Synergistic anti-inflammatory effect of methyl protocatechuate and isorhamnetin from Tetrastigma hemsleyanum Diels et Gilg and application of their composition in preparing anti-inflammatory drugs

Abstract

This application takes the extract of Tetrastigma hemsleyanum Diels et Gilg as the research object, especially the research on methyl protocatechuate and isorhamnetin, to find a component combination with synergistic anti-inflammatory effects. By designing experiments, it is verified that isorhamnetin and methyl protocatechuate have synergistic anti-inflammatory effects, and the mechanism 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 p65, and promoting the expression of antioxidant-related Nrf2 protein. Experiments verify that isorhamnetin and methyl protocatechuate have synergistic anti-inflammatory effects, and their effects occur in the cell nucleus. It is found that methyl protocatechuate in the concentration range of 5 μmol·L⁻¹ - 500 μmol·L⁻¹ has anti-inflammatory activity. Among them, the CI value of 5 μmol·L⁻¹ isorhamnetin + 250 μmol·L⁻¹ methyl protocatechuate is 0.15415, and the synergistic effect is excellent.

Description

Technical Field

[0001] The present invention relates to Tetrastigma hemsleyanum extract, and particularly to the anti-inflammatory activity of the methyl protocatechuate component of Tetrastigma hemsleyanum extract and its application in the preparation of anti-inflammatory drugs, specifically its application of anti-inflammatory activity and its synergistic anti-inflammatory application with rhamnetin. Background Art

[0002] Tetrastigma hemsleyanum is the tuberous root of Tetrastigma hemsleyanum Diels et. Gilg of Vitaceae, also known as Jinxiandiaohulu and Shefuzi. It is mainly produced in Zhejiang, Jiangxi, Hunan and Fujian. It is a precious understory medicinal material. Its whole herb can be used as medicine, and the underground tuberous root has the best medicinal effect. Tetrastigma hemsleyanum is recorded in historical books and is mostly used for high fever and convulsions. The efficacy and treatment of Tetrastigma hemsleyanum were first recorded in "Illustrated Classics of Plants" in the Qing Dynasty, which states that "it is used to treat high fever in children, relieve abdominal pain, and take the juice for oral administration"; "Chinese Medicine Sea" records: "…… clearing heat and relieving convulsions, promoting blood circulation and removing stasis. It is mainly used for sore throat, snake and insect bites, high fever and convulsions in children, infantile convulsions……"; "Chinese Herbal Medicine" records: "Mainly used for high fever and convulsions; pneumonia; hepatitis; nephritis……"; "Compendium of Chinese Herbal Medicines" records: "Clearing heat and detoxifying. It is used to treat high fever and convulsions in children, bronchitis, pneumonia, pharyngolaryngitis, hepatitis and viral meningitis……". According to "Essentials of Chinese Ethnomedicine", Tetrastigma hemsleyanum is used by multiple ethnic groups and has a medicinal history among ethnic groups such as the Lahu, She, Achang, Tujia, and Yao. Tetrastigma hemsleyanum is more commonly used in folk anti-inflammatory and antipyretic clinical applications. "Zhejiang Provincial Traditional Chinese Medicine Processing Specification" (2015 edition) records: It is mild in nature, slightly bitter in taste, clearing heat and detoxifying, reducing swelling and relieving pain, resolving phlegm and dissipating nodules. It is used for high fever and convulsions in children, whooping cough, sores and phlegm nodules, and snake bites. The chemical constituents of Tetrastigma hemsleyanum are diverse, containing rich flavonoids and phenolic acid components, as well as polysaccharides, terpenoids, etc.

[0003] Studies have found that Tetrastigma hemsleyanum extract has good anti-inflammatory, analgesic and antipyretic effects. Tetrastigma hemsleyanum extract can inhibit the inflammatory exudation of capillaries in the abdominal cavity of mice, inhibit the auricle swelling of mice caused by xylene and the paw swelling of rats; reduce the number of writhing times of mice caused by acetic acid, increase the pain threshold of mice by the hot plate method, and reduce the body temperature of the fever model of rats caused by dry yeast and 2,4-dinitrophenol; Tetrastigma hemsleyanum has an inhibitory effect on different tumor cell lines such as gastric cancer, liver cancer and lung cancer. As a phenolic acid substance isolated from Tetrastigma hemsleyanum, the anti-inflammatory activity and anti-inflammatory application of methyl protocatechuate, especially its application in the preparation of anti-inflammatory drugs, still need to be studied. Summary of the Invention

[0004] The object of the present invention is to provide the anti-inflammatory activity of the extract from Tetrastigma hemsleyanum and its application in the preparation of anti-inflammatory drugs. In particular, the combined use of methyl protocatechuate and isorhamnetin in Tetrastigma hemsleyanum has a synergistic anti-inflammatory effect and can reduce the drug toxicity, which has a positive significance for the development of new natural anti-inflammatory drugs. To achieve the above object of the invention, the solution of the present application is realized in the following manner.

[0005] Optionally, the present application provides the application of the extract from Tetrastigma hemsleyanum in the preparation of anti-inflammatory drugs.

[0006] Optionally, the anti-inflammatory activity acts on the cell nucleus.

[0007] Optionally, the anti-inflammatory active ingredients of the extract from Tetrastigma hemsleyanum include flavonoids and phenolic acids.

[0008] Optionally, the phenolic acid active ingredient with anti-inflammatory activity in the extract from Tetrastigma hemsleyanum includes the methyl protocatechuate component, and the flavonoid active ingredient with anti-inflammatory activity in the extract from Tetrastigma hemsleyanum includes the isorhamnetin component.

[0009] Optionally, the application concentration of the methyl protocatechuate component is 5 μmol·L -1 ~500 μmol·L -1 .

[0010] Optionally, the application concentration of the isorhamnetin component is 5 μmol·L -1 ~25 μmol·L -1 .

[0011] Optionally, the combined use of methyl protocatechuate and isorhamnetin in the anti-inflammatory active ingredients of the extract from Tetrastigma hemsleyanum has a synergistic inhibitory effect on anti-inflammatory.

[0012] Optionally, the concentration range in which the combined use of methyl protocatechuate and isorhamnetin in the anti-inflammatory active ingredients of the extract from Tetrastigma hemsleyanum has a synergistic inhibitory effect on anti-inflammatory is 5 μmol·L -1 ~500 μmol·L -1 methyl protocatechuate + 5 μmol·L -1 isorhamnetin, or 5 μmol·L -1 ~250 μmol·L -1 methyl protocatechuate + 5 μmol·L -1 ~25 μmol·L -1 of the isorhamnetin component, and its CI value is less than 1.

[0013] Optionally, the active concentration at which methyl protocatechuate and isorhamnetin have a synergistic effect is 250 μmol·L -1 methyl protocatechuate + 5 μmol·L -1For isorhamnetin, its CI value is 0.15415.

[0014] The beneficial effects of the present invention are as follows: This application takes the extract of Tetrastigma hemsleyanum as the research object, studies the anti-inflammatory activities and action mechanisms of flavonoids and phenolic acids, especially the combination of methyl protocatechuate and isorhamnetin. Through experimental design, its action mechanism is verified, and a component combination with synergistic anti-inflammatory effects is searched for, providing guidance for the development of new natural drugs with anti-inflammatory effects. Brief Description of the Drawings

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

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

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

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

[0019] Figure 5 It is a comparison chart of the effects of different concentrations of methyl protocatechuate on cell activity.

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

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

[0022] Figure 8 It is a comparison chart of the effects of the combination of different drug combinations and LPS on the activity of RAW264.7 cells.

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

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

[0025] Figure 11 It is a Venn diagram of the target genes of the SY combination and the target genes of the disease.

