HMGB1 inhibitor, method for targeting the preparation of HMGB1 inhibitor from extracts of Leontopodium alpinum and applications thereof

Abstract

The present invention belongs to the technical field of analytical chemistry, and provides an HMGB1 inhibitor and a method for targeting the preparation of an HMGB1 inhibitor from the extract of Leontopodium leontopodioides. The inhibitor is apigenin-7-O- β -D-glucoside. The method for targeting the preparation of the HMGB1 inhibitor from the extract of Leontopodium leontopodioides is as follows: incubate immobilized HMGB1 with the extract of Leontopodium leontopodioides to obtain a precipitate, wash the non-specific adsorbents of the precipitate with a buffer solution and then wash it with an inactivator, so as to obtain the HMGB1 inhibitor. Through the established targeting preparation technology, the present invention obtains a new HMGB1 inhibitor, apigenin-7-O- β -D-glucoside from Leontopodium leontopodioides, which enriches the types of direct HMGB1 inhibitors and provides a scientific clue for the subsequent application of Leontopodium leontopodioides in anti-inflammatory aging. In addition, the established targeting preparation technology can target and efficiently prepare HMGB1 inhibitors from the complex extract of Leontopodium leontopodioides, overcoming the low efficiency problem in the prior art that each compound needs to be prepared separately and its activity needs to be screened.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and particularly relates to an HMGB1 inhibitor, a method for targeting the preparation of an HMGB1 inhibitor from the extract of Leontopodium leontopodioides and applications thereof. Background Art

[0002] High mobility group box 1 (HMGB1) is a non-histone DNA-binding protein that plays a key role in DNA bending and transcriptional regulation. Extracellular HMGB1 interacts with the receptor for advanced glycation end products located on the cell surface, leading to the activation of sterile immune responses and causing inflammation. Excessive extracellular HMGB1 is associated with persistent inflammation, and this chronic inflammation caused by HMGB1 is considered to be one of the causes of aging. Inhibiting the pro-inflammatory effect of HMGB1 can delay aging by inhibiting the development of chronic inflammation, which is a new anti-aging approach.

[0003] Leontopodium leontopodioides is a plant of the genus Leontopodium in the family Asteraceae. It is a wild flower growing in plateau areas and has various pharmacological activities such as anti-inflammatory and hypoglycemic effects. Due to its anti-inflammatory and antioxidant effects, Leontopodium leontopodioides has also received extensive attention in the cosmetics industry, and its extract can play soothing, anti-aging and other effects when added to cosmetics. However, there is currently no literature or patent that details the association between the active ingredients in Leontopodium leontopodioides and HMGB1 inhibitory activity. In addition, there is no relevant report on a method for efficiently screening direct HMGB1 inhibitors from such complex plant samples as Leontopodium leontopodioides. Traditional methods for screening HMGB1 inhibitors usually use the HMGB1-induced mouse RAW264.7 cell inflammation model to screen HMGB1 inhibitors one by one for individual samples. This method for screening direct HMGB1 inhibitors requires monomeric compounds, cells in good condition, and HMGB1 protein, which lacks efficiency and is too costly, resulting in no objective results in the study of the correlation between the exact active substance basis in Leontopodium leontopodioides and HMGB1.

[0004] Leontopodium leontopodioides has been used as a raw material for cosmetics in multiple product fields, but its anti-aging effect, especially for sensitive skin, has not been fully developed. The research on anti-aging for sensitive skin has always been a technical difficulty in the cosmetics industry. According to statistics, about 60% of the global population has varying degrees of skin sensitivity symptoms, and the core mechanism of aging of such skin has been proven to be closely related to "inflammatory aging" caused by chronic low-grade inflammation. This special skin condition requires that the anti-aging plan must simultaneously achieve the dual goals of barrier function repair and inflammation response regulation. It is worth noting that HMGB1, a core member of damage-associated molecular patterns, has an expression level in chronic skin inflammation tissues that can reach 3-5 times that of normal skin, and this characteristic makes it a key target for cracking the mechanism of inflammatory aging. However, up to now, anti-aging technologies based on the HMGB1 target have not been effectively transformed and applied in the cosmetics field. Summary of the Invention

[0005] The present invention aims to solve the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes an application of targeting the preparation of HMGB1 inhibitors in Leontopodium leontopodioides in the ligand fishing technology based on immobilized HMGB1, providing a more convenient, efficient and low-cost method for discovering HMGB1 inhibitors, and at the same time providing a scientific basis for the correlation between the substance basis that truly exerts activity in Leontopodium leontopodioides and HMGB1.

[0006] In the first aspect, the present invention provides an HMGB1 inhibitor, which is apigenin-7-O- β -D-glucopyranoside, namely Apigenin-7-O- β -D-glucopyranoside.

[0007] In the second aspect, the present invention provides an application of the above HMGB1 inhibitor in cosmetics.

[0008] In the third aspect, the present invention provides a method for targeting the preparation of the above HMGB1 inhibitor from the extract of Leontopodium leontopodioides, including:

[0009] Incubating the immobilized HMGB1 with the extract of Leontopodium leontopodioides to obtain a precipitate, washing the precipitate with a buffer solution and then with an inactivator to inactivate the immobilized HMGB1, thereby releasing the HMGB1 inhibitor obtained by fishing, namely apigenin-7-O- β -D-glucopyranoside (Apigenin-7-O- β -D-glucopyranoside).

[0010] Preferably, the above method further includes steps such as analyzing, preparing the fishing results by HPLC, and verifying the experimental results. As Figure 1 shown, the specific operation of the method for targeting the preparation of the above HMGB1 inhibitor from the extract of Leontopodium leontopodioides is as follows:

[0011] (1) Incubate the immobilized HMGB1 with the extract of Leontopodium leontopodioides according to a mass ratio of 2:1.

