HMGB1 inhibitor, method for targeted preparation of HMGB1 inhibitor from leontopodium leontopodum extract and application of HMGB1 inhibitor

Abstract

The invention belongs to the technical field of analytical chemistry, and provides an HMGB1 inhibitor and a method for targeted preparation of the HMGB1 inhibitor from a leontopodium leontopodum extract. The inhibitor is apigenin-7-O-beta-D-glucoside, and a method for preparing the HMGB1 inhibitor from the edelweiss extract in a targeting manner comprises the following steps: co-incubating immobilized HMGB1 and the edelweiss extract to obtain a precipitate, washing a non-specific adsorbate of the precipitate with a buffer solution, and then washing with an inactivating agent to obtain the HMGB1 inhibitor. The new HMGB1 inhibitor apigenin-7-O-beta-D-glucoside is obtained from the leontopodium leontopodum through the established targeted preparation technology, the variety of HMGB1 direct inhibitors is enriched, and a scientific clue is provided for subsequent application of the leontopodium leontopodum in resisting inflammatory aging. In addition, the HMGB1 inhibitor can be efficiently prepared from the complex leontopodium leontopodum extract in a targeted mode through the established targeted preparation technology, and the problem that in the prior art, each compound needs to be independently prepared, activity screening needs to be conducted, and the efficiency is low is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and in particular relates to an HMGB1 inhibitor, a method for preparing the HMGB1 inhibitor in a targeted manner from a leontopodium extract, and an application 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 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 is a plant of the genus Leontopodium in the Asteraceae family. It is a wild flower that grows in plateau areas and has multiple pharmacological activities such as anti-inflammatory and hypoglycemic. Due to its anti-inflammatory and antioxidant effects, Leontopodium has also received widespread attention in the cosmetics industry. Its extracts can play a soothing and anti-aging role when added to cosmetics. However, there is currently no literature or patent that elaborates on the relationship between the active ingredients in Leontopodium and the HMGB1 inhibitory activity. In addition, there is no report on the efficient screening of HMGB1 direct inhibitors from complex plant samples such as Leontopodium. Traditional HMGB1 inhibitor screening methods usually use the HMGB1-induced mouse RAW264.7 cell inflammation model to screen HMGB1 inhibitors one by one for single samples. This HMGB1 direct inhibitor screening method requires monomer compounds, cells in good condition, and HMGB1 protein. It lacks efficiency and is too costly, resulting in the lack of objective results in the study of the relationship between the exact material basis of the activity in Leontopodium and HMGB1.

[0004] As a cosmetic raw material, leontopodium alpinum has been used in many product fields, but its anti-aging effect, especially for sensitive skin, has not been fully developed. Anti-aging research on sensitive skin has always been a technical difficulty in the cosmetics industry. According to statistics, about 60% of the world's population has skin sensitivity symptoms to varying degrees, and the core mechanism of this type of skin aging has been confirmed to be closely related to "inflammatory aging" caused by chronic low-grade inflammation. This special skin condition requires that the anti-aging solution must simultaneously achieve the dual goals of barrier function repair and inflammatory response regulation. It is worth noting that the expression level of HMGB1, a core member of the damage-associated molecular pattern, in chronic skin inflammation tissue can reach 3-5 times that of normal skin. This feature makes it a key target for cracking the inflammatory aging mechanism. However, to date, anti-aging technology based on the HMGB1 target has 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 the application of the ligand fishing technology based on immobilized HMGB1 in the targeted preparation of HMGB1 inhibitors in leontopodium, 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 material basis for the exact activity in leontopodium and HMGB1.

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

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

[0008] In a third aspect, the present invention provides a method for preparing the above HMGB1 inhibitor from a leontopodium extract in a targeted manner, comprising: The immobilized HMGB1 was co-incubated with the extract of Leontopodium edulis to obtain a precipitate, which was then washed 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-glucoside (Apigenin-7-O- β -D-glucopyranoside).

[0009] Preferably, the method further comprises the steps of analyzing and preparing the fishing results using HPLC, and verifying the experimental results, such as Figure 1 As shown, the specific operation of the method for preparing the above HMGB1 inhibitor from the extract of Leontopodium leontopodium is as follows: (1) Immobilized HMGB1 was co-incubated with Leontopodium leontopodium extract at a mass ratio of 2:1.

[0010] (2) After centrifugation, the precipitate was washed three times with Tris-HCl buffer to obtain the precipitate, and then washed with 95% methanol ( v / v ) to inactivate HMGB1 and release the HMGB1 inhibitor into the above-mentioned methanol solution to obtain the methanol solution of the fishing results. The methanol solution of the fishing results is then dried using a nitrogen blower and a certain amount of 100 uL of methanol is used for re-dissolution to increase the concentration of the fishing results in the methanol solution for detection by HPLC.

[0011] (3) In the HPLC spectrum, the detected compound is an HMGB1 inhibitor, and the undetected compound is not an HMGB1 inhibitor. The detected HMGB1 inhibitors are collected by HPLC for subsequent identification and activity verification experiments.