[0026] Figure 12 It is a schematic diagram of the GO enrichment analysis of the target genes of the SY combination.

[0027] Figure 13It is the analysis diagram of KEGG annotation results of SY combination target genes.

[0028] Figure 14 It is the schematic diagram of SY combination component - target - pathway network.

[0029] Figure 15 It is the schematic diagram of SY combination target gene protein - protein interaction network.

[0030] Figure 16 It is the schematic diagram of the visualization analysis result of the PPI network of SY combination.

[0031] Figure 17 It is the schematic diagram of the docking mode of isorhamnetin and 1NSI protein.

[0032] Figure 18 It is the schematic diagram of the visualization analysis of the binding cavity of isorhamnetin and 1NSI protein.

[0033] Figure 19 It is the schematic diagram of the docking mode of methyl protocatechuate and 1NSI protein.

[0034] Figure 20 It is the schematic diagram of the visualization analysis of the binding cavity of methyl protocatechuate and 1NSI protein.

[0035] Figure 21 It is the schematic diagram of isorhamnetin binding to amino acid residues GLU - 377 and TRP - 372 by hydrogen bonds.

[0036] Figure 22 It is the schematic diagram of methyl protocatechuate forming two hydrogen bonds with amino acid residue ARG - 381 for connection.

[0037] Figure 23 It is the comparison diagram of the inhibitory effects of different components and component combinations on the TNF - α factor level.

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

[0039] Figure 25 It is the comparison diagram of the effects of different concentrations of drugs and drug combinations on the IL - 10 factor level.

[0040] Figure 26 It is the comparison diagram of the effects of components and component combinations on the iNOS expression in mouse RAW264.7 cells.

[0041] Figure 27 It is the comparison diagram of the effects of components and component combinations on the relative expression level of iNOS in mouse RAW264.7 cells.

[0042] Figure 28It 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] Figure 29 It is a comparison chart of the effects of components and component combinations on the relative expression levels of p-ERK / ERK in mouse RAW264.7 cells.

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

[0045] Figure 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] Figure 32 It 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] Figure 33 It is a comparison chart of the effects of components and component combinations on the expression of Nrf2 protein in LPS-induced RAW264.7 cells.

[0048] Figure 34 It is a comparison chart of the effects of components and component combinations on the relative expression levels of Nrf2 in mouse RAW264.7 cells. Detailed implementation manners

[0049] To make the objectives, technical solutions and advantages of this application clearer, the technical solutions of the present invention will be 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 components in Tetrastigma hemsleyanum are flavonoids and phenolic acids. Methyl protocatechuate is a phenolic acid compound newly isolated from Tetrastigma hemsleyanum by the applicant's laboratory in the early stage. Methyl protocatechuate, namely methyl 3,4-dihydroxybenzoate, has strong antioxidant activity and is distributed in plants such as Coptis chinensis, Fagopyrum dibotrys, Dianthus superbus, and Hedyotis diffusa. The applicant first isolated this compound from Tetrastigma hemsleyanum in the early stage, and this isolation is the first in the industry. Isorhamnetin is a chemical component with a relatively high content among the flavonoid components of Tetrastigma hemsleyanum and also has certain anti-inflammatory activity. Isorhamnetin, namely 3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)benzopyran-4-one, has the chemical formula C 16 H 12 O 7, with the English name Isorhamnetin, is a plant flavonoid. Isorhamnetin can have a synergistic effect when used in combination with other components, and is used to reduce drug toxicity and enhance the anti-tumor effect. Further research is still needed on the single component and its combined use for synergistic anti-inflammatory research and application.

[0051] The inventors believe that, based on the characteristics of traditional Chinese medicine with multiple components and multiple targets, the current research on the anti-inflammatory effect of Tetrastigma hemsleyanum is still insufficient. Especially for the methyl protocatechuate component newly isolated from Tetrastigma hemsleyanum, the research mainly focuses on content determination and relatively limited pharmacological activity research. Methyl protocatechuate has significant antioxidant stress capacity. When it is used as an anti-inflammatory component, its anti-inflammatory effect requires further research. In view of this, the applicant designed an experimental plan to study the anti-inflammatory application of the methyl protocatechuate component newly isolated from Tetrastigma hemsleyanum, and verified the anti-inflammatory activity of methyl protocatechuate. At the same time, the applicant found through experiments the optimal dosage of the methyl protocatechuate component and isorhamnetin as anti-inflammatory components and their dosage for synergistic anti-inflammatory effect.

[0052] The inventors found that the anti-inflammatory effect of Tetrastigma hemsleyanum and its good curative effect on viral infection and inflammatory diseases may be related to the ability of its extract to regulate antioxidant-related enzymes and the immune system against liver injury and immune imbalance. Through research, the inventors found that the flavonoids in the extract of Tetrastigma hemsleyanum have obvious anti-inflammatory and analgesic effects in vivo, can relieve low-level inflammation of vascular endothelial cells, significantly up-regulate the expression of Nrf2 gene and protein in cells, down-regulate the expression of Keap1 protein, activate Nrf2, and play a strong anti-inflammatory effect by binding to Keap1 protein to activate the NF-κB pathway activity.

[0053] It can be understood that the anti-inflammatory mechanisms of common traditional Chinese medicines include inhibiting the secretion of inflammatory mediators, inhibiting the expression of inflammatory signaling pathways, regulating oxidative stress injury, and regulating the function of the hypothalamic-pituitary-adrenal cortex axis. Inflammatory signals will activate the secretion of inflammatory mediators by macrophages, and the inflammatory mediators will then activate the inflammatory pathway and trigger the inflammatory response. Inhibiting the secretion of inflammatory mediators can block the process of inflammation to a certain extent and inhibit the generation of inflammation. To study the changes in the body during the inflammatory process, researchers have established a variety of inflammatory models, and common inducers include IFN-γ, TNF-α, HSP60 / 70, etc. Among them, LPS is a highly efficient, safe, and most widely used inducer. The RAW264.7 inflammatory cells induced by LPS are the most commonly used inflammatory model. LPS (lipopolysaccharide, with the English name Lipopolysaccharide, abbreviated as LPS, is a component of the outer wall of the cell wall of Gram-negative bacteria, a substance composed of lipids and polysaccharides, that is, a glycolipid substance) binds to the TLR receptor on the cell membrane, activates macrophages, induces the cells to secrete a large amount of NO and inflammatory factors and activates the inflammatory pathway, resulting in macrophages being in an inflammatory state.

[0054] Based on this, this application uses the RAW264.7 cell inflammation model as the object to study the anti-inflammatory activity and mechanism of drug components. At present, the mechanism of the LPS-induced inflammation model is mainly as follows: LPS recognizes and binds to TLR4, transmits signals into the cell, activates the cascade reactions of signal pathways such as MAPK and NF-κB, thereby generating a large amount of cytokines such as IL-1β, IL-6, TNF-α and inflammatory mediators such as NO. Thus, people can evaluate the anti-inflammatory effect of drugs by detecting the contents of cytokines and inflammatory mediators, the expression levels of channel proteins, etc.

[0055] It can be understood that the binding of LPS to the TLR receptor can activate macrophages and release 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 stage of inflammation, which can indicate the occurrence and progress of inflammation and has a positive guiding significance for inflammation judgment. Therefore, by measuring the effect of drugs on the NO concentration in the inflammation model, it can be preliminarily judged whether the drugs have anti-inflammatory activity.

[0056] To illustrate this application, the specific experiments, characterizations and analyses are as follows.

[0057] Reagents used in the experiment: high-glucose DMEM culture medium, MTT, fetal bovine serum, penicillin and streptomycin, DMSO, LPS, PBS buffer, nitric oxide detection kit.

