Application of clover extract in the preparation of anti-inflammatory drugs

Abstract

This application uses the extract of Tripterygium wilfordii as the research object, especially the study of protocatechuic acid methyl ester and isovitexin, looking for a combination of components with synergistic anti-inflammatory effects. The designed experiment verifies that isovitexin and protocatechuic acid methyl ester have synergistic anti-inflammatory effects. The mechanism of action is related to inhibiting the excessive secretion of inflammatory factors such as TNF‑α, IL‑6, and IL‑10, inhibiting the phosphorylation of inflammatory pathway proteins such as ERK, JNK, and conclusion 53p65, and promoting the expression of antioxidant-related Nrf2 protein. The experiment verifies that isovitexin and protocatechuic acid methyl ester have synergistic anti-inflammatory effects, and their effects occur in the cytoplasm. The experiment found that 5μmol·L ‑1 ‑250μmol·L ‑1 Protocatechuic acid methyl ester has anti-inflammatory activity within a concentration range of 5 μmol·L ‑1 Isovitexin + 250μmol·L ‑1 The CI value of protocatechuic acid methyl ester is 0.13377, and the synergistic effect is excellent.

Description

Technical Field

[0001] The invention relates to a cloverleaf green extract, in particular to the anti-inflammatory activity of a protocatechuic acid methyl ester component of the cloverleaf green extract and its application in the preparation of anti-inflammatory drugs, and specifically to its anti-inflammatory activity application and its synergistic anti-inflammatory application with isovitexin. Background Art

[0002] Tetrastigma hemsleyanum Diels et.Gilg., also known as golden thread hanging gourd, snake aconite, stone mouse, belongs to the Rhamnosae order, the grape family herbaceous vine plant, the whole plant can be used as medicine, is a precious Chinese medicinal material unique to my country. The medicinal parts of the Chinese medicinal material Tetrastigma hemsleyanum are the tubers, fruits or whole plants of the three-leaf green, which can be harvested throughout the year and can be used dried or fresh. The "National Compendium of Chinese Herbal Medicines" records: It is flat in nature and slightly bitter in taste. It has the effects of clearing away heat and detoxifying, dispelling wind and resolving phlegm, promoting blood circulation and relieving pain. It is mainly used to treat snake bites, tonsillitis, lymph node tuberculosis, traumatic injuries, children's high fever convulsions and other diseases. The chemical composition of the three-leaf green is diverse, containing rich flavonoids, polyphenols, polysaccharides, terpenes, etc.

[0003] Pharmacological studies have shown that Tripterygium wilfordii has an inhibitory effect on different tumor cell lines such as gastric cancer, liver cancer, and lung cancer. It can regulate antioxidant-related enzymes and the immune system to fight liver damage and immune imbalance, and has a good therapeutic effect on viral infection and inflammatory diseases. Researchers have found that Tripterygium wilfordii extract can improve swelling in inflammatory models of mice and rats. Among them, the flavonoid components in Tripterygium wilfordii extract have a certain theoretical basis as the anti-inflammatory material basis of Tripterygium wilfordii. Protocatechuic acid methyl ester, as a phenolic acid substance isolated from Tripterygium wilfordii, its anti-inflammatory activity and anti-inflammatory application, especially in the preparation of anti-inflammatory drugs, still need to be studied. Summary of the invention

[0004] The purpose of the present invention is to provide the anti-inflammatory activity of the extract from Tripterygium wilfordii and its application in the preparation of anti-inflammatory drugs, especially the anti-inflammatory application of protocatechuic acid methyl ester in Tripterygium wilfordii and isovitexin in combination, the synergistic use can reduce the toxicity of the drug, and has guiding significance for the development of new natural anti-inflammatory drugs.

[0005] To achieve the above-mentioned purpose of the invention, the solution of the present application is implemented in the following way.

[0006] Optionally, use of the Tripterygium wilfordii extract in the preparation of anti-inflammatory drugs.

[0007] Optionally, the anti-inflammatory activity acts in the cytoplasm.

[0008] Optionally, the anti-inflammatory active ingredients of the Tripterygium wilfordii extract include flavonoids and phenolic acids.

[0009] Optionally, the flavonoid active ingredients with anti-inflammatory activity in the clover extract include isovitexin component, and the phenolic acid active ingredients with anti-inflammatory activity in the clover extract include protocatechuic acid methyl ester component.

[0010] Optionally, the application concentration of the protocatechuic acid methyl ester component is 5 μmol·L -1 ~250μmol·L -1 .

[0011] Optionally, the application concentration of the isovitexin component is 5 μmol·L -1 ~25μmol·L -1 .

[0012] Optionally, the protocatechuic acid methyl ester in the anti-inflammatory active ingredients of the clover extract and the isovitexin are used in combination to have a synergistic anti-inflammatory effect.

[0013] Optionally, the concentration range of the protocatechuic acid methyl ester and the isovitexin in the anti-inflammatory active ingredients of the clover extract having a synergistic anti-inflammatory effect is 5 μmol·L -1 ~25μmol·L -1 Isovitexin component + 5μmol·L -1 ~250μmol·L -1 The CI values ​​of protocatechuic acid methyl ester components are all less than 1.

[0014] Optionally, the active concentration of the protocatechuic acid methyl ester and the isovitexin having a synergistic effect is 250 μmol·L -1 Protocatechuic acid methyl ester + 5μmol·L -1 The CI value of isovitexin is 0.13377. The beneficial effects of the present invention are as follows: the present application uses the extract of Tripterygium wilfordii as the research object to study the anti-inflammatory activity and mechanism of action of flavonoids and phenolic acids, especially protocatechuic acid methyl ester and isovitexin, verifies their mechanism of action through designing experiments, searches for component combinations with synergistic anti-inflammatory effects, and provides guidance for the development of new natural medicines with anti-inflammatory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a comparison chart of the effects of different concentrations of LPS on the activity of RAW264.7 cells.

[0016] Figure 2 This is a comparison chart of the effects of different concentrations of LPS on NO secretion.

[0017] Figure 3 This is a comparison chart of the effects of the same LPS concentration and different induction times on NO secretion.

[0018] Figure 4This is a comparison chart of the effects of different concentrations of isovitexin on cell activity.

[0019] Figure 5 This is a comparison chart of the effects of different concentrations of protocatechuic acid methyl ester on cell activity.

[0020] Figure 6 This is a comparison chart of the effects of different concentrations of drug combinations on the activity of RAW264.7 cells.

[0021] Figure 7 This is a comparison chart of the effects of a single drug and LPS combined on the activity of RAW264.7 cells.

[0022] Figure 8 This is a comparison chart of the effects of different drug combinations combined with LPS on the activity of RAW264.7 cells.

[0023] Fig. 9 This is a comparison chart of the inhibitory effects of single drugs on LPS-induced NO release from RAW264.7 cells.

[0024] Fig.10 This is a comparison chart of the inhibitory effects of different drug combinations on LPS-induced NO release from RAW264.7 cells.

[0025] Fig.11 It is a Venn diagram of MY combination target genes and disease target genes.

[0026] Fig.12 This is a schematic diagram of GO enrichment analysis of MY combination target genes.

[0027] Fig.13 This is the KEGG annotation result analysis diagram of MY combination target genes.

[0028] Fig.14 This is a schematic diagram of the MY combination component-target-pathway network.

[0029] Fig.15 This is a schematic diagram of the MY combination target gene protein interaction network.

[0030] Fig.16 This is a schematic diagram of the PPI network visualization analysis results of the MY combination.

[0031] Fig.17 This is a schematic diagram of the docking mode of isovitexin and 5F19 protein.

[0032] Fig.18 This is a schematic diagram of the visual analysis of the binding cavity of isovitexin to the 5F19 protein.

[0033] Fig.19 Schematic diagram of the docking mode of protocatechuic acid methyl ester and 1NSI protein.

[0034] Fig. 20 This is a schematic diagram of the visualization analysis of the binding cavity of protocatechuic acid methyl ester and 1NSI protein.