[0012] (4) The prepared HMGB1 inhibitor was qualitatively analyzed by HPLC-Q-TOF-MS / MS and compared with a standard.

[0013] (5) The HMGB1-induced mouse RAW246.7 inflammation model was used to verify the activity of the prepared HMGB1 inhibitor.

[0014] (6) Molecular docking technology was used to further investigate the binding site between the prepared HMGB1 inhibitor and HMGB1.

[0015] The present invention provides a targeted acquisition of HMGB1 inhibitors in leontopodium using immobilized HMGB1, and utilizes the two characteristics of specific binding between protein and its ligand and solid-liquid separation of immobilized enzyme to achieve rapid and targeted preparation of HMGB1 inhibitors. 1 mg of leontopodium extract can be obtained by this method. 3 μg of Apigenin-7-O- β -D-glucopyranoside. Compared with traditional methods, it is faster, more efficient and less costly.

[0016] Preferably, the method for preparing synthetic immobilized HMGB1 comprises the following steps: Step SA-1, using protein engineering technology, recombinantly expressing recombinant human HMGB1 fused with N-terminal His-tag in an E. coli system to obtain a cell lysate containing recombinant human HMGB1 fused with N-terminal His-tag.

[0017] Step SA-2, using solvothermal method to synthesize the metal organic framework material UiO-66-NH 2 Afterwards, glutaraldehyde was used as a connecting arm and its surface was modified by tyrosine to make the surface of the material have free carboxyl groups. Nickel ions were then added for chelation to synthesize NT-MOF.

[0018] Step SA-3, a certain amount of NT-MOF is incubated with the above cell lysate, and immobilized HMGB1 is obtained after multiple washings, and a series of experiments are performed to verify the immobilized HMGB1.

[0019] The immobilized HMGB1 prepared by the above method is simple to operate, and an immobilized HMGB1 with a good protein loading capacity can be obtained in just one step, which can be used to establish the ligand fishing technology of HMGB1 inhibitors. Compared with other immobilized enzyme synthesis methods, the recombinant human HMGB1 with N-terminal fusion His-tag can fix HMGB1 on the NT-MOF surface in a way that does not affect the exposure of the HMGB1 inflammatory active domain to the solution, ensuring the accuracy of the ligand fishing technology.

[0020] Preferably, the mass ratio of immobilized HMGB1 to Leontopodium leontopodium extract is 2:1, the incubation temperature is 37° C., and the pH is 8.0.

[0021] More preferably, an Agilent ZORBAX SB-Aq column is used for HPLC analysis, with a column size of 3.0 mm×100 mm, 3.5 μm; the mobile phase is phase A of 0.01% formic acid aqueous solution, and phase B of acetonitrile; the elution program is: 0-30 min, the volume fraction of mobile phase B in the mobile phase is 10%-50%.

[0022] Preferably, when the immobilized HMGB1 is co-incubated with the leontopodium extract, the immobilized HMGB1 is diluted with a Tris-HCl buffer having a pH of 8.0 and a concentration of 50 mM.

[0023] Preferably, the preparation method of the leontopodium extract comprises the following steps: step S0-1, drying and crushing the leontopodium raw material at 70°C~80°C and then passing it through a 30~50 mesh sieve; step S0-2, mixing the leontopodium obtained in step S0-1 with an ethanol extraction solvent with a mass ratio of 60%~95%, extracting at room temperature for 24h~48h, extracting 1~3 times, filtering the extract through a 100~400 mesh sieve, and then concentrating under reduced pressure to obtain a leontopodium extract; step S0-3, drying the leontopodium extract obtained in step S0-2 to obtain a leontopodium extract.

[0024] Preferably, the deactivating agent is a 95% by volume methanol aqueous solution, referred to as 95% methanol solution.

[0025] Preferably, the buffer is Tris-HCl with a pH of 8.0 and a concentration of 50 mM.

[0026] The present invention provides the use of the above-mentioned immobilized HMGB1 in ligand fishing technology, which can be used for a method for preparing a direct inhibitor of HMGB1 in leontopodium leontopodium, and is verified by the following method, including the following steps: (1) Through literature research, glycyrrhizic acid was selected as the positive compound, and gallic acid and chlorogenic acid were selected as negative compounds to prepare a mixed model solution.

[0027] (2) Take 2 mg of immobilized HMGB1 and incubate it with the mixed model solution.

[0028] (3) After centrifugation, the precipitate was washed three times with PBS to obtain the precipitate, and then washed with 95% methanol ( v / v ) to inactivate HMGB1 and release the HMGB1 inhibitor to obtain the fishing result. Then, a nitrogen blower was used to dry the mixture and a certain amount of methanol was used to re-dissolve the mixture for subsequent HPLC detection.

[0029] (4) In the fishing results, the detected compounds are determined to be HMGB1 inhibitors, and those not detected are not HMGB1 inhibitors. The feasibility and accuracy of the ligand fishing technology established by immobilized HMGB1 are verified 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 mixed samples, while non-specific compounds are not recognized, which proves the feasibility and accuracy of the immobilized enzyme in the ligand fishing technology, and can be applied to the targeted preparation of HMGB1 direct inhibitors in edelweiss.