[0058] The cells used in the experiment are mouse mononuclear macrophage RAW264.7 cells. The methods of cell culture, passage, cryopreservation and resuscitation are as follows. In this application, unless otherwise clearly specified and limited, the cells used in the experiment are all mouse mononuclear macrophage RAW264.7 cells, and their methods of cell culture, passage, cryopreservation and resuscitation are the same as the following methods.

[0059] Specifically, cell culture includes: injecting 5 mL of cell suspension with a certain cell density into a T25 cell culture flask. The culture medium consists of DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody (composed of 100 U·mL-1 penicillin and 0.1 mg·mL-1 streptomycin). It is placed in an incubator for culture under the conditions of 37 °C and 5% CO2. The whole process is carried out under sterile conditions, and the cell status and density are observed daily. Cell passage includes: preheating PBS and the culture medium at 37 °C, aspirating the old culture medium, and carefully adding PBS to wash three times. Aspirate the PBS, add 5 mL of the culture medium, scrape the cells with a cell scraper, then add fresh culture medium and blow evenly, passage the cells, shake well crosswise, and place them in the incubator for culture. The cycle is about one to two days. Cell cryopreservation includes: when RAW 264.7 cells grow to a high density, select cells in good condition for cryopreservation. Preheat PBS and the culture medium at 37 °C, aspirate the old culture medium, and carefully add PBS to wash three times. Aspirate the PBS, add 5 mL of fresh culture medium, carefully scrape the bottom of the cell flask with a cell scraper, and after scraping off most of the cells, blow evenly the cell suspension. Collect every five bottles into a 50 mL centrifuge tube. Centrifuge at low speed to collect the cells. Carefully pour off the supernatant and add cryopreservation solution. Among them, the cryopreservation solution contains 10% dimethyl sulfoxide to protect the cells. Aliquot an appropriate amount of the cryopreservation solution containing cells into cryopreservation tubes, and store them in liquid nitrogen after cooling. Cell recovery includes: melting the solidified cell suspension at 37 °C, mixing it with fresh culture medium. Centrifuge at low speed to collect the cells. Discard the supernatant, mix with the culture medium, and culture in a culture flask and an incubator.

[0060] Among them, cell viability is measured by the MTT method, including the steps: (1) Add the set culture medium for each group, mix and culture with cells for 24 h, and record the cell status of each group. (2) Keep in the dark, add 20 μL of MTT to each well, and continue to incubate for 4 h. (3) After 4 h, terminate the incubation, carefully aspirate the culture medium completely with a pipette gun, avoiding touching the bottom of the well and sucking away the purple crystals. Add 100 μL of DMSO to each well and incubate at room temperature for 30 min. (4) Measure the OD490 nm value of each well. (5) Result processing: Use the group containing only the culture medium as the blank group, and the group containing only the culture medium and cells as the control group.

[0061]

[0062] Experiment 1: Establish a RAW264.7 cell inflammation model

[0063] Among them, the grouping and treatment methods of the cells are as follows: (1) Collect the cells, count them, adjust the cell density, and inoculate them into a 96-well culture plate, 100 μL per well, and 1×104 cells per well. Culture overnight in an incubator. (2) The experiment is divided into a blank group, a control group, different concentration LPS groups, and different LPS induction time groups. Among them, in the blank group, only the culture medium is added; in the control group, no treatment is performed; in the LPS group, LPS solutions with concentration gradients are added to the cell culture medium, and the LPS concentration gradients are set to 10 ng·mL -1 , 100 ng·mL -1 , 500 ng·mL -1 , 1 μg·mL -1 , 10 μg·mL -1 for five final concentrations; in the different LPS induction time groups, with the optimal induced LPS concentration as the condition, the LPS induction time gradient is set to 6, 12, and 24 h. Each group treatment is repeated 5 wells. (3) After the culture is completed, aspirate the old culture medium in each well, add 100 μL of the culture medium set according to the experimental conditions to each well, and continue to incubate in the incubator for 24 h for subsequent experiments. In this application, unless otherwise clearly specified and limited, for the RAW264.7 cell inflammation model established in this experiment, the grouping and treatment methods of the cells are the same as above.

[0064] In addition, the detection of the expression of the inflammatory factor NO is as above, and the grouping and treatment operations are the same as above. After incubation for 24 h, centrifuge the supernatant at 12000 rpm for 10 min, immediately detect it or store it short-term at 4 °C, and measure the NO concentration in the supernatant according to the instructions of the nitric oxide detection kit.

[0065] The analysis of the synergistic anti-inflammatory effect with the NO inhibition rate as the index calculates the NO inhibition rate of different concentration drugs according to the NO concentration of each group. Among them:

[0066]

[0067] Specifically, the Compusyn software used in this application is a software that calculates the combination index (CI) using the Chou-Talalay formula and is used to evaluate the interaction between two drugs. Optionally, import the NO inhibition rates corresponding to the single application and combined application of the 3 components into the Compusyn software, and select the non-continuous combination analysis to obtain the CI value. Select the combination with a CI value < 1 as the combination with a synergistic effect. For the results and analysis in this application, unless otherwise stated, the combination index is calculated by the Compusyn software. Optionally, each group of experiments is repeated 5 times and expressed as Mean±SD. All results are processed by SPSS 20.0 software, and P < 0.05 indicates a significant difference. The pictures are drawn by Graphpad Prism8.

[0068] Specifically, as Figure 1 shown in the figure is the characterization of the effects of different concentrations of LPS on the viability of RAW264.7 cells, where cell viability was detected using the MTT method. The experiment was divided into a blank group and different concentrations of LPS groups. Among them, in the blank group, only the culture medium was added; in the LPS groups, LPS solutions with concentration gradients were added to the cell culture medium, and the LPS concentration gradients were set to 10 ng·mL -1 , 100 ng·mL -1 , 500 ng·mL -1 , 1 μg·mL -1 , 10 μg·mL -1 at five final concentrations. Each treatment was repeated in 5 wells. The experimental results showed that when the LPS concentration ≥ 10 μg·mL -1 , the viability of RAW264.7 cells was less than 90%, and it can be considered that LPS at concentrations higher than this had certain toxic side effects on the cells; LPS at a concentration of 100 ng·mL -1 had the ability to promote cell proliferation. Therefore, when using the LPS-induced inflammation model, the LPS concentration used in the experiment was not higher than 10 μg·mL -1 .

[0069] As Figure 2 , 3 shown is the characterization and analysis of the experimental results of screening the LPS modeling stimulation conditions. Specifically, as Figure 2 shown is the effect of different concentrations of LPS on NO secretion. The experimental results showed that adding LPS could stimulate RAW264.7 cells to secrete the inflammatory mediator NO, and there was a significant difference compared with the CK group (i.e., control check, representing the blank control group) (P < 0.05), and this effect was dose-dependent in the range of 10 ng·mL -1 to 1 μg·mL -1 . As the LPS concentration increased, the secretion of the inflammatory mediator NO continuously increased and reached the peak at 1 μg·mL -1 . When LPS reached the toxic concentration of 10 μg·mL -1 , the NO concentration decreased, and it can be considered that the optimal LPS concentration for use was 1 μg·mL -1 .

[0070] As Figure 3 shown, under the condition of the optimal LPS induction concentration, that is, when the LPS concentration for use was 1 μg·mL -1 , the experimental results of exploring the optimal induction time of LPS were analyzed. It can be found from the figure that when the LPS induction time was 24 h, the NO secretion reached the peak, and there was a highly significant difference (P < 0.01). Therefore, it can be considered that the optimal induction condition of LPS was a concentration of 1 μg·mL-1 , act for 24 h.

