Desmodium styracifolium extract as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of plant extracts, and discloses a desmodium styracifolium extract as well as a preparation method and application thereof. The herba desmodii styracifolii extract disclosed by the invention comprises quercetin, luteolin and kaempferol. The analysis method for chemical components of the desmodium styracifolium is established by utilizing a UPLC-Q-TOF-MS technology, the material basis of the desmodium styracifolium is clarified, the potential action mechanism of the desmodium styracifolium for treating inflammation is discussed by adopting a network pharmacology means, and verification is carried out by utilizing an in-vitro cell experiment; and a basis is provided for further systematic development of in-vivo pharmacodynamic research on inflammation treatment of desmodium styracifolium and reasonable use and clinical popularization of the medicine.

Description

Technical Field

[0001] The invention relates to the technical field of plant extracts, and in particular to a Herba Lysimachiae extract and a preparation method and application thereof. Background Art

[0002] Inflammatory response is the basis for many diseases. Currently, two major types of anti-inflammatory drugs, steroidal and non-steroidal, are mainly used clinically. Although they have significant effects, they often cause a series of complications after use. Traditional Chinese medicine has good efficacy and low toxicity and side effects. Its anti-inflammatory immune mechanism is to regulate the hypothalamus-pituitary-adrenal axis, inhibit the release of inflammatory mediators and the production of related factors, free radical substances such as NO. Traditional Chinese medicine has significant effects in anti-inflammatory and immune enhancement. It has been used in disease prevention and treatment since ancient times. The advantages of traditional Chinese medicine resources, small side effects and high safety have attracted widespread attention in the medical field at home and abroad.

[0003] Traditional Chinese medicine has many ingredients and different effects, and its metabolic process in the human body is more complicated. It is difficult to clarify its material basis and molecular mechanism of action in the body. Network pharmacology follows the TCM theory of holistic approach and syndrome differentiation and treatment, and can establish a research model for the multi-component, multi-target, and multi-pathway characteristics of traditional Chinese medicine. It can reveal the complex interaction network relationship between "drug-target-gene-disease" and systematically explore the efficacy and mechanism of drug action.

[0004] Desmodium styracifolium (Osb.) Merr. is the aerial part of the leguminous plant Desmodium styracifolium (Osb.) Merr., and is a commonly used traditional Chinese medicine in my country. It has the effects of removing dampness and jaundice, diuresis and relieving stranguria, and is used for jaundice, red urine, hot stranguria, stranguria with stones, painful urination, edema and oliguria. The chemical composition of traditional Chinese medicine is complex and diverse, and the absorption and metabolic process in the body is unclear. However, the current research on Desmodium styracifolium mainly focuses on the analysis of its components and the investigation of its extracts or single components on choleretic and diuretic effects. Therefore, clarifying the active ingredients of traditional Chinese medicine and identifying the target proteins targeted by the active ingredients can more accurately guide the quality control and clinical use of traditional Chinese medicine. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a Herba Lysimachiae extract and a preparation method and application thereof.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a Desmodium strychnifolium extract, comprising quercetin (MOL000098), luteolin (MOL000006), and kaempferol (MOL000422).

[0008] As a preferred embodiment of the Desmodium Styrax Extract of the present invention, it includes isovitexin (MOL002322), isorhamnetin (MOL000354), quercetin (MOL000098), chryseriol (MOL003044), kaempferol (MOL000422), luteolin (MOL000006), acacetin (MOL001689), and diosmetin (MOL002881).

[0009] As a further preferred embodiment of the Herba Lysimachiae extract of the present invention, it includes isovitexin, isorhamnetin, quercetin, chryseriol, kaempferol, luteolin, acacetin, diosmetin, soyasaponin I, astragaloside, hesperidin, nicotiflorin, vicenin-2, naringin or hesperidin, daucostero, stigmasterol glucoside. glucoside), Schaftoside, Isoschaftoside, Vicenin-1, Vicenin-3, Isoquercitrin, Astragalin, Trifolin, Isoorientin, Lupenone, Chlorogenicacid, Apigenin, Genistein, Daidzein, Ferulic acid, Caffeic acid, Vanillic acid, Protocatechuic acid, Salicylic acid, Daidzin.

