Rheumatoid arthritis resisting effect of xanthoceras sorbifolia-4-flavor decoction

Abstract

The invention provides an anti-rheumatoid arthritis effect of xanthoceras sorbifolia 4-flavor decoction. According to the invention, a CCK-8 method is adopted to detect the inhibition rate of MH7A cells, flow cytometry is adopted to detect cell apoptosis, and an ELISA method is adopted to detect the expression levels of TNF-a, IL-6, RF, MMP-9, CRP and ASO in the MH7A cells. The xanthoceras sorbifolia-4-flavor decoction and the effective components thereof have a certain inhibition effect on proliferation of synovial fibroblasts MH7A of rheumatoid arthritis patients, and except for the xanthoceras sorbifolia-4-flavor decoction, the effective components of gallic acid, dihydromyricetin, ellagic acid, berberine and the like can cause apoptosis of the MH7A cells, so that the effect of inhibiting the proliferation of the synovial fibroblasts MH7A can be achieved. The expression levels of CRP, IL-6, ASO and RF factors indicate that the xanthoceras sorbifolia 4-flavor decoction is one of the mechanisms of the xanthoceras sorbifolia 4-flavor decoction for resisting the RA effect.

Description

Technical Field

[0001] The invention relates to the application field of Mongolian medicine Xanthoceras sorbifolia-4-flavor decoction, and in particular to an anti-rheumatoid arthritis effect of Xanthoceras sorbifolia-4-flavor decoction. Background Art

[0002] The four-flavor soup of Xanthoceras sorbifolia is composed of Xanthoceras sorbifolia, Terminalia chebula, Gentiana macrophylla, and Phellodendron amurense. The medicinal parts are the dried wood of the stems or branches, the dried mature fruits, the dried roots, and the dried bark. In the "Selection of Common Prescriptions of Mongolian Medicine" published in 1975, it is recorded that the compatibility ratio of Xanthoceras sorbifolia is 5 qian, Terminalia chebula 2 qian, Gentiana macrophylla root 2 qian, and Phellodendron amurense is decocted in water and taken warm. No relevant research on this prescription has been found in modern literature reports, but the research on single herbs Xanthoceras sorbifolia, Terminalia chebula, Gentiana macrophylla, and Phellodendron amurense is relatively rich, mainly in chemistry, pharmacology, and clinical research, which provides valuable ideas for clinical and basic scientific research.

[0003] The quality markers of TCM for anti-rheumatoid arthritis can be obtained in a variety of ways, including chemistry, medicinal properties, pharmacokinetics, measurable components, intestinal flora and network pharmacology. In addition, according to the concepts related to quality markers, the structural, chemical, biological and ecological characteristics that affect the quality of Chinese medicinal materials and their marked components can be regarded as quality markers to predict the effectiveness of preventing and treating rheumatoid arthritis. For example, the Inner Mongolian specialty Mongolian medicine Naru Sanwei Pills can effectively prevent and treat bone and joint degenerative diseases such as lumbar disc herniation, but the drug mechanism still needs to be further studied. In recent years, as an emerging discipline, network pharmacology can explore the pharmacological mechanism of drugs from multiple angles, multiple methods and multiple targets.

[0004] Although the future development of network pharmacology faces many challenges, its emergence has also created new opportunities for TCM research, which can more scientifically understand the pharmacological mechanisms and compatibility of TCM or TCM compound prescriptions, thus proposing new ideas and methods for the development of TCM. Practice has proved that the organic combination of traditional medicine and modern medical technology can greatly improve the efficacy of TCM, thus playing an irreplaceable role.

[0005] According to the latest data in 2019, the prevalence of RA of all ages is 1%. Rheumatoid arthritis is a severe autoimmune disease characterized by symmetrical joint pain and inflammation, local bone tissue and cartilage damage, and vascular hyperplasia. In the occurrence and development of RA, a variety of cells are involved in the occurrence and development of RA. Immortalized human rheumatic joint synovial fibroblasts (MH7A) are a type of macrophage with immunoregulatory function. It can abnormally proliferate, migrate, and invade blood vessels, joints and other parts, and can produce abnormal energy metabolism in the inflammatory microenvironment, thereby causing self-activation and the occurrence of synovitis. Therefore, the present invention studies the effects of the 4-flavor decoction and its main active ingredients on the proliferation and apoptosis of MH7A cells, and is of great significance for the prevention and treatment of RA and the development of new drugs. Summary of the invention

[0006] The invention aims to provide a 4-flavor decoction of Xanthoceras sorbifolia for anti-rheumatoid arthritis effect.