[0030] The present invention provides a ligand fishing technology established by using immobilized HMGB1, which efficiently and conveniently prepares HMGB1 inhibitors from edelweiss in a targeted manner. Compared with the prior art, the present invention has at least the following beneficial effects: The present invention uses the established ligand fishing technology based on immobilized HMGB1 to prepare a new HMGB1 inhibitor Apigenin-7-O- β -D-glucopyranoside enriches the types of direct HMGB1 inhibitors and provides scientific clues for the subsequent development of Leontopodium. This technology can screen and prepare HMGB1 inhibitors from complex Leontopodium extracts in a targeted and efficient manner, overcoming the inefficiency of the existing technology that requires each compound to be prepared separately and screened for activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the process of directional immobilization of HMGB1 and application of ligand fishing technology to targeted preparation of HMGB1 direct inhibitor from Edelweiss in the present invention; Figure 2 The Coomassie blue staining and immunoblotting images of the recombinant HMGB1 in Example 1 of the present invention are shown; Figure 3 Various characterization diagrams of immobilized HMGB1 in Example 1 of the present invention; Figure 4This is an HPLC spectrum of the results of targeted preparation of HMGB1 inhibitors from a mixed model solution using immobilized HMGB1 in Example 2 of the present invention; Figure 5 The blank material UiO-66-NH3 containing no HMGB1 in Comparative Example 1 of the present invention is used. 2 HPLC profile of the results obtained by fishing the mixed model solution; Figure 6 HPLC spectrum of the results of fishing for Leontopodium leontopodium using the extract of Leontopodium leontopodium in Example 3 of the present invention and the results of fishing for Leontopodium leontopodium using immobilized HMGB1 (fishing results) and blank materials (blank control); Figure 7 Apigenin-7-O- is the HMGB1 inhibitor Apigenin-7-O- β -Qualitative analysis of D-glucopyranoside; Figure 8 Apigenin-7-O- β -D-glucopyranoside cell survival rate and HMGB1 inhibitory activity assay results; Fig. 9 Apigenin-7-O- β -D-glucopyranoside binding site map for HMGB1.

[0032] Fig.10 This is a fluorescence staining image of the inhibition of HMGB1 release by Leontopodium alba in Example 6 of the present invention. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0034] Unless otherwise specified, the raw materials and materials used in the examples of the present invention were purchased from general commercial sources. All chemical reagents used in the experiments were of analytical grade and were used directly without further purification.

[0035] The sources of the raw materials, materials and instruments involved in the following examples or comparative examples are as follows: 2-Aminoterephthalic acid (NH 2 -BDC), zirconium chloride (ZrCl 4 ), nickel sulfate (NiSO 4) and glutaraldehyde (50% aqueous solution) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China); Tyrosine, kanamycin, isopropyl- β -D-thiogalactoside, glycyrrhizic acid, gallic acid, scutellariae flavonoids, and chlorogenic acid were provided by Medicilon Prime Pharmaceutical Technology Co., Ltd. (Shanghai, China); N , N -Dimethylformamide (DMF), acetic acid (HAc), hydrochloric acid, methanol, dimethyl sulfoxide (DMSO), ethanol, and acetonitrile were provided by TEDIA (Ohio, USA); Recombinant human high-mobility group protein B1 plasmid (pET-24d) was purchased from GenScript Biotech Co., Ltd. (Jiangsu, China); Escherichia coli BL21 (DE3) strain was purchased from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China); Mouse RAW246.7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China); Edelweiss was purchased from Zhaotong City, Yunnan Province; Apigenin-7-O- β -D-glucoside (Apigenin-7-O- β -D-glucopyranoside) standard was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0036] Instruments: High-performance liquid chromatography (HPLC) analysis was performed using an Agilent 1260 series HPLC; field emission scanning electron microscopy characterization and EDS spectroscopy of metal organic frameworks (MOFs) were performed on a GeminiSEM 360 operating at 10.0 kV; Thermo Scientific Multiskan FC microplate reader.

[0037] The leontopodium extract of the present invention is prepared by itself, and the preparation method is as follows: Step S0-1, drying and crushing the raw material of edelweiss at 70°C-80°C and then passing through a 30-50 mesh sieve.

[0038] Step S0-2, mixing the edelweiss obtained in step S0-1 with an ethanol extraction solvent having a mass ratio of 60% to 95%, extracting at room temperature for 24 h to 48 h, and extracting 1 to 3 times; filtering the extract using a 100 to 400 mesh screen, and then concentrating under reduced pressure to obtain an edelweiss extract. The solid-liquid ratio (mass ratio) of edelweiss to the extraction solvent is 1:5 to 8.

[0039] Step S0-3, drying the leontopodium extract obtained in step S0-2 to obtain a leontopodium extract. The drying treatment is any one or more of freeze drying, vacuum drying or spray drying.

[0040] <Preparation Example> This preparation example prepares a leontopodium extract.