[0010] The present invention adopts the UPLC-Q-TOF-MS method to comprehensively characterize the chemical components of the water extract of Desmodium strychnifolium, can simultaneously identify the active component compounds of the water extract of Desmodium strychnifolium, and clarifies the material basis of the water extract of Desmodium strychnifolium.

[0011] The identified compounds were screened and targets were acquired through multiple databases, and the network pharmacology method was used to construct the medicinal material-active ingredient-intersection target network diagram and target PPI network diagram. It was finally determined that isovitexin, isorhamnetin, quercetin, Chryseriol, kaempferol, luteolin, Acacetin, and Diosmetin are the key ingredients of Desmodium vulgare for treating inflammation, and TNF, AKT1, IL1B, TP53, CASP3, MMP9, PTGS2, BCL2, MYC, etc. are key targets.

[0012] In a second aspect, the present invention provides a method for preparing the Desmodium sibiricum extract, comprising the following steps:

[0013] (1) Take St. John's wort herb, dry it, and grind it to obtain coarse powder;

[0014] (2) weighing the coarse powder, soaking it in ethanol, and subjecting it to ultrasonic treatment;

[0015] (3) filtration, and ultrasonic extraction of the residue with ethanol; filtration again, and combining the two filtrates;

[0016] (4) The filtrate is concentrated under reduced pressure to a thick paste, and then dried under vacuum to obtain a Herba Lysimachiae extract.

[0017] As a preferred embodiment of the method for preparing the Desmodium Styrax extract of the present invention, in step (1), the drying temperature is 40° C. to 60° C., and the drying time is 1 h to 2 h.

[0018] As a preferred embodiment of the method for preparing the Desmodium Styracifolium extract of the present invention, in step (1), the pulverization is carried out by a pulverizer for 0.5 h to 1.5 h.

[0019] As a preferred embodiment of the method for preparing the Desmodium Styrax extract of the present invention, in step (2), the concentration of the ethanol is 65% to 75%; the mass volume ratio of the coarse powder to the ethanol is 1:(10 to 15); and the soaking time is 20 minutes to 1 hour.

[0020] As a preferred embodiment of the method for preparing the Desmodium sibiricum extract of the present invention, in steps (2) and (3), the power of the ultrasound is 800W to 1200W, the frequency is 30kHz to 50kHz, the temperature is 40°C to 60°C, and the time is 20min to 1h;

[0021] As a preferred embodiment of the method for preparing the Desmodium Styrax extract of the present invention, in step (3), the concentration of ethanol is 65% to 75%, and the volume is 500 mL to 700 mL; the time of ultrasonic extraction is 10 min to 30 min.

[0022] In a third aspect, the present invention uses the Desmodium sibiricum extract in the preparation of anti-inflammatory drugs.

[0023] The network pharmacology and cell experiment research results of the present invention show that the extract of Desmodium sibiricum may jointly play the role of treating inflammation through relevant targets and pathways in anti-inflammatory and immune regulation.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention uses UPLC-Q-TOF-MS technology to establish an analytical method for the chemical components of Desmodium sibiricum, clarifies its material basis, uses network pharmacology to analyze the main anti-inflammatory bioactive components of Desmodium sibiricum extract and its pharmacological mechanism, and explains the complex network interaction relationship between multiple targets and multiple components and multiple pathways. The potential mechanism of action for treating inflammation is explored, and preliminary verification is performed using in vitro cell experiments, providing a theoretical basis for further systematic in vivo pharmacodynamic research on Desmodium sibiricum for treating inflammation, as well as the rational use and clinical promotion of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the VENNY diagram of the corresponding targets and disease targets of the active ingredients of Desmodium strychnifolia.

