Cornus officinalis extract, its preparation method, and its application in the preparation of anti-inflammatory drugs

Abstract

This invention relates to the application of Cornus officinalis extract in the preparation of anti-inflammatory drugs, and provides a method for extracting and separating Cornus officinalis: using dried Cornus officinalis pulp as raw material, extracting with ethanol to obtain an extract, and recovering the solvent under reduced pressure to obtain an extract paste; purifying the extract using macroporous resin column, silica gel column, and gel column, etc., to obtain pure Cornus officinalis extract, which was identified as 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene. Experiments show that this invention can inhibit the secretion of NO in LPS-stimulated RAW264.7 cells, and inhibit the secretion of TNF-α and IL-6, as well as their mRNA and IL-1. β It exerts its anti-inflammatory effect by regulating mRNA expression levels and inhibiting the phosphorylation levels of P38, ERK, and JNK in RAW264.7 cells, and also through the MAPK signaling pathway. It can be used to prepare anti-inflammatory drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a Cornus officinalis extract, its preparation method, and its application in the preparation of anti-inflammatory drugs. Background Technology

[0002] Inflammation is a complex defensive response of the body to external stimuli, and most diseases are related to it. Excessive inflammatory responses can lead to a series of complications. Macrophages are central cells regulating inflammatory responses, playing a crucial role in the occurrence, maintenance, and recovery of inflammation. Activation of pattern recognition receptors on macrophages can induce a series of intracellular signaling cascades, such as the NF-κB and MAPK signaling pathways. Activation of these pathways leads to the production of related inflammatory factors, such as nitric oxide (NO), interleukin-6 (IL-6), interleukin-1β (IL-1β), and TNF-α (tumor necrosis factor-α). These inflammatory factors can indirectly reflect the degree of inflammatory response.

[0003] This invention is the first to extract Cornus officinalis extract from Cornus officinalis (family Cornaceae), and further purify and separate a natural compound 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene, with the molecular formula C 15 H 18 O4, m / z: 262, belongs to the sesquiterpenoid class of compounds. Sesquiterpenoids exhibit diverse biological activities, including antitumor and anticardiovascular disease activities. Currently, research on 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene is limited. It has only been extracted and isolated from the root of the Miao medicine Alangium chinense (Lour.) Harms, and its effect on COX-2 has been verified. However, a systematic study of the anti-inflammatory activity of this compound has not been conducted. This invention delves into its mechanism of action, which is of certain value for the development of new anti-inflammatory drugs. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the present invention aims to provide a Cornus officinalis extract with significant anti-inflammatory effects, which can be used to prepare anti-inflammatory drugs.

[0005] The present invention also provides a method for preparing the above-mentioned Cornus officinalis extract and its application in the preparation of anti-inflammatory drugs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing Cornus officinalis extract, comprising the following steps:

[0008] 1) Using dried Cornus officinalis pulp as raw material, extract was obtained by immersion in ethanol-water solution, and solvent was recovered under reduced pressure to obtain extract paste; the extract paste was dissolved in methanol, loaded onto a D101 macroporous resin column, and eluted sequentially with 20% ethanol, 40% ethanol, 60% ethanol and 95% ethanol, and the 60% ethanol eluent was collected.

[0009] 2) The 60% ethanol eluent was mixed with silica gel and packed into a column. Gradient elution was performed with dichloromethane-methanol at a volume ratio of 30:1 to 2:1. Thin-layer chromatography was used for detection. Identical components were combined and the solvent was recovered under reduced pressure to obtain 17 components. The obtained components were labeled as Fr.1-1 to Fr.1-17 in ascending order of polarity.

[0010] 3) After mixing Fr.1-1 with silica gel, it was loaded onto a Flash column and eluted with a gradient of petroleum ether-ethyl acetate at a volume ratio of 15:1 to 1:1. Thin-layer chromatography was used for detection. Identical components were combined and the solvent was recovered under reduced pressure to obtain Fr.1-1-1, which is the extract of Cornus officinalis.

[0011] Specifically, in step 1), the volume fraction of the ethanol aqueous solution can be 70-80%. The ethanol aqueous solution is used for extraction 2-4 times at room temperature, each time for 6-8 days. The extracts are combined, and the solvent is recovered under reduced pressure to obtain the extract paste.