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention relates to a 4-flavor decoction for the treatment of rheumatoid arthritis, which comprises the following specific steps:

[0009] (1) Drug preparation

[0010] The preparation of the decoction of the 4-flavor decoction of Sorbus sorbifolia was carried out by decoction (the optimal process conditions of the 4-flavor decoction of Sorbus sorbifolia were decoction time of 35 min, water addition amount of 40 times, and soaking time of 30 min); the extract of the 4-flavor decoction of Sorbus sorbifolia was freeze-dried and sealed in the dark and refrigerated; the dosage of the positive drug Tripterygium wilfordii polyglycosides tablets was 5 mg / L. The corresponding drug concentration was prepared using a special culture medium. 10 mg of the 4-flavor decoction of Sorbus sorbifolia, Tripterygium wilfordii polyglycosides tablets, dihydromyricetin, epicatechin, berberine, gentiopicroside, gallic acid, and ellagic acid were weighed respectively and added to a special culture medium to make a fixed volume of 10 mL, and the mother solution was prepared for standby use after ultrasonication for 20 min to completely dissolve.

[0011] (2) Cell culture

[0012] Use T25 culture flasks to place MH7A cells for recovery, culture in a carbon dioxide incubator, and change the medium every other day. When the cells grow to more than 85%, they can be subcultured. After the cells are subcultured for 3 generations, the logarithmic growth phase is selected for the experiment.

[0013] (3) Detection

[0014] (3.1) CCK-8 assay to detect the effects of Xanthoceras sorbifolia 4-flavor decoction and its main active ingredients on MH7A cell proliferation

[0015] Take the MH7A cells cultured in (2.2) and press 1x10 5The cells were counted at a density of 100 μL / mL and plated in a 96-well plate, with 100 μL per well. A control blank group and different drug concentrations were set. After 24 hours of cell culture, the corresponding concentrations of the drug were added according to the above grouping and cultured for another 24 hours. The CCK-8 method was used, and the sample type was set for detection when the absorbance was at a wavelength of 450 nm. Calculate the cell inhibition rate (%) = (blank group-experimental group) / blank group × 100%. Calculate the inhibition rate of each group of drugs on cells to observe the effects of the Crownwood-4 decoction and its main active ingredients on the proliferation of MH7A cells, and screen out the best concentrations for cell apoptosis experiments.

[0016] (3.2) Flow cytometry to detect the effects of Xanthoceras sorbifolia-4-flavor decoction and its main active ingredients on apoptosis of MH7A cells

[0017] Take the cultured MH7A cells and press 1x10 5 The cells were counted at a density of 100 / mL and plated in a 24-well plate, with 1 mL in each well. A control blank group and different drug concentrations were set up. Different drug concentrations in each group were obtained through the detection experiment (3.1): 12.5 mg / L, 25, and 50 mg / L of Wenguanmu-4 decoction; 5 mg / L of positive drug; 0.02 mg / L, 0.1 mg / L, and 0.5 mg / L of epicatechin; 0.1 mg / L, 0.5 mg / L of ellagic acid; 5 mg / L, 10 mg / L, and 20 mg / L of berberine; 10 mg / L, 20 mg / L, and 40 mg / L of dihydromyricetin; 250 mg / L, 500 mg / L, and 1000 mg / L of gentiopicroside; 3 mg / L, 5 mg / L, and 10 mg / L of gallic acid; each group was set up with 3 replicate wells and placed in a carbon dioxide incubator for 24 hours. After that, the cells were taken out of the incubator, and different concentrations of drug dilutions were added, and then placed for another 24 hours. Then the experiment was performed according to the instructions of the cell apoptosis detection kit, from collecting cells, washing cells, grouping, staining to testing on the machine within the specified 1 hour.