[0012] (2) After centrifugation, wash the precipitate three times with Tris-HCl buffer solution to obtain a precipitate, and use 95% methanol ( v / v ) to inactivate HMGB1 and release the HMGB1 inhibitor into the above methanol solution to obtain a methanol solution of the fishing results. Subsequently, use a nitrogen blower to dry the methanol solution of the fishing results, and re-dissolve it with a certain amount of 100 μL methanol to increase the concentration of the fishing results in the methanol solution for convenient detection by HPLC.

[0013] (3) In the HPLC chromatogram, the detected compound is an HMGB1 inhibitor, and the undetected one does not belong to the HMGB1 inhibitor. The detected HMGB1 inhibitor is collected by HPLC for subsequent identification and activity verification experiments.

[0014] (4) Qualitative analysis of the prepared HMGB1 inhibitor is carried out by HPLC-Q-TOF-MS / MS and compared with the standard.

[0015] (5) The activity of the prepared HMGB1 inhibitor is verified using the HMGB1-induced murine RAW246.7 inflammation model.

[0016] (6) The binding site between the prepared HMGB1 inhibitor and HMGB1 is further investigated by molecular docking technology.

[0017] The present invention provides a method for targeted acquisition of HMGB1 inhibitors in Leontopodium leontopodioides using immobilized HMGB1. By utilizing the specific binding between a protein and its ligand and the property that an immobilized enzyme can be separated by solid-liquid separation, the rapid and targeted preparation of HMGB1 inhibitors is achieved. Using this method, 3 μg of Apigenin-7-O- β -D-glucopyranoside can be obtained from 1 mg of Leontopodium leontopodioides extract. Compared with traditional methods, it is more rapid, efficient, and has lower costs.

[0018] Preferably, the preparation method of synthetic immobilized HMGB1 includes the following steps:

[0019] Step SA-1: Using protein engineering technology, recombinant human HMGB1 with an N-terminal fusion His-tag is recombinantly expressed in the Escherichia coli system to obtain a cell lysate containing recombinant human HMGB1 with an N-terminal fusion His-tag.

[0020] Step SA-2: After synthesizing the metal-organic framework material UiO-66-NH2 by the solvothermal method, using glutaraldehyde as a linker arm, its surface is modified by tyrosine to make the surface of the material carry free carboxyl groups. Then nickel ions are added for chelation to synthesize NT-MOF.

[0021] Step SA-3: Take a certain amount of NT-MOF and incubate it with the above cell lysate. After washing multiple times, immobilized HMGB1 is obtained, and a series of experiments are carried out on the immobilized HMGB1 for verification.

[0022] The immobilized HMGB1 prepared by the above method is simple to operate, and the immobilized HMGB1 with good protein loading can be obtained in only one step, which can be used to establish the ligand fishing technology for HMGB1 inhibitors. Compared with other methods for synthesizing immobilized enzymes, recombinant human HMGB1 with an N-terminal fused His-tag immobilizes HMGB1 on the surface of NT-MOF in a way that does not affect the exposure of the pro-inflammatory active domain of HMGB1 to the solution, ensuring the accuracy of the ligand fishing technology.

[0023] Preferably, the mass ratio of the immobilized HMGB1 to the extract of Leontopodium leontopodioides is 2:1, the incubation temperature is 37 °C, and the pH is 8.0.

[0024] More preferably, HPLC analysis is carried out using an Agilent ZORBAX SB-Aq column with a column specification of 3.0 mm × 100 mm and 3.5 μm; the mobile phase is 0.01% formic acid aqueous solution for phase A and acetonitrile for phase B; the elution program is: the volume fraction of mobile phase B in the mobile phase is 10% - 50% from 0 to 30 min.

[0025] Preferably, when co-incubating the immobilized HMGB1 with the extract of Leontopodium leontopodioides, the immobilized HMGB1 is diluted with a Tris-HCl buffer solution with pH = 8.0 and a concentration of 50 mM.

[0026] Preferably, the preparation method of the extract of Leontopodium leontopodioides includes the following steps: Step S0-1, drying and pulverizing the raw material of Leontopodium leontopodioides at 70 °C - 80 °C and then passing through a 30 - 50 mesh sieve; Step S0-2, mixing the Leontopodium leontopodioides obtained in Step S0-1 with an ethanol extraction solvent with a mass ratio of 60% - 95% and carrying out extraction at room temperature for 24 h - 48 h, extracting 1 - 3 times, filtering the extract with a 100 - 400 mesh sieve, and then obtaining the extract of Leontopodium leontopodioides after concentration under reduced pressure; Step S0-3, drying the extract of Leontopodium leontopodioides obtained in Step S0-2 to obtain the extract of Leontopodium leontopodioides.

[0027] Preferably, the inactivator is an aqueous methanol solution with a volume ratio of 95%, abbreviated as 95% methanol solution.

[0028] Preferably, the buffer solution is Tris-HCl with pH = 8.0 and a concentration of 50 mM.

[0029] The present invention provides the application of the above immobilized HMGB1 in the ligand fishing technology, which can be used for the method of targeting the preparation of direct inhibitors of HMGB1 in Leontopodium leontopodioides, and the following method was used for verification, including the following steps:

[0030] (1) Through literature research, glycyrrhizic acid was selected as the positive compound, and gallic acid and chlorogenic acid were selected as the negative compounds to prepare a mixed model solution.

[0031] (2) Take 2 mg of immobilized HMGB1 and co-incubate it with the mixed model solution.

[0032] (3) After centrifugation, wash the precipitate three times with PBS to obtain the precipitate, and inactivate HMGB1 with 95% methanol ( v / v ) to release the HMGB1 inhibitor and obtain the fishing result. Then, dry it with a nitrogen blower and re-dissolve it with a certain amount of methanol for subsequent HPLC detection.