[0012] Furthermore, in step 2), gradient elution can be performed using dichloromethane-methanol at volume ratios of 30:1, 20:1, 10:1, 5:1, 3:1, and 2:1; in step 3), gradient elution can be performed using petroleum ether-ethyl acetate at volume ratios of 15:1, 12:1, 10:1, 5:1, and 1:1.

[0013] Further preferably, in step 3), Fr.1-1-1 can be dissolved, purified using an LH-20 gel chromatography column, eluted with methanol, detected by thin-layer chromatography, and the same components can be combined and the solvent recovered under reduced pressure to obtain the pure Cornus officinalis extract. Identification revealed that the pure Cornus officinalis extract is a sesquiterpene compound 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene, with the following structural formula:

[0014]

[0015] This invention provides a Cornus officinalis extract prepared using the aforementioned preparation method.

[0016] The present invention also provides the application of the Cornus officinalis extract in the preparation of anti-inflammatory drugs.

[0017] Specifically, in the aforementioned applications, the Cornus officinalis extract exerts its anti-inflammatory effect by inhibiting LPS-stimulated NO secretion in RAW264.7 cells, inhibiting the secretion and mRNA expression levels of TNF-α and IL-6, as well as IL-1β mRNA expression levels, and inhibiting the phosphorylation levels of P38, ERK, and JNK in RAW264.7 cells. Furthermore, the Cornus officinalis extract exerts its anti-inflammatory effect by inhibiting the MAPK signaling pathway.

[0018] The present invention also provides an anti-inflammatory drug comprising the aforementioned Cornus officinalis extract.

[0019] Furthermore, the aforementioned anti-inflammatory drugs are formulated into tablets, granules, pills, capsules, or injections using conventional pharmaceutical excipients in the art.

[0020] The present invention conducted the following verification experiments on the anti-inflammatory application of Cornus officinalis extract:

[0021] The effect of MTT assay on the viability of RAW264.7 cells was detected.

[0022] The secretion of NO in RAW264.7 cells after LPS stimulation was detected using a nitric oxide kit.

[0023] The effects of LPS stimulation on the secretion of inflammatory factors in RAW264.7 cells were detected by ELISA and RT-PCR.

[0024] Western blotting was used to analyze the phosphorylation levels of P38, ERK, and JNK proteins in RAW264.7 cells after LPS stimulation.

[0025] After verification through routine experiments, this invention revealed that Cornus officinalis extract can inhibit the secretion of NO in LPS-stimulated RAW264.7 cells, inhibit the secretion of TNF-α and IL-6 and their mRNA and IL-1β mRNA expression levels, and inhibit the phosphorylation levels of P38, ERK, and JNK in RAW264.7 cells. Furthermore, it can inhibit the overactivation of the MAPK signaling pathway, thus exerting an anti-inflammatory effect. This indicates that Cornus officinalis extract possesses anti-inflammatory properties and can be used in the preparation of anti-inflammatory agents or drugs, showing potential value in the development of novel anti-inflammatory drugs.

[0026] This invention relates to the preparation and application of Cornus officinalis extract, specifically the application of 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene in the preparation of anti-inflammatory drugs. Compared with the prior art, the beneficial effects of this invention are as follows:

[0027] Currently, there are no reports on the anti-inflammatory mechanism of Cornus officinalis extract. This invention analyzes the effects of 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene on the secretion of pro-inflammatory factors, the expression of iNOS and COX-2 proteins, and the MAPK signaling pathway of LPS-induced RAW264.7 cells to determine the significance and mechanism of the compound's effect on anti-inflammatory activity. The experimental results show that 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene has significant anti-inflammatory activity and exerts its effect through the MAPK signaling pathway, and can be used to prepare anti-inflammatory drugs. Attached Figure Description

[0028] Figure 1 The compound 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene 1 H spectrum (400MHz, CD3OD);

[0029] Figure 2 The compound 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene 13 C spectrum (400MHz, CD3OD);

