Spirulina phycocyanin polypeptide for relieving inflammatory response as well as preparation method and application of spirulina phycocyanin polypeptide

Abstract

The invention belongs to the technical field of active peptides, and particularly relates to spirulina phycocyanin polypeptide capable of relieving inflammatory response and a preparation method and application thereof.The sequence of the spirulina phycocyanin polypeptide is shown as SEQ ID NO.1, a polypeptide sequence structure is obtained through enzymolysis, separation and structural identification, and the spirulina phycocyanin polypeptide is obtained through enzymolysis, separation and structural identification. A macrophage in-vitro anti-inflammatory experiment verifies that the active peptide has anti-inflammatory ability.

Description

Technical Field

[0001] The present application relates to the technical field of active peptides, and more specifically, to a Spirulina phycocyanin polypeptide for relieving inflammatory response, and a preparation method and application thereof. Background Art

[0002] Inflammation is a defensive reaction of the animal body to adverse stimuli. It is also the basis for the occurrence and development of many diseases and can easily cause damage to the body. Glucocorticoids with strong and rapid anti-inflammatory effects are often used clinically to deal with inflammation, among which dexamethasone is the most used. However, relevant reports point out that long-term use of glucocorticoids may lead to adverse reactions such as anaphylactic shock, mental abnormalities, and allergic rashes. Therefore, it is imperative to develop and research special medical foods with anti-inflammatory effects that are easily absorbed by the human body and have no toxic side effects. Summary of the invention

[0003] The present application is provided to solve the above defects in the prior art. A Spirulina phycocyanin polypeptide for relieving inflammatory response and a preparation method and application thereof are needed, and a small molecule peptide with anti-inflammatory effect is provided.

[0004] In a first aspect of the present application, a Spirulina phycocyanin polypeptide for relieving inflammatory response is provided, wherein the Spirulina phycocyanin polypeptide sequence is shown as SEQ ID NO.1.

[0005] In a second aspect of the present application, a method for preparing the Spirulina phycocyanin polypeptide described in any embodiment of the present application is provided, the preparation method comprising: soaking Spirulina platensis in water to obtain a mixture A, freezing the mixture A, and then melting the mixture A; centrifuging the melted mixture A, and taking the supernatant as a crude phycocyanin extract; adjusting the crude phycocyanin extract to be alkaline, adding alkaline protease for enzymatic hydrolysis to obtain an enzymatic solution; after centrifuging the enzymatic solution, taking the supernatant, filtering and drying the supernatant to obtain a freeze-dried powder; separating and analyzing the freeze-dried powder by ultra-performance liquid chromatography to obtain the Spirulina phycocyanin polypeptide shown in SEQ ID NO.1.

[0006] The third aspect of the present application provides a use of the Spirulina phycocyanin polypeptide described in any embodiment of the present application in the preparation of an anti-inflammatory product.

[0007] The fourth aspect of the present application provides a use of the Spirulina phycocyanin polypeptide described in any embodiment of the present application in the preparation of an antioxidant product.

[0008] In a fifth aspect of the present application, a drug is provided, wherein the active ingredient of the drug at least comprises the Spirulina phycocyanin polypeptide described in any embodiment of the present application.

[0009] The Spirulina phycocyanin polypeptides for relieving inflammatory reactions, as well as their preparation methods and uses, provided in various embodiments of the present application, have been verified to have anti-inflammatory capabilities through in vitro anti-inflammatory experiments on macrophages. This indicates that the novel active peptide MGHP can be used as a functional ingredient in foods, health products, cosmetics and medicines with anti-inflammatory effects, and has good application prospects. Compared with dexamethasone, it can avoid adverse reactions such as anaphylactic shock, mental disorders, allergic rashes, etc. caused by long-term use. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In the drawings, which are not necessarily drawn to scale, the same reference numerals may describe similar parts in different views. The same reference numerals with letter suffixes or different letter suffixes may represent different instances of similar parts. The accompanying drawings generally illustrate various embodiments by way of example and not limitation, and together with the specification and claims, are used to illustrate the embodiments applied for. When appropriate, the same reference numerals are used throughout the drawings to refer to the same or similar parts. Such embodiments are illustrative and are not intended to be exhaustive or exclusive embodiments of the present apparatus or method.