[0071] Experiment 2. Determination of the concentrations of methyl protocatechuate and isorhamnetin

[0072] The experimental treatment methods were as follows: (1) Grouping and treatment of cells: Collect cells, count them, adjust the cell density, inoculate them in a 96-well culture plate, 100 μL per well, 1×104 cells per well, and culture overnight in an incubator; (2) The experiment was divided into a blank group, a control group, and a treatment group. Among them, the blank group only contained the culture medium; the control group was not treated; the treatment group was set with gradient concentrations of isorhamnetin and methyl protocatechuate, with the final concentrations being 5, 25, 50, 100, 250, and 500 μmol·L -1 , and each treatment was repeated in 5 wells; (3) After overnight culture until the cells adhered, aspirate the old culture medium in each well, add 100 μL of the culture medium set under the experimental conditions to each well, incubate in an incubator for 24 h, and conduct subsequent experiments; (4) The cell viability of each group was measured by the MTT method.

[0073] As Figure 4 , Figure 5 shown is the analysis of the effects of different concentrations of isorhamnetin and methyl protocatechuate on cell viability. As Figure 4 shown is the result analysis of the effects of different concentrations of isorhamnetin on cell viability. Specifically, after grouping and treating the cells, they were divided into a blank group and a treatment group. The blank group was not treated, and the treatment group was treated with 5 μmol·L -1 , 25 μmol·L -1 , 50 μmol·L -1 , 100 μmol·L -1 , 250 μmol·L -1 , and 500 μmol·L -1 of isorhamnetin respectively. It can be seen from the figure that when RAW264.7 cells were treated with 5 μmol·L -1 and 25 μmol·L -1 concentrations of isorhamnetin, the cell viability > 90%. As Figure 5 shown is the result analysis of the effects of different concentrations of methyl protocatechuate on cell viability. Specifically, after grouping and treating the cells, they were divided into a blank group and a treatment group. The blank group was not treated, and the treatment group was treated with 5 μmol·L -1 , 25 μmol·L -1 , 50 μmol·L -1 , 100 μmol·L -1 , 250 μmol·L -1 , and 500 μmol·L -1 of methyl protocatechuate respectively. It can be seen from the figure that from 5 μmol·L -1 to 250 μmol·L-1 When RAW264.7 cells were treated with methyl protocatechuate at a concentration, the cell viability was > 90%.

[0074] In summary, regarding the effects of methyl protocatechuate and isorhamnetin at different concentrations on the viability of RAW264.7 cells, it was found that when -1 ~250 μmol·L -1 methyl protocatechuate within the concentration range and 5 μmol·L -1 , 25 μmol·L -1 isorhamnetin were used to treat RAW264.7 cells, the cell viability was > 90%. Thus, the screening concentrations for verifying the anti-inflammatory activity of methyl protocatechuate and its synergistic anti-inflammatory combination with isorhamnetin were determined.

[0075] Experiment 3: Test the effects of the combination of methyl protocatechuate and isorhamnetin components with LPS on cell viability, and select safe concentrations of methyl protocatechuate and isorhamnetin for combination.

[0076] The experimental treatment methods were as follows: (1) Grouping and treatment of cells: Collect cells, count them, adjust the cell density, inoculate them into 96-well culture plates, 100 μL per well, 1 × 104 cells per well, and culture overnight in an incubator; (2) The experiment was divided into a blank group, a control group, and a treatment group. Among them, the blank group only contained the culture medium; the control group was not treated; the treatment group was set with different drug combinations as shown in Table 1 below, and each group treatment was repeated 5 wells; (3) After overnight culture until the cells adhered, aspirate the old culture medium in each well, add 100 μL of the culture medium set under the experimental conditions to each well, incubate in an incubator for 24 h, and conduct subsequent experiments; (4) Use the MTT method to measure the cell viability of each group above.

[0077]

Table 1

[0078]

[0079] As Figure 6 The results showed that the combination of 25 μmol·L -1 isorhamnetin with 100 μmol·L -1 and 250 μmol·L -1 methyl protocatechuate had a negative impact on the viability of RAW264.7 cells, and the other combinations had no effect on the viability of RAW264.7 cells.

[0080] Experiment 4: Effects of single components or component combinations combined with LPS on cell viability

[0081] Specifically, the experimental treatment methods were the same as those in Experiment 3, the difference being that 1 μmol·L -1Under the condition of LPS, isorhamnetin, methyl protocatechuate or different component combinations (the combination method is the same as in Table 1) were added respectively and incubated together for 24 h to investigate the effect of co-incubation of LPS and components on cell viability. As Figure 7 , Figure 8 For the analysis of experimental results, the results showed that the single component or component combination under the experimental conditions had no effect on the viability of RAW264.7 cells.

[0082] Experiment 5: Anti-inflammatory activity of methyl protocatechuate and inhibitory effect of component combination on NO release

[0083] Specifically, the experimental treatment method and grouping method were the same as in Experiment 3, and the inhibitory effect of a single drug on the release of NO from LPS-induced RAW264.7 cells was measured. As Figure 9 Shown in the analysis of 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 shows concentration dependence. The results showed that methyl protocatechuate has the ability to inhibit NO release to varying degrees.

[0084] After clarifying the anti-inflammatory activity of methyl protocatechuate in this way, methyl protocatechuate and isorhamnetin were used in combination. The experimental treatment method and grouping method were the same as in Experiment 3, and the experimental results were as Figure 10 Shown in the inhibitory effect of the drug combination on the release of NO from LPS-induced RAW264.7 cells. As shown in the figure, as the concentration of methyl protocatechuate in the combination increased, the NO inhibition rate also increased and finally tended to saturate. Comparing Figure 9 , Figure 10 , it was found that the addition of methyl protocatechuate had the effect of enhancing NO inhibition by comparing the NO inhibition rates before and after the combination.

[0085] After calculating the NO inhibition rates of the single component and component combination, the concentration of the single component, the corresponding concentration of the component combination and the corresponding NO inhibition rate were imported into Compusyn software to calculate the combination index CI value of the component combination. 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 in each group, 5 groups of drug combinations had CI < 1, which could be regarded as having a synergistic effect. It can be seen that the addition of methyl protocatechuate has positive significance. Among them, the CI value of the B5 combination was the lowest, that is, the combination of 5 μmol·L -1 isorhamnetin + 250 μmol·L -1 methyl protocatechuate had the lowest CI value. Table 2 is the drug combination with the synergistic inhibitory effect on the release of NO from RAW264.7 cells.

[0086]

Table 2

[0087]

[0088] It is understandable that MTT can distinguish the survival status of cells. The generated crystals can be dissolved with DMSO, and the absorption wavelength at 490 nm can be measured. By comparing the obtained results with those of the control group, the cell viability of the drug at this concentration can be obtained to judge the toxicity of the drug. In the efficacy study of drugs, it is first necessary to ensure that the drug is at a safe concentration and has no effect on cell viability before further exploring the efficacy and mechanism of action of the drug.

[0089] In the cytotoxicity experiment, it can be found that isorhamnetin exhibits certain cytotoxicity at low concentrations; while methyl protocatechuate shows cytotoxicity only at a concentration of 500 μmol·L -1 and has a high cell tolerance concentration. This phenomenon is related to the small molecular weight and low toxicity of methyl protocatechuate. Based on the NO inhibition rate results of the combined treatment group, it is speculated that the addition of methyl protocatechuate can achieve the same anti-inflammatory effect with a reduced dosage. Compared with the single application of the three components, the NO inhibition rates after combined treatment all have a synergistic effect and can better reduce the NO content. After adding methyl protocatechuate, the proportion of drug combinations with synergistic effects among all combinations reaches 66.7%. It can be seen that the combination with methyl protocatechuate has a positive significance for inhibiting NO secretion, and thus it is determined that methyl protocatechuate has a positive effect on the synergistic anti-inflammatory effect.