[0033] (4) In the fishing result, the detected compound is determined as an HMGB1 inhibitor, and if not detected, it does not belong to the HMGB1 inhibitor. Verify whether the ligand fishing technology established using immobilized HMGB1 is feasible and accurate in the above manner. It can be seen that the use of immobilized HMGB1 provided by the present invention can accurately obtain HMGB1 inhibitors in a mixed sample without recognizing non-specific compounds, proving the feasibility and accuracy of the application of this immobilized enzyme in the ligand fishing technology, and it can be applied to the targeted preparation of direct HMGB1 inhibitors in Leontopodium leontopodioides.

[0034] The present invention provides a ligand fishing technology established using immobilized HMGB1, which efficiently and conveniently targets and prepares HMGB1 inhibitors from Leontopodium leontopodioides. Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] The present invention uses the established ligand fishing technology based on immobilized HMGB1 to target and prepare a new HMGB1 inhibitor, Apigenin-7-O- β -D-glucopyranoside from Leontopodium leontopodioides, enriching the types of direct HMGB1 inhibitors and providing a scientific clue for the subsequent development of Leontopodium leontopodioides. This technology can target and efficiently screen and prepare HMGB1 inhibitors from complex Leontopodium leontopodioides extracts, overcoming the low efficiency problem in the prior art that requires individual preparation and activity screening of each compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a schematic flow chart of the present invention for the directional immobilization of HMGB1 and its application to the ligand fishing technology to target and prepare direct HMGB1 inhibitors from Leontopodium leontopodioides;

[0037] Figure 2 is the gel Coomassie brilliant blue staining and immunoblotting images of the recombinant HMGB1 in Example 1 of the present invention;

[0038] Figure 3 is a variety of characterization images of the immobilized HMGB1 in Example 1 of the present invention;

[0039] Figure 4 This is the HPLC chromatogram of the results obtained by using immobilized HMGB1 to target the preparation of HMGB1 inhibitors from the mixed model solution in Example 2 of the present invention;

[0040] Figure 5 This is the HPLC chromatogram of the results obtained by using the blank material UiO-66-NH2 without HMGB1 to fish the mixed model solution in Comparative Example 1 of the present invention;

[0041] Figure 6 This is the HPLC chromatogram of the results obtained by using the extract of Leontopodium leontopodioides and fishing Leontopodium leontopodioides with immobilized HMGB1 (fishing results) and blank material (blank control) respectively in Example 3 of the present invention;

[0042] Figure 7 This is the qualitative analysis of the HMGB1 inhibitor Apigenin-7-O- β -D-glucopyranoside prepared by targeting in Leontopodium leontopodioides in Example 4 of the present invention;

[0043] Figure 8 This is for Apigenin-7-O- in Example 5 of the present invention β Graph of the cell survival rate of -D-glucopyranoside and the measurement results of the inhibitory activity of HMGB1;

[0044] Figure 9 This is for Apigenin-7-O- in Example 6 of the present invention β Binding site map of -D-glucopyranoside and HMGB1.

[0045] Figure 10 This is the fluorescence staining map of Leontopodium leontopodioides inhibiting the release of HMGB1 in Example 6 of the present invention. Detailed implementation manners

[0046] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation manners. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the protection of the rights of the present invention. The scope of protection of the present invention is subject to the claims.

[0047] Unless otherwise specified, the raw materials and materials used in the embodiments of the present invention are purchased through general commercial channels. All chemical reagents used in the experiments are of analytical grade and are used directly without further purification.

[0048] The source information of the relevant raw materials, materials, and instruments involved in the following examples or comparative examples is as follows:

[0049] 2-Aminoterephthalic acid (NH2-BDC), zirconium chloride (ZrCl4), nickel sulfate (NiSO4), and glutaraldehyde (50% aqueous solution) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China);

[0050] Tyrosine, kanamycin, isopropyl- β -D-thiogalactoside, glycyrrhizic acid, gallic acid, sciadopitysin, and chlorogenic acid were provided by Medicilon Primedic Pharmaceutical Technology Co., Ltd. (Shanghai, China);

[0051] N , N -dimethylformamide (DMF), acetic acid (HAc), hydrochloric acid, methanol, dimethyl sulfoxide (DMSO), ethanol, and acetonitrile were provided by TEDIA (Ohio, USA);

[0052] The recombinant human high mobility group protein B1 plasmid (pET-24d) was purchased from GenScript Biotech Corporation (Jiangsu, China);

[0053] The Escherichia coli BL21(DE3) strain was purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China);

[0054] Mouse RAW246.7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China);

[0055] Leontopodium leontopodioides was purchased from Zhaotong City, Yunnan Province;

[0056] Apigenin-7-O- β -D-glucopyranoside (Apigenin-7-O- β -D-glucopyranoside) standard was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0057] Instruments: High-performance liquid chromatography (HPLC) analysis was performed using an Agilent 1260 series high-performance liquid chromatograph; Field emission scanning electron microscope characterization and EDS energy spectrum determination of metal-organic frameworks (MOFs) were performed on a GeminiSEM 360 at a working voltage of 10.0 kV; Thermo Scientific Multiskan FC microplate reader.

[0058] The Leontopodium leontopodioides extract of the present invention was prepared by itself, and the preparation method is as follows:

[0059] Step S0-1, drying and pulverizing the Leontopodium leontopodioides raw material at 70 °C to 80 °C and then passing through a 30-50 mesh sieve.