[0018] (3.3) Enzyme-linked immunosorbent assay (ELISA) to detect TNF-a, CRP, IL-6, RF, and MMP-9

[0019] The expression levels of TNF-a, CRP, IL-6, ASO, RF, and MMP-9 were detected, and the experiment was repeated 3 times. The data were calculated by ELISA regression analysis to compare the significant levels of different concentrations.

[0020] The present invention has the following advantages:

[0021] The present invention screened the results of cell proliferation inhibition rate and found that compared with the blank control group, when the drug was administered for 24 hours, the concentration increased and the cell inhibition rate increased, which was statistically significant (P<0.01, P<0.0001). Flow cytometry was used to detect the effects of the 4-flavor decoction of wenguanmu and its main active ingredients on MH7A cell apoptosis. The experimental results showed that compared with the blank control group, after 24 hours of administration, the concentration of each group increased and obvious apoptosis occurred. The calculation of early apoptosis and late apoptosis rates showed that MH7A cell apoptosis could be induced after administration. Triptolide can effectively prevent the reproduction, infection and metastasis of human synovial cells MH7A, and inhibit the expression of MMP-2 and MMP-9. In addition, 20 μg / L TNF-α treatment can significantly increase the proliferation, infection and metastasis of MH7A cells and reduce cell apoptosis, while isochlorogenic acid a can effectively inhibit these processes. Compared with TNF-α in vitro induction, it has a stronger inhibitory effect, thereby effectively improving the condition of cell apoptosis. By increasing the release of ROS, the apoptosis rate of cells can be significantly improved. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of microRNA-193a-3p in the synovial tissues of 30 RA patients and healthy controls. MH7A cells were induced by TNF-α to construct an in vitro RA model, and TNF-α induction significantly inhibited the apoptosis of MH7A cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a flow chart of the in vitro experiment of the present invention;

[0023] Figure 2 This is a data graph showing the effects of the control blank group and different drug concentrations on the proliferation of MH7A cells;

[0024] Figure 3 is a comparison chart of cell apoptosis rates in each group;

[0025] Figure 4 This is the first picture of early and late apoptosis of cells in each group;

[0026] Figure 5 The second picture shows the early and late apoptosis of cells in each group;

[0027] Figure 6 The third picture shows the early and late apoptosis of cells in each group;

[0028] Figure 7 The fourth picture shows the early and late apoptosis of cells in each group;

[0029] Figure 8 The fifth picture shows the early and late apoptosis of cells in each group;

[0030] Fig. 9 This is the sixth picture of early and late apoptosis of cells in each group;

[0031] Fig.10 This is the seventh picture of early and late apoptosis of cells in each group;

[0032] Fig.11 The eighth picture shows the early and late apoptosis of cells in each group;

[0033] Fig.12 is the expression graph of group cytokine ASO concentration;

[0034] Fig.13 is the expression graph of group cytokine CRP concentration;

[0035] Fig.14 is the expression graph of cytokine IL-6 concentration in the group;

[0036] Fig.15 is the expression graph of the cytokine MMP-9 concentration in the group;

[0037] Fig.16 is the expression graph of group cytokine RF concentration;

[0038] Fig.17 This is a graph showing the expression of cytokine TNF-a concentration. DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments are only further descriptions of the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0040] Example

[0041] The present embodiment relates to the anti-rheumatoid arthritis effect of Xanthoceras sorbifolia-4-flavor decoction, and the CCK-8 method is used to detect the inhibition rate of MH7A cells, the flow cytometry method is used to detect cell apoptosis, and the ELISA method is used to detect the expression levels of TNF-a, IL-6, RF, MMP-9, CRP, and ASO in MH7A cells.