[0011] Figure 1 The HPLC spectrum of the polypeptide sample containing MGHP according to the embodiment of the present application is shown; Figure 2 The MS spectrum of the polypeptide MGHP according to an example of the present application is shown. DETAILED DESCRIPTION

[0012] In order to enable those skilled in the art to better understand the technical solution of the present application, the present application is described in detail below in conjunction with the accompanying drawings and specific implementation examples. The embodiments of the present application are further described in detail below in conjunction with the accompanying drawings and specific implementation examples, but are not intended to limit the present application.

[0013] The words "first", "second" and similar words used in this application do not indicate any order, quantity or importance, but are only used to distinguish. The words "include" or "comprise" and similar words mean that the elements before the word include the elements listed after the word, and do not exclude the possibility of including other elements.

[0014] According to an embodiment of the present application, a spirulina phycocyanin polypeptide for relieving inflammatory response is provided, and the sequence of the spirulina phycocyanin polypeptide is shown in SEQ ID NO. 1. The amino acid sequence of the spirulina phycocyanin polypeptide is MGHP, i.e., Met-Gly-His-Pro. The activity of the peptide is usually higher than that of its parent protein, and the molecular weight of the peptide is smaller than that of the protein, which is conducive to human absorption.

[0015] According to an embodiment of the present application, a method for preparing the Spirulina phycocyanin polypeptide described in any embodiment of the present application is provided, and the preparation method comprises: soaking Spirulina platensis in water to obtain a mixture A, freezing the mixture A, and then melting the mixture A; centrifuging the melted mixture A, and taking the supernatant as a crude phycocyanin extract; adjusting the crude phycocyanin extract to be alkaline, adding alkaline protease for enzymatic hydrolysis to obtain an enzymatic hydrolyzate; after centrifuging the enzymatic hydrolyzate, taking the supernatant, filtering and drying the supernatant to obtain a freeze-dried powder; separating and analyzing the freeze-dried powder by ultra-performance liquid chromatography-tandem mass spectrometry to obtain the Spirulina phycocyanin polypeptide shown in SEQ ID NO.1.

[0016] The supernatant obtained after centrifugation of the thawed mixture A contains C-phycocyanin and allophycocyanin.

[0017] In some embodiments, the preparation method includes: soaking platensis in water, the volume ratio of platensis to water is 1:18-22; the freezing treatment temperature is -18~-22°C, and the freezing time is 20-26h; the temperature of centrifuging the melted mixture A is 3-5°C.

[0018] In some embodiments, the conditions for centrifuging the melted mixture A include: a rotation speed of 5000-7000 rpm and a centrifugal time of 5-30 min. Specifically, the rotation speed may be 6000-6500 rpm and a centrifugal time of 10-20 min.

[0019] In some embodiments, the melting temperature is 30-45° C. The specific melting temperature may be 35-40° C.

[0020] In some embodiments, the phycocyanin crude extract is adjusted to an alkaline pH of 7.5-8.5; the enzymatic activity of the added alkaline protease is (4-6)×10 5 ; The enzymatic hydrolysis time is 3-5h, the enzymatic hydrolysis temperature is 35-45℃; the temperature of the enzymatic hydrolysate centrifugation is 3-5℃.

[0021] In some embodiments, the supernatant is filtered and dried to obtain a lyophilized powder, which is then ultrafiltered using a 3 kDa ultrafiltration membrane to obtain a peptide solution with a molecular weight of <3 kDa.