[0035] Fig.21 This is a schematic diagram of the hydrogen bonding of isovitexin to amino acid residues GLY-135, GLN-327, MET-48, CYS-4, and CYS-36.

[0036] Fig. 22 This is a schematic diagram of the connection between protocatechuic acid methyl ester and the amino acid residue ARG-381 by forming two hydrogen bonds.

[0037] Fig.23 It is a comparison chart of the inhibitory effects of different components and component combinations on TNF-α factor levels.

[0038] Fig.24 It is a comparison chart of the inhibitory effects of different components and component combinations on the IL-6 factor level.

[0039] Fig.25 It is a comparison chart of the effects of different concentrations of drugs and drug combinations on IL-10 factor levels.

[0040] Fig.26 This is a comparison chart of the effects of components and component combinations on iNOS expression in mouse RAW264.7 cells.

[0041] Fig. 27 This is a comparison chart of the effects of components and component combinations on the relative expression of iNOS in mouse RAW264.7 cells.

[0042] Fig.28 This is a comparison chart of the effects of components and component combinations on the expression of MAPK pathway proteins in mouse RAW264.7 cells.

[0043] Fig.29 This is a comparison chart of the effects of components and component combinations on the relative expression of p-ERK / ERK in mouse RAW264.7 cells.

[0044] Fig.30 It is the effect of components and component combinations on the relative expression of p-JNK / JNK in mouse RAW264.7 cells.

[0045] Fig.31 It is a comparison chart of the effects of components and component combinations on the expression of p-p65 / p65 in mouse RAW264.7 cells.

[0046] Fig.32 This is a comparison chart of the effects of components and component combinations on the relative expression levels of p65 / p-p65 in mouse RAW264.7 cells.

[0047] Fig.33 This is a comparison chart of the effects of components and component combinations on LPS-induced Nrf2 protein expression in RAW264.7 cells.

[0048] Fig.34 This is a comparison chart of the effects of components and component combinations on the relative expression of Nrf2 in mouse RAW264.7 cells. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the present application clearer, the technical solution of the present invention is described in detail below in combination with specific experiments, data processing and characterization analysis.

[0050] Pharmacological research and chemical composition analysis show that the main anti-inflammatory active ingredients in Sanyeqing are flavonoids and phenolic acids. Methyl protocatechuate is a phenolic acid compound newly isolated from Sanyeqing by the laboratory of the applicant. Methyl protocatechuate, i.e. methyl 3,4-dihydroxybenzoate, has strong antioxidant activity and is distributed in plants such as Coptis chinensis, Fagopyrum scabra, Dianthus superbus, and Hedyotis diffusa. The applicant isolated this compound from Sanyeqing for the first time in the early stage, and this separation is the first in the industry. Isovitexin and isorhamnetin are chemical components with higher content in the flavonoid components of Sanyeqing, and they also have certain anti-inflammatory activity. Isovitexin, i.e. isovitexin, has a molecular formula of C 21 H 20 O 10 The English name is 6-(β-D-Glucopyranosyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one. The inventors believe that the combination of isovitexin and other ingredients can have a synergistic effect, which can be used to reduce drug toxicity and enhance anti-tumor effects. The research and application of single components and combined use for synergistic anti-inflammatory effects need to be further studied.

[0051] The inventor(s) believe that, according to the multi-component and multi-target action characteristics of traditional Chinese medicine, the current research on the anti-inflammatory effect of Sanyeqing is still insufficient, especially the newly isolated protocatechuic acid methyl ester component in Sanyeqing. The research on it mainly focuses on content determination and relatively limited pharmacological activity research. Protocatechuic acid methyl ester has significant antioxidant stress resistance. When it is used as an anti-inflammatory component, its anti-inflammatory effect needs to be studied in depth. In view of this, the applicant designed an experimental scheme to study the anti-inflammatory application of the newly isolated protocatechuic acid methyl ester component in Sanyeqing, and verified the anti-inflammatory activity of protocatechuic acid methyl ester. At the same time, the applicant discovered through experiments that the optimal usage dosage of protocatechuic acid methyl ester and isovitexin as anti-inflammatory components and their synergistic anti-inflammatory dosage.

[0052] It is understandable that the anti-inflammatory mechanisms of common Chinese medicines include inhibiting the secretion of inflammatory mediators, inhibiting the expression of inflammatory signaling pathways, regulating oxidative stress damage, and regulating the function of the hypothalamus-pituitary-adrenal cortex axis. Inflammatory signals activate the secretion of inflammatory mediators by macrophages, which in turn activate inflammatory pathways and activate inflammatory responses. Inhibiting the secretion of inflammatory mediators can block the process of inflammation to a certain extent and inhibit the occurrence of inflammation. In order to study the changes in the body during the inflammatory process, researchers have established a variety of inflammatory models. Commonly used inducers include IFN-γ, TNF-α, HSP60 / 70, etc., among which LPS is the most efficient, safe and widely used inducer. LPS-induced RAW264.7 inflammatory cells are the most commonly used inflammation model. LPS (lipopolysaccharide, abbreviated as LPS in English, is a component of the outer wall of the cell wall of Gram-negative bacteria. It is a substance composed of lipids and polysaccharides, that is, a glycolipid substance) activates macrophages by binding to TLR receptors on the cell membrane, inducing cells to secrete a large amount of NO and inflammatory factors and activate inflammatory pathways, causing macrophages to be in an inflammatory state.

[0053] Based on this, this application uses the RAW264.7 cell inflammation model as the object, and uses this model to study the anti-inflammatory activity and mechanism of drug components. At present, the mechanism of the LPS-induced inflammation model is mainly: LPS recognizes and binds to TLR4, transmits signals to the cell, activates the cascade reaction of signaling pathways such as MAPK and NF-κB, thereby producing a large amount of IL-1β, IL-6, TNF-α and other cytokines and inflammatory mediators such as NO. Therefore, people can achieve the purpose of evaluating the anti-inflammatory effect of drugs by detecting the content of cytokines and inflammatory mediators, the expression of channel proteins, etc.

[0054] It is understandable that LPS binds to TLR receptors, activates macrophages, and releases inflammatory factors. It is an ideal and commonly used in vitro inflammation model and is often used to evaluate the anti-inflammatory effect of drugs. NO is a signal molecule that appears in the early stages of inflammation and can indicate the occurrence and progression of inflammation. It has a positive guiding significance for the judgment of inflammation. Therefore, measuring the effect of drugs on the NO concentration in the inflammation model can preliminarily determine whether the drug has anti-inflammatory activity.

[0055] To illustrate the present application, specific experiments, characterizations and analyses are as follows.

[0056] Reagents used in the experiment: DMEM high-glucose culture medium, MTT, fetal bovine serum, penicillin and streptomycin, DMSO, LPS, PBS buffer, and nitric oxide detection kit.

[0057] The cells used in the experiment are mouse mononuclear macrophages-RAW264.7 cells, and the cell culture, passaging, freezing and recovery methods are as follows. In this application, unless otherwise clearly specified and limited, the cells used in the experiment are mouse mononuclear macrophages-RAW264.7 cells, and the cell culture, passaging, freezing and recovery methods are the same as the following methods.