[0041] Step S0-1, drying and crushing the raw material of edelweiss at 75°C and then passing through a 40-mesh sieve; Step S0-2, mixing the edelweiss obtained in step S0-1 with an ethanol extraction solvent having a mass ratio of 75%, extracting at room temperature for 36 hours, extracting twice; filtering the extract using a 200-mesh screen, and then concentrating under reduced pressure to obtain an edelweiss extract. The solid-liquid ratio (mass ratio) of edelweiss to the extraction solvent is 1:6.

[0042] Step S0-3, freeze-drying the leontopodium extract obtained in step S0-2 to obtain a leontopodium extract for later use.

[0043] <Example 1> This embodiment provides an immobilized HMGB1, which is a directional immobilization method using His-tag and Ni 2+ The preparation method is based on the inter-specific adsorption principle, and the preparation method comprises the following steps: Step SA-1: clone the full-length HMGB1 gene into the pET-24d(+) vector and fuse the His-tag at its N-terminus to obtain a recombinant plasmid. E. coli BL21(DE3) competent cells were selected and monoclonal strains were obtained. Monoclonal colonies were selected and inoculated into LB medium for amplification until OD 600 The value reaches 0.6~0.8. At this time, 0.5 mM isopropyl β-D-thiogalactoside was used to induce expression at 18 °C for 20 hours. After the induction of expression, ultrasonic disruption was performed. After ultrasonic treatment, the supernatant after centrifugation was collected to obtain a cell lysate containing recombinant HMGB1. The 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 loaded with Ni-NTA agarose beads. During purification, the column was washed with 20 mM imidazole and 250 mM imidazole in Tris-HCl (50 mM, pH=8.0) buffer at a flow rate of 1 mL / min until no protein appeared in the eluate. The collected eluate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to find the component mainly containing recombinant HMGB1 protein, and then the eluate was filtered through an Amicon Ultra centrifugal filter (Millipore, USA) and concentrated with storage buffer (50 mMTris, 100 mM NaCl, 50 mM KCl, 5% glycerol, pH=8.0) to obtain purified recombinant HMGB1 protein for subsequent HMGB1 inhibitor activity verification experiments.

[0044] Step SA-2, take 191 mg ZrCl 4 and 146 mg NH 2 -BDC was dissolved in 40 mL DMF, and then 45 μL HCl and 4.2 mL HAc were added under ultrasonication for 15 min. The molar mass ratio of the substances in the mixed solution was ZrCl 4 :NH 2 -BDC:HAc:HCl:DMF =1:1:90:2:640. Subsequently, the mixed solution was transferred to a 100 mL reactor and reacted at 120 °C for 24 hours. After the reaction, the reactor 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 DMF and 10 mL anhydrous ethanol, respectively, and the precipitate was vacuum dried at 80 °C overnight. The obtained light yellow powder is UiO-66-NH 2 .

[0045] 20 mg UiO-66-NH 2Add to a NaOH aqueous solution (0.4 M, 200 μL) containing 10 mg of tyrosine, add 720 μL of PBS (50 mM, pH 6.8) buffer, stir magnetically for 5 min, then add 80 μL (424.68 mM) glutaraldehyde solution. After stirring at room temperature for 2 h, centrifuge at 8000 rpm for 5 min, then wash 5 times with PBS buffer (50 mM, pH 7.6, 1 mL). Discard the supernatant and wash the precipitate with 2 mL of 0.1 M NiSO 4 After being resuspended evenly and placed at room temperature for 30 min, the suspension was centrifuged (8000 rpm, 5 min) and washed 5 times with PBS buffer, 1 mL each time, to obtain Ni@Tyr@UiO-66-NH 2 , referred to as NT-MOF.

[0046] Step SA-3, synthesis of immobilized HMGB1: 100 μL of the cell lysate in step SA-1 was mixed with 5 mg of NT-MOF and placed at 4 °C for 30 min, then washed three times with pH=8.0, 50 mM Tris-HCl buffer, and the supernatant of each wash was collected to determine the protein content to calculate the protein loading capacity of NT-MOF for HMGB1. The resulting precipitate was the immobilized HMGB1 used in subsequent experiments, named HMGB1@NT-MOF.

[0047] The cell lysate and the samples purified by chromatography column loaded with Ni-NTA agarose beads were analyzed by SDS-PAGE and western blotting. Figure 2 .in, Figure 2 A in the middle is the Coomassie blue staining image of the SDS-PAGE gel of all samples; Figure 2 Middle B is the spectrum obtained by immunoblotting analysis using HMGB1 antibody; Figure 2 The C in the middle is His 6 The spectrum obtained by immunoblotting analysis with antibodies. According to the experimental results, after the recombinant plasmid was transferred and induced by IPTG, the recombinant HMGB1 fused with His-tag was successfully expressed in E. coli, that is, the corresponding protein band was observed at a molecular weight of 25kDa. After nickel column purification, the recombinant HMGB1 was eluted in Tris-HCl buffer containing 250 mM imidazole ( Figure 2 After obtaining the purified recombinant HMGB1 protein, the anti-HMGB1 antibody ( Figure 2 B) with anti-His 6 Antibody( Figure 2C) Western blot analysis was performed, and it can be observed from the results that recombinant HMGB1 with N-terminal fusion His-tag was successfully obtained through protein engineering. The cell lysate containing the protein can be used for the subsequent synthesis of immobilized HMGB1.

