Preparation method of polypeptide of arnebia euchroma, polypeptide of arnebia euchroma and application thereof

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a preparation method of a polypeptide of Arnebia euchroma, the polypeptide of Arnebia euchroma and application thereof. The preparation method comprises the following steps: extracting Arnebia euchroma after CO2 supercritical extraction, adding water to obtain an extract; performing enzyme hydrolysis in cooperation with Bacillus subtilis, neutral protease and amino peptidase to obtain an enzyme hydrolysate; performing preliminary filtration on the enzyme hydrolysate, and sequentially separating the obtained filtrate through an ultrafiltration membrane and a nanofiltration membrane to collect a nanofiltration concentrated solution; and drying to obtain the polypeptide of Arnebia euchroma. The polypeptide of Arnebia euchroma prepared by the method has high polypeptide content, strong anti-inflammatory, anti-aging, anti-oxidation activities, and can be applied to functional cosmetics or medicines, and is helpful to the development and utilization of Arnebia euchroma resources.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing Xinjiang Lithospermum peptides with anti-inflammatory, repairing and anti-aging effects, as well as the Xinjiang Lithospermum peptides and their applications. Background Technology

[0002] Xinjiang Lithospermum ( Arnebia euchroma This plant, belonging to the Boraginaceae family, is a perennial medicinal plant mainly produced in Northwest and North China. It is sweet, salty, and cold in nature, and enters the heart and liver meridians. It has the effects of clearing heat and cooling blood, detoxifying and promoting rash eruption. Clinically, it is used for rashes, sores, and skin lesions caused by excessive blood heat and toxins. Modern research has confirmed that plants in this genus are rich in naphthoquinones (such as Xinjiang shikonin) and polysaccharides, exerting antitumor, antibacterial, and antiviral activities through multiple mechanisms including antioxidant, anti-inflammatory, and immunomodulatory effects. Its derivatives have wide applications in the pharmaceutical, functional food, and cosmetic fields.

[0003] Currently, the main methods for extracting active ingredients from Lithospermum erythrorhizon in Xinjiang include organic solvent extraction, ultrasound-assisted extraction, and supercritical fluid extraction. Among these, supercritical CO2 extraction technology is widely used for extracting fat-soluble naphthoquinones from Lithospermum erythrorhizon due to its advantages such as high extraction efficiency, no solvent residue, and low-temperature operation. However, the residue from supercritical CO2 extraction is usually treated as waste, resulting in a significant waste of resources. Studies have shown that the residue still contains a large number of water-soluble components (such as polysaccharides, polyphenols, and some proteins), which have the potential for further development and utilization.

[0004] Polypeptides, due to their small molecular weight, easy absorption, and diverse biological activities, have become a hot topic in the field of natural product research. Currently, research on the preparation methods of peptides from Lithospermum erythrorhizon is limited. Existing technologies mostly focus on the total extract or single components of Lithospermum erythrorhizon, lacking targeted enzymatic hydrolysis and biotransformation technologies for protein resources in Lithospermum erythrorhizon residues. Furthermore, conventional enzymatic hydrolysis methods often suffer from low hydrolysis efficiency, low yield of active peptides, and unsatisfactory anti-inflammatory activity of the products. Although microbial fermentation technology can produce abundant enzyme systems through microbial metabolism, promoting the transformation and release of active ingredients, research on its synergistic application with enzymatic hydrolysis technology is still insufficient.

[0005] Therefore, how to make full use of the supercritical extraction residue of Lithospermum erythrorhizon in Xinjiang, develop an efficient and green method for preparing Lithospermum erythrorhizon peptides, obtain peptide products with better anti-inflammatory, repairing and anti-aging effects, and realize the high-value comprehensive utilization of Lithospermum erythrorhizon resources is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments.