[0027] Figure 2 This is the Chinese medicine-disease-target network diagram of Desmodium sibiricum;

[0028] Figure 3 It is the PPI network diagram of intersection targets and key targets;

[0029] Figure 4 Three-in-one graph for GO enrichment analysis;

[0030] Figure 5 It is a dual-axis diagram of KEGG enriched pathways;

[0031] Figure 6 It is a network diagram of disease-pathway-target-Gentlemannia glutinosa medicine;

[0032] Figure 7 Effect of Desmodium sibiricum extract on the survival rate of RAW264.7 cells ( n=3);

[0033] Figure 8 Effects of Herba Lysimachiae Extract on NO Release from RAW264.7 Cells Induced by LPS ( n=3); compared with the blank group, ***P<0.001; compared with the model group, # P<0.1, ## P<0.01, ### P < 0.001;

[0034] Fig. 9 Effect of Herba Lysimachiae Extract on IL-6 Secretion from RAW 264.7 Cells Induced by LPS ( n=3); compared with the blank group, ***P<0.001; compared with the model group, # P<0.1, ## P<0.01, ### P < 0.001;

[0035] Fig.10 Effects of Herba Lysimachiae Extract on IL-1β Secretion in RAW 264.7 Cells Induced by LPS ( n=3); compared with the blank group, ***P<0.001; compared with the model group, # P<0.1, ## P<0.01, ### P < 0.001;

[0036] Fig.11 Effects of Herba Lysimachiae Extract on TNF-α Secretion in RAW 264.7 Cells Induced by LPS ( n=3); compared with the blank group, ***P<0.001; compared with the model group, # P<0.1, ## P<0.01, ### P < 0.001;

[0037] Fig.12 Effects of Herba Lysimachiae Extract on the Expression of NF-κB Signaling Pathway Proteins in RAW 264.7 Cells Stimulated by LPS n=3); compared with the blank group, ***P<0.001; compared with the model group, # P<0.1, ## P<0.01, ### P < 0.001;

[0038] Fig.13Effects of Herba Lysimachiae Extract on the Expression of MAPK Signaling Pathway Proteins in RAW 264.7 Cells Induced by LPS n=3); compared with the blank group, ***P<0.001; compared with the model group, # P<0.1, ## P<0.01, ### P<0.001. DETAILED DESCRIPTION

[0039] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0040] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0041] Example 1: Determination of chemical components of Desmodium strychnifolium

[0042] (1) The Chinese medicine system pharmacology database and analysis platform TCMSP (https: / / tcmspw.com / tcmsp.php) and the NPAI natural product research and development platform were used to search for "Gentlemansia chinensis" as the keyword, but the Chinese medicine was not included.

[0043] (2) The applied chemistry database was searched using the keyword "Desmodium strychnifolium". The results showed that only 5 compounds of Desmodium strychnifolium were included.

[0044] (3) By searching relevant literature from 2008 to 2023, 520 compound information including flavonoids, alkaloids, terpenes, phenolic acids and volatiles were found.

[0045] (4) Preparation and analysis of Desmodium sibiricum extract

[0046] The herb was dried in a 50°C oven for 1.5 hours, and then crushed with a grinder for 1 minute. Weigh 60 g of the dried and crushed herb coarse powder (not sieved), add 720 mL of 65% ethanol, soak for 0.5 hours, extract with ultrasound (power 1000 W, frequency 40 kHz, 50°C) for 30 minutes, filter, add 65% ethanol 600 mL to the residue and extract with ultrasound for 20 minutes, filter, and combine the two filtrates. Concentrate under reduced pressure to a thick paste, vacuum dry to obtain the herb extract, store at -20°C for standby use.

