Px. SOD catalyzes O2 - Converted into H2O2, CAT further reduces H2O2 to water. In addition, GSH-Px can convert H2O2 into water or react with GSH to generate water. Non-enzymatic and enzymatic reactions maintain the dynamic balance between the generation and removal of free radicals in cells by directly or indirectly participating in intracellular redox reactions, thereby maintaining the normal life activities of red blood cells. However, when red blood cells are attacked by free radicals for a long time, the defense system in the cells is not enough to remove excess free radicals, leading to the destruction of red blood cells. The addition of yellow eel antioxidant peptides can control the oxidative damage of free radicals to red blood cells, maintain the balance between enzymes and non-enzymatic antioxidants, and thus inhibit the oxidative damage of AAPH to red blood cells. Among these twelve antioxidant peptides, VLYPW showed superior activity in chemical antioxidant activity and protection of human red blood cell morphology.

[0119] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The application of eel peptide in the preparation of antioxidant products is characterized in that: The amino acid sequence of the eel peptide is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The antioxidant products include antioxidant drugs, food antioxidants or cosmetics.

3. A method for preparing an eel peptide having an antioxidant effect, characterized in that: The method comprises the following steps: hydrolyzing the eel with Alcalase alkaline protease to obtain the eel hydrolysate; separating and purifying the eel hydrolysate with gel column chromatography, collecting the eluate according to the peak type, and selecting the eluate with the best antioxidant activity for UPLC-Q-TOF-MS / MS identification; predicting the biological activity score of the peptide obtained by identification with the PeptideRanker database, predicting the potential allergenicity, potential toxicity and physicochemical properties of the peptide obtained by identification with the AllerTOP and ToxinPred databases, searching the BIOPEP and Uniprot databases for predicting the antioxidant activity of the peptide obtained by identification, and selecting the eel peptide with a predicted value higher than 0.5, no allergenicity, no toxicity, a ratio of antioxidant amino acids Y and W greater than 30%, and a biological activity score higher than 0.6; the amino acid sequence of the eel peptide is shown in SEQ ID NO.

1.

4. The preparation method according to claim 3, characterized in that: The temperature of the enzymolysis is 50-60° C., the pH of the enzymolysis is 7.5-8.5, and the time of the enzymolysis is 2.5-5 hours.

5. The preparation method according to claim 3, characterized in that: The dosage of the Alcalase alkaline protease is 3-5g of Alcalase alkaline protease per 100g of rice field eel.

6. The preparation method according to claim 3, characterized in that: The gel column is a Sephadex G-25 gel chromatography column.

7. The preparation method according to claim 3, characterized in that: The chromatographic column model used for UPLC-Q-TOF-MS / MS identification was ACQUITYUPLC HSS T3, and the column temperature was 40-50°C.

8. The preparation method according to claim 3, characterized in that: The mobile phase A for UPLC-Q-TOF-MS / MS identification was an aqueous solution containing 0.1% formic acid, the mobile phase B was an 80% acetonitrile solution, the flow rate was set at 0.4 mL / min, and the gradient elution program was: 0-2.0 min, 92.0% A; 2-45 min, 92.0-72.0% A; 45.0-55.0min, 72.0-60.0%A; 55.0-56.0min, 60.0-5.0%A; 56.0-66.0min, 5.0%A.

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