[0058] Specifically, cell culture includes: 5 mL of cell suspension containing a certain cell density is injected into a T25 cell culture flask, and the culture medium consists of DMEM high-glucose culture medium containing 10% fetal bovine serum and 1% double antibody (consisting of 100U·mL-1 penicillin and 0.1mg·mL-1 streptomycin). Place in an incubator for culture, and the culture conditions are 37°C and 5% CO2. The whole process is aseptic, and the cell state and density are observed daily. Cell passaging includes: preheating PBS and culture medium at 37°C, aspirating and discarding the old culture medium, and carefully adding PBS to wash three times. Aspirate and discard PBS, add 5 mL of culture medium, scrape the cells with a scraper, add fresh culture medium and blow evenly, passage, cross-shake evenly, and place in an incubator for culture. The cycle is about one to two days. Cell freezing includes: when RAW 264.7 cells grow to a high density, select cells in better condition for freezing operation. Preheat PBS and culture medium at 37°C, aspirate and discard the old culture medium, and carefully add PBS to wash three times. Aspirate and discard PBS, add 5mL of fresh culture medium, use a cell scraper to carefully scrape the bottom of the cell bottle, scrape off most of the cells and blow the cell suspension evenly, collect every five bottles in a 50mL centrifuge tube. Centrifuge at low speed to collect the cells. Carefully pour off the supernatant and add freezing solution. The freezing solution contains 10% dimethyl sulfoxide to protect the cells. Aliquot an appropriate amount of cell-containing freezing solution into cryopreservation tubes, cool down and store in liquid nitrogen. Cell recovery includes: melting the solidified cell suspension at 37°C and mixing it with fresh culture medium. Centrifuge at low speed to collect the cells. Discard the supernatant, mix it with the culture medium, and culture it in a culture bottle or incubator.

[0059] Among them, cell activity was determined by MTT method, including the steps: (1) adding the culture medium set for each group, mixing with cells for 24 hours, and recording the cell status of each group. (2) Protect from light, add 20μMTT to each well, and continue incubation for 4 hours. (3) After 4 hours, terminate the incubation, use a pipette to carefully aspirate the culture medium, avoid touching the bottom of the well and aspirating purple crystals. Add 100μLDMSO to each well and incubate at room temperature for 30 minutes. (4) Determine the OD490 nm value of each well. (5) Result processing: The group containing only culture medium is used as the blank group, and the group containing only culture medium and cells is used as the control group.

[0060]

[0061] Experiment 1: Establishment of RAW264.7 cell inflammation model

[0062] The cells were grouped and processed as follows: (1) Collect cells, count, adjust cell density, and inoculate them into 96-well culture plates, 100 μL per well, 1×104 cells per well. Culture in an incubator overnight. (2) The experiment was divided into a blank group, a control group, groups with different LPS concentrations, and groups with different LPS induction times. In the blank group, only culture medium was added; in the control group, no treatment was performed; in the LPS group, a concentration gradient of LPS solution was added to the cell culture medium, and the LPS concentration gradient was set to 10 ng·mL -1 , 100ng·mL -1 , 500ng·mL -1 , 1μg·mL -1 , 10μg·mL -1 Five final concentrations; different LPS induction time groups, with the optimal induced LPS concentration as the condition, set the LPS induction time gradient to 6, 12, and 24 hours. Each group of treatments was repeated for 5 wells. (3) After the culture was completed, the old culture medium in each well was discarded, and 100 μL of the culture medium under the experimental setting conditions was added to each well, and the incubator was continued to incubate for 24 hours for subsequent experiments. In this application, unless otherwise clearly specified and limited, the RAW264.7 cell inflammation model established in this experiment, and the cell grouping and treatment methods were the same as above.

[0063] In addition, the expression of inflammatory factor NO was detected as above, and the grouping and processing operation was the same as above. After incubation for 24 hours, the supernatant was centrifuged at 12000 rpm for 10 minutes, and immediately detected or stored at 4°C for a short period of time, and the NO concentration in the supernatant was determined according to the instructions of the nitric oxide detection kit.

[0064] The NO inhibition rate was used as an indicator for the synergistic anti-inflammatory effect analysis. The NO inhibition rate of drugs at different concentrations was calculated based on the NO concentration of each group.

[0065]

[0066] Specifically, the Compusyn software used in this application is a software that uses the Chou-Talalay formula to calculate the combination index (CI), which is used to evaluate the interaction between two drugs. Optionally, the NO inhibition rates corresponding to the three components used alone and in combination are imported into the CompuSyn software, and the non-continuous combination analysis is selected to obtain the CI value. The combination with a CI value of <1 is taken as a synergistic combination. Unless otherwise stated, the results and analysis in this application are all calculated by Compusyn software. Optionally, the experiment is repeated 5 times for each group and expressed as Mean±SD. All results are processed by SPSS2 software, and P<0.05 indicates a significant difference. The picture is drawn by GraphpadPrism8.

[0067] Specifically, Figure 1 The results of the effect of different concentrations of LPS on the activity of RAW264.7 cells are shown in the figure, where the cell activity is detected by MTT method. The experiment was divided into a blank group and different concentrations of LPS groups. In the blank group, only the culture medium was added; in the LPS group, LPS solutions with different concentration gradients were added to the cell culture medium, where the LPS concentration gradient was set to 10 ng·mL -1 , 100ng·mL -1 , 500ng·mL -1 , 1μg·mL -1 , 10μg·mL -1 Five final concentrations. Each group of treatments was repeated 5 times. The experimental results showed that when the LPS concentration was ≥10μg·mL -1 When the concentration of LPS was 100 ng·mL, the activity of RAW264.7 cells was less than 90%. It can be considered that LPS above this concentration had certain toxic side effects on cells. -1 LPS at high concentrations can promote cell proliferation. Therefore, when using LPS to induce inflammation models, the LPS concentration used in the experiment is not higher than 10 μg mL -1 .

[0068] like Figure 2 , 3 The figure shows the characterization and analysis of the experimental results of LPS modeling stimulation conditions screening. Figure 2 The experimental results show that the addition of LPS can stimulate RAW264.7 cells to secrete inflammatory mediator NO, which is significantly different from the CK group (i.e., control check, indicating the blank control group) (P < 0.05), and the LPS concentration is 10ng·mL -1 To 1 μg mL -1 This effect was dose-dependent. As the concentration of LPS increased, the secretion of inflammatory mediator NO continued to increase, and at 1 μg mL -1 Reach the highest peak. When LPS reaches a toxic concentration of 10 μg·mL -1 When the NO concentration decreased, it can be considered that the optimal LPS concentration is 1 μg·mL -1 .

[0069] like Figure 3 As shown, under the optimal LPS induction concentration condition, that is, the LPS concentration was 1 μg mL -1 The results of the experiment were analyzed to explore the optimal induction time of LPS. It can be seen from the figure that NO secretion reached the highest peak when LPS was induced for 24 hours, and there was a very significant difference (P < 0.01). Therefore, it can be considered that the optimal induction condition of LPS is a concentration of 1 μg mL -1, effective for 24 hours.

[0070] Experiment 2: Determination of the concentration of protocatechuic acid methyl ester and isovitexin components

[0071] The experimental treatment method is as follows: (1) Cell grouping treatment: collect cells, count, adjust cell density, inoculate in 96-well culture plates, 100 μL per well, 1×104 cells per well, and culture in an incubator overnight; (2) The experiment is divided into a blank group, a control group, and a treatment group. Among them, the blank group only contains culture medium; the control group is not treated; the treatment group is set with a gradient concentration of isovitexin protocatechuic acid methyl ester, so that the final concentrations are 5, 25, 50, 100, 250, and 500 μmol·L -1 , each group of treatments was repeated for 5 wells; (3) After overnight culture, the cells adhered to the wall, the old culture medium of each well was aspirated, and 100 μL of the culture medium of the experimental setting conditions was added to each well, and the cells were incubated in the incubator for 24 h before the subsequent experiments; (4) The cell activity of each group was determined by MTT method.

[0072] like Figure 4 , Figure 5 The figure shows the effect of different concentrations of isovitexin and protocatechuic acid methyl ester on cell activity. Figure 4 The results of the analysis of the effects of different concentrations of isovitexin on cell activity were shown in Figure 2. Specifically, the cells were divided into a blank group and a treatment group after group treatment. The blank group was not treated, and the treatment groups were treated with 5 μmol·L -1 , 25μmol·L -1 , 50μmol·L -1 , 100 μmol·L -1 , 250μmol·L -1 , 500μmol·L -1 The figure shows that 5μmol·L -1 , 25μmol·L -1 When RAW264.7 cells were treated with isovitexin at a concentration of 1.5, the cell viability was >90%. Figure 5 The results of the analysis of the effects of different concentrations of protocatechuic acid methyl ester on cell activity are shown. Specifically, the cells were divided into a blank group and a treatment group after group treatment. The blank group was not treated, and the treatment group was treated with 5 μmol·L -1 , 25μmol·L -1 , 50μmol·L -1 , 100 μmol·L -1 , 250μmol·L -1 , 500μmol·L -1 The figure shows that 5μmol·L -1 ~250μmol·L -1When RAW264.7 cells were treated with methyl protocatechuate at a concentration of 1:1, the cell viability was >90%.