[0030] Figure 3 The effect of compound 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene on the cell viability of RAW264.7 cells; Note: Compared with the control group, **P<0.01, labeled as compound 18 in the figure, and the same applies below;

[0031] Figure 4 The effect of compound 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene on NO secretion in RAW264.7 cells stimulated by LPS; Note: Compared with the blank group, the figure shows the effect of compound 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene on NO secretion in RAW264.7 cells stimulated by LPS. ### P<0.001; compared with the LPS group, *** P<0.001;

[0032] Figure 5 The effects of compound 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene on the levels of TNF-α (A) and IL-6 (B) secreted by LPS-induced RAW264.7 cells and the expression of TNF-α (C), IL-6 (D), and IL-1β mRNA (E); Note: Compared with the blank group, ### P<0.001; compared with the LPS group, * P<0.05, ** P<0.01, *** P<0.001;

[0033] Figure 6 The image shows the effect of compound 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene on the protein bands (left) and protein expression (right) of iNOS and COX-2 induced by LPS in RAW264.7 cells; Note: Compared with the control group, ### P<0.001; compared with the LPS group, * P<0.05, ** P<0.01, *** P<0.001;

[0034] Figure 7 The image shows the effect of compound 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene on the protein bands (left) and phosphorylated protein expression levels (right) of P38, ERK, and JNK in RAW264.7 cells stimulated by LPS. Note: Compared with the control group:### P<0.001; compared with the LPS group: *** P<0.001. Detailed Implementation

[0035] To make the technical objectives, technical solutions, and beneficial effects of the present invention clearer, the technical solutions of the present invention will be further described below in conjunction with specific embodiments. However, the embodiments are intended to explain the present invention and should not be construed as limiting the present invention.

[0036] Where specific techniques or conditions are not detailed in the embodiments or experiments, they shall be carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0037] In the following examples, the dried fruits of Cornus officinalis used as raw materials were collected from Luanchuan County, Luoyang City, Henan Province. Unless otherwise specified, the concentrations of ethanol and methanol are volume concentrations. Room temperature refers to 25±5℃.

[0038] Example 1

[0039] A method for preparing a Cornus officinalis extract, which specifically includes the following steps:

[0040] 1) Using 20 kg of dried Cornus officinalis pulp as raw material, it was extracted with 75% ethanol aqueous solution. The extraction was carried out three times at room temperature for 7 days each time. The extracts were combined and the solvent was recovered under reduced pressure to obtain an extract paste. The extract paste was dissolved in methanol, loaded onto a D-101 macroporous resin column, and eluted sequentially with 20% ethanol, 40% ethanol, 60% ethanol and 95% ethanol. The 60% ethanol eluent was collected.

[0041] 2) The 60% ethanol eluent was mixed with silica gel and packed into a column. Gradient elution was performed using dichloromethane-methanol solutions at volume ratios of 30:1, 20:1, 10:1, 5:1, 3:1, and 2:1. Thin-layer chromatography was used for detection. Identical fractions were combined, and the solvent was recovered under reduced pressure, yielding 17 fractions. These fractions were labeled Fr.1-1 to Fr.1-17 according to their polarity from lowest to highest.

[0042] 3) The first component Fr.1-1 was mixed with silica gel and loaded onto a Flash column. It was eluted with a gradient of petroleum ether-ethyl acetate at volume ratios of 15:1, 12:1, 10:1, 5:1, and 1:1. Thin-layer chromatography was used for detection. Components with the same composition were combined and the solvent was recovered under reduced pressure to obtain Fr.1-1-1.

[0043] Fr.1-1-1 was dissolved in methanol, purified by LH-20 gel chromatography column, eluted with methanol, detected by thin-layer chromatography, and the same components were combined and the solvent was recovered under reduced pressure to obtain 21 mg of pure Cornus officinalis extract.