[0007] As one aspect of the present invention, the present invention provides a method for preparing Xinjiang Lithospermum peptides with anti-inflammatory, repairing and anti-aging effects, which includes the following steps: (1) Extraction: Take the residue of Xinjiang Lithospermum after CO2 supercritical extraction, add water for extraction, and obtain the extract; (2) Synergistic enzymatic hydrolysis: Add Bacillus subtilis powder, neutral protease and aminopeptidase to the extract obtained in step (1) to carry out enzymatic hydrolysis to obtain the hydrolysate; (3) Separation and purification: The enzyme hydrolysate is initially filtered, and the resulting filtrate is separated by passing it through an ultrafiltration membrane and a nanofiltration membrane in sequence, and the nanofiltration concentrate is collected; (4) Drying: Dry the nanofiltration concentrate to obtain Xinjiang Lithospermum polypeptide; the Xinjiang Lithospermum polypeptide contains one or more of the following peptides: FN, PF, FA, FR, WG, QF, FYP, GPF, RF, FP, GQF, DGF, DPF, PPP, SPF, DW, FSP, PNF, FNP, FQP, GGPF, FDP, PDF, PGWPLFGH, PPPP, PGWPVFGH, PGGW.

[0008] As a preferred embodiment of the preparation method described in this invention, in step (2), the temperature of the enzymatic hydrolysis reaction is 40-50℃ and the pH is 7.5-8.0; the amount of Bacillus subtilis powder added is 1%-3% of the volume of the extract, the amount of neutral protease added is 1%-3% of the volume of the extract, and the amount of aminopeptidase added is 0.5%-1.5% of the volume of the extract.

[0009] As a preferred embodiment of the preparation method described in this invention, in step (1), the preparation method of the residue after supercritical CO2 extraction of Xinjiang Lithospermum includes: pre-treating the root powder of Xinjiang Lithospermum with high pressure instantaneous explosion, using GTCC as an entrainer, extracting under supercritical CO2 extraction conditions, collecting the residue after extraction, repeating the extraction 2-3 times, and combining the residues.

[0010] As a preferred embodiment of the preparation method described in this invention, the pressure of the high-pressure instantaneous explosion pretreatment is 0.6-1.0 MPa, and the holding time is 20-30 seconds; the temperature of the supercritical CO2 extraction is 40-50℃, and the pressure is 35-45 MPa.

[0011] As a preferred embodiment of the preparation method described in this invention, in step (2), the preparation method of the Bacillus subtilis powder solution is as follows: dissolve Bacillus subtilis powder in a culture medium to prepare a Bacillus subtilis powder solution with a mass concentration of 0.2%-0.5%.

[0012] As a preferred embodiment of the preparation method described in this invention, in step (3), the preliminary filtration is performed using a 0.45 μm filter plate; the molecular weight cutoff of the ultrafiltration membrane is 5000 Da, and the permeate is collected; the molecular weight cutoff of the nanofiltration membrane is 200 Da, and the concentrate is collected.

[0013] As a preferred embodiment of the preparation method described in this invention, in step (4), the drying is spray drying; hydroxypropyl methylcellulose is added to the nanofiltration concentrate before drying; the inlet air temperature of the spray drying is 140-160℃, the outlet air temperature is 80-90℃, and the feed flow rate is 150-250 mL / h.

[0014] As a preferred embodiment of the preparation method described in this invention, in step (1), the ratio of the residue of Xinjiang Lithospermum erythrorhizon after CO2 supercritical extraction to water is 1:20-1:40 (g / mL); in step (2), the enzymatic hydrolysis reaction takes 4-6 hours.

[0015] The present invention also provides the application of the aforementioned Xinjiang purple gromwell polypeptide in the preparation of drugs, cosmetics or special medical foods with anti-inflammatory, repairing, anti-aging and / or antioxidant effects.

[0016] The beneficial effects of this invention are as follows: The Xinjiang Lithospermum polypeptide of this invention is obtained by extracting the residue from Xinjiang Lithospermum using CO2 supercritical extraction, followed by enzymatic hydrolysis. The Xinjiang Lithospermum polypeptide of this invention has a molecular weight between 200-5000 Da and a uniform molecular weight distribution. The Xinjiang Lithospermum polypeptide prepared by this invention has a high polypeptide content and strong anti-inflammatory, repairing, anti-aging, and antioxidant activities, making it suitable for use in functional cosmetics or pharmaceuticals and contributing to the development and utilization of Xinjiang Lithospermum resources. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein: Figure 1 This is a graph showing the evaluation results of anti-aging efficacy. Detailed Implementation

[0018] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0019] Raw material information: Root of Lithospermum erythrorhizon from Yili, Xinjiang, produced by Bozhou Huikangtang Traditional Chinese Medicine Technology Co., Ltd.