[0047] In order to confirm the compounds in the extract of Desmodium strychnifolium, the extract of Desmodium strychnifolium was analyzed by ultra-performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) technology in combination with the literature search results. The quasi-molecular ion peaks and fragment peak information of each chromatographic peak were extracted. Combined with the reference substances and literature data, the compounds were analyzed and structurally identified. A total of 35 chemical components of Desmodium strychnifolium were identified (see Table 1).

[0048] The chromatographic conditions are as follows: the chromatographic column is Agilent 5TC-C18 (250 mm×4.6 mm, 5 μm), the mobile phase is methanol (A)-2.5 mmol / L ammonium acetate solution (B), gradient elution (0-20 min, 30%→40% A; 20-35 min, 40%→55% A; 35-45 min, 55%→70% A), the detection wavelength is 340 nm, the column temperature is 40°C; the flow rate is 0.3 mL·min-1. Injection volume: 2 μL.

[0049] The mass spectrometry conditions were as follows: the ion source was an electrospray ion source (ESI), in positive and negative ion modes: mass scanning range 50-1000 m / z; spray voltage 5500 V, -4500 V; nebulizer gas temperature 550°C; curtain gas 30 psi; auxiliary gas, nebulizer gas 50 psi, 60 psi; collision energy (CE) ±35 V; collision activation scan (CES) 15 V, 10 V.

[0050] Table 1 Chemical components of Desmodium sibiricum extract (UPLC-Q-TOF-MS)

[0051]

[0052]

[0053] Example 2: Analysis of components, targets and pathways of Desmodium strychnifolium based on network pharmacology

[0054] It includes the collection and screening of active ingredients and related targets of Desmodium sibiricum extract, the collection of disease targets, the construction of a traditional Chinese medicine-disease-target network, the construction of a key target PPI network for Desmodium sibiricum in treating inflammatory responses, and enrichment analysis for treating inflammatory responses.

[0055] (1) Collection and screening of active ingredients and related targets of Desmodium sibiricum

[0056] The above 35 related components were imported into the TCMSP database, and the oral availability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 were used as screening conditions, and a total of 8 effective components were screened. The specific information is shown in Table 2. A total of 179 targets corresponding to the effective components were screened using TCMSP, and the Uniprot database (https: / / www.uniprot.org) was used to convert the gene names of the screened targets.

[0057] Table 2 Information on the active ingredients of Desmodium strychnifolium

[0058] Mol ID Molecule Name MW OB(%) DL MOL002322 isovitexin 432.41 31.29 0.72 MOL000354 isorhamnetin 316.28 49.6 0.31 MOL000098 quercetin 302.25 46.43 0.28 MOL003044 Chryseriol 300.28 35.85 0.27 MOL000422 kaempferol 286.25 41.88 0.24 MOL000006 Luteolin 286.25 36.16 0.25 MOL001689 acacetin 284.28 34.97 0.24 MOL002881 Diosmetin 300.28 31.14 0.27

[0059] (2) Collection of disease targets

[0060] Using the OMIM, TTD, and GeneCards databases, we searched for inflammation targets using the keyword "inflammation" and removed duplicates to obtain 4512 disease targets. We drew a VENNY graph and obtained 137 intersection targets, see Figure 1 .

[0061] (3) Construction of the Herba Lysimachiae TCM-Disease-Target Network

[0062] The information of effective ingredients of traditional Chinese medicine, ingredients and disease intersection targets was integrated to construct an interaction relationship table, and the information was imported into Cytoscape 3.10.1 for visualization analysis to construct a "Gentlemansia strychnifolia traditional Chinese medicine-disease-target" network diagram, see Figure 2 Using CytoNCA auxiliary software and based on the degree obtained by analysis, the main key active ingredients were screened as quercetin (MOL000098), luteolin (MOL000006), and kaempferol (MOL000422).