[0048] The immobilized HMGB1 synthesized was characterized in multiple dimensions. Figure 3 .in, Figure 3 A is made of NT-MOF and UiO-66-NH 2 The protein loading capacity of synthetic immobilized HMGB1; Figure 3 Middle B 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 the middle is UiO-66-NH 2 Scanning electron microscope image of Figure 3 Center D is a scanning electron microscopy image of immobilized HMGB1; Figure 3 E in the middle is UiO-66-NH 2 EDS spectrum analysis; Figure 3 F in the figure is the EDS spectrum analysis of immobilized HMGB1.

[0049] From the characterization results, we can know that ( Figure 3 ), 1 g NT-MOF can adsorb 110.22 mg recombinant HMGB1, while the blank material without nickel ions has almost no adsorption on HMGB1 ( Figure 3 In addition, in order to verify whether NT-MOF specifically adsorbs HMGB1, the same method as the purification of cell lysate by Ni-NTA agarose gel column was used for analysis. The results showed that NT-MOF only adsorbed recombinant HMGB1 fused with His-tag, and had little adsorption to other proteins ( Figure 3 B). This proves that the recombinant HMGB1 is accurately fixed on NT-MOF. Subsequently, the morphology of the immobilized HMGB1 synthesized above was observed using a scanning electron microscope. The results showed that the immobilized HMGB1 ( Figure 3 C) than UiO-66-NH 2 ( Figure 3 D) has a larger particle size and a rougher surface. At the same time, the EDS spectrum shows that compared with the unmodified UiO-66-NH 2 ( Figure 3 Middle E), the presence of nickel and sulfur was observed in immobilized HMGB1 ( Figure 3 (F) further confirmed the successful preparation of immobilized HMGB1.

[0050] <Example 2> This example uses the immobilized HMGB1 prepared in Example 1 to provide a model establishment process for the targeted preparation of HMGB1 inhibitor ligand fishing technology from leontopodium extract, which specifically includes the following steps: (1) Take 1 mg of the immobilized HMGB1 in Example 1, add 490 μL of 50 mM Tris-HCl buffer at pH = 8.0 and mix well, then add 10 μL of mixed model solution (100 mM glycyrrhizic acid, 100 mM chlorogenic acid and 100 mM gallic acid, solvent is DMSO). After the above mixed solution is mixed well, shake it at 37 °C for 60 min, centrifuge (8000 rpm, 5 min) to 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 ) The precipitate was washed with methanol solution to inactivate the immobilized HMGB1 and release the ligand obtained by fishing, i.e., the HMGB1 inhibitor. The methanol solution was collected and the methanol solution was the fishing result. The methanol solution was dried and then re-dissolved with 100 μL of methanol, i.e., concentrated and analyzed by HPLC. The results are shown in Figure 4 .

[0051] The above chromatographic separation was performed 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 in water (A), and the elution program was: 10%-50% B from 0 to 30 min. The column temperature was maintained at 30 °C, the flow rate was 0.4 mL / min, and the UV detection wavelength was set to 254 nm.

[0052] The mixed model solution contains nonspecific compounds of equal molar mass, gallic acid and chlorogenic acid, as well as glycyrrhizic acid, a known HMGB1 inhibitor. According to the experimental results, ( Figure 4 ), the ligand fishing technology established in this example successfully prepared HMGB1 inhibitors from the mixed model solution by using immobilized HMGB1, but had no recognition for non-specific compounds such as gallic acid and chlorogenic acid. It was 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 targeted mining of active components with HMGB1 inhibition in Leontopodium alpinum.

[0053] <Comparative Example 1> Only blank material UiO-66-NH without HMGB1 was used 2 The experimental process of Example 2 was carried out, and the final methanol solution sample obtained was named blank control. The HPLC conditions were the same as those of Example 2, and the HPLC results were shown in Figure 5 .

[0054] According to the experimental results, it can be seen that ( Figure 5 ), using blank material UiO-66-NH without HMGB1 2 , neither the positive compound glycyrrhizic acid nor the negative compounds gallic acid and chlorogenic acid can be recognized in the mixed solution. This further proves that the immobilized HMGB1 synthesized in Example 1 can be used to establish a ligand fishing technique to prepare HMGB1 inhibitors from Leontopodium edulis in a targeted manner.