[0020] Reagent Information: Peptone, Catalog No. 01-001, Beijing Aoboxing Biotechnology Co., Ltd.; D-Anhydrous Glucose, Beijing Bio-Top Technology Co., Ltd.; Yeast Extract, Beijing Solarbio Technology Co., Ltd.; Neutral Protease, CAS No. 9068-59-1, Beijing Bio-Top Technology Co., Ltd.; Aminopeptidase, Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.; Hydroxypropyl Methylcellulose, Type 2910, Shandong Liaocheng Ahua Pharmaceutical Co., Ltd.

[0021] Preparation of the supercritical extraction residue of Lithospermum erythrorhizon used in this invention: Step 1: Pretreatment: Crush the dried Xinjiang purple gromwell root into powder using a pulverizer, sieve (0.355mm aperture), retain the sieved portion, and load it into a high-pressure steam explosion reactor. Use high-pressure instantaneous explosion technology to maintain pressure at 0.8 MPa for 30 seconds. After the pressure holding is completed, open the explosion valve to rupture the cell walls and release the active ingredients, thus obtaining the pretreated material.

[0022] Step 2: Supercritical CO2 Extraction: 1 kg of the material obtained in Step 1 is loaded into the extraction vessel, and 0.5 kg of GTCC is added and stirred until homogeneous. The extraction vessel is heated to 45 ℃, the separation vessel is heated to 45 ℃, and the storage tank is heated to 35 ℃. Then, the CO2 cylinder is turned on, and the CO2 flow rate is controlled at 50 L / h. The system is pressurized by a high-pressure pump. When the pressure in the extraction vessel is 40 MPa and the pressure in the separation vessel is 5 MPa, the circulation extraction begins, and the extraction vessel is kept at a constant temperature and pressure. After extraction for 1 h, the material is discharged from the outlet of the separation vessel, the residue is retained, and the extract is discarded. After the first extraction is completed, 1 kg of GTCC is pumped in through the entrainer pump, and the above experimental operation is repeated. The second extraction is completed after 1 h, the residue is retained, and the extract is discarded. After the second extraction is completed, 1 kg of GTCC is pumped in through the entrainer pump, and the above experimental operation is repeated. The residue is retained, the extract is discarded, and the residues are combined.

[0023] Comparative Example 1: (1) The dried Xinjiang purple gromwell root was pulverized into powder by a pulverizer and sieved (0.355 mm aperture). The sieved part was retained. 100g of powder was added to 3000mL of deionized water and extracted at 90℃ for 2h.

[0024] (2) Adjust the temperature to 45℃, adjust the pH to 7.5, add 2% neutral protease, and enzymatically hydrolyze for 2h; then add 1% aminopeptidase and enzymatically hydrolyze for 2h; after the enzymatic hydrolysis is completed, keep at 90℃ for 10min to inactivate the enzyme.

[0025] (3) After cooling to room temperature, filter with a 0.45μm filter plate, then use a 5000Da membrane core for ultrafiltration, collect the permeate, then replace with a 200Da membrane core for nanofiltration, and collect the concentrate.

[0026] (4) Use a rotary evaporator to concentrate the liquid at 60°C to 500mL, add 0.8% hydroxypropyl methylcellulose, set the inlet air temperature to 150°C, the outlet air temperature to 85°C, and the flow rate to 200mL / h for spray drying.

[0027] Comparative Example 2: (1) Take 100g of supercritical extraction residue of Lithospermum erythrorhizon from Xinjiang and add it to 3000mL of deionized water. Extract at 90℃ for 2h.

[0028] (2) Adjust the temperature to 45℃, adjust the pH to 7.5, add 2% neutral protease, and enzymatically hydrolyze for 2h; then add 1% aminopeptidase and enzymatically hydrolyze for 2h; after the enzymatic hydrolysis is completed, keep at 90℃ for 10min to inactivate the enzyme.