[0022] In some embodiments, the method of analyzing using ultra-high performance liquid chromatography-tandem mass spectrometry includes: filtering the lyophilized powder using an aqueous filter membrane, performing gradient elution on the filtrate through ultra-high performance liquid chromatography, and then analyzing it using tandem mass spectrometry; the tandem mass spectrometry uses a positive ion mode.

[0023] The freeze-dried powder is dissolved in water and then filtered using a water filter membrane.

[0024] In some embodiments, the pore size of the water filter membrane is 0.22 microns.

[0025] In some embodiments, the mobile phase A of the ultra-high performance liquid chromatography gradient elution is a 0.1% by mass aqueous formic acid solution, and the mobile phase B is an acetonitrile solution containing 0.1% by mass formic acid; the elution time is 55-65 min.

[0026] According to the embodiments of the present application, there is provided a use of the Spirulina phycocyanin polypeptide described in any embodiment of the present application in the preparation of an anti-inflammatory product.

[0027] The product can be a medicine, food, cosmetics or experimental reagent, etc. The experimental reagent is used for basic research.

[0028] Furthermore, when the product is a drug, the drug can be used to prevent or treat diseases related to inflammation through its anti-inflammatory effect.

[0029] It also includes pharmaceutically necessary carriers, of course, the pharmaceutically acceptable carrier dosage should be harmless to the subject, specifically including but not limited to buffers, antioxidants, preservatives, bactericides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, surfactants, salt-forming counterions and metal complexes. Moreover, according to the usual method, it can be made into oral preparations, external preparations, suppositories and sterile injection solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc.

[0030] In some embodiments, the anti-inflammatory effect is manifested as: inhibiting the secretion of macrophage inflammatory factors and inflammatory mediators. Inflammatory factors include IL-1B and TNF-a, and inflammatory mediators include PGE2.

[0031] According to the embodiments of the present application, there is provided a use of the Spirulina phycocyanin polypeptide described in any embodiment of the present application in the preparation of a product with antioxidant efficacy.

[0032] The product can be a medicine, food, cosmetics or experimental reagent, etc. The experimental reagent is used for basic research.

[0033] According to an embodiment of the present application, a medicine is provided, wherein the active ingredient of the medicine at least comprises the Spirulina phycocyanin polypeptide described in any embodiment of the present application.

[0034] Example 1: Extraction of Spirulina Phycocyanin Repeated freeze-thaw method to break Spirulina: Take Spirulina powder and Polysiphonium and place them in two 500ml reagent bottles, add distilled water at a ratio of 1:20 (w / v), soak overnight, freeze at -20℃ for 24 hours, and then thaw in a 37℃ water bath. Repeat the operation 3 times. Centrifuge at 6000 rpm for 15 minutes at 4℃, and the supernatant is the crude extract of phycobiliprotein (containing C-phycocyanin and allophycocyanin).

[0035] Example 2: Preparation of Spirulina Phycocyanin Peptide 1) The pH of the crude extract of Spirulina phycocyanin prepared in Example 1 was adjusted to 8, and then 5×10 5 U / g of alkaline protease, mix well and place in a shaker for enzymolysis at a speed of 100-200 rpm, for 4 h at a temperature of 40°C. Boil the enzymolysis solution in a boiling water bath for 10 min to inactivate the enzyme and terminate the enzymolysis.

[0036] 2) Centrifuge the enzymatic hydrolysate at 10000-12000rpm for 15min at 4℃, collect the supernatant and discard the precipitate. The supernatant is the Spirulina phycocyanin peptide solution. Then use a 3kDa ultrafiltration membrane for ultrafiltration to obtain a peptide solution with a molecular weight of <3kDa, freeze-dry, and obtain the Spirulina phycocyanin peptide freeze-dried powder.