[0090] Experiment 6: Analysis of its synergistic anti-inflammatory mechanism based on network pharmacology and molecular docking technology

[0091] It is understandable that the network pharmacology method uses various means such as network databases, high-throughput omics data analysis, and computer simulation to construct a "compound-target-pathway" network for research, which is in line with the holistic concept of traditional Chinese medicine. It can analyze the connections between different components of traditional Chinese medicine and make the research more efficient and reasonable. Molecular docking is a technology that virtually binds proteins and small molecule ligands, and the magnitude 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 isorhamnetin and methyl protocatechuate (SY for short) screened in the previous stage to preliminarily analyze the combined anti-inflammatory mechanism.

[0092] Specific method: (1) Obtaining the structural files of methyl protocatechuate and isorhamnetin. Search for methyl protocatechuate and isorhamnetin using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Download the.mol2 format corresponding to the compound, import it into Pymol software to adjust the compound conditions, and output it as a.pdbqt file. (2) Obtaining the target proteins and target genes of methyl protocatechuate and isorhamnetin. Obtain the target genes of methyl protocatechuate and isorhamnetin from the TCMSP database, Pubchem database, and Uniprot database, with the species being "HOMO sapiens". (3) Obtaining the potential anti-inflammatory target genes of the component combination. Search the Genecards database (https: / / www.genecards.org / ) using "inflammation" as the keyword, download the.tsv format file, and screen the genes in the table with the relevant score > 5.0 and P < 0.01 as the conditions. Import the screened disease genes and the target genes of the component combination into the Venny 2.1 platform, and consider the common genes of the two as the potential anti-inflammatory target genes of the component combination. (4) Annotation of the potential anti-inflammatory target genes of the component combination. Upload the obtained potential anti-inflammatory target genes of the component combination to the DAVID platform for analysis, with the species being "HOMO sapiens". Download the tables of GO enrichment analysis and KEGG annotation results, and sort the tables based on Count. (5) Construction of the ingredient-target gene-pathway network diagram. Classify and organize the component combination ingredients, potential anti-inflammatory target genes, and KEGG pathway annotation results, and import them into Cytoscape software in sequence. Select the Network analyze function to analyze the network and arrange it according to the degree value. Use the Merge function to merge, and finally obtain the component combination compound-target-pathway diagram. (6) Construction of the PPI network. Correct the target proteins corresponding to the potential anti-inflammatory target genes, construct a protein-protein interaction network, and arrange and plot it according to the degree value. (7) Molecular docking. Download the protein structure from the PDB website, determine the binding cavity according to the bound ligand; import the.pdb file into 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 Autodock 4.2.6 software for operations such as adding hydrogen and charges, define the range of the Gridbox according to the binding cavity, and conduct a molecular docking experiment.

[0093] Analysis of the potential anti-inflammatory target genes of the component combination: Search the Genecards database using "inflammation" as the keyword, and a total of 1647 inflammation-related genes were obtained. Import the combined target genes and inflammation-related genes into the Venny platform, and after taking the intersection, the SY combination obtained 36 common anti-inflammatory target genes, accounting for 36.36%. For example Figure 11The Venn diagram of the SY combination target gene and the disease target gene is shown, where the left side represents the combination of isorhamnetin and methyl protocatechuate. Table 3 shows the common target genes of the anti-inflammatory target genes of the SY combination.

[0094]

Table 3

[0095]

[0096] The obtained potential anti-inflammatory target genes were imported into the DAVID platform for GO enrichment and KEGG annotation, as Figure 12 shown in the GO enrichment analysis diagram of the SY combination target gene, which represents GOBP, GOCC, and GOMF from left to right. As Figure 13 shown in the KEGG annotation result of the SY combination target gene.

[0097] The GOBP enrichment result of the SY combination suggests that the target genes are related to transcription, redox, inflammatory response, signal transduction, apoptosis, etc.; the GOCC enrichment result shows that it mostly acts on the nuclear part; the GOMF enrichment result shows that the target genes are related to protein binding, transcription factor activity, transcription factor binding, etc. The KEGG pathway annotation shows that the SY combination can act on pathways such as TNF and MAPK to exert anti-inflammatory effects.

[0098] Analyzing the GO enrichment and KEGG annotation results, it was found that the SY combination can antagonize the inflammatory response by regulating apoptosis, regulating transcription factor activity, and inflammatory response. Pathways such as PI3K-Akt, TNF, TLR, and MAPK are all closely related to inflammation. Thus, it can be seen that the SY combination exerts a synergistic anti-inflammatory effect through multiple targets, multiple methods, and multiple pathways.

[0099] Constructing a component-target-pathway network: After importing the component combination, target genes, and KEGG annotation results into the Cytoscape software for processing, the corresponding component-target-pathway network of the combination was obtained, as Figure 14 shown in the SY combination component-target-pathway network. In the figure, the colored rhombus represents the compounds in the combination, the colored ellipse represents the potential anti-inflammatory target genes, and the colored square represents the pathways with relatively high KEGG annotation enrichment, enabling a more intuitive view of the target genes and pathways involved in the combination.

[0100] Analyzing the PPI network: The target genes obtained from the previous screening were imported into the String platform, and a protein-protein interaction (PPI) network was constructed with medium confidence. Visual analysis was performed using Cytoscape and arranged according to the degree value. As Figure 15 shown in the SY combination target gene protein interaction network. As Figure 16 shown in the visualization analysis result of the PPI network of the SY combination.

[0101] The PPI results of the SY combination found that the target genes with relatively high degree values and contributions were: PTGS2, PPARG, RELA, NOS3, MAPK3, MAPK8, MAPK14, MAPK1, etc. 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. The above pathways are all closely related to inflammation; IL-1B, TNF, NOS3, and PTGS2 are related to the secretion of inflammatory factors and the process of inflammatory response. Combining the functional analysis of the target genes, it can be verified that the SY combination can play an anti-inflammatory role by regulating the inflammatory pathway, mediating immune function, and regulating the secretion of inflammatory factors.

[0102] Combined with molecular docking technology, 9 common inflammatory proteins were selected: CCL2, IL-6, NOS2, PTGS2, TNF, Akt1, ERK, JNK, NF-κB as the target proteins for molecular docking. The corresponding PDBIDs were 1D0K, 1ALU, 1NSI, 5F19, 3UP3, 6CCY, 2G01, 5BVE, 1IKN. The molecular docking scoring results are shown in Table 4, that is, Table 4 is the scoring of the molecular docking results of isorhamnetin and methyl protocatechuate with common inflammatory proteins.

[0103]

Table 4

[0104]

[0105] Analysis of the binding diagrams of different components with the protein with the optimal scoring value: such as Figure 17 is the schematic diagram of the docking mode of isorhamnetin with 1NSI protein. Such as Figure 18 is the visualization analysis schematic diagram of the binding cavity of isorhamnetin with 1NSI protein. Such as Figure 19 is the schematic diagram of the docking mode of methyl protocatechuate with 1NSI protein. Figure 20 is the visualization analysis schematic diagram of the binding cavity of methyl protocatechuate with 1NSI protein.

[0106] Analyzing the molecular docking results, it was found that the docking scores of the above components with 8 common proteins were mostly > -5 KJ·mol-1, showing good binding activity. Among them, the binding activity of isorhamnetin with inflammatory proteins was better than that of methyl protocatechuate. Isorhamnetin and methyl protocatechuate both had good binding with 1NSI protein, and it was the NOS2 gene, related to NO synthesis.