[0029] (3) After cooling to room temperature, filter using a 0.45 μm filter plate. Then perform ultrafiltration using a 5000 Da membrane element and collect the permeate. Then replace with a 200 Da membrane element for nanofiltration and collect the concentrate.

[0030] (4) Use a rotary evaporator to concentrate the liquid at 60°C to 500mL; add 0.8% hydroxypropyl methylcellulose to the liquid, set the inlet air temperature to 150°C, the outlet air temperature to 85°C, and the flow rate to 200mL / h for spray drying.

[0031] Comparative Example 3: (1) Take 100g of supercritical extraction residue of Lithospermum erythrorhizon from Xinjiang and mix it with 10g of water. Put it into a 2500ml conical flask, stir it evenly, seal it with plastic wrap, sterilize it at 121℃ for 20 minutes, and obtain solid fermentation culture medium after cooling. Inoculate it with Bacillus subtilis and place it in a constant temperature incubator at 28℃ for solid fermentation for 3 days. Finally, sterilize it at 121℃ for 20 minutes.

[0032] (2) Take 100g of fermented powder and add it to 3000mL of deionized water, and cook at 90℃ for 2h.

[0033] (3) Adjust the temperature to 45℃, adjust the pH to 7.5, add 2% neutral protease, and enzymatically hydrolyze for 2h; then add 1% aminopeptidase and enzymatically hydrolyze for 2h; after the enzymatic hydrolysis is completed, keep at 90℃ for 10min to inactivate the enzyme.

[0034] (4) After cooling to room temperature, filter using a 0.45 μm filter plate. Then perform ultrafiltration using a 5000 Da membrane element and collect the permeate. Then replace with a 200 Da membrane element for nanofiltration and collect the concentrate.

[0035] (5) Use a rotary evaporator to concentrate the liquid at 60°C to 500mL; add 0.8% hydroxypropyl methylcellulose to the liquid, set the inlet air temperature to 150°C, the outlet air temperature to 85°C, and the flow rate to 200mL / h for spray drying.

[0036] Example 1: (1) Take 100g of supercritical extraction residue of Lithospermum erythrorhizon from Xinjiang and add it to 3000mL of deionized water. Extract at 90℃ for 2h.

[0037] (2) Preparation of Bacillus subtilis powder solution: Dissolve 3g of Bacillus subtilis powder (purchased from Henan Xinyangshao Biotechnology Co., Ltd., batch number KC20250609) in 1kg of culture medium; the culture medium composition is: 10g / L peptone, 10g / L glucose, 5g / L yeast powder, and deionized water as solvent; the mass concentration of Bacillus subtilis powder solution is 0.3%.

[0038] (3) Adjust the temperature of the extract obtained in step (1) to 45℃, adjust the pH to 7.5, add 2% (volume percentage) of Bacillus subtilis powder solution prepared in step (2), add 2% neutral protease, enzymatically hydrolyze for 2h, then add 1% aminopeptidase, enzymatically hydrolyze for 2h; after the enzymatic hydrolysis is completed, keep at 90℃ for 10min to inactivate the enzyme.

[0039] (4) After cooling to room temperature, filter using a 0.45 μm filter plate. Then perform ultrafiltration using a 5000 Da membrane element and collect the permeate. Then replace with a 200 Da membrane element for nanofiltration and collect the concentrate.

[0040] (5) Use a rotary evaporator to concentrate the liquid at 60°C to 500mL; add 0.8% hydroxypropyl methylcellulose to the liquid, set the inlet air temperature to 150°C, the outlet air temperature to 85°C, and the flow rate to 200mL / h for spray drying.

[0041] Experimental methods and results: Polypeptide content determination experiment (biuret reagent method): 1) Biuret reagent: Reagent A: 0.15g CuSO4, 0.6g potassium sodium tartrate, and 50mL distilled water are mixed evenly; Reagent B: 30mL 10% NaOH solution (mix A and B evenly before use).

[0042] 2) Preparation of bovine serum albumin standard curve: (Standard range 0-5 mg / mL) First, prepare a 10 mg / mL bovine serum albumin solution (water as solvent). Take 0, 50, 100, 150, 200, and 250 μL into EP tubes respectively, add water to 0.5 mL, add 2 mL of biuret reagent (A and B mixed before use), react at room temperature for 30 min, and measure its absorbance at 540 nm to prepare the standard curve.