[0037] Example 3: Structural Identification of Spirulina Phycocyanin Peptide The lyophilized powder of Spirulina phycocyanin peptide obtained in Example 2 was re-dissolved in ultrapure water, filtered through a 0.22 micron water filter membrane, and samples were collected according to the peak time by liquid chromatography to separate the polypeptide samples, and then the sequence structure of Spirulina phycocyanin peptide was analyzed and identified by ultra-high performance liquid chromatography tandem mass spectrometry. In ultra-high performance liquid chromatography, mobile phase A (0.1% formic acid) aqueous solution, mobile phase B (0.1% formic acid) acetonitrile solution, elution time was 60 min, gradient elution conditions were shown in Table 1, injection volume was 5 μL, and flow rate was set to 300 μL / min.

[0038] Table 1 ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY ELUTATION CONDITIONS The peptide samples separated by ultra-high performance liquid chromatography were analyzed by mass spectrometry. The tandem mass spectrometry adopted positive ion mode and secondary mass spectrometry analysis. The full MS scan of 100-2000 m / z was performed at a resolution of 120000. The specific mass spectrometry parameter settings are shown in Table 2.

[0039] Table 2 Tandem mass spectrometry analysis conditions Mass spectrometry data interpretation: The original mass spectrometry files were converted into MGF format files using MM File Conversion software, and then the MASCOT mass spectrometry data analysis platform was used to retrieve protein data in the uniport database. The specific search parameters are shown in Table 3.

[0040] Table 3 Mascot search parameters Note: Search the comparison database http: / / www.uniprot.org / taxonomy / 8139, Search software http: / / www.matrixscience.com / .

[0041] Example 4: Bioinformatics prediction of Spirulina phycocyanin peptides 1) Prediction of potential biological activity and toxicity of Spirulina phycocyanin peptides The potential biological activities of multiple polypeptide sequences obtained in Example 3 were analyzed using the PeptideRanker online platform (http: / / distilldeep.ucd). The polypeptides were ranked according to their predicted probability of biological activity. The preset threshold of the PeptideRanker prediction model was 0.5, and any polypeptide with a threshold exceeding 0.5 was marked as having biological activity. This application selected a threshold of >0.8 as a screening condition for the biological activity of the polypeptide.

[0042] Toxicity and sensitization are also concerns in the research and development of peptides used in the field of skin care products. The ToxinPrep (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php) platform is used to predict the potential toxicity of structurally resolved peptide sequences based on the SVM (Swiss-Port) algorithm.

[0043] The results showed that the multiple polypeptides separated in Example 3 were non-toxic.

[0044] 2) Prediction of antioxidant activity of Spirulina phycocyanin peptides The antioxidant activity of multiple peptides screened in Example 3 with no toxicity and potential biological activity PeptideRanker>0.8 was predicted using the AnOxPePred (https: / / services.healthtech.dtu.dk / service.php AnOxPePred-1.0) platform, and the analysis model selected Peptide Mode, and Minimumpeptide length and Maximum peptide length were both set to the peptide chain length of the predicted polypeptide. This application selects the prediction results FRS score>0.5 or CHEL score>0.25 as a sign that the polypeptide has antioxidant activity.

[0045] The results showed that only some of the multiple peptides isolated from 3 had antioxidant activity.

[0046] 3) Prediction of anti-inflammatory and immune activities of Spirulina phycocyanin peptides During an inflammatory response, cells release a variety of inflammatory mediators, such as interleukins, tumor necrosis factor, prostaglandins, leukotrienes, etc. These mediators can cause vasodilation and increased permeability, thereby exacerbating inflammation. Anti-inflammatory drugs such as cyclooxygenase inhibitors can inhibit the production of these mediators, thereby reducing inflammation. The IL-1 and TNF-β inhibitory secretion peptide prediction module of the PHI platform was used to predict and analyze anti-inflammatory peptides.

[0047] The anti-inflammatory and immune activities of the peptides with antioxidant activity after the above 2) screening were predicted by bioinformatics, and finally the peptide MGHP with good antioxidant and anti-inflammatory properties was screened out, with a score of 0.81 and a molecular weight of 440.57. It can significantly inhibit the biological activities of IL-1 and TNF-β, and has never appeared in the peptide database. Therefore, it can be used as a new anti-inflammatory peptide for subsequent research, and then its efficacy can be further verified by macrophage anti-inflammatory experiments.

