Bioactive peptide and application

Abstract

The invention belongs to the technical field of bioactive peptides, and discloses a bioactive peptide and application thereof. The bioactive peptide provided by the invention is extracted from white snakeheaded fish, and the amino acid sequence is as shown in SEQ ID NO.1. The bioactive peptide provided by the invention can obviously reduce the generation amount of NO and inhibit the generation of inflammatory factors such as TNF-alpha, IL-6 and the like, and has no obvious cytotoxicity in a cellular level experiment. The compound has a strong anti-inflammatory effect and can be used for preparing anti-inflammatory agent products.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptides and relates to bioactive peptides and applications. Background Art

[0002] Inflammation is a very complex process and one of the earliest diseases to be identified and diagnosed. Inflammation occurs when the body responds to external harmful stimuli and infections. Excessive inflammatory responses of the immune system can cause the accumulation of inflammatory mediators (such as NO, cellular inflammatory factors and chemokines, etc.), leading to aggravated inflammatory responses and damage to the body and tissues, resulting in local inflammatory diseases. Although inflammatory diseases have multiple causes, they have common pathological mechanisms in inflammatory responses, including the cellular pathways of activated immune cells regulating inflammatory mediators. Therefore, studying the important cellular pathways in the inflammatory response process can develop anti-inflammatory active substances for inflammation-related diseases. Therefore, the development of low-side effect anti-inflammatory drugs is urgent. In clinical practice, there are many drugs with anti-inflammatory properties such as aspirin, but these drugs usually have great side effects. The public prefers to obtain effective anti-inflammatory ingredients from natural products and their derivatives, and apply them to the market in the form of anti-inflammatory agents and their products. Food-derived anti-inflammatory peptides can regulate inflammation by regulating the secretion of pro-inflammatory and anti-inflammatory cytokines by immune cells, and have shown good anti-inflammatory effects in both in vivo and in vitro studies.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide a bioactive peptide and its application. The bioactive peptide provided by the present invention has the function of regulating inflammatory factors and improving inflammation.

[0005] To achieve the above object, the first technical solution adopted by the present invention is: The bioactive peptide is selected from one of the peptides whose amino acid sequences are shown in SEQ ID NO.1-3.

[0006] Preferably, the bioactive peptides are extracted from white mullet.

[0007] Preferably, the extraction method is: homogenizing the white mullet, simulating human in vitro digestion and enzymatic hydrolysis to obtain an enzymatic solution; using ultrafiltration to separate the enzymatic solution to retain ultrafiltration components with a molecular weight cutoff of less than 3000Da; using gel chromatography to chromatograph the ultrafiltration components; and separating and purifying the components obtained after chromatography.

[0008] The second technical solution adopted in the present invention is: Application of bioactive peptides in the preparation of anti-inflammatory products.

[0009] The third technical solution adopted in the present invention is: An anti-inflammatory agent comprising the bioactive peptide of the first technical solution.

[0010] The above-mentioned bioactive peptides can also be synthesized by chemical methods using amino acids as raw materials, which is well known in the art.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The bioactive peptide provided by the present invention can significantly reduce the production of NO, and inhibit the production of inflammatory factors such as TNF-α and IL-6, and has no obvious cytotoxicity in cell level experiments. It has strong anti-inflammatory activity and has good application prospects in the field of drugs with anti-inflammatory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the chromatogram of the ultrafiltration component with a molecular weight below 3000Da after being separated by Sephadex G-15 gel chromatography; Figure 2 is the cell survival rate at different concentrations of chromatography groups F1, F2, and F3; Figure 3 is the NO generation at different concentrations of chromatography groups F1, F2, and F3; Figure 4 is the HPLC separation spectrum of the chromatographic component F1; Figure 5 is the HPLC separation spectrum of the chromatographic component F2; Figure 6 is the HPLC separation spectrum of the chromatographic component F3; Figure 7 It is the secondary mass spectrum of sequence SEQ ID NO.1; Figure 8 is the secondary mass spectrum of sequence SEQ ID NO.2; Fig. 9 is the secondary mass spectrum of sequence SEQ ID NO.3; Fig.10 Ultra-high performance liquid chromatography separation spectrum for purity verification of the synthetic compound of sequence SEQ ID NO.1; Fig.11 Ultra-high performance liquid chromatography separation spectrum for purity verification of the synthetic compound of sequence SEQ ID NO.2; Fig.12 Ultra-high performance liquid chromatography separation spectrum for purity verification of the synthetic compound of sequence SEQ ID NO.3; Fig.13 The effect of the synthetic compounds of SEQ ID NO.1~3 on cell survival rate; Fig.14 The effect of the compounds of SEQ ID NO.1~3 on the NO production; Fig.15 The effect of the compounds of SEQ ID NO. 1 to 3 on the production of inflammatory cytokine TNF-α; Fig.16 The effect of the compounds of sequence SEQ ID NO.1~3 on the production of inflammatory cytokine IL-6. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and accompanying drawings. It should be understood that these descriptions are exemplary only, and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. In the embodiment, those who do not indicate specific conditions are carried out according to the conditions recommended by normal conditions or manufacturers. Those who do not indicate manufacturers for reagents or instruments used are conventional products that can be obtained by commercial purchase.