[0043] 3) Determination of sample solution: Take the sample, add 1.0 mL of 10% trichloroacetic acid to dissolve it, centrifuge to precipitate the protein, take 0.5 mL of supernatant, add 2 mL of biuret reagent (A and B mixed before use), react at room temperature for 30 min, measure its absorbance at 540 nm, and calculate the sample peptide content according to the standard curve.

[0044] 4) Formula for calculating polypeptide content: The standard curve for polypeptide content is y = Ax + B(R). 2 >0.99); Polypeptide content (%) = Protein or polypeptide concentration (mg / mL) / Sample solution concentration (mg / mL) × 100%.

[0045] 5) The results of the polypeptide content determination experiment are shown in Table 1.

[0046] Table 1

[0047] In vitro antioxidant effect experiment (DPPH free radical inhibition experiment): (1) Preparation of DPPH ethanol solution: Weigh 20 mg of DPPH, dissolve it in anhydrous ethanol, and dilute to a final volume of 250 mL in a volumetric flask. The DPPH concentration is prepared to be 2 × 10⁻⁶ mg / mL. -4 mol / L; store protected from light at 0-4 ℃, prepare and use immediately, effective within 4 hours (positive control: vitamin C: 1 mg / mL).

[0048] (2) Preparation of the test solution: The polypeptide samples were prepared into test solutions with a polypeptide concentration of 5 mg / mL.

[0049] (3) Experimental steps: Add the reagents according to Table 2.

[0050] 1) Take 1 mL of the test solution and 1 mL of 2×10⁻⁶ solution. -4 Mix the mol / L DPPH solution thoroughly (tube A). 2) Take 1 mL of solvent and 1 mL of 2×10 -4 Mix the mol / L DPPH solution thoroughly (tube B). 3) Mix 1 mL of solvent with 1 mL of the test solution (tube C). 4) After reacting in the dark for 30 minutes, measure the absorbance values ​​of tubes A, B, and C at 517 nm.

[0051] Table 2 Reagent Proportioning Table

[0052] (4) Calculation formula for DPPH free radical inhibition rate: DPPH inhibition rate (%) = (B+CA) / B×100%.

[0053] (5) The results of the DPPH free radical inhibition experiment are shown in Table 3.

[0054] Table 3 Results of DPPH free radical scavenging rate determination

[0055] Evaluation of anti-inflammatory efficacy (COX-2 inhibition experiment): (1) Sample preparation: Dilute the sample to the concentration to be tested.

[0056] (2) Reagent preparation: 1) Dissolve all reagents except rhCOX-2 at room temperature, centrifuge briefly to allow the solution to settle to the bottom of the tube, then mix well for later use. COX-2 Probe, COX-2 Cofactor (50X), and COX-2 Substrate (50X) are prepared in DMSO and can be dissolved in a 37°C water bath for 0.5-2 minutes to promote dissolution. After use, store immediately at -20°C protected from light.

[0057] 2) Preparation of COX-2 Cofactor working solution: Prepare an appropriate amount of COX-2 Cofactor working solution according to the ratio of 5 μL of COX-2 Cofactor working solution required for each sample. Take an appropriate amount of COX-2 Cofactor (50X) and dilute it with COX-2 Assay Buffer at a ratio of 1:49. For example, add 4 μL of COX-2 Cofactor (50X) to 196 μL of COX-2 Assay Buffer to prepare 200 μL of COX-2 Cofactor working solution. The prepared COX-2 Cofactor working solution can be stored at 4°C and should only be used on the same day.

[0058] 3) Preparation of COX-2 working solution: Prepare an appropriate amount of COX-2 working solution according to the ratio of 5 μL of COX-2 working solution required for each sample. Take an appropriate amount of rhCOX-2 (25X) and dilute it with COX-2 Assay Buffer at a ratio of 1:24. For example, add 8 μL of rhCOX-2 (25X) to 192 μL of COX-2 Assay Buffer to prepare 200 μL of COX-2 working solution. The prepared COX-2 working solution can be temporarily stored on ice; the enzyme activity is basically stable within 1 hour. All operations involving COX-2 should be performed on ice.