[0055] <Example 3> This example uses the ligand fishing technology established by immobilizing HMGB1 to prepare HMGB1 inhibitors from Edelweiss in a targeted manner, which specifically includes the following steps: Take 1 mg of the immobilized HMGB1 in Example 1, dilute it with 490 μL of pH=8.0, 50 mM Tris-HCl buffer, add 1 mg of Leontopodium extract, incubate at 37°C for 60 min, collect the precipitate by centrifugation (8000 rpm, 5 min), 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 was used to wash the precipitate, inactivate the immobilized HMGB1, and release the ligand obtained by fishing, i.e., the HMGB1 inhibitor, and the methanol solution was collected. The methanol solution was the fishing result. After the methanol solution was dried, it was re-dissolved in 100 μL of methanol, i.e., concentrated and analyzed by HPLC. During HPLC analysis, 20 μL of the fishing result methanol solution was injected, and the tail liquids of 5.0-7.5 min and 12.5 min-14.0 min in the fishing result were collected. After comparing the fishing result with the blank control, the impurities introduced by the immobilized HMGB1, i.e., the tail liquid of 5.0 min-7.5 min, were discarded. The above process was repeated until all the tail liquids of 5.0 min-7.5 min in the 100 μL fishing result methanol solution were collected. The tail liquid of 12.5 min-14.0 min in the fishing result was concentrated to dryness under reduced pressure at 40 °C to obtain the HMGB1 inhibitor and used for subsequent identification and verification experiments. The yield of HMGB1 inhibitors was calculated by the relative content of the substances detected in the fishing results. Figure 6 .

[0056] The above HPLC separation was performed 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 in water (A), and the elution program was: 10%-50% B from 0 to 30 min. The column temperature was maintained at 30°C, the flow rate was 0.4 mL / min, and the UV detection wavelength was set to 254 nm.

[0057] <Example 4> The HMGB1 inhibitor prepared in the targeted form of Leontopodium in Example 2 was qualitatively analyzed by HPLC-Q-TOF-MS / MS. The test sample was consistent with the HPLC test sample. The measurement conditions were as follows: Agilent ZORBAX SB-Aq column (2.1×250 mm, 3.5 μm) was used for chromatographic separation, with a flow rate of 0.3 mL / min and an injection volume of 1 μL. The column temperature was set to 35°C. A quadrupole time-of-flight mass spectrometer was used and operated in negative ion mode. The main parameters were set as follows: 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; and 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 A in the formula is Apigenin-7-O- β -HPLC spectrum of qualitative analysis of D-glucopyranoside standard and leontopodium extract; Figure 7 Center B shows the mass spectrometry data of the HMGB1 inhibitor prepared by immobilizing HMGB1.

[0058] From the experimental results, it can be seen that there are a total of 21 compounds in leontopodium alpinum. After fishing with immobilized HMGB1, a potential HMGB1 inhibitor was prepared from leontopodium alpinum extract ( Figure 6 ). Analyzed by HPLC-QTOF-MS / MS ( Figure 7 B), and standard control ( Figure 7 A) The potential HMGB1 inhibitor identified by immobilized HMGB1 was apigenin-7-O- β -D-glucoside (Apigenin-7-O- β -D-glucopyranoside) (peak 14).

[0059] <Example 5> Purchase the standard of potential HMGB1 inhibitor obtained by fishing (Apigenin-7-O- β -D-glucopyranoside), and then its HMGB1 inhibitory activity was verified by HMGB1-induced mouse RAW 264.7 cell inflammation model. Compared with the model group (HMGB1), the compound can inhibit the expression of NO and TNF- α expression level, it has direct HMGB1 inhibitory activity. The results are shown in Figure 8 . Figure 8 A in the middle is Apigenin-7-O- β - Cytotoxicity assay results of D-glucopyranoside (abbreviated as AG); Figure 8 B is Apigenin-7-O- β -D-glucopyranoside and the positive compound glycyrrhizic acid (Gly) acted in parallel on HMGB1-induced RAW264.7 cells, and the NO content in the cell supernatant was measured. The control group was RAW264.7 cells without any treatment (i.e., no purified HMGB1 was added to induce the expression of inflammatory factors, and no test samples were added); Figure 8 C in the middle is Apigenin-7-O- β -D-glucopyranoside and the positive compound Gly acted in parallel on HMGB1-induced RAW264.7 cells, and TNF- α The results of content determination showed that the control group consisted of RAW264.7 cells without any treatment (i.e., no purified HMGB1 was added to induce the expression of inflammatory factors, and no test samples were added).

[0060] The targeted HMGB1 direct inhibitors were further verified and analyzed, and the activity of the potential HMGB1 inhibitors was measured in the HMGB1-induced RAW264.7 cell inflammation model. β -D-glucopyranoside (AG) and positive compound Gly were tested in parallel. Using Thermo Scientific Multiskan FC microplate reader, CCK-8 method was first used to determine the expression of AG ( Figure 8 The concentration of A) was selected as 50 μM, which was not cytotoxic. Figure 8 B) and TNF- α ( Figure 8C) The results of the determination of the expression of these two inflammatory factors show that the prepared AG has good HMGB1 inhibitory activity, which is equivalent to that of Gly. The above data once again prove the feasibility and accuracy of the application of the fishing technique established in Example 2 in the targeted preparation of HMGB1 inhibitors in Leontopodium, and also illustrate the material basis of HMGB1 inhibitory activity in Leontopodium.