[0014] The first embodiment of the present invention provides a bioactive peptide selected from one of the peptides having amino acid sequences as shown in SEQ ID NO. 1 to 3.

[0015] Among them, SEQ ID NO.1 is FRALPFWDQ; SEQ ID NO. 2 is LLGGLEE; SEQ ID NO. 3 is MHHDRFK.

[0016] In the embodiment of the present invention, the bioactive peptides are extracted from white mullet. In fact, amino acids can also be used as raw materials to synthesize peptides with amino acid sequences of SEQ ID NO. 1 to 3 by chemical methods, which is well known in the art.

[0017] In a specific embodiment, the specific method for extracting the bioactive peptides is as follows: removing the by-products of white mullet, adding ultrapure water 2-6 times the mass of the fish meat for homogenization to obtain a homogenous material; adjusting the pH of the homogenous material to 2.0 with HCL, adding pancreatic enzyme at 1%-4% of the mass of the homogenous material, simulating intestinal digestion for 2.5 hours under 37°C water bath conditions, placing it in boiling water for 10 minutes to terminate digestion, and cooling it to room temperature to obtain an enzymatic solution; centrifuging the above enzymatic solution to obtain the supernatant and vacuum freeze-drying to obtain a crude peptide powder of white mullet; ultrafiltration separation of the above crude peptide powder, retaining the ultrafiltration component with a molecular weight of less than 3000Da, freeze-drying and storing it at -18°C; preparing the above ultrafiltration component into a solution with a concentration of 10-50 mg / mL, chromatographing it using Sephadex G-15 dextran gel chromatography, and freeze-drying the obtained components and storing them at -18°C; selecting the components with anti-inflammatory activity after chromatography through anti-inflammatory activity determination, and separating and purifying the selected components using RP-HPLC method.

[0018] In a further embodiment, the centrifugation conditions are 4°C, 8000xg, and time 20 min.

[0019] In a further embodiment, the Sephadex G-15 dextran gel chromatography method has a sample loading volume of 2-4 mL, an elution rate of 1-2 mL / min, and a detection wavelength of 220 nm.

[0020] Furthermore, the sample loading volume is 4 mL.

[0021] In the following specific implementation of the present invention, the ultrafiltration fraction is chromatographed by Sephadex G-15 dextran gel chromatography, and the fraction having anti-inflammatory activity after chromatography is selected and further separated by RP-HPLC.

[0022] Among them, the anti-inflammatory activity uses the determination of cell viability, and the effect of the polypeptide components on the secretion of cellular NO (A), TNF-α (B), and IL-6 (C). The specific determination method is as follows: (1) Cell culture RAW264.7 macrophages were maintained in DMEM medium, and then the cells were cultured in a 37°C incubator containing 5% CO2 and passaged every 24 h.

[0023] (2) Determination of cell viability The cytotoxicity of bioactive peptides to RAW264.7 macrophages was evaluated by MTT colorimetry. RAW264.7 cells (4.5×10 4100 μL of complete medium containing peptides of different concentrations (0, 62.5, 125, 250, 500, 1000, 2000 ug / mL) was added. After culturing for 20 hours, the culture medium was discarded, and 20 μL (5 mg / mL) of tetrazolium dye (MTT) solution was added to each well. The well was placed in an incubator containing 5% CO2 for 4 hours, and the supernatant was discarded again. Finally, 100 μL of dimethyl sulfoxide (DMSO) was added. After 10 minutes, the absorbance was detected at 570 nm. The calculation formula of cell survival rate is: Cell survival rate (%) = A2 / A1×100%; where A2 is the absorbance of the group with added peptides, and A1 is the absorbance of the group without added peptides.