[0059] 4) Preparation of COX-2 Substrate Working Solution: Prepare an appropriate amount of COX-2 Substrate working solution according to the ratio of 5 μL of COX-2 Substrate working solution required for each sample. Take an appropriate amount of COX-2 Substrate (50X), add an equal volume of Substrate Buffer, and vortex thoroughly. Dilute this mixture with Milli-Q grade pure water or redistilled water at a ratio of 1:24, and vortex thoroughly. For example, add 20 μL of COX-2 Substrate (50X) to 20 μL of Substrate Buffer, vortex thoroughly, then add 960 μL of Milli-Q grade pure water or redistilled water, and vortex thoroughly again to obtain 1 mL of COX-2 Substrate working solution. The prepared COX-2 Substrate working solution can be temporarily stored on an ice bath and is relatively stable for up to 1 hour. Note: The COX-2 Substrate working solution can also be prepared during the sample detection process by incubating at 37°C for 10 minutes.

[0060] 5) Preparation of the positive control inhibitor Celecoxib solution: The positive control inhibitor Celecoxib provided in this kit is at a concentration of 100 µM, prepared in DMSO. It can be diluted to the desired concentration or concentration gradient using the same solvent as the test inhibitor, as needed. Typically, the IC50 of Celecoxib is approximately 10 nM to 100 nM.

[0061] (3) Sample testing 1) Referring to Table 4, set up control wells and sample wells using a 96-well blackboard, and add the sample and each solution in the order shown in the table below. After adding the sample to be tested, mix well and incubate at 37°C for 10 minutes.

[0062] Table 4 Reagent Proportioning Table

[0063] 2) Add 5 μL of COX-2 Probe to each well.

[0064] 3) Quickly add 5 μL of COX-2 Substrate working solution to each well and mix well. Note: The reaction will start immediately after adding the COX-2 Substrate working solution. If there are many wells, you can operate at a low temperature or use a multi-pipette to reduce the error caused by the time difference in adding the COX-2 Substrate working solution between wells. Mixing can also be done on a culture plate shaker.

[0065] 4) After incubating at 37℃ in the dark for 5 minutes, perform fluorescence measurement. The excitation wavelength is 560nm and the emission wavelength is 590nm. If the fluorescence reading is low, the incubation time can be appropriately extended to 10-20 minutes.

[0066] (4) Calculation 1) Calculate the average fluorescence value of each sample well and blank control well, which can be recorded as RFU blank control, RFU 100% enzyme activity control, RFU positive inhibitor control, and RFU sample, respectively. RFU stands for Relative Fluorescence Unit.

[0067] 2) Calculate the inhibition percentage for each sample. The calculation formula is as follows: Inhibition rate (%) = (RFU 100% enzyme activity control - RFU sample) / (RFU 100% enzyme activity control - RFU blank control) × 100%.

[0068] (5) The results of the COX-2 inhibition experiment are shown in Table 5.

[0069] Table 5 Results of COX-2 inhibition rate determination

[0070] The product obtained in Example 1 was analyzed by liquid chromatography-mass spectrometry (LC-MS). Sample processing: Peptide solution treatment: Dissolve the sample from Example 1 thoroughly in 0.1% TFA. Centrifuge at 4°C and 12,000 rpm, and transfer the supernatant to a new EP tube.

[0071] Desalting using a BKMAMLAB C18 1mL SPE column: Load 2ml of 100% ACN into the SPE column and slowly elute to activate it. Load 2ml of 0.1% TFA into the SPE column and slowly elute to equilibrate it. Load the sample into the SPE column and slowly expel the liquid. Load 3ml of 5% ACN and 0.1% TFA into the SPE column and slowly elute to wash it. Load 1ml of 90% ACN and 0.1% TFA into the SPE column, elute the peptides into a new EP tube, and vacuum dry.

[0072] Analytical testing: RPLC-MS: Dissolve the peptide in 100 μL of dissolving buffer (0.1% formic acid), vortex thoroughly, centrifuge at 17000 rpm and 4°C for 20 min, transfer the supernatant to a sample tube, and take 1 μL for mass spectrometry identification.