[0063] (4) Construction of key target PPI network for treating inflammatory response with Desmodium sibiricum

[0064] The PPI network diagram of the intersection targets was constructed, and Centiscape 2.2 was used for auxiliary analysis. The network analysis was performed with "Closense", "Betweenness" and "Degree" as topological parameters, and the thresholds were "Closense>0.0041207783423973", "Betweenness>113.338235294117" and "Degree>33.3970588235294", respectively. Targets with topological parameters greater than the thresholds were screened out, and a total of 32 key targets were finally screened out. The specific information is shown in Table 3. The PPI network diagram of key targets was also constructed, see Figure 3 .

[0065] Table 3 Topological parameters related to key targets

[0066]

[0067]

[0068] (5) Enrichment analysis of Desmodium sibiricum in treating inflammatory response

[0069] Using DAVID to analyze the gene ontology, a total of 677 biological processes, 73 cellular components, and 123 molecular functions were obtained. The top ten items were visualized with a P ≤ 0.01 threshold. The results are shown in Figure 4 The biological processes mainly involve positive regulation of gene expression, negative regulation of apoptotic process, inflammatory response, etc.; the cell composition includes extracellular space, extracellular region, macromolecular complex, etc.; the molecular functions are mainly identical protein binding, enzyme binding, protein binding, etc. KEGG enrichment pathway analysis was performed on the intersection targets, and a total of 152 pathways were enriched. With P less than or equal to 0.01 as the screening condition, 20 pathways were selected for visualization analysis, see Figure 5 Enrichment analysis showed that pathways in cancer, lipid and atherosclerosis, prostate cancer, fluid shear stress and atherosclerosis, and hepatitis B may be important pathways for the treatment of inflammation by Desmodium strychnifolium. Cytoscape 3.10.1 was used to draw a disease-pathway-target-Desmodium strychnifolium network diagram, see Figure 6 .

[0070] Example 3: Study on the effect and mechanism of Desmodium sibiricum extract in inhibiting LPS-induced production of inflammatory factors in RAW264.7 cells

[0071] This includes the effects of Desmodium sibiricum water extract on LPS-induced NO release, inflammatory factor secretion, protein expression and signaling pathways in RAW264.7 macrophages.

[0072] (1) LPS-induced RAW264.7 macrophage model

[0073] 1) Cell culture and grouping

[0074] RAW267.4 cells were cultured in a 37°C, 5% CO2 incubator with DMEM complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. When the cells in the culture dish grew to cover 70%-80% of the culture dish, they were passaged or split. The cells were divided into a control group, a model group, and an experimental group with different ELP concentrations.

[0075] 2) Cell treatment

[0076] After 24 h of cell culture, the control group continued to culture with normal DMEM medium, and the experimental groups were added with different concentrations of ELP for incubation for 1 h. Except for the control group, LPS (final concentration of 1.5 μg / mL) was added to the model group and the experimental group for incubation for 30 min or 24 h for subsequent index detection.

[0077] (2) Effect of Desmodium sibiricum extract on LPS-induced survival rate of RAW264.7 cells

[0078] The CCK-8 method was used to examine the effect of the Desmodium Lysimachia extract prepared in Example 1 on the survival rate of LPS-induced RAW264.7 macrophages.

[0079] RAW264.7 cells were seeded in 96-well plates at 5 × 10 3 After 24 hours of culture, different concentrations of Herba Lysimachiae extract (40 μg / mL-320 μg / mL) were added to the experimental groups. Except for the control group, LPS (final concentration of 1 μg / mL) was added to each group for 24 hours, and then 10 μL of CCK8 solution (final concentration of 0.5 mg / mL) was added to each well and incubated at 37°C for 3 hours. The supernatant was discarded and 100 μL of DMSO was used to dissolve the crystals. The absorbance of each well was measured using an ELISA reader at a wavelength of 450 nm.

[0080] Results Figure 7 Compared with the blank control group, the extract of Desmodium sibiricum had no effect on the survival rate of RAW264.7 macrophages at the set concentrations (40μg / mL~280μg / mL).