[0060] Step S0-2: Mix the edelweiss obtained in Step S0-1 with an ethanol extraction solvent with a mass ratio of 60% - 95%, and perform extraction at room temperature for 24 h - 48 h, extracting 1 - 3 times; filter the extract using a 100 - 400 mesh sieve, and then obtain the edelweiss extract after concentration under reduced pressure. Among them, the material-liquid ratio (mass ratio) of edelweiss to the extraction solvent is 1:5 - 8.

[0061] Step S0-3: Dry the edelweiss extract obtained in Step S0-2 to obtain the edelweiss extract. The drying treatment is any one or more of freeze-drying, vacuum drying, or spray drying.

[0062] <Preparation Example>

[0063] This preparation example prepares the edelweiss extract.

[0064] Step S0-1: Dry and crush the edelweiss raw material at 75 °C and then pass through a 40-mesh sieve.

[0065] Step S0-2: Mix the edelweiss obtained in Step S0-1 with an ethanol extraction solvent with a mass ratio of 75%, and perform extraction at room temperature for 36 h, extracting 2 times; filter the extract using a 200-mesh sieve, and then obtain the edelweiss extract after concentration under reduced pressure. Among them, the material-liquid ratio (mass ratio) of edelweiss to the extraction solvent is 1:6.

[0066] Step S0-3: Freeze-dry the edelweiss extract obtained in Step S0-2 to obtain the edelweiss extract for standby.

[0067] <Example 1>

[0068] This example provides an immobilized HMGB1, which is a directional immobilization and is prepared using the specific adsorption principle between His-tag and Ni 2+ The preparation method includes the following steps:

[0069] Step SA-1: Clone the full-length HMGB1 gene into the pET-24d(+) vector and fuse His-tag at its N-terminus to obtain a recombinant plasmid. Transfer this recombinant plasmid into E.coli BL21(DE3) competent cells and perform screening to obtain monoclonal strains. Select monoclonal colonies and inoculate them into LB medium for amplification until the OD 600 value reaches 0.6 - 0.8. At this time, use 0.5 mM isopropyl β-D-thiogalactoside was induced to express for 20 hours at 18 °C. After the induction of expression was completed, ultrasonic disruption was carried out. After ultrasonic treatment, the supernatant after centrifugation was collected to obtain a cell lysate containing recombinant HMGB1. This cell lysate was divided into two parts. One part was used for the synthesis of immobilized HMGB1 without any purification steps, and the other part was purified by a chromatography column packed with Ni-NTA agarose beads. During purification, the column was rinsed with Tris-HCl (50 mM, pH = 8.0) buffer containing 20 mM imidazole and 250 mM imidazole at a flow rate of 1 mL / min until no more protein appeared in the eluate. The collected eluate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to find the fraction mainly containing recombinant HMGB1 protein. Subsequently, an Amicon Ultra centrifugal filter (Millipore, USA) was used to replace the buffer and concentrate it with a storage buffer (50 mM Tris, 100 mM NaCl, 50 mM KCl, 5% glycerol, pH = 8.0) to obtain purified recombinant HMGB1 protein for subsequent activity verification experiments of HMGB1 inhibitors.

[0070] In step SA-2, 191 mg of ZrCl4 and 146 mg of NH2-BDC were dissolved in 40 mL of DMF. After ultrasonic treatment for 15 min, 45 μL of HCl and 4.2 mL of HAc were added. The molar mass ratio of substances in the mixed solution was ZrCl4:NH2-BDC:HAc:HCl:DMF = 1:1:90:2:640. Subsequently, the above mixed solution was transferred to a 100 mL reaction kettle and reacted at 120 °C for 24 hours. After the reaction, the reaction kettle was cooled to room temperature, and the mixed solution after the reaction was centrifuged (8000 rpm, 5 min). The precipitate was washed 3 times with 10 mL of DMF and 10 mL of absolute ethanol respectively, and the precipitate was vacuum dried at 80 °C overnight. The obtained pale yellow powder was UiO-66-NH2.

[0071] 20 mg of UiO-66-NH2 was added to an aqueous NaOH solution (0.4 M, 200 μL) containing 10 mg of tyrosine, and 720 μL of PBS (50 mM, pH 6.8) buffer was added. After magnetic stirring for 5 min, 80 μL (424.68 mM) of glutaraldehyde solution was added. After stirring at room temperature for 2 h, centrifugation was performed at 8000 rpm for 5 min. Subsequently, washing was carried out 5 times with PBS buffer (50 mM, pH 7.6, 1 mL). The supernatant was discarded, and the precipitate was resuspended evenly with 2 mL of 0.1 M NiSO4 and placed at room temperature for 30 min. Then, centrifugation (8000 rpm, 5 min) was performed, and washing was carried out 5 times with PBS buffer, 1 mL each time, to obtain Ni@Tyr@UiO-66-NH2, abbreviated as NT-MOF.

[0072] Step SA-3, synthesis of immobilized HMGB1: 100 μL of the cell lysate in Step SA-1 was mixed evenly with 5 mg of NT-MOF and placed at 4 °C for 30 min. Then, washing was carried out 3 times with Tris-HCl buffer at pH = 8.0 and 50 mM. The supernatant of each washing was collected to measure the protein content for calculating the protein loading capacity of NT-MOF for HMGB1. The resulting precipitate was the immobilized HMGB1 used in subsequent experiments, named HMGB1@NT-MOF.