[0073] Liquid chromatography settings parameters: Table 6

[0074] Mobile phase parameters: Table 7

[0075] Peptides with a bioactivity probability of 90% or higher were screened from the peptide sequencing results. These peptides were then docked with TRPV4 molecules. The docking results were analyzed to determine whether the candidate peptides were bioactive peptides with actual functional activity.

[0076] Peptide ligand preparation: The 3D structures of the 33 peptides used were all drawn by PyMol 3.1 and optimized using the MMFF94s force field in Avogadro 1.2.0 software. Polar hydrogen atoms were used in AutoDockTools 1.5.7.

[0077] Preparation of target protein structure: Download the TRPV4 (PDB ID: 8FC7) structure from the RCSB PDB database, and use PyMol to remove the XPW, GDP, and MG ligands from TRPV4 (PDB ID: 8FC7). Use AutoDockTools 1.5.7 to remove water and add polar hydrogen atoms and charges.

[0078] Conduct virtual docking: The TRPV4 binding site of 8FC7 and XPW was determined using PyMol software, and was used as its inhibitor binding site. The TRPV4 binding site is defined as center_x 112.1 --center_y 112.4 --center_z 156.4 --size_x20.3 --size_y 16.6 --size_z 18.3. The 5D3I binding site of 8FC7 and PCW was determined, and was used as its inhibitor binding site.

[0079] Vina's ability to combine: criteria for judging Vina's ability to combine Weak binding: ΔG>-5 kcal / mol, generally considered to have limited binding capacity, which may require further optimization.

[0080] Moderate binding: -7 kcal / mol ≤ ΔG ≤ -5 kcal / mol. These ligands are often used as candidates for initial screening.

[0081] Strong binding: ΔG ≤ -9 kcal / mol suggests that the ligand and acceptor may form stable interactions such as hydrogen bonds, salt bridges or hydrophobic clusters, which can be a key research focus.

[0082] ΔG ≤ -7 kcal / mol was used as the screening threshold for potential inhibitors.

[0083] The predicted binding energies of 33 Xinjiang Lithospermum peptides to TRPV4 are ranked as follows: MM > GF > SF > FG > AF > DF (ΔG = -6.834) > FN (ΔG = -7.113) > PF > FA > FR > WG > QF > FYP > GPF > RF > FP > GQF > DGF > DPF > PPP > SPF > DW > FSP > PNF > FNP > FQP > GGPF > FDP > PDF > PGWPLFGH (ΔG = -8.721) > PPPP (ΔG = -9.002) > PGWPVFGH > PGGW. Sequences with four or more amino acids are listed in SEQ ID NO. 1-5.

[0084] Evaluation of repair efficacy: Human immortalized keratinocytes (HaCaT) in the logarithmic growth phase were harvested, digested, resuspended, and seeded into 24-well cell culture plates containing scabs, with three replicates per group. The cells were cultured at 37°C in a 5% CO2 environment. When the cells reached approximately 50% confluence, culture medium containing the sample from the example (with a peptide concentration of 50 μg / mL) was added. Groups BC served as the control group and did not receive the sample. After 3 days of further culture, the supernatant was discarded, and the cells were fixed. The relative fluorescence intensity of the inner lining protein (IVL) in each group was detected by immunofluorescence antibody incubation and fluorescence microscopy.

[0085] Table 8

[0086] As can be seen from Table 8, the Xinjiang Lithospermum peptide in Example 1 can significantly promote the expression of IVL in keratinocytes and has a good repair effect.

[0087] Evaluation of anti-aging efficacy: Human skin fibroblasts in the logarithmic growth phase were harvested, digested, resuspended, and seeded into 6-well cell culture plates, with three replicates per group. Cells were incubated at 37°C in a 5% CO2 environment. When the cells reached approximately 80% confluence, the model and sample groups were treated with H2O2 to induce cell senescence. The sample group was supplemented with culture medium containing the peptide of the sample from the example study (peptide concentration 50 μg / mL). The BC group served as the blank control group, and the NC group served as the model group. Cells were cultured for another 3 days, then the supernatant was discarded, and the cells were fixed and observed under a microscope after staining with β-galactosidase (β-gal). Figure 1 At least 100 cells were collected in each field of view, and the β-gal positivity rate of each group was calculated.