[0073] In summary, the effects of different concentrations of protocatechuic acid methyl ester and isovitexin on the activity of RAW264.7 cells showed that when 5 μmol·L -1 ~250μmol·L -1 Protocatechuic acid methyl ester in the concentration range of 5μmol·L -1 , 25μmol·L -1 When RAW264.7 cells were treated with isovitexin at a concentration of 1:1, the cell viability was >90%. This determined the screening concentration for studying the anti-inflammatory activity of protocatechuic acid methyl ester and its synergistic anti-inflammatory combination with isovitexin.

[0074] Experiment 3: Test the effects of protocatechuic acid methyl ester, isovitexin components and LPS on cell activity, and select safe concentrations of protocatechuic acid methyl ester and isovitexin for combination.

[0075] The experimental treatment method is as follows: (1) Cell grouping treatment: collect cells, count, adjust cell density, inoculate in 96-well culture plates, 100 μL per well, 1×104 cells per well, and culture in an incubator overnight; (2) The experiment is divided into a blank group, a control group, and a treatment group. Among them, the blank group contains only culture medium; the control group is not treated; the treatment group is set with different concentrations of drug combinations, as shown in Table 1 below, and each group is treated with 5 replicates; (3) After overnight culture, wait for the cells to adhere to the wall, remove the old culture medium from each well, add 100 μL of the culture medium under the experimental setting conditions to each well, incubate in an incubator for 24 hours, and conduct subsequent experiments; (4) Use the MTT method to determine the cell activity of the above groups.

[0076]

Table 1

[0077]

[0078] like Figure 6 The results showed that each combination had no effect on the activity of RAW264.7 cells.

[0079] Experiment 4: Effects of single components or combinations of components combined with LPS on cell activity

[0080] Specifically, the experimental treatment method is the same as that of Experiment 3, except that 1 μmol·L -1 Under the condition of LPS, isovitexin, protocatechuic acid methyl ester or different component combinations (the combination method is the same as Table 1) were added and incubated for 24 hours to investigate the effect of LPS and component co-incubation on cell activity. Figure 7 , Figure 8 For the analysis of experimental results, the results showed that single component or combination of components under experimental conditions had no effect on the activity of RAW264.7 cells.

[0081] Experiment 5: Anti-inflammatory activity of protocatechuic acid methyl ester and inhibitory effect of component combination on NO release

[0082] Specifically, the experimental treatment and grouping method were the same as those in Experiment 3, and the inhibitory effect of a single drug on LPS-induced NO release in RAW264.7 cells was determined. Fig. 9 The figure shows the analysis of the experimental results. It can be seen from the figure that methyl protocatechuate has the ability to inhibit NO secretion within the experimental concentration range and is concentration-dependent. The results show that methyl protocatechuate has the ability to inhibit NO release to varying degrees.

[0083] After confirming the anti-inflammatory activity of protocatechuic acid methyl ester, protocatechuic acid methyl ester was used in combination with isovitexin. The experimental treatment and grouping method were the same as those in Experiment 3. The experimental results are as follows: Fig.10 The inhibitory effect of the drug combination on LPS-induced NO release in RAW264.7 cells. As shown in the figure, as the concentration of protocatechuic acid methyl ester in the combination increases, the NO inhibition rate also increases and eventually tends to saturation. Fig. 9 , Fig.10 , that is, the NO inhibition rate before and after the combination. It was found that the addition of methyl protocatechuate has an enhanced NO inhibition effect.

[0084] After calculating the NO inhibition rate of a single component and a combination of components, the single component concentration, the corresponding concentration of the component combination and the corresponding NO inhibition rate were imported into the Compusyn software to calculate the combined index CI value of the component combination, and the component combination with CI < 1 and the best score was selected for the next experiment. The specific CI value analysis is shown in Table 2. Among the experimental combinations, there are 7 drug combinations with CI < 1, which can be regarded as having a synergistic effect. It can be seen that the addition of protocatechuic acid methyl ester has a positive significance. Among them, the CI value of the A5 combination is the lowest, that is, 5μmol·L -1 Isovitexin + 250μmol·L -1 The CI value of the protocatechuic acid methyl ester combination is the lowest. Table 2 shows the drug combinations that have the synergistic effect of inhibiting the release of NO from RAW264.7 cells.

[0085]

Table 2

[0086]

[0087] It is understandable that MTT can distinguish the survival state of cells. The generated crystals can be dissolved in DMSO and the absorption wavelength at 490nm can be measured. The results obtained are compared with those of the control group to obtain the cell activity of the drug at this concentration, which is used to determine the toxicity of the drug. In the study of drug efficacy, it is first necessary to ensure that the drug is at a safe concentration and has no effect on cell activity before further exploring the drug's efficacy and mechanism of action.

[0088] In the cytotoxicity experiment, it was found that isovitexin showed certain cytotoxicity at low concentrations; while protocatechuic acid methyl ester showed certain cytotoxicity at 500 μmol·L -1 The cytotoxicity is only revealed at a high concentration, and the cells tolerate a high concentration. This phenomenon is related to the small molecular weight and low toxicity of methyl protocatechuate. Combined with the results of the NO inhibition rate of the combined treatment group, it can be seen that the addition of methyl protocatechuate can achieve the same anti-inflammatory effect on the basis of reducing the dosage. Compared with the application of the components alone, the NO inhibition rate of the combined treatment has a synergistic effect and can better reduce the NO content. After the addition of methyl protocatechuate, the proportion of drug combinations with synergistic effects in all combinations reached 66.7%, which shows that the combination with methyl protocatechuate has a positive significance for inhibiting NO secretion, thus determining that methyl protocatechuate has a positive effect on synergistic anti-inflammatory effects.

[0089] Experiment 6: Analysis of synergistic anti-inflammatory mechanism based on network pharmacology and molecular docking technology

[0090] It is understandable that the network pharmacology method uses a variety of means such as network databases, high-throughput omics data analysis and computer simulation to construct a "compound-target-pathway" network for research, which is consistent with the overall concept of traditional Chinese medicine and can analyze the relationship between different components of traditional Chinese medicine, making the research more efficient and reasonable. Molecular docking is a technology that virtually combines proteins with small molecule ligands, and the size of the binding energy reflects the quality of the binding activity. Therefore, network pharmacology and molecular docking technology can be used to study the combination of isovitexin and protocatechuic acid methyl ester (MY, the same below) obtained in the early screening, and preliminarily analyze the anti-inflammatory mechanism of the combination.

[0091] Specific methods: (1) Obtaining the structural files of protocatechuic acid methyl ester and isovitexin. The TCMSP platform, a database and analysis platform for Chinese medicine system pharmacology, was used to search for protocatechuic acid methyl ester and isovitexin, and the corresponding .mol2 format of the compound was downloaded. The compound conditions were adjusted by importing the Pymol software and output as .pdbqt files. (2) Obtaining the target proteins and target genes of protocatechuic acid methyl ester and isovitexin. The target genes of protocatechuic acid methyl ester and isovitexin were obtained from the TCMSP database, Pubchem database, and Uniprot database, and the species was "HOMOsapiens". (3) Obtaining the potential anti-inflammatory target genes of component combination. The Genecards database (https: / / www.genecards.org / ) was searched with inflammation as the keyword, and the .tsv format file was downloaded. The genes in the table were screened with the correlation score >5.0 and P <0.01 as the conditions. The disease genes and target genes of the component combination obtained by the screening were imported into the Venny2.1 platform, and the common genes of the two were regarded as the potential anti-inflammatory target genes of the component combination. (4) Annotation of potential anti-inflammatory target genes of component combinations. The obtained potential anti-inflammatory target genes of component combinations were uploaded to the DAVID platform for analysis, and the species was "HOMOspaiens". The tables of GO enrichment analysis and KEGG annotation results were downloaded, and the tables were sorted by Count. (5) Construction of component-target gene-pathway network diagram. The component combination components, potential anti-inflammatory target genes, and KEGG pathway annotation results were classified and sorted, and imported into Cytoscape software in turn. The Networkanalyze function was selected to analyze the network, and the degree value was used as the standard for arrangement. The Merge function was used to merge, and finally the component combination compound-target-pathway diagram was obtained. (6) Construction of 6PPI network. Correct the target proteins corresponding to the potential anti-inflammatory target genes, construct a protein interaction network, and arrange the drawing according to the degree value. (7) Molecular docking: Download the protein structure from the PDB website and determine the binding cavity based on the binding ligand. Import the .pdb file into the Pymol software to delete the ligand, water, etc. to purify the protein structure and output it as a .pdbqt file. Then import the protein and small molecule into the Autodock4.2.6 software for hydrogenation and charge addition operations. Define the range of the Gridbox based on the binding cavity and conduct molecular docking experiments.