[0042] The specific steps are:

[0043] Step 1. Preparation of materials and equipment

[0044] (1.1) Cells

[0045] Immortalized human rheumatic joint synovial fibroblasts (MH7A);

[0046] (1.2) Drugs

[0047] Wenguanmu-4 flavor soup (Sendeng-4), referred to as WGM-4 (SD-4), Horqin Left Middle Banner Mongolian Hospital;

[0048] Tripterygium wilfordii glycosides tablets (Positive), Liaoning Xinshanyuan Pharmaceutical Co., Ltd.;

[0049] Dihydromyricetin (batch number: 19031501, content 99.57%), Beijing Putian Tongchuang Biotechnology Co., Ltd.;

[0050] Epicatechin (batch number: 20042012, content 99.65%), Beijing Putian Tongchuang Biotechnology Co., Ltd.;

[0051] Berberine (batch number: DST210420-009, content 99.48%), Chengdu Dester Biotechnology Co., Ltd.;

[0052] Gentianopicroside (batch number: 110770-202219, content 98.1%), China Food and Drug Inspection Institute;

[0053] Gallic acid (batch number: 8338, content 94.5%), Shanghai Shidande Standard Technology Service Co., Ltd.;

[0054] Ellagic acid (batch number: 5432, content 98.9%), Shanghai Shidande Standard Technology Service Co., Ltd.

[0055] (1.3) Reagents

[0056] PBS buffer (0.01 M pH 7.2-7.4), Beijing Solebow Technology Co., Ltd.;

[0057] iCell-008a001b Human rheumatoid arthritis synovial fibroblast immortalized cell culture medium (basal medium 500mL, additive 5mL, fetal bovine serum (FBS) 50mL, double antibody 5mL), Cybio (Shanghai) Biotechnology Co., Ltd.;

[0058] C1062L Annexin V-FITC apoptosis detection kit, Biotime Biotech;

[0059] B51503 CytoFLEX sheath fluid, Beckman, USA;

[0060] Enzyme-linked immunosorbent assay (ELISA) for human tumor necrosis factor alpha (TNF-a), human C-reactive protein (CRP), human interleukin 6 (IL-6), human rheumatoid factor (RF), human matrix metalloproteinase 9 (MMP9), etc., Jiangsu Jingmei Biotechnology Co., Ltd.

[0061] (1.4) Instruments

[0062] A00-1-11102CytoFLEX, Beckman Coulter Biotech (Suzhou) Co., Ltd.;

[0063] Inverted fluorescence microscope, Thermo Fisher Scientific (China) Co., Ltd.;

[0064] BPN-80CW (UV) CO2 incubator, Shanghai Yiheng Scientific Instrument Co., Ltd.;

[0065] Vario-skan LUX multi-function microplate reader, Thermo Fisher Scientific;

[0066] Digital constant temperature water bath, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory;

[0067] Sigma3-18K centrifuge, Changsha Tianheng Scientific Instrument Equipment Co., Ltd.;

[0068] Sartorius electronic scale, Jinan Ailabao Instrument Equipment Co., Ltd.;

[0069] KG SX-500 Steam Sterilizer, KAGOSHIMA SEISAKUSYO IN

[0070] Step 2, Method

[0071] (2.1) Drug preparation

[0072] The preparation of the decoction of the 4-flavor decoction of Sorbus sorbifolia was carried out by decoction (the optimal process conditions of the 4-flavor decoction of Sorbus sorbifolia were decoction time of 35 min, water addition amount of 40 times, and soaking time of 30 min); the extract of the 4-flavor decoction of Sorbus sorbifolia was freeze-dried and sealed in the dark and refrigerated; the dosage of the positive drug Tripterygium wilfordii polyglycosides tablets was 5 mg / L. The corresponding drug concentration was prepared using a special culture medium. 10 mg of the 4-flavor decoction of Sorbus sorbifolia, Tripterygium wilfordii polyglycosides tablets, dihydromyricetin, epicatechin, berberine, gentiopicroside, gallic acid, and ellagic acid were weighed respectively and added to a special culture medium to make a fixed volume of 10 mL, and the mother solution was prepared for standby use after ultrasonication for 20 min to completely dissolve.

[0073] (2.2) Cell culture

[0074] Use T25 culture flasks to place MH7A cells for recovery, culture in a carbon dioxide incubator, and change the medium every other day. When the cells grow to more than 85%, they can be subcultured. After the cells are subcultured for 3 generations, the logarithmic growth phase is selected for the experiment.