[0107] Visualizing the analysis of the above components and the protein with the optimal scoring result, it can be seen that isorhamnetin and methyl protocatechuate can enter the same cavity on the surface of 1NSI protein, but the binding sites of the two are different, thus resulting in a difference in binding energy. Further analyzing the binding mode of the compound with the binding cavity, it was found that the above components could all form hydrogen bonds with the amino acid residues at the binding site, with stable binding.

[0108] As shown Figure 21 in the Figure 18 enlarged view within the box, it is a schematic diagram of the binding of isorhamnetin to amino acid residues GLU-377 and TRP-372 by hydrogen bonds. As Figure 22 shown in the figure, it is a schematic diagram of the connection of methyl protocatechuate to amino acid residue ARG-381 by forming two hydrogen bonds.

[0109] It can be understood that network pharmacology is widely used in virtual screening of active ingredients of traditional Chinese medicines, compound prescriptions, etc., and can efficiently and quickly construct a network diagram of actions and visually display it. Molecular docking can simulate and calculate the optimal binding energy between drugs and proteins, provide references for mechanism research, and provide directions for further experimental research.

[0110] The results of network pharmacology and molecular docking techniques show that the Y combination can regulate the secretion of inflammatory factors by binding to inflammatory factor-secreting proteins, and regulate the MAPK and NF-κB pathways to play a synergistic anti-inflammatory effect, which is in line with the characteristics of traditional Chinese medicines with multiple components and multiple targets.

[0111] The above experiments show that the GO enrichment, KEGG annotation and PPI analysis results of the SY drug combination suggest that the combination of isorhamnetin and methyl protocatechuate is more related to the binding of inflammatory factor proteins and involves redox reactions. The molecular docking results show that isorhamnetin has a higher docking score with common inflammatory proteins, which is consistent with its obvious anti-inflammatory activity. The score of methyl protocatechuate is lower, but it is found by comparison that methyl protocatechuate has good binding activity with NOS2 and PTGS2 and has binding activity with other inflammatory proteins. Combining with the good NO inhibition rate results of methyl protocatechuate in the previous experiments, it can be known that methyl protocatechuate can assist the anti-inflammatory effect in other ways.

[0112] In summary, the SY combination obtained 36 common anti-inflammatory target genes. The SY combination can antagonize the inflammatory response by regulating apoptosis, regulating the activity of transcription factors and inflammatory responses, and acts on pathways such as PI3K-Akt, TNF, TLR, and MAPK. It can be seen that the SY combination exerts a synergistic anti-inflammatory effect through multiple targets, multiple ways and multiple pathways. The target genes of the SY combination are: PTGS2, PPARG, RELA, NOS3, MAPK3, MAPK8, MAPK14, MAPK1, etc. The molecular docking results show that the above components have good binding activity with 9 common inflammatory proteins.

[0113] Experiment 7. The effect of component combination on the secretion of inflammatory factors in the RAW264.7 cell inflammation model

[0114] It is understandable that inflammation can stimulate the production of cytokines, which can exacerbate the inflammatory response. Cytokines and inflammation are interrelated. Therefore, inhibiting cytokines has positive significance for the treatment of inflammation. Cytokines such as TNF-α, IL-1β, and IL-6 are closely related to the development of inflammation and are the key research objects for studying the anti-inflammatory mechanism of drugs. Based on the above experimental results, methyl protocatechuate isolated from Tetrastigma hemsleyanum has anti-inflammatory activity and has a synergistic anti-inflammatory effect when combined with isorhamnetin respectively. Studying the synergistic anti-inflammatory effect of component combinations from the level of inflammatory factor secretion can better confirm the anti-inflammatory mechanism of Tetrastigma hemsleyanum.

[0115] Optionally, cell culture, subculture, cryopreservation and resuscitation are the same as above, and the contents of TNF-α, IL-6, and IL-10 cytokines are detected by ELISA. The method for detecting the contents of TNF-α, IL-6, and IL-10 cytokines by ELISA is as follows: 1) After the cell culture is completed, without touching the bottom of the culture plate, aspirate the cell culture medium and centrifuge at 12,000 rpm and 4°C for 20 min. Without touching the bottom of the centrifuge tube, aspirate the supernatant and store it at low temperature for testing. 2) Sample addition: Set up experimental wells and add samples according to the instructions. 3) Enzyme addition: Add 100 μL 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 60 min. 5) Prepare the washing solution: Dilute the concentrated solution for later use. 6) Washing: Add the washing solution, let it stand for about 1 min, centrifuge off, and pat dry on filter paper until there is no obvious water stain. Repeat 5 times. 7) Color development: Add color developer A and B, mix well, and develop color at 37°C in the dark for about 25 min. 8) Termination: Add 50 μL of termination solution to each well to terminate the reaction. 9) Measurement: Measure the OD450nm value of each well.

[0116] Specifically, the grouping treatment of the effect of component combinations on the secretion of TNF-α, IL-6, and IL-10 factors in the mouse RAW264.7 cell inflammation model includes the following steps: (1) Collect cells, count them, and inoculate them into a 24-well culture plate, with 5×105 cells per well, and culture overnight. (2) The experiment is divided into a control group, an LPS group, and a treatment group. In the control group, no treatment is performed; in the LPS group, the culture medium contains LPS with a final concentration of 1 μg·mL -1 LPS; in the treatment group, after pretreatment with the component or component combination for 2 h, then mix in a final concentration of 1 μg·mL -1Incubate with LPS for 24 h. (3) Discard the old culture medium of each group, replace it with the corresponding culture medium of each experimental group, and incubate in an incubator for the required time. Each treatment is repeated in 3 wells. After RAW264.7 cells are incubated for the set time, aspirate the culture medium supernatant of each well, centrifuge at 12,000 rpm and 4 °C for 20 min, aspirate the supernatant, and detect the contents of TNF-α, IL-6, and IL-10 factors in the supernatant. The data processing method is that each experiment is repeated 3 times, expressed as Mean±SD, and the data is processed by SPSS. P<0.05 is the significant difference level. The pictures are drawn by Graphpadprsim 8.2 software.

[0117] As Figure 23 shown, the inhibitory effects of different concentrations of components and component combinations on the secretion level of TNF-α in the inflammatory model of mouse RAW264.7 cells were analyzed. For better distinction and representation, in the figure, S represents 5 μmol·L -1 isorhamnetin; Y represents 250 μmol·L -1 methyl protocatechuate group; SY represents 5 μmol·L -1 isorhamnetin + 250 μmol·L -1 methyl protocatechuate group. In the LPS group, the content of TNF-α secreted by RAW264.7 cells was extremely significantly different from that of the control group (P<0.01), and an inflammatory model of mouse RAW264.7 cells was successfully constructed. Compared with the LPS group, the selected components and component combinations in the experiment could significantly inhibit the secretion of inflammatory factors (P<0.01).

[0118] As Figure 24 shown is the comparison chart of the inhibitory effects of different components and component combinations on the level of IL-6 factor. The inhibitory effects of different concentrations of components and component combinations on the secretion level of IL-6 in the inflammatory model of mouse RAW264.7 cells are shown in Table 5. After adding 1 μg·mL -1Under the condition of LPS induction, the content of IL-6 in the LPS group was extremely significantly different from that in the control group (P < 0.01). Compared with the LPS group, the selected components and component combinations in the experiment could also significantly inhibit the secretion of IL-6 in the RAW264.7 cell inflammation model (P < 0.05). By analyzing and comparing the inhibitory effects of rhamnetin combined with methyl protocatechuate on the secretion level of the IL-6 inflammatory factor respectively, it can be intuitively seen that the combined application can better inhibit the secretion level of IL-6 and reduce the content of IL-6. There was a statistical difference compared with the single application (P < 0.05). Rhamnetin has the effect of inhibiting the secretion level of inflammatory factors; methyl protocatechuate has antioxidant effects, can counteract the cell damage caused by oxidative burst, and reduce cell apoptosis. It can thus be proved that methyl protocatechuate can, through anti-inflammatory and antioxidant effects, inhibit cell apoptosis, block the connection between oxidative stress and inflammation, and play a synergistic role in inhibiting the secretion of IL-6.