[0092] Analysis of potential anti-inflammatory target genes of the component combination: Searching the Genecards database with inflammation as the keyword, a total of 1,647 inflammation-related genes were obtained. The combined target genes and inflammation-related genes were imported into the Venny platform, and after taking the intersection, 49 common anti-inflammatory target genes were obtained for the MY combination, accounting for 23.22%. Fig.11The Venn diagram of MY combination target genes and disease target genes is shown, wherein the left side represents the combined use of isovitexin and protocatechuic acid methyl ester. Table 3 shows the common target genes of the anti-inflammatory effect target genes of the MY combination.

[0093]

Table 3

[0094]

[0095] The obtained potential anti-inflammatory target genes were imported into the DAVID platform for GO enrichment and KEGG annotation, such as Fig.12 The figure shows the GO enrichment analysis of the target genes of the MY combination, which represent GOBP, GOCC, and GOMF from left to right. Fig.13 This is the KEGG annotation result of the target gene of the MY combination. The GOBP enrichment results of the MY combination suggest that the target genes are related to transcription, inflammatory response, apoptosis regulation, innate immune response, etc.; the GOCC enrichment results show that they mostly act on the cytoplasm; the GOMF enrichment results show that the target genes are related to binding activity, transcription factor activity, etc. The KEGG pathway annotation shows that the MY combination can act on TNF, MAPK, PI3K-Akt, TLR and other pathways to exert anti-inflammatory effects.

[0096] Analysis of GO enrichment and KEGG annotation results revealed that the MY combination can antagonize inflammatory responses by regulating cell apoptosis, regulating transcription factor activity and inflammatory response. PI3K-Akt, TNF, TLR, MAPK and other pathways are closely related to inflammation. This shows that the MY combination exerts a synergistic anti-inflammatory effect through multiple targets, multiple modes and multiple pathways.

[0097] Construct component-target-pathway network diagram: After importing the component combination, target gene, and KEGG annotation results into Cytoscape software for processing, the component-target-pathway network corresponding to the combination is obtained, such as Fig.14 This is the MY combination component-target-pathway network. The diamonds in the figure represent the compounds in the combination, the ovals represent potential anti-inflammatory target genes, and the squares represent pathways with higher KEGG annotation enrichment. This allows us to more intuitively see the target genes and pathways involved in the combination.

[0098] Analysis of PPI network: The target genes obtained in the previous screening were imported into the String platform, and the protein interaction (PPI) network was constructed with medium confidence. The Cytoscape visualization analysis was used and the arrangement was based on the degree value. Fig.15 This is the protein interaction network of MY combination target gene. Fig.16 This is the PPI network visualization analysis result of the MY combination.

[0099] The PPI results of the MY combination showed that the target genes with larger values ​​and higher contributions include: TNF, AKT1, IL1B, TP53, RELA, PTGS2, IKBKB, etc.

[0100] It is easy to understand that MAPK1, 8, and 14 are important components of the MAPK pathway, and RELA and IKBKB are components of the NF-κB pathway. Both pathways are closely related to inflammation; IL-1B, TNF, NOS3, and PTGS2 are related to the secretion of inflammatory factors and the process of inflammatory response. Combined with the functional analysis of the target genes, it can be verified that MY can play an anti-inflammatory role by regulating the inflammatory pathway, mediating immune function, and regulating the secretion of inflammatory factors.

[0101] Combined with molecular docking technology, 9 common inflammatory proteins were selected as target proteins for molecular docking: CCL2, IL-6, NOS2, PTGS2, TNF, Akt1, ERK, JNK, and NF-κB. The corresponding PDBIDs are 1D0K, 1ALU, 1NSI, 5F19, 3UP3, 6CCY, 2G01, 5BVE, and 1IKN. The molecular docking scoring results are shown in Table 4, that is, Table 4 shows the molecular docking results and scores of isovitexin, protocatechuic acid methyl ester and common inflammatory proteins.

[0102]

Table 4

[0103]

[0104] Analysis of protein binding graphs of different components and optimal scoring values: Fig.17 is the docking mode of isovitexin and 5F19 protein, Fig.18 This is a visualization analysis of the binding cavity of isovitexin to 5F19 protein. Fig.19 is the docking mode of protocatechuic acid methyl ester and 1NSI protein, such as Fig. 20 Visual analysis of the binding cavity of protocatechuate methyl ester and 1NSI protein.

[0105] Analysis of molecular docking results showed that the docking scores of the above components with 8 common proteins were mostly >-5KJ·mol-1, showing good binding activity, among which isovitexin had better binding activity with inflammatory proteins than protocatechuic acid methyl ester. Protocatechuic acid methyl ester had better binding with 1NSI protein, and isovitexin had the best binding with 5F19 protein, which was the PTGS2 gene and was related to PGE2 synthesis.

[0106] Visual analysis of the above components and the results of the best scoring protein shows that isovitexin can enter the cavity on the surface of 5F19 protein, and protocatechuic acid methyl ester can enter the cavity on the surface of 1NSI protein. Further analysis of the binding mode of the compound to the binding cavity shows that the above components can form hydrogen bonds with the amino acid residues at the binding site, and the binding is stable.

[0107] like Fig.21 Shown Fig.18 The enlarged picture in the box is a schematic diagram of the hydrogen bonding between isovitexin and amino acid residues GLY-135, GLN-327, MET-48, CYS-4, and CYS-36. Fig. 22 The figure shows a schematic diagram of the connection between protocatechuic acid methyl ester and the amino acid residue ARG-381 by forming two hydrogen bonds.

[0108] Understandably, network pharmacology is widely used in virtual screening of active ingredients of traditional Chinese medicine, compound prescriptions, etc. It can efficiently and quickly construct action network diagrams and display them intuitively. Molecular docking can simulate and calculate the optimal binding energy between drugs and proteins, providing a reference for mechanism research and providing direction for further experimental research.

[0109] The results of network pharmacology and molecular docking technology showed that the MY combination can regulate the secretion of inflammatory factors by binding to the secretory proteins of inflammatory factors, regulating the MAPK and NF-κB pathways to exert a synergistic anti-inflammatory effect, which is in line with the characteristics of traditional Chinese medicine with multiple components and multiple targets.

[0110] The above experiments show that the GO enrichment, KEGG annotation and PPI analysis results of the MY drug combination suggest that the combination of isovitexin and protocatechuic acid methyl ester can regulate more inflammation-related pathways, involving redox reactions. The molecular docking results found that isovitexin had a high docking score with common inflammatory proteins, which is consistent with its more obvious anti-inflammatory activity. Protocatechuic acid methyl ester scored lower, but comparison found that protocatechuic acid methyl ester had good binding activity with NOS2 and PTGS2, and had binding activity with other inflammatory proteins. Combined with the good NO inhibition rate results of protocatechuic acid methyl ester in previous experiments, it can be seen that protocatechuic acid methyl ester can assist in anti-inflammatory effects in other ways.