[0024] (3) Determination of NO generation RAW264.7 cells (4.5×10 4 The cells were inoculated into 96-well plates, and the old culture medium was discarded after continuous culture for one day and one night. Samples of different concentrations were added, and a blank group, an induced group induced by lipopolysaccharide (LPS), and a positive control group were set up as follows: Blank group: normal culture for 2h+24h; Induction group: normal culture for 2 h + LPS (1 ug / mL) stimulation culture for 24 h; Experimental group: normal culture of samples for 2 h + LPS (1 ug / mL) stimulation culture for 24 h; Positive culture group: normal culture for 2h with dexamethasone added to the positive control group (100uL) + LPS (1ug / mL) stimulated culture for 24h; Three replicates were set up for each well, and the supernatant was collected after the culture was completed.

[0025] The NO concentration in the supernatant was determined by the Griess method. The specific steps were as follows: 50uL of cell supernatant was mixed with 50uL Griess I reagent and 50uL Griess II reagent, and the absorbance was measured at 540nm after 5 minutes. The NO content of the cell fluid in each sample was calculated based on the drawn nitrite standard curve.

[0026] (4) Determination of cytokines TNF-α and IL-6 RAW264.7 cells (5 × 10 4 The cells were inoculated into 96-well plates, with 3 replicates per well. The experimental group, blank group, induced group, and positive control group were set up in the same way as the determination of NO production. After the culture was completed, the supernatant was collected, and the ELISA kit was used to determine the effect of the polypeptide on the production of inflammatory factors (TNF-α, IL-6) after induction.

[0027] Example 1 The specific steps of the extraction, separation and purification of bioactive peptides are as follows: The by-products of white mullet are removed, and ultrapure water with an amount 2-6 times the weight of the fish meat is added for homogenization to obtain a homogenous material; the pH of the homogenous material is adjusted to 2.0 with HCL, and pancreatic enzyme is added at a rate of 1%-4% of the weight of the homogenous material, and intestinal digestion is simulated for 2.5 hours in a water bath at 37° C., and the material is placed in boiling water for 10 minutes to terminate the digestion, and then cooled to room temperature to obtain an enzymolysis solution; the enzymolysis solution is centrifuged to obtain the supernatant, and vacuum freeze-dried to obtain a crude peptide powder of white mullet; the crude peptide powder is separated by ultrafiltration, and the ultrafiltration component with a molecular weight of less than 3000Da is retained, and the ultrafiltration component is freeze-dried and stored at -18° C.

[0028] The above ultrafiltration components were prepared into a solution with a concentration of 25 mg / mL and filtered through a 0.22 um aqueous microporous filter membrane. Sephadex G-15 dextran gel chromatography was used for chromatography. Double distilled water was used as the eluent. The chromatography method was as follows: the elution flow rate was 1 mL / min, the detection wavelength was 220 nm, the sample volume was 4 mL, and the chromatographic spectrum results were as follows: Figure 1 As shown, the horizontal axis represents the elution time (min), and the vertical axis represents the absorbance A. During the chromatography collection and elution process, three separated components are obtained, which are labeled F1, F2, and F3 in turn, and freeze-dried and stored at -18°C. The obtained components are freeze-dried and stored at -18°C.

[0029] The F1, F2, and F3 fractions obtained after chromatography were selected for anti-inflammatory activity determination. Figure 2-3 As shown, Figure 2-3 The horizontal axis represents the concentration of each component. Figure 2-3 It can be seen that components F1, F2, and F3 all have good anti-inflammatory activity, and these three components were subjected to the next step of RP-HPLC separation and purification.

[0030] The above three components were separated and purified by RP-HPLC. The separation conditions of RP-HPLC were as follows: mobile phase A was water containing 0.1% TFA; mobile phase B was acetonitrile containing 0.1% TFA; gradient elution, i.e. 0min2%B, 0-18min2%-10%B, 18-36min10%-20B, 36-55min 20%-30B, 55-70min 30%-40%B, analytical column was C18 (3μm, 100A), injection volume was 10μL; flow rate was 1mL / min.

[0031] The RP-HPLC separation profiles of the chromatographic components F1-F3 are as follows: Figure 4-6 In the figure, the horizontal axis represents the elution time (min), and the vertical axis represents the response value (AU).

[0032] The three purified components were identified by liquid chromatography-mass spectrometry. Figure 4 The component at 5-6min of the F1 component, Figure 5 The component at 15-20min of the F2 component, Figure 6 The components at 45-55min of the F3 component were identified. The liquid phase conditions were: mobile phase A was water containing 0.1% formic acid; mobile phase B was water containing 0.1% formic acid and 80% CAN; gradient elution, i.e. 0min 4% B, 0-2min 4%-8% B, 2-45min 8%-28% B, 45-55min 28%-40% B, 55-56min 40%-95% B, 56-66min 95% B, analytical column was packed with Acclaim PepMap RPLC C18 (3μm, 150μm i.d.×150mm); injection volume was 10μL; flow rate was 600nL / min. Mass spectrometry conditions: primary mass spectrometry parameters were Resolution 70000, AGCtarget 3e6, MaximumIT 100 ms, Scanrange 100 to 1500 m / z; secondary mass spectrometry parameters were Resolution 17500, AGCtarget 1e5 MaximumIT 50 ms, TopN20, nce / steppedNCE28.