[0119]

Table 5

[0120]

[0121] As Figure 25 shown is the comparison chart of the inhibitory effects of different concentrations of components and component combinations on the secretion level of IL-10 in the RAW264.7 cell inflammation model of mice. Compared with the LPS group, the selected component combinations in the experiment could significantly reduce the secretion of IL-10 in the RAW264.7 cell inflammation model (P < 0.05). Compared with the CK group, rhamnetin, methyl protocatechuate and component combinations promoted the secretion of the IL-10 factor; compared with the model group, rhamnetin, methyl protocatechuate and component combinations inhibited the high-concentration state of IL-10. By analyzing the inhibitory effects of single components and component combinations on the IL-10 inflammatory factor, it was found that the combined use of drugs had a synergistic effect, and there was a significant difference between the effect of using rhamnetin alone and the effect of combined use of drugs (P < 0.05).

[0122] In summary, controlling the overproduction of inflammatory factors is of positive significance for controlling inflammation. The drug alone can effectively inhibit the synthesis of inflammatory factors. The combined application of isorhamnetin and methyl protocatechuate shows a more effective inhibitory effect on IL-6 factor. Isorhamnetin and methyl protocatechuate have a good synergistic inhibitory effect on IL-10 factor. Considering the well-studied antioxidant stress effect of methyl protocatechuate, it can be known that when combined with isorhamnetin, the antioxidant 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 peak expression is later than that of pro-inflammatory factors. Moreover, IL-10 has a bidirectional regulatory effect. When at a low concentration, it can inhibit the secretion of inflammatory factors; but when at a high concentration, IL-10 shows immunosuppressive effects. The secretion amount of IL-10 in the isorhamnetin group is lower than that in the model group, indicating that this drug can inhibit the inflammatory process to a certain extent and acts on the whole stage of the inflammatory process; it can be known that methyl protocatechuate and the component combination can act on the early stage of the inflammatory process to inhibit inflammatory factors.

[0123] Experiment 8: The component combination affects the MAPK and NF-κB pathway proteins in the RAW264.7 cell inflammation model. In the downstream signaling pathways activated by LPS induction, the NF-κB pathway and the MAPK pathway are important research indicators. It can be understood 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 the cell to defend against oxidative stress. The Nrf2 / ARE pathway plays an important role in maintaining the cellular redox homeostasis. The previous experiments showed that methyl protocatechuate, isorhamnetin and the SY combination can regulate the secretion of inflammatory factors. The effects of the component combination on the signaling pathways, and the effects of isorhamnetin, methyl protocatechuate and the combination on the NF-κB, MAPK and Nrf2 pathway proteins are used to better characterize the anti-inflammatory molecules and the mechanism of action.

[0124] Specifically, the methods of cell culture, passage, cryopreservation and resuscitation are the same as above.

[0125] Preparation of proteins separated by SDS-PAGE electrophoresis, where the reagents include: 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 buffer, mix well, and store at 4°C for later use. When lysing cells, add 100 μL - 150 μL of cell lysis buffer per well according to 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 fresh before use. When measuring protein concentration by the BCA method, add 200 μL per well. 5×SDS-PAGE electrophoresis buffer concentrate: Weigh 15.1 g of Tris, 94 g of glycine, and 5 g of SDS, add appropriate distilled water, and dissolve while ultrasonically agitating gently. After the solids dissolve and the solution becomes clear, add distilled water to make up to 1 L, mix well, and store at room temperature. When using, dilute one part of 5×SDS-PAGE electrophoresis buffer concentrate with four parts of distilled water, mix well, and set aside. TBST washing buffer: Dilute one part of 10×TBST washing buffer concentrate with nine parts of distilled water, mix well, and store at 4°C for later use. Prepare fresh before use 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 10 mL of 10×transfer buffer concentrate, 10 mL of methanol, and 80 mL of water, mix well, and store at 4°C for later use; for other proteins: Add 10 mL of 10×transfer buffer concentrate, 20 mL of methanol, and 70 mL of water, mix well, and store at 4°C for later use. Blocking solution: The blocking solution contains 5% non-fat milk powder, mix well, store at 4°C for later use, and prepare fresh before use. Add 20 mL 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% non-fat milk powder and the primary antibody at the set concentration, mix well, and store at 0°C for later use. When the primary antibody is a rabbit polyclonal antibody, additionally add 0.01% sodium chloride. Add 10 mL of primary antibody working solution to each PVDF membrane. Secondary antibody working solution: The secondary antibody working solution contains 0.5% non-fat milk powder and the secondary antibody at the set concentration, mix well, and store at 0°C for later use. Add 10 mL of secondary antibody working solution to each PVDF membrane. ECL developing solution: Mix the developing solution and the stabilizing solution at a ratio of 1:1, store at 4°C in the dark for later use. Add approximately 1.2 mL of ECL developing solution to each PVDF membrane.

[0126] The preparation of SDS-PAGE gel includes: 1) Wash the glass plates, rinse with distilled water, and air dry for later use. Place the glass plates according to the marks, assemble them, and clip them into the gel-making bracket. Add distilled water to check for leakage. If there is no leakage, pour out the distilled water and blot the excess water with filter paper. 2) The concentrations of the separating gel are 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), Gelbuffer A, 10% APS, and TEMED to prepare the separating gel. Mix well and carefully add an appropriate amount of the separating gel liquid into the glass plates. Note that after adding APS and TEMED, mix well and add it as soon as possible to avoid gel solidification. 3) After adding an appropriate amount of the separating gel, carefully and evenly add distilled water slowly above the separating gel to flatten the interface of the separating gel. Let it solidify naturally at room temperature for 20 min - 30 min. 4) After the separating gel solidifies, carefully pour out the distilled water and blot 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), Gelbuffer B, 10% APS, and TEMED to prepare the stacking gel. Mix well and carefully add an appropriate amount of the stacking gel liquid above the separating gel. Note that after adding APS and TEMED, mix well and add it as soon as possible to avoid gel solidification. 5) After adding an appropriate amount of the stacking gel, carefully insert the comb, avoiding the appearance of bubbles. Let it solidify naturally at room temperature for 20 min - 30 min. 6) After the stacking gel solidifies, place it in a plastic bag containing a small amount of electrophoresis buffer and store it at 4°C.

[0127] Protein sample extraction and concentration determination include: Group and treat the cells. After incubating for a predetermined period of time, aspirate the culture medium. Add 1 mL of PBS pre-cooled at 4°C to each well, gently shake for 1 min, and then discard the washing solution. Repeat this process three times to wash away the culture medium. Place the 6-well plate on ice and add 100 μL - 150 μL of cell lysate pre-cooled at 4°C to each well. Lyse for 30 min, gently shaking the 6-well plate from time to time. After lysis is completed, use a pipette to transfer the cell debris and lysate to a 1.5 mL centrifuge tube at the same time. Centrifuge the suspension at 4°C and 12,000 rpm for 20 min, and 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 determination kit. Add 2 μL of the sample solution to each well, and add 2 μL of cell lysate to the blank well. Add 18 μL of cell lysate to each well again and mix well. Add 200 μL of the BCA working solution to each well, carefully pipette to mix evenly, place it in an incubator at 37°C, and react for 30 min. After the reaction is completed, measure the absorbance value at 562 nm. Calculate the sample protein concentration according to the bovine serum albumin standard curve.