[0048] 4) Chemical synthesis of Spirulina phycocyanin peptide The peptide sequence of MGHP finally selected in Example 3 was chemically synthesized using Fmoc amino acid solid phase synthesis technology. The chemical synthesis was completed by Nanjing Peptide Valley Biotechnology Co., Ltd. The purity was ≥95%. The purity and molecular weight of the synthesized phycocyanin-derived active peptide identified by RP-HPLC and MS met the requirements. The HPLC spectrum of MGHP is shown in Figure 1 As shown, through Figure 1 It can be seen that the detection peak appears during the elution process from 6 to 8 minutes. The MS spectrum of MGHP is shown in Figure 2 As shown, through Figure 2The mass spectrum peaks can be found to be at 441.5-442.57.

[0049] Example 5: Cellular anti-inflammatory experiment This experiment is divided into two parts: the first part is based on macrophages, cytotoxicity detection is carried out to determine the dosage concentration of the chemically synthesized polypeptide MGHP in Example 4 on macrophages; the second part uses lipopolysaccharide (LPS) to stimulate macrophages, and the anti-inflammatory effect of the test sample is evaluated by detecting the changes in the content of inflammatory factors (IL-1β, TNF-α) and inflammatory mediators (PGE2).

[0050] The cells used in this test are macrophages, batch number: 210622-1, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0051] Cytotoxicity test: 1) Cell inoculation: After thawing the cells, when the plating rate reaches about 60%, inoculate the cells into a 96-well plate and incubate overnight in a CO2 incubator (37°C, 5% CO2).

[0052] 2) Experimental grouping: The experiment set up a zero adjustment group, a solvent control group, a positive control group and a sample group. In the sample group, each sample was set with 8 concentration gradients, and 3 replicate wells were set under each concentration gradient.

[0053] 3) Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table (Table 4).

[0054] 4) Administration: Administration was performed when the cell plating rate in the 96-well plate reached 50%-60%. The solvent control group (corresponding to the control in Table 4, 200 microliters of DMEM culture medium (Gibco) were added to each well), the positive control group (corresponding to the PC in Table 4, 200 microliters of culture medium containing 10% DMSO were added to each well); 200 microliters of culture medium containing the corresponding concentration were added to each well of the sample group (the culture medium contained the polypeptide MGHP and DMEM culture medium); the zero adjustment group had no cell inoculation, and only 200 mL of cell culture medium was added. After the administration was completed, the 96-well plate was placed in a CO2 incubator (37°C, 5% CO2) and cultured for 24 hours.

[0055] 5) Detection: After the cells were incubated for 24 h, the supernatant was discarded, and MTT working solution (0.5 mg / mL) was added. The cells were incubated at 37° C. in the dark for 4 h. After the incubation, the supernatant was discarded, 150 μL of DMSO was added to each well, and the OD value was read at 490 nm.

[0056] Calculation of relative cell viability: Relative cell viability (%) .

[0057] Table 4 MTT test results of samples Mean in Table 4 represents the average relative cell viability of three replicate wells under each concentration gradient; SD represents the standard deviation of the average relative cell viability. According to the MTT results, when the volume concentration of the sample group of the peptide MGHP was less than 5%, the relative cell viability reached 90% or more. Therefore, the peptide MGHP did not show obvious cytotoxicity based on macrophages within the concentration range of 5% (v / v).

[0058] Immuno-inflammatory tests: 1) Cell seeding: After thawing the cells, when the plating rate reaches about 60%, seed the cells into a 6-well plate and incubate overnight in a CO2 incubator (37°C, 5% CO2).

[0059] 2) Solution preparation: Prepare the working solution of the test substance according to the test grouping (Table 5), wherein the sample group is the corresponding group in Table 4 with a volume concentration of 2.5% of the polypeptide MGHP.