[0081] (3) Effect of Desmodium sibiricum extract on NO release from RAW264.7 cells induced by LPS

[0082] The Griess method was used to investigate the effect of the Desmodium sibiricum extract prepared in Example 1 on the NO release from RAW 264.7 macrophages induced by LPS.

[0083] The cells were seeded in 24-well culture plates, with 25 × 10 4 , overnight culture, different concentrations of Desmodium sibiricum extract (40μg / mL-160μg / mL) pretreatment for 1h, except for the control group, LPS (final concentration of 1μg / mL) was added to each well and cultured for 24h, then the cell culture fluid was collected and stored at -20℃. 100μL culture fluid was added to each well of the 96-well plate, and then 100μL Griess reagent was added. After incubation at room temperature for 10min, the absorbance at 540nm was measured. Different concentrations of NaNO2 solution were gradiently prepared, the absorbance was measured at 540nm and a standard curve was drawn, and the NO concentration corresponding to the absorbance of each well was calculated based on the standard curve.

[0084] Results Figure 8 Compared with the blank control group, the NO level in the model group (LPS stimulation group) increased significantly (P<0.001), indicating that the RAW 264.7 cell inflammation model was successfully established. Compared with the model group, the NO content in the supernatant of cells treated with Desmodium sibiricum extract and LPS was significantly reduced (P<0.01, P<0.001) in a dose-dependent manner.

[0085] (4) Effects of Herba Lysimachiae Extract on LPS-induced IL-6, IL-1β and TNF-α secretion in RAW264.7 cells

[0086] The effect of the extract of Desmodium sibiricum prepared in Example 1 on the secretion of inflammatory factors IL-6, IL-1β and TNF-α by RAW264.7 cells induced by LPS was investigated by ELISA kit.

[0087] The collected culture medium was used as the sample to be tested. Set up standard wells and sample wells to be tested respectively, and perform the following operations according to the instructions of the ELISA kit: add the coating solution to the test plate and coat it overnight at 4°C. After washing the plate, add the working solution of the kit and block it at room temperature for 1 hour. After washing the plate, add the standard or sample to be tested, cover it with a plate sticker, and incubate it at room temperature for 2 hours. After washing the plate three times, add 100 μL of biotin-labeled antibody working solution to each well and incubate it at room temperature for 1 hour. After washing the plate again, add horseradish peroxidase-labeled avidin working solution to each well and incubate it at room temperature for 30 minutes. After washing the plate 5 times, add the substrate solution, protect it from light and color it for 5min-15min, then add 50 μL of the stop solution to each well, use an enzyme reader to measure the absorbance at a wavelength of 450nm, and calculate the concentration of inflammatory factors corresponding to the absorbance of each well according to the standard curve.

[0088] The experimental results showed that compared with the blank control group, the levels of inflammatory factors IL-6, IL-1β and TNF-α in the model group were significantly increased (P<0.001), and LPS promoted the secretion of inflammatory factors IL-6 and TNF-α by RAW264.7 cells; compared with the model group, the levels of inflammatory factors IL-6, IL-1β and TNF-α in the group treated with Desmodium sibiricum extract were significantly decreased (P<0.01, P<0.001), showing an inhibitory effect in a dose-dependent manner. Figure 9-11 .

[0089] (5) Effects of Desmodium sibiricum extract on LPS-induced iNOS, COX-2 and GAPDH protein expressions in RAW264.7 cells

[0090] Western blot was used to investigate the effects of Desmodium sibiricum extract on iNOS, COX-2 and GAPDH proteins in RAW264.7 cells induced by LPS.