[0075] Step 3, Detection

[0076] (3.1) CCK-8 assay to detect the effects of Xanthoceras sorbifolia 4-flavor decoction and its main active ingredients on MH7A cell proliferation

[0077] Take the MH7A cells cultured in (2.2) and press 1x10 5The cells were counted at a density of 100 μL / mL and spread in a 96-well plate, with 100 μL per well. A control blank group and different drug concentrations were set (see 2). After 24 hours of cell culture, the corresponding concentrations of the drug solution were added according to the above grouping and cultured for another 24 hours. The CCK-8 method was used, and the sample type was set for detection when the absorbance was at a wavelength of 450 nm. Calculate the cell inhibition rate (%) = (blank group-experimental group) / blank group × 100%. Calculate the inhibition rate of each group of drugs on cells to observe the effects of the Crownwood-4 decoction and its main active ingredients on the proliferation of MH7A cells, and screen out the best concentrations for cell apoptosis experiments.

[0078] (3.2) Flow cytometry to detect the effects of Xanthoceras sorbifolia-4-flavor decoction and its main active ingredients on apoptosis of MH7A cells

[0079] Take the cultured MH7A cells and culture them at 1x10 5 The cells were counted at a density of 100 / mL and plated in a 24-well plate, with 1 mL in each well. A control blank group and different drug concentrations were set up. Different drug concentrations in each group were obtained through the detection experiment (3.1): 12.5 mg / L, 25, and 50 mg / L of Wenguanmu-4 decoction; 5 mg / L of positive drug; 0.02 mg / L, 0.1 mg / L, and 0.5 mg / L of epicatechin; 0.1 mg / L, 0.5 mg / L of ellagic acid; 5 mg / L, 10 mg / L, and 20 mg / L of berberine; 10 mg / L, 20 mg / L, and 40 mg / L of dihydromyricetin; 250 mg / L, 500 mg / L, and 1000 mg / L of gentiopicroside; 3 mg / L, 5 mg / L, and 10 mg / L of gallic acid; each group was set up with 3 replicate wells and placed in a carbon dioxide incubator for 24 hours. After that, the cells were taken out of the incubator, and different concentrations of drug dilutions were added, and then placed for another 24 hours. Then the experiment was performed according to the instructions of the cell apoptosis detection kit, from collecting cells, washing cells, grouping, staining to testing on the machine within the specified 1 hour.

[0080] (3.3) Enzyme-linked immunosorbent assay (ELISA) to detect TNF-a, CRP, IL-6, RF, and MMP-9

[0081] The expression levels of TNF-a, CRP, IL-6, ASO, RF, and MMP-9 were detected, and the experiment was repeated 3 times. The data were calculated by ELISA regression analysis to compare the significant levels of different concentrations.

[0082] Step 4, test results

[0083] (4.1) Effects of Xanthoceras sorbifolia-4-flavor decoction and its main active ingredients on MH7A cell proliferation

[0084] The results of cell proliferation inhibition rate screening showed that compared with the blank control group, after 24h of drug treatment, the effects of 12.5mg / L, 25mg / L, 50mg / L, and 100mg / L of Wenguanmu-4 decoction, 5mg / L of positive drug, 0.02mg / L, 0.1mg / L, 0.5mg / L, and 1mg / L of epicatechin, 0.1mg / L, 0.5mg / L, and 1mg / L of ellagic acid, and 5mg / L of berberine were significantly inhibited. L, 10mg / L, 20mg / L, 40mg / L; dihydromyricetin 10mg / L, 20mg / L, 40mg / L, 60mg / L; gentiopicroside 250mg / L, 500mg / L, 1000mg / L; gallic acid 3mg / L, 5mg / L, 10mg / L, 20mg / L; the increase in concentration led to an increase in cell inhibition rate, which was statistically significant (P<0.01, P<0.0001).

