Polypeptide of Fuchuan aconitum carmichaeli in Greenzhou city and application of polypeptide

Abstract

The invention relates to the technical field of biological polypeptides, in particular to a polypeptide of Fuchuan aconitum carmichaeli in the Greenzhou city and application of the polypeptide. The polypeptide is prepared by a compound enzymolysis method, and the polypeptide contains three characteristic sequences, namely SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3, through third-party LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) identification. Functional tests show that the polypeptide SEQ ID No: 1 has strong antioxidation, antibiosis, anti-inflammation and fibroblast proliferation promotion functions; the polypeptide SEQ ID No: 2 or the polypeptide SEQ ID No: 3 is weak in function. The invention provides a new way for high-value utilization of Fuchuan characteristic resources, and can be applied to the fields of medicines, cosmetics and food additives.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological polypeptides, in particular, the present application relates to a kind of Hezhou Fuchuan Wuniuzi polypeptide and its application. BACKGROUND

[0002] Plant-derived active polypeptides have become the focus of research in the field of biological medicine and functional food due to their green safety and multiple functions. However, the existing technology has significant limitations: the extraction process relies on acid / alkali hydrolysis, which easily destroys the activity of the peptide segment, and the enzymatic method is high in cost and difficult to control the purity of the product; the functional level is mainly focused on single activity (such as antioxidant), and there are few cases with multiple effects such as antibacterial, anti-inflammatory and immune regulation, and there is a lack of systematic mining of characteristic resource polypeptides.

[0003] Hezhou Fuchuan Wuniuzi is a local traditional Chinese medicinal material, which belongs to the dried mature seeds of Lagenaria siceraria (Molina) Standl. of Cucurbitaceae, mainly produced in the karst topography selenium-rich soil area. Traditional medicine records that it has the effects of promoting water flow and reducing swelling, expelling parasites and relieving pain. The book "Compendium of Materia Medica" and other classics mention its "promoting water flow and reducing swelling, expelling parasites and relieving pain". Modern research shows that Fuchuan Wuniuzi is rich in high-quality protein and polypeptide components, and its unique geographical environment may endow the polypeptide with special spatial conformation and biological activity. However, existing researches mainly focus on oil extraction, and the high-efficiency preparation technology, structure-activity relationship and multi-scene application of the core functional component-polypeptide have not been involved, which leads to the long-term stagnation of the high-value utilization of this characteristic traditional Chinese medicinal resource, and it is urgent to break through the technical bottleneck to release its medicinal and health potential. SUMMARY

[0004] The present application provides a kind of Hezhou Fuchuan Wuniuzi polypeptide, which is derived from Wuniuzi produced in Hezhou Fuchuan, prepared by complex enzymolysis, and contains three characteristic sequences: SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3.

[0005] The present application also provides a preparation method of the above-mentioned polypeptide, which comprises: (1) Wuniuzi pretreatment; (2) complex protease enzymolysis; (3) centrifugation to obtain supernatant, then 3 kDa ultrafiltration, and then Sephadex G-25 chromatography; (4) freeze-drying to obtain a polypeptide mixture containing SEQ ID No: 1-3.

[0006] In some embodiments, the complex protease comprises trypsin and papain.

[0007] In some embodiments, the mass fraction ratio of the trypsin and papain is 1:2.

[0008] In some embodiments, the enzymolysis conditions are pH 7.5, 50℃ and 4 h.

[0009] In some embodiments, the enzyme dosage is 0.5% (w / w), the enzyme inactivation condition is 90℃ for 10 min, and the enzyme hydrolysis liquid ratio is 1:5 (w / v).

[0010] The application also provides the application of the polypeptide of SEQ ID No: 1 in preparing an antioxidant.

[0011] The application also provides the application of the polypeptide of SEQ ID No: 1 in preparing an antibacterial drug.

[0012] The application also provides the application of the polypeptide of SEQ ID No: 1 in preparing an anti-inflammatory preparation.

[0013] The application also provides the application of the polypeptide of SEQ ID No: 1 in preparing a skin repair-promoting cosmetic.

[0014] Compared with the prior art, the application has at least the following beneficial effects: (1) Multi-functional synergy: the polypeptide of SEQ ID No: 1 has four activities of antioxidant, antibacterial, anti-inflammatory and proliferation promotion, which is superior to most plant polypeptides; (2) Green and efficient preparation: composite enzymatic hydrolysis instead of chemical hydrolysis, without solvent pollution; (3) Clear structure: three characteristic sequences are identified by a third party LC-MS / MS, and the polypeptide of SEQ ID No: 1 is a core functional peptide; (4) Wide application: the polypeptide of SEQ ID No: 1 can be developed into an antioxidant, an antibacterial agent, an anti-inflammatory preparation and a skin repair-promoting cosmetic, and promote the high-value utilization of rich river characteristic resources. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The figure is an antioxidant activity test result graph of the polypeptide. ** indicates a significant difference from the VC group P <0.01, *** indicates a significant difference from the VC group P <0.001.