[0073] Analysis of SDS-PAGE and Western blot was performed on the cell lysate and the sample purified by passing through a chromatography column filled with Ni-NTA agarose beads. The results are shown in Figure 2 . Among them, Figure 2 in A is the Coomassie brilliant blue staining map of the SDS-PAGE gel of all samples; Figure 2 in B is the map obtained by Western blot analysis using the HMGB1 antibody; Figure 2 in C is the map obtained by Western blot analysis using the His6 antibody. According to the experimental results, after transferring the recombinant plasmid and inducing with IPTG, recombinant HMGB1 fused with His-tag was successfully expressed in Escherichia coli, that is, the corresponding protein band was observed at the molecular weight of 25 kDa. Through nickel column purification, recombinant HMGB1 was eluted in Tris-HCl buffer containing 250 mM imidazole ( Figure 2 in A). After obtaining the purified recombinant HMGB1 protein, Western blot analysis was performed respectively with the anti-HMGB1 antibody ( Figure 2 in B) and the anti-His6 antibody ( Figure 2 in C). It can be observed from the results that through protein engineering, recombinant HMGB1 with His-tag fused at the N-terminus was successfully obtained. The cell lysate containing this protein can be used for the synthesis of subsequent immobilized HMGB1.

[0074] Multi-dimensional characterization of the synthesized immobilized HMGB1 was carried out, and the results are shown in Figure 3 . Among them, Figure 3 A in it is the protein loading of immobilized HMGB1 synthesized using NT-MOF and UiO-66-NH2; Figure 3 B in it is the SDS-PAGE analysis of the eluate obtained by purifying recombinant HMGB1 using NT-MOF, and the gel was stained with Coomassie Brilliant Blue; Figure 3 C in it is the scanning electron microscope image of UiO-66-NH2; Figure 3 D in it is the scanning electron microscope image of immobilized HMGB1; Figure 3 E in it is the EDS energy spectrum analysis of UiO-66-NH2; Figure 3 F in it is the EDS energy spectrum analysis of immobilized HMGB1.

[0075] It can be known from the characterization results ( Figure 3 ) that 1 g of NT-MOF can adsorb 110.22 mg of recombinant HMGB1, while the blank material without bonded nickel ions has basically no adsorption of HMGB1 ( Figure 3 A in it). In addition, in order to verify whether NT-MOF specifically adsorbs HMGB1, the same method of purifying cell lysate using Ni-NTA agarose gel column was used for analysis. The results show that NT-MOF only adsorbs recombinant HMGB1 fused with His-tag and has basically no adsorption of other proteins ( Figure 3 B in it). It is proved that recombinant HMGB1 is accurately immobilized on NT-MOF. Subsequently, the morphology of the above-synthesized immobilized HMGB1 was observed by scanning electron microscopy. The results show that the immobilized HMGB1 ( Figure 3 C in it) has a larger particle size than UiO-66-NH2 ( Figure 3 D in it), and its surface is also rougher. At the same time, the EDS energy spectrum shows that compared with UiO-66-NH2 without any modification ( Figure 3 E in it), the presence of nickel element and sulfur element was observed in immobilized HMGB1 ( Figure 3 F in it), further confirming the successful preparation of immobilized HMGB1.

[0076] <Example 2>

[0077] In this example, the immobilized HMGB1 prepared in Example 1 was used to provide a process for establishing a model of the ligand fishing technology for targeting the preparation of HMGB1 inhibitors from the extract of Leontopodium leontopodioides, which specifically includes the following steps:

[0078] (1) Take 1 mg of the immobilized HMGB1 in Example 1, add it to 490 μL of 50 mM Tris-HCl buffer with pH = 8.0 and mix well. Then add 10 μL of the mixed model solution (100 mM glycyrrhizic acid, 100 mM chlorogenic acid, and 100 mM gallic acid, with DMSO as the solvent). After the above mixed solution is mixed well, place it in a shaker at 37 °C for 60 min, then centrifuge (8000 rpm, 5 min) to collect the precipitate, and wash the precipitate three times with 1 mL of 50 mM Tris-HCl buffer with pH = 8.0 each time. Finally, take 1 mL of 95%( v / v ) methanol solution to wash the precipitate to inactivate the immobilized HMGB1 and release the ligand obtained by fishing, that is, the HMGB1 inhibitor. Collect the methanol solution, and this methanol solution is the fishing result. After drying the methanol solution, redissolve it with 100 μL of methanol, that is, concentrate it and then perform HPLC analysis. The results are shown in Figure 4 .

[0079] The above chromatographic separation was carried out using an Agilent ZORBAX SB-Aq column (3.0 × 100 mm, 3.5 μm). The mobile phase was acetonitrile (B) and 0.01% formic acid aqueous solution (A), and the elution program was: 10% - 50% B from 0 - 30 min. The column temperature was maintained at 30 °C, the flow rate was 0.4 mL / min, and the ultraviolet detection wavelength was set at 254 nm.

[0080] The mixed model solution contains equimolar amounts of the non-specific compounds gallic acid and chlorogenic acid, and a known HMGB1 inhibitor, glycyrrhizic acid. According to the experimental results ([[]] Figure 4 ), the ligand fishing technology established in this example uses immobilized HMGB1 to successfully target and prepare HMGB1 inhibitors from the mixed model solution, without recognizing the non-specific compounds gallic acid and chlorogenic acid. It is proved that the immobilized HMGB1 synthesized in Example 1 can be used for the targeted preparation of HMGB1 inhibitors and can be used for the directional mining of HMGB1 inhibitory active components in Leontopodium leontopodioides.

[0081] <Comparative Example 1>

[0082] Only use the blank material UiO-66-NH2 without HMGB1 to carry out the experimental procedure of Example 2 above. The final methanol solution sample obtained is named the blank control. The HPLC conditions are the same as in Example 2, and the HPLC results are shown in Figure 5 .

[0083] According to the experimental results ([[]] Figure 5), Using the blank material UiO-66-NH2 without HMGB1, it can neither recognize the positive compound glycyrrhizic acid nor the negative compounds gallic acid and chlorogenic acid in the mixed solution. It further proves that the immobilized HMGB1 synthesized in Example 1 can be used to establish a ligand fishing technology for targeted preparation of HMGB1 inhibitors from Leontopodium leontopodioides.