[0061] <Example 6> The specific action sites were further explored by molecular docking technology, and the protein structure files were obtained from the protein database (PDB number: 2YRQ). The AutoDock Vina software (version 1.1.2) was used to dock the ligand with the corresponding protein. The docking results were subsequently visualized using Pymol software (version 2.5.5). Fig. 9 . Fig. 9 A in the middle is Apigenin-7-O- β -Three-dimensional molecular docking diagram of the complex formed by the interaction between D-glucopyranoside and HMGB1 (PDB 2YRQ); Fig. 9 B is Apigenin-7-O- β -2D molecular docking diagram of the complex formed by the interaction between D-glucopyranoside and HMGB1. Further molecular docking analysis of the binding mode between AG and HMGB1 showed that ( Fig. 9 AG can bind to the inflammatory active domain B-box of HMGB1 with a binding energy of -28.7092 kcal / mol. At the same time, AG can 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 thus exerting an anti-inflammatory effect.

[0062] 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 aggravate inflammatory responses. Under external stimulation, HMGB1 in the nucleus will transfer to the extracellular space and release HMGB1 into the intercellular matrix through exocytosis, thereby causing or aggravating a series of inflammatory responses. In order to further explore the HMGB1 inhibitory activity of leontopodium alpinum, 2 O 2 In the induced NIH / 3T3 cell inflammation model, we tested whether Leontopodium alba could inhibit the release of HMGB1 from cells to investigate whether Leontopodium alba has the activity of inhibiting the release of HMGB1. Fig.10 , Fig.10 A is H 2 O 2 Fluorescence staining of HMGB1 in induced NIH / 3T3 cells; Fig.10 B in the middle is the H after the treatment of edelweiss2 O 2 Figure 1. Fluorescence staining of HMGB1 in NIH / 3T3 cells induced by HMGB1. 2 O 2 Induced NIH / 3T3 cells have a large amount of HMGB1 transferred to the nucleus ( Fig.10 A), while Leontopodium alba can significantly inhibit the transfer of HMGB1 from the nucleus to the extranuclear part of NIH / 3T3 cells, indicating that Leontopodium alba inhibits the release of HMGB1 under external stimulation ( Fig.10 In summary, the scientific association between Leontopodium and HMGB1 inhibitory activity was established from two dimensions: direct HMGB1 inhibitory activity and inhibition of HMGB1 release.

[0063] <Example 7> Use purchased Apigenin-7-O- β -D-glucopyranoside is used in cosmetic facial creams. The proportions of the various components of the facial creams are shown in Table 1 below: Table 1. Cream ingredients

[0064] The preparation method of the above-mentioned facial cream is as follows: 1. Add phase A raw materials into the main mixing tank, mix evenly, then add phase B raw materials, start stirring and heating, the target temperature is 80℃-85℃.

[0065] 2. Add Phase C raw materials into the oil phase tank, start stirring and heating, the target temperature is 80℃-85℃.

[0066] 3. After reaching the target temperature, start the homogenization in the main preparation tank, and then add the raw materials in the oil phase tank into the main preparation tank, with a homogenization speed of 6000rpm and a homogenization time of 5min.

[0067] 4. Stop homogenization, start stirring (150rpm-200rpm), start cooling, and the target temperature is 75℃.

[0068] 5. When the temperature of the main preparation tank drops to 75°C, add the phase D raw materials into the main preparation tank, stir and mix evenly, and continue to cool down. The target temperature is 45°C.

[0069] 6. When the temperature drops to 45°C, add the E phase raw materials and stir to mix evenly; cool to room temperature and discharge the material.

[0070] <Example 8> In this example, the cosmetics of Example 7 are subjected to performance tests.

[0071] Testing method: Under normal circumstances, adult subjects use the test product continuously for 56 days according to the following usage method to evaluate whether the test product has anti-wrinkle effect and whether it is suitable for sensitive skin (non-irritating). Adult subjects use the evaluation product continuously for 56 days according to the instructions and then stop using it for 7 days to evaluate whether the product can continue to improve the skin condition after stopping use for 7 days.

[0072] Subjects: A total of 34 valid subjects completed the assessment, including healthy Chinese men and women with sensitive skin (lactic acid stinging screening), including 6 males and 28 females, aged 42 to 60 years old, with an average age of 52.76±5.23 years old, who met the voluntary inclusion and exclusion criteria.

[0073] Area assessed: Face.

[0074] How to use: Apply an appropriate amount of this product evenly on the face, avoiding the skin around the eyes, and massage gently in circles in the same direction until absorbed. Frequency of use: once in the morning and once in the evening.

[0075] Use period: 63 days.

[0076] 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).

[0077] Evaluation method: The subjects' faces were photographed using the skin fast three-dimensional imaging system PRIMOS CR, and wrinkle changes were analyzed through images; a questionnaire survey was conducted to ask the subjects to self-assess whether the test product was suitable for sensitive skin.