[0091] RAW264.7 cells were seeded in 6 wells (2×10 5 The cells were plated in a 37°C, 5% CO2 incubator (100 μg / ml) culture plate, 3 mL per well, and 6 replicates per group. The grouping and administration were the same as (2). After 24 h of culture in a 37°C, 5% CO2 incubator, the culture medium was discarded, and the total RNA of each group of cells was extracted by Trizol method, the protein was quantified, and the Evo M-MLV premix was used for reverse transcription. The SYBR Green fluorescence method was used for quantitative PCR amplification and determination. The reaction system was: 2×SYBR Green QPCR Master Mix 10 μL, RNase Free H2O 3.7 μL, control fluorescent dye 0.3 μL, forward primer 2 μL, reverse primer 2 μL, reverse transcription product cDNA 2 μL. Reaction conditions: 95°C pre-denaturation for 3 min; 95°C denaturation for 5 s, 60°C annealing for 1 min, and then the fluorescence signal was collected, and repeated 40 cycles. The primer sequences are shown in Table 4.

[0092] Table 4 Primer sequences

[0093]

[0094]

[0095] RAW264.7 cells were seeded in 60 mm culture dishes at 2 × 10 5After 24 hours of culture, the experimental group was pretreated with different concentrations of ELP for 1 hour. Except for the control group, LPS (final concentration of 1 μg / mL) was added to each plate for incubation for 30 minutes or 24 hours. The cells were collected, centrifuged at 4000r / min and 4℃ for 10 minutes, the supernatant was discarded, a certain amount of RIPA lysis buffer was added, lysed in an ice bath for 20 minutes, centrifuged at 12000r / min and 4℃, and the supernatant was aspirated. The sample protein concentration was determined by Bradford method and quantified. 4× loading buffer containing bromophenol blue was added, heated at 95℃ for 8 minutes for denaturation, and stored at -20℃. After gel preparation, the sample was loaded, and after 1 hour of 45V constant voltage, the voltage was changed to 90V constant voltage for 40 minutes until bromophenol blue ran to the bottom of the separation gel. Ice bath electrotransfer membrane at 300mA constant current for 70min-100min, PVDF membrane was incubated with 5% skim milk powder blocking solution at room temperature for 1 hour, the corresponding primary antibody was added, and incubated overnight at 4℃. The PVDF membrane was washed 3 times with 1×TBST solution, 5 min each time. The secondary antibody was added, incubated for 1 h, and washed 3 times with 1×TBST solution, 15 min each time. The bands were incubated with ECL chemiluminescent colorimetric solution and exposed and developed using the TANON 5200 multi-channel chemiluminescent imaging system. The protein band images were analyzed by grayscale using Image J software and divided by the grayscale value of the corresponding internal reference protein β-actin / GAPDH / Sp1 to obtain the relative expression level of the protein to be tested.

[0096] The results are as follows Fig.12 As shown in the figure, compared with the blank group, the expression of iNOS, COX-2 and GAPDH proteins in the model group after LPS stimulation was significantly increased (P<0.01). Compared with the model group, the medium and high doses of Herba Lysimachiae extract in the group could significantly inhibit the expression of iNOS, COX-2 and GAPDH proteins in the cells, and the effect was dose-dependent. The experimental results showed that Herba Lysimachiae extract may exert anti-inflammatory effects by inhibiting the protein expression of iNOS, COX-2 and GAPDH in RAW264.7 cells induced by LPS.

[0097] (6) Effect of Desmodium sibiricum extract on TLR4 signaling pathway in RAW264.7 cells stimulated by LPS

[0098] Western blot was used to further investigate the effect of Desmodium sibiricum extract on the expression of TLR4_GAPDH and p-NF-κB p65 / NF-κB p65 related proteins of TLR4 signaling pathway in RAW264.7 cells induced by LPS.

[0099] The results are as follows Fig.12The results showed that compared with the blank control group, the expression of TLR4_GAPDH and p-NF-κB p65 / NF-κB p65 proteins in LPS-stimulated cells in the model group was significantly increased (P<0.001). Compared with the model group, the expression of p-NF-κB p65 / NF-κB p65 proteins in the medium and high dose groups of Desmodium sibiricum extract administration group was significantly decreased (P<0.01, P<0.001), showing an inhibitory effect. The experimental results showed that DG2 may exert anti-inflammatory effects by inhibiting the protein expression of TLR4_GAPDH and p-NF-κB p65 / NF-κB p65 in the MAPK signaling pathway of RAW264.7 cells induced by LPS.