[0111] In summary, MY combination obtained 49 common anti-inflammatory target genes. MY combination can antagonize inflammatory response by regulating cell apoptosis, regulating transcription factor activity and inflammatory response, and acting on pathways such as PI3K-Akt, TNF, TLR, MAPK, etc. It can be seen that MY combination exerts synergistic anti-inflammatory effects through multiple targets, multiple modes and multiple pathways. The target genes of MY combination are: TNF, AKT1, IL1B, TP53, RELA, PTGS2, IKBKB, etc. Molecular docking results show that the above components have good binding activity with 9 common inflammatory proteins.

[0112] Experiment 7: Effects of component combination on the secretion of inflammatory factors in the RAW264.7 cell inflammation model

[0113] It is understandable that inflammation can stimulate the production of cytokines, cytokines can aggravate inflammatory reactions, and cytokines and inflammation are interconnected, so inhibiting cytokines has a positive significance for the treatment of inflammation. Cytokines such as TNF-α, IL-1β, and IL-6 are inextricably linked to the development of inflammation and are the key research objects for studying the anti-inflammatory mechanism of drug action. Based on the above experimental results, the protocatechuic acid methyl ester isolated from Tripterygium wilfordii has anti-inflammatory activity and has a synergistic anti-inflammatory effect in combination with isovitexin. Studying the synergistic anti-inflammatory effect of the component combination from the level of inflammatory factor secretion can better confirm the anti-inflammatory mechanism of Tripterygium wilfordii.

[0114] Optionally, cell culture, passage, freezing and resuscitation are the same as above, and the content of TNF-α, IL-6, and IL-10 cytokines is detected by ELISA. The ELISA method for detecting the content of TNF-α, IL-6, and IL-10 cytokines is as follows: 1) After the cell culture is completed, the cell culture medium is aspirated without touching the bottom of the culture plate, and centrifuged at 12000rpm and 4°C for 20min. Without touching the bottom of the centrifuge tube, the supernatant is aspirated and stored at low temperature for testing. 2) Sample addition: set up the experimental wells and add samples according to the instructions. 3) Add enzyme: add 100μL of HRP-labeled antibody to each well, except for the blank well. 4) Incubation: seal the plate with a sealing film and incubate at 37°C for 60min. 5) Prepare washing solution: dilute the concentrate for use. 6) Washing: add washing solution, let stand for about 1min, spin dry, and pat dry on filter paper until there is no obvious water stain. Repeat 5 times. 7) Color development: add color developers A and B, mix well, and color at 37°C in the dark for about 25min. 8) Stop: Add 50 μL of stop solution to each well to stop the reaction. 9) Determination: Determine the OD450nm value of each well.

[0115] Specifically, the component combination for the grouping treatment of cells secreting TNF-α, IL-6, and IL10 factors in the mouse RAW264.7 cell inflammatory model includes the following steps: (1) Collecting cells, counting them, inoculating them in a 24-well culture plate, 5×105 cells per well, and culturing them overnight. (2) The experiment is divided into a control group, an LPS group, and a treatment group. The control group was not treated; the LPS group contained a final concentration of 1 μg mL -1 LPS culture medium; treatment group, component or component combination was pretreated for 2 h and then mixed with a final concentration of 1 μg mL -1LPS was co-incubated for 24 hours. (3) The old culture medium of each group was discarded and replaced with the corresponding culture medium of each experimental group. The cells were placed in an incubator and incubated for the required time. Each group was treated with 3 replicates. Detection of the expression of inflammatory factors TNF-α, IL-6, and IL-10 After the RAW264.7 cells were incubated for the set time, the supernatant of the culture medium of each well was aspirated, centrifuged at 12000rpm and 4℃ for 20 minutes, the supernatant was aspirated, and the content of TNF-α, IL-6, and IL-10 factors in the supernatant was detected. The data were processed by SPSS, and P<0.05 was considered a significant difference. The picture was drawn by Graphpadprsim8.2 software.

[0116] like Fig.23 Analysis of the inhibitory effects of different concentrations and combinations of components on the secretion level of TNF-α in the mouse RAW264.7 cell inflammatory model. In order to better distinguish the representation, in the figure, M represents 5μmol·L -1 Isovitexin group Y represents 250 μmol·L -1 Protocatechuic acid methyl ester group; MY represents 5 μmol·L -1 Isovitexin + 250μmol·L -1 Protocatechuic acid methyl ester group. In the LPS group, the secretion of TNF-α by RAW264.7 cells was significantly different from that in the control group (P < 0.01), and the mouse RAW264.7 cell inflammation model was successfully established. Compared with the LPS group, the components and component combinations selected in the experiment could significantly inhibit the secretion of inflammatory factors (P < 0.01).

[0117] like Fig.24 The figure shows the inhibitory effects of different components and component combinations on the IL-6 factor level. The inhibitory effects of different concentrations of components and component combinations on the IL-6 secretion level of the mouse RAW264.7 cell inflammation model are shown in Table 5. -1Under LPS-induced conditions, the IL-6 content in the LPS group was significantly different from that in the control group (P < 0.01). Compared with the LPS group, the experimentally selected components and component combinations can also significantly inhibit the secretion of IL-6 in the RAW264.7 cell inflammatory model (P < 0.05). Analysis and comparison of the inhibitory effects of isovitexin and protocatechuic acid methyl ester on the secretion level of IL-6 inflammatory factors, it can be intuitively seen that the combined use can better inhibit the secretion level of IL-6 and reduce the content of IL-6. Compared with the use of isovitexin alone, there is a statistical difference (P < 0.05). Isovitexin has the effect of inhibiting the secretion level of inflammatory factors; protocatechuic acid methyl ester has an antioxidant effect, which can resist the cell damage caused by oxidative burst and reduce cell apoptosis. It can be proved that isovitexin and protocatechuic acid methyl ester can inhibit cell apoptosis through anti-inflammatory and antioxidant effects, block the connection between oxidative stress and inflammation, and play a synergistic role in inhibiting IL-6 secretion.

[0118]

Table 5

[0119]

[0120] like Fig.25 The figure shows the inhibitory effect of different concentrations of components and component combinations on the IL-10 secretion level of the mouse RAW264.7 cell inflammatory model. Compared with the LPS group, the component combination selected in the experiment can significantly reduce the secretion of IL-10 in the RAW264.7 cell inflammatory model (P < 0.05). Compared with the CK group, isovitexin, protocatechuic acid methyl ester and the component combination promoted the secretion of IL-10 factors; compared with the model group, isovitexin, protocatechuic acid methyl ester and the component combination inhibited the high concentration state of IL-10. The inhibitory effect of single components and component combinations on IL-10 inflammatory factors was analyzed, and it was found that the combined use of drugs had a synergistic effect, and the effect of isovitexin alone was significantly different from that of the combined use (P < 0.05).

[0121] In summary, controlling the overproduction of inflammatory factors has a positive significance for controlling inflammation. The drug alone can effectively inhibit the synthesis of inflammatory factors. The combined use of isovitexin and protocatechuic acid methyl ester showed a more effective inhibitory effect on IL-6 factors. Isovitexin and protocatechuic acid methyl ester had a good synergistic inhibitory effect on IL-10 factors. In connection with the more studied anti-oxidative stress effect of protocatechuic acid methyl ester, it can be seen that when combined with isovitexin, the anti-oxidative stress effect has a positive significance for the anti-inflammatory effect of the drug combination. LPS stimulation can induce the expression of IL-10, and the expression peak is later than that of pro-inflammatory factors. IL-10 has a bidirectional regulatory effect. When it is at a low concentration, it can inhibit the secretion of inflammatory factors; but when it is at a high concentration, IL-10 shows an immunosuppressive effect. The secretion of IL-10 in the isovitexin group was lower than that in the model group, indicating that the drug can inhibit the inflammatory process to a certain extent and act on the whole stage of the inflammatory process; it can be seen that protocatechuic acid methyl ester and the combination of components can act on the early stage of the inflammatory process to inhibit inflammatory factors.