[0084] <Example 3>

[0085] In this example, a ligand fishing technology established using immobilized HMGB1 was used to target the preparation of HMGB1 inhibitors from Leontopodium leontopodioides, which specifically includes the following steps:

[0086] Take 1 mg of the immobilized HMGB1 in Example 1, dilute it with 490 μL of pH = 8.0, 50 mM Tris-HCl buffer, then add 1 mg of Leontopodium leontopodioides extract and incubate at 37 °C for 60 min. After centrifugation (8000 rpm, 5 min), collect the precipitate and wash the precipitate three times with pH = 8.0, 50 mM Tris-HCl buffer, 1 mL each time. Finally, take 1 mL of 95% ( v / v ) methanol solution to wash the precipitate, inactivate the immobilized HMGB1 to release the fished ligand, that is, the HMGB1 inhibitor, collect the methanol solution, and this methanol solution is the fishing result. After drying the methanol solution, redissolve it with 100 μL of methanol, that is, concentrate it and then perform HPLC analysis. When performing HPLC analysis, inject 20 μL of the fishing result methanol solution, and collect the tailing solution at 5.0 - 7.5 min and 12.5 min - 14.0 min in the fishing result. After comparing the fishing result with the blank control, discard the impurities introduced by the immobilized HMGB1, that is, the tailing solution at 5.0 min - 7.5 min. Repeat the above process until all the tailing solution at 5.0 min - 7.5 min in 100 μL of the fishing result methanol solution is collected. Concentrate the tailing solution at 12.5 min - 14.0 min in the fishing result to dryness under reduced pressure at 40 °C to obtain the HMGB1 inhibitor and use it for subsequent identification and verification experiments. The yield of the HMGB1 inhibitor is calculated by the relative content of the substances detected in the fishing result. The HPLC results are shown in Figure 6 .

[0087] The above HPLC chromatographic separation was carried out using an Agilent ZORBAX SB-Aq column (3.0 × 100 mm, 3.5 μm). The mobile phase was acetonitrile (B) and 0.01% formic acid aqueous solution (A), and the elution program was: 10% - 50% B for 0 - 30 min. The column temperature was maintained at 30 °C, the flow rate was 0.4 mL / min, and the ultraviolet detection wavelength was set at 254 nm.

[0088] <Example 4>

[0089] Qualitative analysis of the HMGB1 inhibitor prepared by targeting in Leontopodium leontopodioides in Example 2 was carried out by HPLC-Q-TOF-MS / MS. The test sample was the same as the HPLC test sample. The determination conditions were as follows: Chromatographic separation was carried out using an Agilent ZORBAX SB-Aq chromatographic column (2.1×250 mm, 3.5 μm), the flow rate was 0.3 mL / min, and the injection volume was 1 μL. The column temperature was set at 35 °C. A quadrupole time-of-flight mass spectrometer was used and operated in the negative ion mode. The main parameters were set as follows: The gas temperature was 350 °C; the flow rate of the drying gas (nitrogen) was 8 L / min; the nebulizer pressure was 35 psig; the capillary voltage was 4.0 kV; the fragmentation voltage was 170 V; the sampling cone voltage was 65 V. Nitrogen was used as the collision gas for MS / MS scanning. The mobile phase consisted of solvent A (0.1% (v / v) formic acid aqueous solution) and solvent B (acetonitrile). The results are shown in Figure 7 . Figure 7 In A, it is the HPLC chromatogram for qualitative analysis by comparing the standard of Apigenin-7-O- β -D-glucopyranoside with the extract of Leontopodium leontopodioides; Figure 7 In B, it is the mass spectrometry data of the HMGB1 inhibitor prepared by immobilized HMGB1.

[0090] It can be seen from the experimental results that there are a total of 21 compounds in Leontopodium leontopodioides. After fishing with immobilized HMGB1, 1 potential HMGB1 inhibitor ( Figure 6 ) was prepared by targeting from the extract of Leontopodium leontopodioides. Through HPLC-QTOF-MS / MS analysis ( Figure 7 in B), and using the standard for comparison ( Figure 7 in A), it was found that the potential HMGB1 inhibitor fished out with immobilized HMGB1 was apigenin-7-O- β -D-glucoside (Apigenin-7-O- β -D-glucopyranoside) (peak 14).

[0091] <Example 5>

[0092] Purchase the standard of the potential HMGB1 inhibitor obtained by fishing (Apigenin-7-O- β-D-glucopyranoside), and its HMGB1 inhibitory activity was verified through the HMGB1-induced murine RAW 264.7 cell inflammation model after comparison. Compared with the model group (HMGB1), the compound could inhibit NO and TNF- in the cell supernatant α expression levels, then it had direct HMGB1 inhibitory activity, and the results were shown in Figure 8 . Figure 8 In which A is the cytotoxicity assay result of Apigenin-7-O- β -D-glucopyranoside (abbreviated as AG); Figure 8 In which B is the NO content assay result in the cell supernatant when Apigenin-7-O- β -D-glucopyranoside and the positive compound glycyrrhizin (Gly) acted on HMGB1-induced RAW264.7 cells in parallel. The control group was RAW264.7 cells without any treatment (that is, without adding purified HMGB1 to induce the expression of inflammatory factors and without adding any assay samples); Figure 8 In which C is the TNF- β content assay result in the cell supernatant when Apigenin-7-O- α -D-glucopyranoside and the positive compound Gly acted on HMGB1-induced RAW264.7 cells in parallel. The control group was RAW264.7 cells without any treatment (that is, without adding purified HMGB1 to induce the expression of inflammatory factors and without adding any assay samples).