[0085] (4.2) Effects of Xanthoceras sorbifolia-4-flavor decoction and its main active ingredients on apoptosis of MH7A cells

[0086] Flow cytometry was used to detect the effects of the 4-flavor decoction and its main active ingredients on the apoptosis of MH7A cells. The experimental results showed that compared with the blank control group, after 24 hours of administration, the increase in concentrations in each group led to obvious apoptosis. The calculation of early apoptosis and late apoptosis rates showed that the administration of the drug could induce apoptosis of MH7A cells. Figure 3 and Figure 4-11 As shown, Figure 4 -Attached Fig.11 (CL: blank control group; YANG: Tripterygium wilfordii glycosides tablets (positive group); B: epicatechin; X: berberine; L: gentiopicroside; E: dihydromyricetin; R: ellagic acid; SD: Xanthium sorbifolia-4 flavor decoction; M: gallic acid).

[0087] (4.3) Expression of cytokines such as ASO, TNF-a, CRP, IL-6, RF, and MMP-9

[0088] The different concentrations of each group were screened by the above cell apoptosis rate, including 12.5mg / L, 25mg / L, and 50mg / L of sorbitan-4 decoction; 5mg / L of positive drug; 0.02mg / L, 0.1mg / L, and 0.5mg / L of epicatechin; 0.1mg / L, 0.5mg / L of ellagic acid; 5mg / L, 10mg / L, and 20mg / L of berberine; 10mg / L, 20mg / L, and 40mg / L of dihydromyricetin; 250mg / L, 500mg / L, and 1000mg / L of gentiopicroside; 3mg / L, 5mg / L, and 10mg / L of gallic acid;

[0089] ELISA was used to detect the expression of ASO, TNF-a, CRP, IL-6, RF, and MMP-9 cytokines. The results showed that compared with the blank control group, there was no significant difference in the expression of ASO cytokines between gentiopicroside and berberine; there were significant differences in the expression of ASO cytokines between 4-flavor decoction of S. sorbifolia and epicatechin, gallic acid, dihydromyricetin, and ellagic acid (P < 0.05), among which the increase of gallic acid, dihydromyricetin, and 4-flavor decoction down-regulated the level of ASO factor. Compared with the blank control group, there was no significant difference in the expression of CRP cytokines between gentiopicroside and berberine; there were significant differences in the expression of ASO cytokines between 4-flavor decoction of S. sorbifolia and epicatechin, gallic acid, dihydromyricetin, and ellagic acid (P < 0.05), among which the increase of gallic acid, ellagic acid, dihydromyricetin, and 4-flavor decoction down-regulated the level of CRP factor. Compared with the blank control group, gentiopicroside had no significant difference in IL-6 cytokine expression; 4-flavor decoction of sorbitan, berberine, epicatechin, gallic acid, dihydromyricetin, and ellagic acid all had certain significant differences (P < 0.05), among which the increase in the concentration of epicatechin, gallic acid, ellagic acid, and sorbitan-4 flavor decoction down-regulated the level of IL-6 factor and alleviated the inflammatory response. Compared with the blank control group, 4-flavor decoction of sorbitan, epicatechin, gallic acid, ellagic acid, and gentiopicroside had no significant difference in MMP-9 cytokine expression; only the increase in berberine concentration down-regulated the level of MMP-9 factor (P < 0.05); the increase in dihydromyricetin concentration promoted the expression of MMP-9 cytokine. Compared with the blank control group, gentiopicroside had no significant difference in RF cytokine expression; sorbitan-4-flavor decoction and berberine, epicatechin, gallic acid, dihydromyricetin, and ellagic acid all had certain significant differences (P < 0.05), and the increase in concentration downregulated the RF factor level. Compared with the blank control group, sorbitan-4-flavor decoction, gallic acid, ellagic acid, gentiopicroside, and dihydromyricetin had no significant difference in TNF a cytokine expression; berberine and epicatechin at high concentrations downregulated the TNF-a factor level (P < 0.05). Figure 12-17 shown.

[0090] The study of the embodiments of the present invention shows that: the 4-flavor decoction of sorbitol and its effective ingredients have a certain inhibitory effect on the proliferation of synovial fibroblasts MH7A of patients with RA. In addition to the 4-flavor decoction of sorbitol, its effective ingredients such as gallic acid, dihydromyricetin, ellagic acid, berberine, etc. can cause apoptosis of MH7A cells. The expression levels of CRP, IL-6, ASO, and RF factors indicate that the 4-flavor decoction of sorbitol is one of the mechanisms of its anti-RA effect. It will lay a theoretical foundation for the use of 4-flavor decoction of sorbitol for the prevention and treatment of RA. Except for the high-dose administration of berberine and dihydromyricetin, the crude extract 4-flavor decoction of sorbitol and other effective activation agents have no significant effect on the factor levels of TNF-a and MMP-9, and the reduced concentration of the screened drugs may lead to insignificant efficacy.