[0088] The formula for calculating the β-gal positivity rate is: β-gal (%) = number of positive cells in the field of view / total number of cells in the field of view × 100%.

[0089] Table 9

[0090] From Table 9 and Figure 1 As can be seen, the Xinjiang Lithospermum peptides in Example 1 can significantly reduce H2O2-induced cell senescence and have a good anti-aging effect.

[0091] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of a Xinjiang Lithospermum erythrorhizon polypeptide with anti-inflammatory and repairing effects in the preparation of drugs or cosmetics with anti-aging effects, characterized in that, The preparation method of the Xinjiang Lithospermum peptide includes the following steps: (1) Extraction: Take the residue of Xinjiang Lithospermum after CO2 supercritical extraction, add water for extraction, and obtain the extract; (2) Synergistic enzymatic hydrolysis: Add Bacillus subtilis powder, neutral protease and aminopeptidase to the extract obtained in step (1) to carry out enzymatic hydrolysis to obtain the hydrolysate; (3) Separation and purification: The enzymatic hydrolysate is initially filtered using a 0.45 μm filter plate. The resulting filtrate is then separated by passing it through an ultrafiltration membrane and a nanofiltration membrane in sequence. The nanofiltration concentrate is collected. The ultrafiltration membrane has a molecular weight cutoff of 5000 Da, and the permeate is collected. The nanofiltration membrane has a molecular weight cutoff of 200 Da, and the concentrate is collected. (4) Drying: The nanofiltration concentrate is dried to obtain Xinjiang Lithospermum polypeptide; the Xinjiang Lithospermum polypeptide contains the following peptides: FN, PF, FA, FR, WG, QF, FYP, GPF, RF, FP, GQF, DGF, DPF, PPP, SPF, DW, FSP, PNF, FNP, FQP, GGPF, FDP, PDF, PGWPLFGH, PPPP, PGWPVFGH and PGGW; The senescence referred to is H2O2-induced cellular senescence.

2. The application according to claim 1, characterized in that, In step (2), the temperature of the enzymatic hydrolysis reaction is 40-50℃ and the pH is 7.5-8.0; the amount of Bacillus subtilis powder added is 1%-3% of the volume of the extract, the amount of neutral protease added is 1%-3% of the volume of the extract, and the amount of aminopeptidase added is 0.5%-1.5% of the volume of the extract.

3. The application according to claim 1 or 2, characterized in that, In step (1), the method for preparing the residue of Xinjiang Lithospermum after supercritical CO2 extraction includes: pre-treating the root powder of Xinjiang Lithospermum under high pressure instantaneous explosion, using GTCC as an entrainer, extracting under supercritical CO2 extraction conditions, collecting the residue after extraction, repeating the extraction 2-3 times, and combining the residues.

4. The application according to claim 3, characterized in that, The pressure of the high-pressure instantaneous explosion pretreatment is 0.6-1.0 MPa, and the holding time is 20-30 seconds; the temperature of the supercritical CO2 extraction is 40-50℃, and the pressure is 35-45 MPa.

5. The application according to claim 1 or 2, characterized in that, In step (2), the preparation method of the Bacillus subtilis powder solution is as follows: dissolve Bacillus subtilis powder in a culture medium to prepare a Bacillus subtilis powder solution with a mass concentration of 0.2%-0.5%.

6. The application according to claim 1 or 2, characterized in that, In step (4), the drying is spray drying; hydroxypropyl methylcellulose is added to the nanofiltration concentrate before drying; the inlet air temperature of the spray drying is 140-160℃, the outlet air temperature is 80-90℃, and the feed flow rate is 150-250 mL / h.

7. The application according to claim 1 or 2, characterized in that, In step (1), the ratio of the residue of Xinjiang purple gromwell after CO2 supercritical extraction to water is 1:20-1:40 g / mL; in step (2), the enzymatic hydrolysis reaction takes 4-6 hours.

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