[0093] Furthermore, the directly prepared HMGB1 inhibitors were further verified and analyzed, and the activities of the potential HMGB1 inhibitors fished out were measured in the HMGB1-induced RAW264.7 cell inflammation model. Apigenin-7-O- β -D-glucopyranoside (AG) and the positive compound Gly were selected for parallel experiments. Using a Thermo Scientific Multiskan FC microplate reader, first, the CCK-8 method was used to determine the assay concentration of AG ( Figure 8 in A), and the test concentration without cytotoxicity was selected as 50 μM. By measuring NO ( Figure 8 in B) and TNF- α ( Figure 8C) The measurement results of the expression of these two inflammatory factors showed that the prepared AG had good HMGB1 inhibitory activity, and its activity was comparable to that of Gly. The above data further demonstrated the feasibility and accuracy of the ligand fishing technology established in Example 2 for targeted preparation of HMGB1 inhibitors from Leontopodium leontopodioides, and also elucidated the material basis of HMGB1 inhibitory activity in Leontopodium leontopodioides.

[0094] <Example 6>

[0095] The specific binding sites were further explored by molecular docking technology. The protein structure file was obtained from the Protein Data Bank (PDB ID: 2YRQ). The docking of the ligand with the corresponding protein was achieved using AutoDock Vina software (version 1.1.2). Subsequently, the docking results were visualized using Pymol software (version 2.5.5), and the results are shown in Figure 9 . Figure 9 In A is the three-dimensional molecular docking map (PDB 2YRQ) of the complex formed by Apigenin-7-O- β -D-glucopyranoside and HMGB1; Figure 9 In B is the two-dimensional molecular docking map of the complex formed by Apigenin-7-O- β -D-glucopyranoside and HMGB1. Further molecular docking analysis of the binding mode of AG and HMGB1 showed that ( Figure 9 ) AG could bind to the pro-inflammatory active domain B-box of HMGB1, and the binding energy was -28.7092 kcal / mol. At the same time, AG could also form hydrogen bonds with the amino acid residues Lys97 and Arg104 in the B-box, thereby affecting the binding of HMGB1 to receptors such as RAGE and playing an anti-inflammatory role.

[0096] In addition to the pro-inflammatory effect of extracellular HMGB1, the release of HMGB1 from the nucleus to the extracellular space can also cause or exacerbate inflammatory responses. Under external stimuli, nuclear HMGB1 will translocate out of the nucleus and be released into the interstitial space through exocytosis, thereby causing or exacerbating a series of inflammatory reactions. To further explore the HMGB1 inhibitory activity of Leontopodium leontopodioides, whether Leontopodium leontopodioides could inhibit the release of HMGB1 from cells was tested in an H2O2-induced NIH / 3T3 cell inflammation model to investigate whether Leontopodium leontopodioides had the activity to inhibit the release of HMGB1. The results are shown in Figure 10 , Figure 10 In A is the fluorescence staining map of HMGB1 in H2O2-induced NIH / 3T3 cells; Figure 10In Figure B, it is the fluorescence staining map of HMGB1 in NIH / 3T3 cells induced by H2O2 after being treated with Leontopodium leontopodioides. It can be seen from the experimental results that a large amount of HMGB1 in the nuclei of H2O2-induced NIH / 3T3 cells transfers from the nucleus to outside the nucleus ( Figure 10 in Figure A), while Leontopodium leontopodioides can significantly inhibit the transfer of HMGB1 from the nucleus to outside the nucleus in NIH / 3T3 cells, indicating that Leontopodium leontopodioides inhibits the release of HMGB1 under external stimuli ( Figure 10 in Figure B). In summary, a scientific association between Leontopodium leontopodioides and the inhibitory activity of HMGB1 is established comprehensively from two dimensions: the direct inhibitory activity of HMGB1 and the inhibition of HMGB1 release.

[0097] <Example 7>

[0098] The purchased Apigenin-7-O- β -D-glucopyranoside is applied to a cosmetic cream, and the proportion of each component of the cream is shown in Table 1 below:

[0099] Table 1. Component Table of the Cream

[0100]

[0101] The preparation method of the above cream is as follows:

[0102] 1. Add the raw materials of Phase A to the main mixing tank. After mixing evenly, add the raw materials of Phase B, turn on the stirring and heating, and the target temperature is 80°C - 85°C.

[0103] 2. Add the raw materials of Phase C to the oil phase tank, turn on the stirring and heating, and the target temperature is 80°C - 85°C.

[0104] 3. After reaching the target temperature, start homogenization in the main mixing tank, and then add the raw materials of the oil phase tank to the main mixing tank. The homogenization speed is 6000 rpm and the homogenization time is 5 min.

[0105] 4. Stop homogenization, turn on the stirring (150 rpm - 200 rpm), turn on the cooling, and the target temperature is 75°C.

[0106] 5. When the temperature in the main mixing tank drops to 75°C, add the raw materials of Phase D to the main mixing tank, stir and mix evenly, and continue to cool down. The target temperature is 45°C.

[0107] 6. When the temperature drops to 45°C, add the raw materials of Phase E, stir and mix evenly; cool to room temperature and discharge to obtain the product.

[0108] <Example 8>

[0109] In this example, a performance experiment is carried out on the cosmetic of Example 7.

[0110] Detection method: Under normal circumstances, adult subjects continuously use the test product for 56 days according to the following usage method to evaluate whether the test product has anti-wrinkle effects and whether it is suitable for sensitive skin (without irritation). And after adult subjects continuously use the evaluation product for 56 days according to the instructions and then stop using it for 7 days, evaluate whether the product still continuously improves the skin condition 7 days after stopping use.

[0111] Subjects: A total of 34 effective subjects completed the evaluation, including Chinese healthy men and women with sensitive skin (screened by lactic acid stinging), 6 men and 28 women, aged 42 to 60 years old, with an average age of 52.76 ± 5.23 years old, meeting the volunteer inclusion and exclusion criteria for subjects.