[0128] Electrophoresis: After measuring the protein concentration according to the instructions of the BCA kit, prepare the sample by diluting it according to the loading amount of 40 μg total protein 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 5 min to denature it, and then cool it to room temperature. Select an 8% or 12% SDS-PAGE gel for electrophoresis according to the protein molecular weight. Assemble the electrophoresis apparatus, add the electrophoresis buffer, and carefully pull out the comb parallelly. Load the sample, adjust the voltage to 80 V, and when the sample runs to the junction of the stacking gel and the separating gel, change the voltage to 120 V until the electrophoresis is completed. Stop the electrophoresis when the front of the bromophenol blue is 1-2 cm away from the gel boundary.

[0129] Detection of protein expression level by Western Blot includes: (1) Transferring the membrane, that is, taking out the gel, washing it with distilled water, first cutting off the part of the gel front containing the bromophenol blue front. Then cut off the gel area where the target protein is located according to the protein marker, wash it with the electrophoresis buffer, put it into a plastic bag with a small amount of electrophoresis buffer, 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 min in advance, and then soak it in the transfer buffer together with the filter paper for more than 30 min. Make a "membrane transfer sandwich" in the order of filter paper - gel - PVDF membrane - filter paper, put it into the semi-dry membrane transfer apparatus, 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 dried out throughout the process. (2) Blocking After the membrane transfer is completed, soak the PVDF membrane with TBST containing 5% skim milk powder, pay attention to submerging the membrane, and block it on a shaker at room temperature for 3 h. (3) Primary antibody incubation Dilute the corresponding primary antibody with TBST containing 0.5% skim milk powder, submerge the PVDF membrane, and incubate it overnight at 4 °C, paying attention to submerging the membrane. The dilution ratio is adjusted according to the antibody type. (4) Secondary antibody incubation After the primary antibody incubation is completed, rinse the PVDF membrane thoroughly with the TBST washing solution 6 times, 5 min each time. Dilute the HRP-labeled secondary antibody with TBST containing 0.5% skim milk powder at a dilution ratio of 1:2000, submerge the PVDF membrane, and incubate it on a shaker at room temperature for 2 h. (5) ECL development After the secondary antibody incubation is completed, rinse the PVDF membrane thoroughly with the TBST washing solution 6 times, 5 min each time. Mix the ECL developing solution and the stabilizing solution evenly at a ratio of 1:1, and store it at 4 °C for later use. Drop an appropriate amount of the developing solution on each membrane to evenly cover the PVDF membrane, and incubate it in the dark for 5 min. After the incubation is completed, turn on the chemiluminescence instrument and perform exposure. The exposure time is adjusted according to the clarity of the band.

[0130] Statistical analysis The experiment was repeated 3 times. The gray value of the band was analyzed by ImageJ, and significant analysis was performed using SPSS software. P < 0.05 indicates significant difference.

[0131] It should be noted that the reagents, instruments, etc. used in the above experiments, as well as the steps of this experiment, should not be regarded as a limitation to this application.

[0132] As Figure 26 shown is the effect of components and component combinations on the expression of iNOS in mouse RAW264.7 cells. As Figure 27 shown is the effect of components and component combinations on the relative expression level of iNOS in mouse RAW264.7 cells. It can be understood that NO is an important signaling molecule and can participate in physiological activities such as the body's immunity and inflammation by transmitting cell signals. In macrophages, NO is mainly synthesized by inducible nitric oxide synthase (iNOS). Therefore, detecting the effect of drugs on the expression level of iNOS protein can further explain the inhibitory effect of drugs on the release of NO by inflammatory cells. After being stimulated by LPS, RAW264.7 cells express a large amount of iNOS protein, and the difference is extremely significant compared with the CK group (P < 0.01), which is consistent with the phenomenon of a large amount of NO secretion. Methyl protocatechuate, isorhamnetin, and component combinations all inhibited the expression of iNOS protein to varying degrees compared with the LPS group, and the difference was extremely significant (P < 0.01), which was consistent with the trend of NO secretion in the corresponding groups. By analyzing and comparing the inhibitory degree of iNOS in the group with isorhamnetin alone and the combined action group, it was found that the combined combination could better inhibit the secretion of iNOS.

[0133] As Figure 28 shown is the effect of components and component combinations on the expression of MAPK pathway proteins in mouse RAW264.7 cells. As Figure 29 shown is the effect of components and component combinations on the relative expression level of p-ERK / ERK in mouse RAW264.7 cells. As Figure 30 shown is the effect of components and component combinations on the relative expression level of p-JNK / JNK in mouse RAW264.7 cells. It can be understood that MAPK is mainly composed of subfamilies such as ERK, JNK, and p38. The activation of the MAPK pathway will further amplify the inflammatory conduction signal. Inhibiting the expression of MAPK pathway proteins or the phosphorylation of related proteins has a positive significance for inhibiting inflammation. By analyzing and comparing the phosphorylation levels of proteins, it was found that the LPS group showed a high phosphorylation level, and the difference was extremely significant compared with the CK group (P < 0.01); components and component combinations could all inhibit the phosphorylation level of MAPK channel proteins, and the difference was statistically significant (P < 0.05). The results showed that the combination of isorhamnetin and methyl protocatechuate could better inhibit the protein phosphorylation levels of ERK and JNK compared with the drugs alone, P < 0.01. However, for the combination of isorhamnetin + methyl protocatechuate, there was no synergistic inhibitory effect on the MAPK pathway.

[0134] As Figure 31Shown are the effects of components and component combinations on the expression of p-p65 / p65 in mouse RAW264.7 cells, as Figure 32 Shown are the effects of components and component combinations on the relative expression levels of p65 / p-p65 in mouse RAW264.7 cells. It can be understood that the NF-κB pathway is an important link in the inflammatory pathway and can be characterized by detecting the expression level of p-65 protein or the relative expression levels 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 in the LPS group was extremely significantly different from that in the CK group, P < 0.01. Single components and combined components had inhibitory effects on the phosphorylation level of p65 protein to varying degrees, and the SY combination had a synergistic effect on the inhibitory effect of the phosphorylation level of p65 protein compared with single components.

[0135] as Figure 33 Shown are the effects of components and component combinations on the expression of Nrf2 protein in LPS-induced RAW264.7 cells, as Figure 34 Shown are the effects of components and component combinations on the relative expression levels of Nrf2 in mouse RAW264.7 cells. It can be understood that compared with the CK group, the relative expression level of Nrf2 protein in the LPS group was higher, 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 could promote the expression of Nrf2 protein to varying degrees (P < 0.05). Compared with the isorhamnetin group, the component combination group synergistically increased the expression level of Nrf2 protein; however, the promoting effect of the component combination group was not obvious compared with the protocatechuic acid methyl ester group, proving that the component combination better inhibited inflammation, and the ability of protocatechuic acid methyl ester to promote the expression of Nrf2 protein was not fully reflected. In summary, the component combination can assist the anti-inflammatory effect through the oxidative stress pathway.

[0136] 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 the combination of protocatechuic acid methyl ester and SY has 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 the inflammatory pathway MAPK and NF-κB pathways.

[0137] 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.

[0138] 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 isorhamnetin and methyl protocatechuate in the preparation of an anti-inflammatory drug, characterized in that, The concentration of isorhamnetin in the drug is 5 μmol·L -1 , the concentration of protocatechuic acid methyl ester was 250 μmol·L -1 .

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