[0016] Figure 2 The figure is a result graph of the inhibition zone in the antibacterial activity test. * indicates a significant difference from the penicillin group P <0.05, ** indicates a significant difference from the penicillin group P <0.01, *** indicates a significant difference from the penicillin group P <0.001.

[0017] Figure 3 The figure is a result graph of MIC in the antibacterial activity test. ** indicates a significant difference from the penicillin group P<0.01, *** indicates a significant difference from the penicillin group P <0.001.

[0018] Figure 4 Figure is the test result graph for anti-inflammatory activity. * indicates significant difference from the model group P <0.05, *** indicates significant difference from the model group P <0.001.

[0019] Figure 5 Figure is the test result graph for fibroblast proliferation promotion. * indicates significant difference from the blank group P <0.05, *** indicates significant difference from the blank group P <0.001. DETAILED DESCRIPTION

[0020] To make the technical problems, technical solutions and advantages to be solved by the present application clearer, specific embodiments will be described in detail below with reference to the accompanying drawings.

[0021] Example 1, preparation and identification of Ubuqi polypeptide 1. Materials and reagents Raw materials: Fuchuan Ubuqi (collected from Fuchuan County, Hezhou, Guangxi in 2024), 1000 g; Reagents: trypsin (Sigma, 10000 U / mg), papain (Solarbio, 8000 U / mg), Sephadex G-25 (GE Healthcare), BCA protein quantification kit (Thermo Fisher); Equipment: constant temperature water bath shaker (THZ-98A), refrigerated centrifuge (Eppendorf 5430R), tangential flow ultrafiltration system (Millipore Pellicon 2), freeze dryer (Labconco FreeZone 4.5).

[0022] Methods and steps (1) Pretreatment: Hezhou Fuchuan Ubuqi was washed, ground into powder, and added with 5000 mL distilled water, and stirred uniformly; (2) Enzymatic hydrolysis: adjust pH to 7.5 (1 M NaOH), add complex protease (trypsin: papain mass fraction ratio = 1:2, total 0.5% w / w), 50°C oscillation enzymatic hydrolysis for 4 h (150 rpm); (3) Enzyme inactivation and centrifugation: 90°C enzyme inactivation for 10 min, 8000 rpm centrifugation for 20 min (radius 10 cm), and take the supernatant; (4) Ultrafiltration chromatography: the supernatant was filtered through a 3 kDa ultrafiltration membrane, and the filtrate was passed through a Sephadex G-25 column (Φ2.6×100 cm, 0.02 M PBS elution), and the main peak fraction was collected; (5) Drying identification: freeze-dried polypeptide powder, sent to a third-party commercial company for sequence identification (LC-MS / MS).

[0023] 3. Results Polypeptide yield: 12.1 ± 0.8% (w / w, n = 3, BCA method); Sequence identification (LC-MS / MS): SEQ1 (1125.6 Da, peak area 40.2%), SEQ2 (1020.4 Da, 35.1%), SEQ3 (1108.5 Da, 24.7%); SEQ ID No: 1: Glu-Gln-Lys-Met-Phe-Gln-Ile-Phe-Cys-Ser, 1.1 KDa, peak area 40.2%.

[0024] SEQ ID No: 2: His-Gln-Cys-Asp-Phe-Gln-Ile-Asp-Ala-Phe, 1.0 KDa, peak area 35.1%.

[0025] SEQ ID No: 3: Pro-Gln-Lys-Met-Asp-Gln-Cys-Asp-His-Ser, 1.1 KDa, peak area 24.7%.

[0026] Purity: HPLC main peak area accounted for 82.3% (impurities were small molecule sugars). Example 2, antioxidant activity test (DPPH scavenging experiment) 1. Materials and methods Samples: solid-phase synthesis of SEQ ID No: 1 (purity 96.5%), SEQ ID No: 2 (purity 95.2%), and SEQ ID No: 3 (purity 94.8%).

[0027] SEQ ID No: 1-3 (0.1-2.0 mg / mL, dissolved in PBS); control group: VC (0.1-2.0 mg / mL), blank group (PBS); Method: 200 μL sample + 200 μL 0.1 mM DPPH ethanol solution (avoid light 25°C for 30 min), measure 517 nm absorbance, clearance rate = [1-(A sample-A empty) / A pair] x 100%; Statistics: n = 3, IC was calculated by GraphPad Prism 9.0 50 , t-test compared polypeptide with VC (p < 0.05 was significant), the results are shown in Figure 1 .