[0112] Evaluation area: Face.

[0113] Usage method: Take an appropriate amount of this product and evenly apply it on the face, avoiding the skin around the eyes, and gently massage in a circular motion in the same direction until absorbed. Usage frequency: Once in the morning and once in the evening.

[0114] Usage period: 63 days.

[0115] Evaluation period: Before using the product (D0), 15 minutes after using the product (D0T15min), 14 days after using the product (D14), 28 days after using the product (D28), 56 days after using the product (D56), 7 days after stopping using the product (D63).

[0116] Evaluation method: Use the skin rapid three-dimensional imaging system PRIMOS CR to take pictures of the subjects' faces, and analyze the wrinkle changes through images; conduct a questionnaire survey for the subjects to self-evaluate whether the test product is suitable for sensitive skin.

[0117] (1) Reduction amount of skin wrinkle volume (cheek)

[0118] During the detection period, the specific results of the reduction amount of skin wrinkle volume (cheek) of the subjects are shown in Table 2 below:

[0119] Table 2. Reduction amount of skin wrinkle volume (cheek) of the subjects (unit: %)

[0120]

[0121] From the results of Table 2 above, it can be seen that the cosmetic with HMGB1 inhibitor Apigenin-7-O-β-D-glucopyranoside as the only active ingredient has a significant difference in the reduction amount of skin wrinkle volume (cheek), indicating that the HMGB1 inhibitor can improve the skin condition, thus having good anti-wrinkle and anti-aging effects.

[0122] (3) Applicability rate for sensitive skin

[0123] During the detection period, the specific results of the applicable rate for sensitive skin are shown in Table 3 below:

[0124] Table 3. Applicable Rate for Sensitive Skin (Unit: %)

[0125]

[0126] From the results in Table 3 above, it can be seen that the cosmetics in Example 7 all have excellent results in terms of the applicable rate for sensitive skin and have a universal effect on sensitive skin.

[0127] The applicant declares that the above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by any person skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for targeted preparation of HMGB1 inhibitors from extracts of Leontopodium alpinum, characterized in that, It includes the following steps: The immobilized HMGB1 was co-incubated with the extract of Leontopodium alpinum to obtain a precipitate. The precipitate was washed with a buffer to remove non-specific adsorbates and then washed with an inactivator to obtain the HMGB1 inhibitor apigenin-7-O- β -D-glucoside; wherein, The preparation method of the immobilized HMGB1 includes the following steps: Step SA-1: Using protein engineering technology, recombinantly express recombinant human HMGB1 with an N-terminal fused His-tag in an E. coli system to obtain a cell lysate containing recombinant human HMGB1 with an N-terminal fused His-tag; Step SA-2: Synthesize the metal-organic framework material UiO-66-NH2 by the solvothermal method, then use glutaraldehyde as a linker to modify the surface of the UiO-66-NH2 through tyrosine, and then add nickel ions for chelation to synthesize NT-MOF; Step SA-3: Mix the NT-MOF with the cell lysate for incubation, and obtain immobilized HMGB1 after washing.

2. The method for targeted preparation of HMGB1 inhibitor from the extract of Leontopodium alpinum as claimed in claim 1, characterized in that, The mass ratio of the immobilized HMGB1 to the edelweiss extract is 2:1, the incubation temperature is 37 °C, and the pH is 8.0; the inactivator is a methanol solution with a volume ratio of 95%.

3. The method for targeted preparation of HMGB1 inhibitor from Leontopodium alpinum extract according to claim 2, characterized in that, Wash the precipitate with a buffer solution and then inactivate the immobilized HMGB1 with a methanol solution with a volume ratio of 95% to release the ligand into the methanol solution, and this methanol solution is the fishing result; then remove the methanol in the fishing result and redissolve it with a predetermined amount of methanol for HPLC analysis. The detected compound is an HMGB1 inhibitor, and if not detected, it does not belong to an HMGB1 inhibitor. Collect the detected compounds for subsequent identification.

4. The method for targeted preparation of HMGB1 inhibitor from Leontopodium alpinum extract as claimed in claim 3, wherein, Perform HPLC analysis using an Agilent ZORBAX SB-Aq column, with the column specifications of 3.0 mm×100 mm, 3.5 μm; the mobile phase is phase A as 0.01% formic acid aqueous solution and phase B as acetonitrile; the elution program is: from 0 to 30 min, the volume fraction of mobile phase B in the mobile phase is 10% - 50%.

5. The method for targeted preparation of HMGB1 inhibitor from Leontopodium alpinum extract according to claim 1, characterized in that, The buffer solution is Tris-HCl with a pH of 8.0 and a concentration of 50 mM.

6. The method for targeted preparation of HMGB1 inhibitor from Leontopodium alpinum extract according to claim 1, wherein When co-incubating the immobilized HMGB1 with the edelweiss extract, dilute the immobilized HMGB1 with a Tris-HCl buffer solution with a pH of 8.0 and a concentration of 50 mM.

7. The method for targeted preparation of HMGB1 inhibitor from Leontopodium alpinum extract according to claim 1, characterized in that, The preparation method of the edelweiss extract includes the following steps: Step S0-1: Dry and crush the edelweiss raw material at 70 °C - 80 °C and then pass through a 30 - 50 mesh sieve; Step S0-2: Mix the edelweiss obtained in step S0-1 with an ethanol extraction solvent with a mass ratio of 60% - 95% and perform extraction at room temperature for 24 h - 48 h, extract 1 - 3 times, filter the extract using a 100 - 400 mesh sieve, and then obtain the edelweiss extract after concentration under reduced pressure; Step S0-3: Dry the edelweiss extract obtained in step S0-2 to obtain the edelweiss extract.

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