[0033] The amino acid sequences of the identified bioactive peptides are shown below: Component F1: Phe-Arg-Ala-Leu-Pro-Phe-Trp-Asp-Gln (FRALPFWDQ); Component F2: Leu-Leu-Gly-Gly-Leu-Glu-Glu (LLGGLEE); Fraction F3: Met-His-His-Asp-Arg-Phe-Lys (MHHDRFK).

[0034] Table 1 shows the information of the above bioactive peptides.

[0035] Table 1 Bioactive peptide information .

[0036] Example 2 In order to further verify the anti-inflammatory activity of the above bioactive peptides, the above sequences were synthesized using solid phase synthesis to obtain the synthetic compounds of sequences SEQ ID NO.1~3 respectively. Figure 7-9The purity of the synthetic compound was tested by the separation and purification method in Example 1. The test results are as follows: Figure 10-12 As shown, they are the ultra-high performance liquid chromatography separation patterns of purity verification of the synthetic compounds of sequences FRALPFWDQ, LLGGLEE, and MHHDRFK, respectively. The purity of the synthetic compounds of sequences SEQ ID NO.1~3 are all ≥98%.

[0037] Using dexamethasone as a positive control, the anti-inflammatory activities of the above-mentioned compounds of SEQ ID NO. 1 to 3 and dexamethasone at different concentrations (1 mg / mL, 20 mg / mL, 50 mg / mL, 100 mg / mL) were measured. Fig.13 The effect of different concentrations of SEQ ID NO.1~3 on cell survival rate, Figure 14-16 The effects of sequences SEQ ID NO.1~3 on the production of NO, inflammatory factors TNF-α, and IL-6 are shown respectively, wherein the positive control group is the dexamethasone group, FRALPFWDQ is the synthetic sample of sequence SEQ ID NO.1, LLGGLEE is the synthetic sample of sequence SEQ ID NO.2, and MHHDRFK is the synthetic sample of sequence SEQ ID NO.3.

[0038] according to Figure 13-16 The results show that the higher the concentration of the sample, the higher the inhibition rate of inflammatory cytokines. When the concentration was 100 mg / mL, under the action of dexamethasone in the positive group, the NO production, inflammatory cytokine TNF-α, and IL-6 production were 17 pg / mL, 160 pg / mL, and 110 pg / mL, respectively; the NO production, inflammatory cytokine TNF-α, and IL-6 production corresponding to the FRALPFWDQ sequence were 22 pg / mL, 206 pg / mL, and 210 pg / mL, respectively; the NO production, inflammatory cytokine TNF-α, and IL-6 production corresponding to the LLGGLEE sequence were 20 pg / mL, 203 pg / mL, and 208 pg / mL, respectively; the NO production, inflammatory cytokine TNF-α, and IL-6 production corresponding to the MHHDRFK sequence were 18 pg / mL, 198 pg / mL, and 203 pg / mL, respectively, indicating that the sequences FRALPFWDQ, LLGGLEE, and MHHDRFK have strong anti-inflammatory activity.

[0039] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content of the present invention by a person skilled in the art should be considered as the protection scope of the claims of the present invention.

Claims

1. A bioactive peptide, characterized in that: The peptide is selected from the group consisting of the peptides with amino acid sequences as shown in SEQ ID NOs. 1 to 3.

2. The bioactive peptide according to claim 1, characterized in that The bioactive peptide is extracted from white mullet.

3. The bioactive peptide according to claim 2, characterized in that: The extraction method comprises the following steps: homogenizing the white mullet, subjecting the homogenized white mullet to an enzymatic hydrolysis treatment simulating human in vitro digestion to obtain an enzymatic hydrolyzate; using ultrafiltration to separate the enzymatic hydrolyzate to obtain an ultrafiltration component with a molecular weight cutoff of less than 3000 Da; performing chromatography on the ultrafiltration component by gel chromatography; and separating and purifying the components obtained after chromatography.

4. Use of the bioactive peptide according to any one of claims 1 to 3 in the preparation of anti-inflammatory products.

5. An anti-inflammatory agent, characterized in that Contains the bioactive peptide according to any one of claims 1 to 3.

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