[0060] Table 5 Test plan 3) Administration: According to the test grouping, when the cell plating rate in the 6-well plate reaches 40%~60%, group administration is carried out, and 3 replicate wells are set up for each group. The blank control group (abbreviated as NC) and the negative control group (abbreviated as BC, 1.8mL of DMEM culture medium (Gibco) is added to each well), the positive control group (abbreviated as PC, 1.8mL of culture medium containing dexamethasone is added to each well), and 1.8mL of culture medium containing the corresponding concentration of the sample to be tested shown in Table 5 is added to each well of the sample group (the culture medium contains polypeptide MGHP and DMEM culture medium). After the administration is completed, the 6-well plate is placed in a CO2 incubator (37℃, 5% CO2) for 24h. 4) LPS stimulation: 2 hours after administration, 200 μl of LPS-containing working solution was added to each well of the remaining groups except the blank control group, and the cells were placed in a CO2 incubator (37°C, 5% CO2) and cultured for 22 hours.

[0061] 5) Collect cells: After incubation, discard the old solution, wash twice with PBS, add 1mL RNAiso Plus to each well, pipette and lyse the cells, and collect the samples. 6) ELISA test: Collect the cell culture supernatant and perform the test analysis according to the operating instructions of the ELISA kit. 7) Calculation of inhibition rate: .

[0062] 8) Statistical analysis of results: The results are expressed as Mean ± SD. The comparison between groups was analyzed by t-test. All statistical analyses were two-tailed. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different. Test results: See Table 6-8.

[0063] Table 6 Summary of IL-1β content detection results Note: When using t-test method for statistical analysis, compared with the BC group, the significance is indicated by #, P-value<0.05 is indicated by #, and P-value<0.01 is indicated by ##. Compared with the NC group, the significance is indicated by *, P-value<0.05 is indicated by *, and P-value<0.01 is indicated by **.

[0064] It can be seen from Table 6 that compared with the BC group, the IL-1β content of the NC group increased significantly, indicating that the stimulation conditions of this test were effective. Compared with the NC group, the IL-1β content of the PC group decreased significantly, indicating that the positive control of this test was effective. Compared with the NC group, the IL-1β content of the sample peptide MGHP-2.5% decreased significantly, and the inhibition rate was 82.31%.

[0065] Table 7 Summary of TNF-α content test results It can be seen from Table 7 that compared with the BC group, the TNF-α content of the NC group increased significantly, indicating that the stimulation conditions of this test were effective. Compared with the NC group, the TNF-α content of the PC group decreased significantly, indicating that the positive control of this test was effective. Compared with the NC group, the TNF-α content of the sample polypeptide MGHP-2.5% decreased significantly, and the inhibition rate was 31.75%.

[0066] Table 8 Summary of PGE2 content test results It can be seen from Table 8 that compared with the BC group, the PGE2 content of the NC group increased significantly, indicating that the stimulation conditions of this test were effective. Compared with the NC group, the PGE2 content of the PC group decreased significantly, indicating that the positive control of this test was effective. Compared with the NC group, the PGE2 content of the sample polypeptide MGHP-2.5% decreased significantly, and the inhibition rate was 87.17%.

[0067] In summary, after LPS stimulation, compared with the negative control group, the contents of inflammatory factors (IL-1β, TNF-a) and inflammatory mediators (PGE2) in the sample polypeptide MGHP at a concentration of 2.5% (v / v) were significantly decreased, and the inhibition rates were 82.31%, 31.75%, and 87.17%, respectively. This shows that the sample can inhibit the secretion of macrophage inflammatory factors (IL-1B, TNF-a) and inflammatory mediators (PGE2) at this concentration, thereby having the effect of alleviating the inflammatory response.