[0028] Result: IC 50 (Half-inhibitory concentration) is a key indicator for measuring antioxidant activity. 50 The lower the value, the stronger the antioxidant activity. Based on the experimental results: SEQ ID No: 1's IC50 value... 50 Significantly lower than VC ( p <0.01), indicating that the antioxidant activity of SEQ ID No: 1 is superior to that of the commonly used antioxidant VC; the IC50 of SEQ ID No: 2 is <0.01. 50 No significant difference from VC, moderate activity; IC50 of SEQ ID No: 3 50 Significantly worse than VC ( p <0.001), indicating weak activity. This suggests that SEQ ID No: 1 is the main antioxidant peptide. Example 3: Antibacterial activity test (Staphylococcus aureus) 1. Materials and Methods Bacterial strain: Staphylococcus aureus ATCC 25923 (0.5 McFarland turbidity); Control group: penicillin (10 U / tablet), blank group (sterile PBS); Methods: The diameter of the inhibition zone (mm) was measured using the disk diffusion method (10 μL peptide 10 mg / mL / disc), and the MIC was determined using the microbroth dilution method. Statistical analysis: n=3; t-test was used to compare the peptide with penicillin (p<0.05 was considered significant). Results are as follows: Figures 2-3 As shown.

[0029] As a result, SEQ ID No: 1, although significantly lower than penicillin (p<0.001), had a much lower MIC value than most existing plant peptides (literature values ​​are often >1 mg / mL), showing excellent performance; SEQ ID No: 2 had moderate activity (p<0.01); SEQ ID No: 3 had the weakest activity (p<0.05). Example 4: Anti-inflammatory activity test (inhibition of TNF-α release) 1. Materials and Methods Cells: RAW264.7 macrophages (1×10⁻⁶) 5 cells / well); Control group: dexamethasone (0.1 μM), model group (LPS 1 μg / mL), blank group (no LPS); Methods: TNF-α was measured by ELISA 24 h after LPS stimulation. Inhibition rate = [1 - (experimental group - blank) / (model group - blank)] × 100%; Statistics: n=3, ANOVA analysis (p<0.05 was considered significant).

[0031] The results are as follows Figure 4The inhibition rate of SEQ ID No: 1 was 72.7±2.2%, close to dexamethasone (84.3±1.8%), and significantly better than the model group (p<0.001); the inhibition rate of SEQ ID No: 3 was 66.8±2.8%, the effect was second (p<0.001); the inhibition rate of SEQ ID No: 2 was only 47.6±3.1%, significantly lower than SEQ ID No: 1 and SEQ ID No: 3 (p<0.05). It is illustrated that SEQ ID No: 1 and SEQ ID No: 3 can inhibit inflammatory factors. Example 5, fibroblast proliferation test (CCK-8 method) 1. Materials and methods Cells: human dermal fibroblasts HDF (5×10 3 cells / well); control group: EGF (10 ng / mL), blank group (culture medium); Method: CCK-8 method was used to measure OD 450 24h after administration, proliferation rate=(OD real-OD blank) / (OD blank-OD zero)×100%; Statistics: n=3, t-test to compare polypeptides and blank group (p<0.05 is significant), and the results are shown in Table 1. Figure 5

[0032] Conclusion: SEQ ID No: 1 can significantly promote fibroblast proliferation.

[0033] In summary, the present application isolates the polypeptide of richuan bull testis containing SEQ ID No: 1-3 three sequences, and the comprehensive activity of SEQ ID No: 1 is the best (antioxidant, antibacterial, anti-inflammatory, and proliferation promotion are significant), and the functions of SEQ ID No: 2 / 3 are weaker. The present application provides technical support for efficient preparation and multifunctional application of polypeptides.

[0034] The above is the preferred embodiment of the present application, and it should be pointed out that for ordinary skilled persons in the technical field, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A polypeptide from black ox seeds in Fuchuan County, Hezhou City, characterized in that: It is derived from black ox antler seeds produced in Fuchuan, Hezhou City, and is prepared by compound enzymatic hydrolysis. It contains three characteristic sequences: SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No:

3.

2. The method for preparing the polypeptide according to claim 1, characterized in that: include: (1) Pretreatment of black ox seeds; (2) Enzymatic hydrolysis by complex protease; (3) Centrifuge to collect the supernatant, then perform 3 kDa ultrafiltration, and then Sephadex G-25 chromatography; (4) Freeze-dry to obtain a polypeptide mixture containing SEQ ID No: 1-3.

3. The method as described in claim 2, characterized in that: The complex protease contains trypsin and papain.

4. The method as described in claim 3, characterized in that: The mass fraction ratio of trypsin and papain is 1:

2.

5. The method as described in claim 2, characterized in that: The enzymatic hydrolysis conditions were pH 7.5, 50°C, and 4 h.

6. The method according to claim 2, wherein the enzymatic hydrolysis material-to-liquid ratio is 1:5 (w / v), the enzyme addition amount is 0.5% (w / w), and the enzyme inactivation conditions are 90℃ for 10 min.

7. The use of the polypeptide described in claim 1 (SEQ ID No: 1) in the preparation of antioxidants.

8. The use of the polypeptide described in claim 1 (SEQ ID No: 1) in the preparation of antibacterial drugs.

9. The use of the polypeptide described in claim 1 (SEQ ID No: 1) in the preparation of anti-inflammatory agents.

10. The use of the polypeptide of claim 1 (SEQ ID No: 1) in the preparation of cosmetics that promote skin repair.

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