[0100] In summary, the prediction results of network pharmacology show that the treatment of inflammation by Desmodium sibiricum is related to signaling pathways such as TNF, IL, and PI3K-Akt, which indicates that the effect of Desmodium sibiricum on inflammation may also involve cell apoptosis, etc. The results of in vitro anti-inflammatory tests show that the extract of Desmodium sibiricum can significantly inhibit the levels of NO, IL-6, IL-1β and TNF-α secreted by RAW264.7 cells induced by LPS, inhibit the expression of iNOS, COX-2 and GAPDH proteins, and downregulate the TLR4 signaling pathway, thereby exerting an anti-inflammatory effect. The present invention can provide a basis for the application of Desmodium sibiricum in anti-inflammatory drugs.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. 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 can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A Herba Lysimachiae extract, characterized in that Including quercetin, luteolin, and kaempferol.

2. The Herba Lysimachiae extract according to claim 1, characterized in that Including isorhamnetin, isorhamnetin, quercetin, kaempferol, luteolin, acacetin, and dioscorea lignin.

3. The Herba Lysimachiae extract according to claim 2, characterized in that Including isovitexin, isorhamnetin, quercetin, golden grass yellow, kaempferol, luteolin, acaciatin, diosgenin, soybean saponin I, astragalus total saponins, hesperidin, kaempferol-3-O-rutinoside, vecin-2, naringin or hesperidin, carotene, stigmasterol glucoside, schaftoside, isoschaftoside, vecin-1, vecin-3, isoquercitrin (glycoside), astragaloside, trifoliate bean glycoside, isoorientin (element), lupine ketone, chlorogenic acid, apigenin, genistein, daidzein, ferulic acid, caffeic acid, vanillic acid, protocatechuic acid, salicylic acid, and daidzein.

4. A method for preparing the Desmodium sibiricum extract according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Take St. John's wort herb, dry it, and grind it to obtain coarse powder; (2) weighing the coarse powder, soaking it in ethanol, and subjecting it to ultrasonic treatment; (3) filtration, and ultrasonic extraction of the residue with ethanol; filtration again, and combining the two filtrates; (4) The filtrate is concentrated under reduced pressure to a thick paste, and then dried under vacuum to obtain a Herba Lysimachiae extract.

5. The method for preparing the Desmodium Styracifolium extract according to claim 4, characterized in that: In step (1), the drying temperature is 40°C to 60°C, and the drying time is 1h to 2h.

6. The method for preparing the Desmodium Styracifolium extract according to claim 4, characterized in that: In step (1), the pulverization is carried out using a pulverizer for 0.5 h to 1.5 h.

7. The method for preparing the Desmodium Styracifolium extract according to claim 4, characterized in that: In step (2), the concentration of the ethanol is 65% to 75%; the mass volume ratio of the coarse powder to the ethanol is 1:(10 to 15); and the soaking time is 20 minutes to 1 hour.

8. The method for preparing the Desmodium Styracifolium extract according to claim 4, characterized in that: In steps (2) and (3), the power of the ultrasound is 800W to 1200W, the frequency is 30kHz to 50kHz, the temperature is 40°C to 60°C, and the time is 20min to 1h.

9. The method for preparing the Desmodium Styracifolium extract according to claim 4, characterized in that: In step (3), the concentration of ethanol is 65% to 75%, and the volume is 500 mL to 700 mL; the time of ultrasonic extraction is 10 min to 30 min.

10. Use of the Herba Lysimachiae extract according to any one of claims 1 to 3 in the preparation of anti-inflammatory drugs.

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