[0078] (1) Reduction in skin wrinkle volume (cheeks) During the test period, the specific results of the reduction in the volume of skin wrinkles (cheeks) of the subjects are shown in Table 2 below: Table 2. Reduction in the volume of skin wrinkles of the subjects (cheeks) (unit: %)

[0079] From the results in Table 2 above, it can be seen that cosmetics with the HMGB1 inhibitor Apigenin-7-O-β-D-glucopyranoside as the only active ingredient have a significant difference in the reduction of skin wrinkle volume (cheeks), indicating that HMGB1 inhibitors can improve skin conditions and thus have good anti-wrinkle and anti-aging effects.

[0080] (3) Applicability for sensitive skin During the testing period, the specific results of the sensitive skin applicability are shown in Table 3 below: Table 3. Applicability rate for sensitive skin (unit: %)

[0081] It can be seen from the results in Table 3 above that the cosmetics of Example 7 have excellent results in terms of applicability to sensitive skin and have a universal effect on sensitive skin.

[0082] The applicant declares that the above is only a specific implementation mode 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 those skilled in the art within the technical scope disclosed by the present invention are within the protection scope and disclosure scope of the present invention.

Claims

1. An HMGB1 inhibitor, characterized in that The inhibitor is apigenin-7-O- β -D-Glucoside.

2. Use of the HMGB1 inhibitor according to claim 1 in cosmetics.

3. A method for preparing a targeted HMGB1 inhibitor from an extract of Leontopodium leontopodium, characterized in that: The following steps are involved: Immobilized HMGB1 was co-incubated with leontopodium extract to obtain a precipitate, and the precipitate was washed with a buffer solution for nonspecific adsorption and then with an inactivating agent to obtain the HMGB1 inhibitor apigenin-7-O- β -D-Glucoside.

4. The method for targeted preparation of HMGB1 inhibitor from Leontopodium leontopodium extract according to claim 3, characterized in that: The preparation method of immobilized HMGB1 comprises the following steps: Step SA-1, using protein engineering technology to recombinantly express recombinant human HMGB1 fused with an N-terminal His-tag in an Escherichia coli system to obtain a cell lysate containing recombinant human HMGB1 fused with an N-terminal His-tag; Step SA-2, synthesizing the metal organic framework material UiO-66-NH2 by a solvothermal method, then using glutaraldehyde as a connecting arm, modifying the surface of the UiO-66-NH2 by tyrosine, and then adding nickel ions for chelation to synthesize NT-MOF; Step SA-3, mixing the NT-MOF with the cell lysate for incubation, and washing to obtain immobilized HMGB1.

5. The method for targeted preparation of HMGB1 inhibitor from Leontopodium leontopodium extract according to claim 3, characterized in that: The mass ratio of the immobilized HMGB1 to the leontopodium extract is 2:1, the incubation temperature is 37° C., and the pH is 8.0; the inactivating agent is a methanol solution with a volume ratio of 95%.

6. The method for targeted preparation of HMGB1 inhibitor from Leontopodium leontopodium extract according to claim 5, characterized in that: The precipitate is washed with buffer and then the immobilized HMGB1 is inactivated with a 95% by volume methanol solution to release the ligand into the methanol solution, which is the fishing result. The methanol in the fishing result is then removed and the compound is re-dissolved with a predetermined amount of methanol and then analyzed by HPLC. The detected compound is an HMGB1 inhibitor, while the undetected compound is not an HMGB1 inhibitor. The detected compounds are collected and used for subsequent identification.

7. The method for preparing a HMGB1 inhibitor from a leontopodium extract as claimed in claim 6, characterized in that: HPLC analysis was performed using an Agilent ZORBAX SB-Aq column with a column size of 3.0 mm × 100 mm, 3.5 μm. The mobile phases were 0.01% formic acid aqueous solution in phase A and acetonitrile in phase B. The elution procedure was: 0–30 min, with the volume fraction of mobile phase B in the mobile phase being 10%–50%.

8. The method for targeted preparation of HMGB1 inhibitor from Leontopodium leontopodium extract according to claim 3, characterized in that: The buffer solution is Tris-HCl with a pH of 8.0 and a concentration of 50 mM.

9. The method for targeted preparation of HMGB1 inhibitor from Leontopodium leontopodium extract according to claim 3, characterized in that: When the immobilized HMGB1 is co-incubated with the leontopodium extract, the immobilized HMGB1 is diluted with a Tris-HCl buffer having a pH of 8.0 and a concentration of 50 mM.

10. The method for targeted preparation of HMGB1 inhibitor from Leontopodium leontopodium extract according to claim 3, characterized in that: The preparation method of the edelweiss extract comprises the following steps: Step S0-1, drying and crushing the raw material of edelweiss at 70°C to 80°C and then passing through a 30-50 mesh sieve; Step S0-2, mixing the edelweiss obtained in step S0-1 with an ethanol extraction solvent having a mass ratio of 60% to 95%, extracting at room temperature for 24 h to 48 h, extracting 1 to 3 times, filtering the extract using a 100 to 400 mesh sieve, and then concentrating under reduced pressure to obtain an edelweiss extract; Step S0-3, drying the leontopodium extract obtained in step S0-2 to obtain a leontopodium extract.

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