[0068] In addition, although exemplary embodiments have been described herein, the scope includes any and all embodiments based on the present application with equivalent elements, modifications, omissions, combinations (e.g., various embodiments intersecting schemes), adaptations or changes. The elements in the claims are to be interpreted broadly based on the language adopted in the claims, and are not limited to the examples described in this specification or during the practice of this application, and the examples are to be interpreted as non-exclusive. Therefore, this specification and examples are intended to be considered as examples only, and the true scope and spirit are indicated by the following claims and the full scope of their equivalents.

[0069] The above description is intended to be illustrative rather than restrictive. For example, the above examples (or one or more schemes thereof) may be used in combination with each other. For example, a person of ordinary skill in the art may use other embodiments when reading the above description. In addition, in the above-mentioned specific embodiments, various features may be grouped together to simplify the present application. This should not be interpreted as an intention that a feature of an application that does not require protection is necessary for any claim. On the contrary, the subject matter of the present application may be less than all the features of an embodiment of a particular application. Thus, the following claims are incorporated herein in the specific embodiments as examples or embodiments, wherein each claim is independently a separate embodiment, and it is considered that these embodiments may be combined with each other in various combinations or arrangements. The scope of the present invention should be determined with reference to the appended claims and the full scope of equivalent forms granted by these claims.

[0070] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. A Spirulina phycocyanin polypeptide for relieving inflammatory response, wherein the Spirulina phycocyanin polypeptide sequence is shown in SEQ ID NO.

1.

2. The method for preparing the Spirulina phycocyanin polypeptide according to claim 1, characterized in that: The preparation method comprises: soaking Spirulina platensis in water to obtain a mixture A, freezing the mixture A, and then melting the mixture A; centrifuging the melted mixture A, and taking the supernatant as a crude phycocyanin extract; adjusting the crude phycocyanin extract to be alkaline, adding alkaline protease for enzymolysis to obtain an enzymolysis solution; centrifuging the enzymolysis solution to obtain a supernatant, filtering and drying the supernatant to obtain a freeze-dried powder; separating and analyzing the freeze-dried powder by ultra-high performance liquid chromatography-tandem mass spectrometry to obtain a Spirulina phycocyanin polypeptide as shown in SEQ ID NO.

1.

3. The preparation method according to claim 2, characterized in that: The preparation method comprises: soaking platen Spirulina in water, wherein the volume ratio of platen Spirulina to water is 1:18-22; freezing treatment is performed at a temperature of -18 to -22°C for 20 to 26 hours; and centrifuging the melted mixture A at a temperature of 3 to 5°C.

4. The preparation method according to claim 2, characterized in that: The pH of the phycocyanin crude extract was adjusted to be alkaline at 7.5-8.5; the activity of the alkaline protease added was 4-6×10 5 ; The enzymatic hydrolysis time is 3-5h, the enzymatic hydrolysis temperature is 35-45℃; the temperature of the enzymatic hydrolysate centrifugation is 3-5℃.

5. The preparation method according to claim 2, characterized in that: The method for analyzing using ultra-high performance liquid chromatography-tandem mass spectrometry includes: filtering the freeze-dried powder using a water filter membrane, performing gradient elution on the filtrate through ultra-high performance liquid chromatography, and then analyzing it using tandem mass spectrometry; the tandem mass spectrometry uses a positive ion mode.

6. The preparation method according to claim 5, characterized in that: The mobile phase A of the ultra-high performance liquid chromatography gradient elution is a 0.1% by mass formic acid aqueous solution, and the mobile phase B is an acetonitrile solution containing 0.1% by mass formic acid; the elution time is 55-65 minutes.

7. Use of the Spirulina phycocyanin polypeptide according to claim 1 in the preparation of anti-inflammatory products.

8. The use according to claim 7, characterized in that: The anti-inflammatory effect is manifested in: inhibiting the secretion of macrophage inflammatory factors and inflammatory mediators.

9. Use of the Spirulina phycocyanin polypeptide according to claim 1 in the preparation of a product with antioxidant efficacy.

10. A drug, characterized in that The active ingredient of the medicine at least comprises the Spirulina phycocyanin polypeptide according to claim 1.

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