[0122] Experiment 8. The combination of components affects the MAPK and NF-κB pathway proteins in the RAW264.7 cell inflammation model. Among the downstream signaling pathways induced by LPS, the NF-κB pathway and the MAPK pathway are important research indicators. It is understandable that the MAPKs pathway is a bridge connecting the outside world and the inside of the cell nucleus, and the NF-κB pathway is a key activation node in the inflammatory signaling pathway. Nrf2 is the main transcriptional regulator of the cellular antioxidant stress program, which can coordinate the activation of protective genes in cells to defend against oxidative stress. The Nrf2 / ARE pathway plays an important role in maintaining cellular redox homeostasis. The above experiments show that the combination of methyl protocatechuate with isovitexin and MY can regulate the secretion of inflammatory factors. The effect of component combination on signaling pathways, the effect of isovitexin, methyl protocatechuate and combination on NF-κB, MAPK, and Nrf2 pathway proteins, in order to better characterize anti-inflammatory molecules and activity mechanisms.

[0123] Specifically, the methods of cell culture, passaging, freezing and thawing are the same as above.

[0124] Preparation of protein separation by SDS-PAGE electrophoresis, including the following reagents: Cell lysis buffer: Add protease inhibitors (1:100), PMSF (1:100), EDTA (1:100), and phosphatase inhibitors (1:50) to Western and IP cell lysis buffers, mix well, and store at 4°C for later use. When lysing cells, add 100μL-150μL of cell lysis buffer to each well according to the cell density. BCA working solution: Mix BCAA solution and BCAB solution at a ratio of 50:1, store at room temperature for later use, and prepare and use immediately. When determining protein concentration by BCA method, add 200μL to each well. 5×SDS-PAGE electrophoresis fluid concentrate: Weigh 15.1gTris, 94gglycine, and 5gSDS, add appropriate amount of distilled water, and gently stir to dissolve while ultrasonicating. After the solid is dissolved and the solution is clarified, add distilled water to 1L, mix well, and store at room temperature. When using, add one 5×SDS-PAGE electrophoresis buffer concentrate to four parts of distilled water to dilute, mix well, and set aside. TBST rinse solution: add one 10×TBST rinse solution concentrate to nine parts of distilled water to dilute, mix well, and store at 4℃ for later use. Prepare and use immediately, and store for no more than one day. Transfer buffer: adjust the transfer buffer according to the molecular weight of the target protein. iNOS: add 10mL10× transfer buffer concentrate to 10mL methanol and 80mL water, mix well, and store at 4℃ for later use; other proteins: add 20mL methanol and 70mL water to 10mL10× transfer buffer concentrate, mix well, and store at 4℃ for later use. Blocking solution: The blocking solution contains 5% skim milk powder, mix well, store at 4℃ for later use, and prepare and use immediately. Add 20mL of blocking solution to each PVDF membrane. Primary antibody working solution: adjust the primary antibody working solution according to the type and concentration of the primary antibody. The primary antibody working solution contains 0.5% skim milk powder and the set concentration of the primary antibody, mix well, and store at 0℃ for later use. When the primary antibody is a rabbit polyclonal antibody, add an additional 0.01% sodium chloride. Add 10mL of primary antibody working solution to each PVDF membrane. Secondary antibody working solution: The secondary antibody working solution contains 0.5% skim milk powder and a set concentration of secondary antibody, mix well, and store at 0℃ for later use. Add 10mL of secondary antibody working solution to each PVDF membrane. ECL developer: Mix the developer and stabilizer in a 1:1 ratio, store at 4℃ away from light for later use. Add about 1.2mL of ECL developer to each PVDF membrane.

[0125] The preparation of SDS-PAGE gel includes: 1) Clean the glass plate, rinse with distilled water, and dry it for later use. Place the glass plate according to the mark, assemble it, and insert it into the gel holder. Add distilled water to check whether it leaks. If it does not leak, pour out the distilled water and absorb the excess water with filter paper. 2) The concentration of the separation gel is 8% and 12% respectively. According to the instructions of the SDS-PAGE denaturing acrylamide gel rapid preparation kit, add the specified volume of distilled water, 30% Acr-Bis (29:1), GelbufferA, 10% APS, and TEMED to make the separation gel, mix well, and carefully add an appropriate amount of separation gel liquid to the glass plate. Note that after adding APS and TEMED, add them as soon as possible after mixing well to avoid colloid solidification. 3) After adding an appropriate amount of separation gel, carefully and evenly add distilled water slowly and evenly on top of the separation gel to flatten the separation gel interface. Naturally solidify at room temperature for 20min-30min. 4) After the separation gel solidifies, carefully pour out the distilled water and absorb the excess water with filter paper. According to the instructions of the SDS-PAGE denaturing acrylamide gel rapid preparation kit, add the specified volume of distilled water, 30% Acr-Bis (29:1), GelbufferB, 10% APS, and TEMED to make concentrated gel, mix well, and carefully add an appropriate amount of concentrated gel liquid on top of the separation gel. Note that after adding APS and TEMED, add them as soon as possible after mixing well to avoid colloid solidification. 5) After adding an appropriate amount of concentrated gel, carefully insert the comb to avoid bubbles. Naturally solidify at room temperature for 20min-30min. 6) After the concentrated gel solidifies, put it in a plastic bag containing a small amount of electrophoresis solution and store it at 4℃.

[0126] Protein sample extraction and concentration determination include: grouping and processing cells, incubating for a predetermined time, aspirating the culture medium, adding 1 mL of PBS pre-cooled at 4°C to each well, shaking carefully for 1 min, then discarding the wash solution, and repeating the process three times to wash away the culture medium. Place the 6-well plate on ice, add 100 μL-150 μL of cell lysis solution pre-cooled at 4°C to each well, lyse for 30 min, and gently shake the 6-well plate from time to time. After the lysis is completed, use a pipette to transfer the cell fragments and lysis solution to a 1.5 mL centrifuge tube at the same time. Centrifuge the suspension at 4°C and 12000 rpm for 20 min, transfer the supernatant after centrifugation to a 0.2 mL sterile centrifuge tube, aliquot, and store at -80°C. Prepare the BCA working solution according to the instructions of the BCA protein concentration assay kit. Add 2 μL of sample solution to each well and 2 μL of cell lysis solution to the blank well. Add 18 μL of cell lysis solution to each well and mix well. Add 200 μL of BCA working solution to each well, mix carefully by pipetting, and place in a 37°C incubator for 30 minutes. After the reaction is completed, measure the absorbance at 562 nm. Calculate the sample protein concentration based on the bovine serum albumin standard curve.

[0127] Electrophoresis: After determining the protein concentration according to the instructions of the BCA kit, dilute and prepare the sample according to the total protein loading amount of 40μg per lane, add an appropriate amount of SDS-PAGE protein loading buffer to each sample and mix well. Preheat the metal bath to 95°C, heat the protein sample for denaturation for 5 minutes, and cool to room temperature. Select 8% or 12% SDS-PAGE gel for electrophoresis according to the molecular weight of the protein. Assemble the electrophoresis device, add the electrophoresis solution, and carefully pull out the comb in parallel. Spot the sample, adjust the voltage to 80V, wait for the sample to run to the junction of the concentrated gel and the separation gel, change the voltage to 120V, and stop the electrophoresis when the bromophenol blue front is 1-2cm away from the gel boundary.