[0091] The present invention mainly relates to a study on the effects of the 4-flavor decoction of sorbifolia and its main active ingredients on the proliferation and apoptosis of MH7A cells: human rheumatoid arthritis fibroblast-like synoviocytes (MH7A) are selected, and different concentrations of 12.5 mg / L, 25 mg / L, and 50 mg / L of sorbifolia-4 decoction; 5 mg / L of positive drug; 0.02 mg / L, 0.1 mg / L, and 0.5 mg / L of epicatechin; 0.1 mg / L, 0.5 mg / L of ellagic acid; 5 mg / L, 10 mg / L, and 20 mg / L of berberine; 10 mg / L, 20 mg / L, and 40 mg / L of dihydromyricetin; 250 mg / L, 500 mg / L, and 1000 mg / L of gentiopicroside; 3 mg / L, 5 mg / L, and 10 mg / L of gallic acid are screened; CCK-8 (Cell Counting Kit-8) method was used to detect the cell inhibition rate, flow cytometry was used to detect cell apoptosis, and ELISA was used to detect the expression levels of TNF-a, IL-6, RF, MMP-9, CRP and ASO in MH7A cells.

[0092] Results: There was no significant difference in the expression of ASO cytokines detected by ELISA between gentiopicroside and berberine; there were significant differences between 4-flavor decoction of sorbitan and epicatechin, gallic acid, dihydromyricetin, and ellagic acid. There was no significant difference in the expression of CRP cytokines between gentiopicroside and berberine; there were significant differences between 4-flavor decoction of sorbitan and epicatechin, gallic acid, dihydromyricetin, and ellagic acid. There was no significant difference in the expression of IL-6 cytokines between gentiopicroside; there were significant differences between 4-flavor decoction of sorbitan and berberine, epicatechin, gallic acid, dihydromyricetin, and ellagic acid. There was no significant difference in the expression of MMP-9 cytokines between 4-flavor decoction of sorbitan, epicatechin, gallic acid, dihydromyricetin, and ellagic acid. Only the increase of berberine concentration downregulated the level of MMP-9 factor; the increase of dihydromyricetin concentration promoted the expression of MMP-9 cytokines. There was no significant difference in RF cytokine expression with gentiopicroside, while there was significant difference with 4-flavor decoction of G. sorbifolia, berberine, epicatechin, gallic acid, dihydromyricetin, and ellagic acid.

[0093] Conclusion: The extract of Xanthoceras sorbifolia-4-flavor decoction and its index monomer components (gallic acid, gentiopicroside, epicatechin, dihydromyricetin, ellagic acid, berberine) can induce apoptosis of MH7A cells and alleviate inflammatory response. Its mechanism of action is related to the down-regulation of TNF-a, CRP, IL-6 and RF factor levels.

[0094] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which will not affect the essence of the present invention.

Claims

1. A 4-flavor decoction for the treatment of rheumatoid arthritis, characterized in that: The inhibition rate of MH7A cells was detected by CCK-8 method, cell apoptosis was detected by flow cytometry, and the expression levels of TNF-a, IL-6, RF, MMP-9, CRP, and ASO in MH7A cells were detected by ELISA.

2. The anti-rheumatoid arthritis effect of the 4-flavor decoction of Xanthoceras sorbifolia according to claim 1, characterized in that: The extract and index monomer components of the Xanthoceras sorbifolia-4-flavor decoction can induce apoptosis of MH7A cells and alleviate inflammatory response.

3. The anti-rheumatoid arthritis effect of the 4-flavor decoction of Xanthoceras sorbifolia according to claim 2, characterized in that: The index monomer components include: gallic acid, gentiopicroside, epicatechin, dihydromyricetin, ellagic acid, and berberine.

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