[0128] Western Blot protein expression measurement includes: (1) Transfer, that is, take out the gel, wash it with distilled water, and first cut off the part of the front of the gel containing the bromophenol blue front. Then cut the gel area where the target protein is located according to the protein marker, rinse it with electrophoresis solution, put it into a plastic bag with a small amount of electrophoresis solution, and store it at 4°C for later use. Cut a PVDF membrane of the same size as the gel and a filter paper slightly smaller than the gel. Soak the PVDF membrane in methanol for 10 minutes in advance, and then soak it in transfer buffer together with the filter paper for more than 30 minutes. Make a "transfer sandwich" in the order of filter paper-gel-PVDF membrane-filter paper, put it into a semi-dry transfer instrument, and clamp it. The transfer time and voltage are adjusted according to the molecular weight of the target protein. Note that the membrane cannot be completely dry during the whole process. (2) After the transfer is completed, soak the PVDF membrane in TBST containing 5% skimmed milk powder, making sure that the membrane is immersed, and block it on a shaker at room temperature for 3 hours. (3) Primary antibody incubation Dilute the corresponding primary antibody with TBST containing 0.5% skim milk powder, immerse the PVDF membrane, and incubate at 4°C overnight. Make sure the membrane is immersed. The dilution ratio is adjusted according to the type of antibody. (4) Secondary antibody incubation After the primary antibody incubation is completed, rinse the PVDF membrane thoroughly with TBST washing solution for 6 times, 5 minutes each time. Dilute the HRP-labeled secondary antibody with TBST containing 0.5% skim milk powder at a dilution ratio of 1:2000, immerse the PVDF membrane, and incubate on a shaker at room temperature for 2 hours. (5) ECL development After the secondary antibody incubation is completed, rinse the PVDF membrane thoroughly with TBST washing solution for 6 times, 5 minutes each time. Mix the ECL developer and stabilizer in a 1:1 ratio and store at 4°C for later use. Add an appropriate amount of developer to each membrane, evenly cover the PVDF membrane, and incubate in the dark for 5 minutes. After incubation, turn on the chemiluminescence instrument and expose. The exposure time is adjusted according to the clarity of the bands.

[0129] Statistical processing The experiment was repeated three times, and the grayscale value of the bands was analyzed by ImageJ. SPSS software was used for significance analysis, and P < 0.05 indicated a significant difference.

[0130] It should be noted that the reagents, instruments, etc. used in the above experiments, as well as the experimental steps should not be regarded as limitations of this application.

[0131] like Fig.26 Shown are the effects of the components and component combinations on iNOS expression in mouse RAW264.7 cells. Fig. 27 The figure shows the effect of components and component combinations on the relative expression of iNOS in mouse RAW264.7 cells. It is understandable that NO is an important signaling molecule that can participate in physiological activities such as immunity and inflammation by transmitting cell signals. In macrophages, NO is mainly synthesized by inducible nitric oxide synthase (iNOS), so detecting the effect of drugs on iNOS protein expression can further explain the inhibitory effect of drugs on the release of NO from inflammatory cells. RAW264.7 cells expressed a large amount of iNOS protein after LPS stimulation, which was significantly different from the CK group (P < 0.01), which was consistent with the phenomenon of large-scale secretion of NO. Compared with the LPS group, protocatechuic acid methyl ester, isovitexin and component combinations all inhibited the expression of iNOS protein to varying degrees, with extremely significant differences (P < 0.01), which was consistent with the trend of NO secretion in the corresponding groups. The iNOS inhibition degree of isovitexin alone and the combined action group was analyzed and compared, and it was found that the combined combination could better inhibit iNOS secretion.

[0132] like Fig.28 Shown are the effects of the components and component combinations on the expression of MAPK pathway proteins in mouse RAW264.7 cells, such as Fig.29 The effects of the components and component combinations shown above on the relative expression of p-ERK / ERK in mouse RAW264.7 cells, such as Fig.30 The figure shows the effect of components and component combinations on the relative expression of p-JNK / JNK in mouse RAW264.7 cells. It is understandable that MAPK is mainly composed of subfamilies such as ERK, JNK, and p38. The activation of the MAPK pathway will further amplify the inflammatory signal. Inhibiting the expression of MAPK pathway proteins or the phosphorylation of related proteins has a positive significance for inhibiting inflammation. Analysis and comparison of the phosphorylation levels of proteins showed that the LPS group showed a high phosphorylation level, which was significantly different from the CK group (P < 0.01); both components and component combinations can inhibit the phosphorylation level of MAPK channel proteins, and the difference is statistically significant (P < 0.05). The results show that the combination of isovitexin + protocatechuic acid methyl ester can better inhibit the protein phosphorylation levels of ERK and JNK compared with the drug alone, P < 0.01.

[0133] like Fig.31 Shown are the effects of the components and component combinations on the expression of p-p65 / p65 in mouse RAW264.7 cells, e.g. Fig.32The effect of components and component combinations on the relative expression of p65 / p-p65 in mouse RAW264.7 cells is shown. It is understandable that the NF-κB pathway is an important link in the inflammatory pathway, which can be characterized by detecting the expression of p-65 protein or the relative expression of phosphorylated p65 protein and p65 protein. Therefore, the effects of components and component combinations on the phosphorylation of p65 protein were detected. It can be seen that the phosphorylation level of the LPS group was significantly different from that of the CK group, P < 0.01. Single components and combined components have different degrees of inhibitory effects on the phosphorylation level of p65 protein, and the inhibitory effect of the MY combination on the phosphorylation level of p65 protein is synergistic compared with the single component.

[0134] like Fig.33 Shown are the effects of the components and component combinations on LPS-induced Nrf2 protein expression in RAW264.7 cells. Fig.34 The figure shows the effect of components and component combinations on the relative expression of Nrf2 in mouse RAW264.7 cells. It is understandable that the relative expression of Nrf2 protein in the LPS group was higher than that in the CK group, indicating that the inflammatory state of the cells promoted the expression of Nrf2 protein. The protocatechuic acid methyl ester group and the component combination group can promote the expression of Nrf2 protein to varying degrees (P < 0.05). Compared with isovitexin, the component combination group synergistically increased the expression of Nrf2 protein; however, the promoting effect of the component combination group relative to the protocatechuic acid methyl ester group was not obvious, proving that the combination of components better inhibited inflammation, so that the ability of protocatechuic acid methyl ester to promote the expression of Nrf2 protein was not fully reflected. In summary, the combination of components can assist the anti-inflammatory effect through the oxidative stress pathway.

[0135] It is important to understand that inhibiting the activation of inflammatory pathways can cut off the cascade reaction of inflammatory-related signaling pathways, stop the transmission of inflammatory signals, and have a positive impact on the control of inflammation. Studying whether a drug has the effect of inhibiting the secretion of NO in an inflammatory model can preliminarily screen out drugs with anti-inflammatory activity. Studying the regulatory effect of drugs on the secretion of inflammatory factors in an inflammatory model can preliminarily explain the anti-inflammatory mechanism of drugs. In-depth research on the inhibitory and regulatory effects of drugs on inflammatory signaling pathways is of great significance for the comprehensive exploration of drug anti-inflammatory effects and drug design. The connection between oxidative stress and inflammation has led researchers to try to explore the anti-inflammatory mechanism of traditional Chinese medicine from an anti-oxidative approach. Nrf2 is an important transcription factor associated with anti-oxidative stress. When cellular oxidation is out of balance, Nrf2 protein dissociates from Keap1 and enters the nucleus to bind to ARE, thereby activating downstream genes related to oxidation balance, regulating the level of cellular oxidative stress, and activating the Nrf2-Keap1 pathway is beneficial to combating inflammatory responses. The above systematic experiments have proved that protocatechuic acid methyl ester and MY combination have the effect of promoting the expression of Nrf2 protein. The addition of protocatechuic acid methyl ester plays an anti-inflammatory role by promoting the expression of Nrf2 protein and inhibiting the activation of inflammatory pathways MAPK and NF-κB pathways.

[0136] In the description of the present application, it should be noted that the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the number or order of the indicated technical features. In the description of the present application, the meaning of "multiple" is two or more, unless otherwise clearly and specifically defined. For ordinary technicians in this field, the specific meanings of the above terms in the present application can be understood according to the specific circumstances. In the absence of conflict, the features in the implementation methods and steps can complement, replace or combine with each other.

[0137] The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. Use of a composition of isovitexin and methyl protocatechuate in the preparation of an anti-inflammatory drug, characterized in that: The concentration of isovitexin in the drug is 5 μmol·L -1 , the concentration of protocatechuic acid methyl ester was 250 μmol·L -1 .

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