[0044] The structure of the pure Cornus officinalis extract prepared above was identified using various spectroscopic techniques, confirming that it is the compound 181-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene, as detailed below:

[0045] Instruments and materials: 1 H, 13 The 10⁻⁶ C NMR spectra were determined using a Bruker am-500MHz NMR spectrometer. TMS was used as an internal standard. See the spectrum below. Figure 1 and Figure 2 The spectral results and structural formulas are as follows:

[0046]

[0047] 1 ¹H-NMR (deuterated methanol, 400MHz) δ H :7.43(1H,s,H-3),7.17(1H,s,H-6),5.90(1H,s,H-9),2.22(3H,s,H-11 ),3.28(1H,m,H-12),1.26(6H,t,J=6.9Hz,H-13,14),1.44(3H,s,H-15). 13 C-NMR (deuterated methanol, 100MHz) δ C :207.29(C-1),147.83(C-2),129.28(C-3),124.77(C-4),159.12(C-5),113.36(C-6),121.60(C-7),165.9 3(C-8),115.91(C-9),77.30(C-10),16.08(C-11),30.06(C-12),22.39(C-13),22.73(C-14),33.16(C-15).

[0048] The compound 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene prepared in Example 1 above was subjected to the following anti-inflammatory test.

[0049] Example 2

[0050] Application Trial

[0051] I. Experimental Cells

[0052] RAW264.7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0053] II. Instruments and Reagents

[0054] Analytical balance (Sartorius Scientific Instruments Ltd.);

[0055] Full-wavelength microplate reader (Thermo Fisher, Model 1510);

[0056] Carbon dioxide incubator (Thermo Scientific, model 3111);

[0057] Centrifuge (Shanghai Surgical Instrument Factory, Model 800);

[0058] Multicolor fluorescence, chemiluminescence and visible light imager (ProteinsiMple FluorcheM Q);

[0059] Miniature benchtop refrigerated centrifuge (D-37520Thermo);

[0060] Electrophoresis apparatus (EPS600 Tanon);

[0061] Mini-Protean Tetra System (BIO-RAD)

[0062] Gradient PCR instrument (MyCycler™ Thermal Cycler);

[0063] Real-time quantitative PCR (Thermo Fisher);

[0064] Inverted microscope (Nikon ECLiPES TS100);

[0065] 96-well, 24-well, and 6-well cell culture plates (Corning);

[0066] DMEM medium (Solarbio);

[0067] Penicillin-streptomycin mixture (Solarbio);

[0068] Australian fetal bovine serum (FBS, Gibco);

[0069] Lipopolysaccharide (LPS, Sigma);

[0070] PBS buffer (Boster Biological);

[0071] Thiazol blue (MTT, Hualan Chemical);

[0072] Dimethyl sulfoxide (DMSO, Shanghai Maclean Biotech Co., Ltd.)

[0073] RIPA cell lysis buffer (weak) (Beyotime Biotechnology);

[0074] Nitric oxide reagent kit (Beyotime Biotechnology);

[0075] Trizol Reagent (Kangwei Century Biotechnology Co., Ltd.);

[0076] PCR primers (Thermo Fisher);

[0077] Prime Script TMRT reagent kit with Gdna Eraser kit (TaKaRa);

[0078] Green Pro Taq HS Premixed qPCR Kit (with Rox) (Aikerui Biotechnology Co., Ltd.);

[0079] BCA Protein Quantitative Kit (Solarbio);

[0080] Protease phosphatase inhibitor (Solarbio);

[0081] PMSF protease inhibitor (Solarbio);

[0082] 5× Protein Loading Buffer (Solarbio);

[0083] Skim milk powder (Amresco);

[0084] PVDF membrane (Solarbio);

[0085] Mouse TNF-α ELISA kit;

[0086] Mouse IL-6 ELISA kit;

[0087] 10×TBST buffer (Wuhan Sewell Biotechnology Co., Ltd.);

[0088] P38, p-P38, ERK, p-ERK, JNK, p-JNK, β-actin primary antibody (Proteintech);

[0089] Horseradish peroxidase-labeled secondary antibody (Cell signaling technology);

[0090] ECL luminescent liquid (Solarbio).

[0091] III. Test Methods

[0092] (1) MTT assay for cell viability

[0093] In the experiment, RAW264.7 cells were cultured in DMEM medium (hereinafter referred to as DMEM complete medium) containing 10% fetal bovine serum and 1% penicillin and streptomycin, at 37°C and 5% CO2. The specific method is as follows:

[0094] Cells in the logarithmic growth phase were taken at a concentration of 1.5 × 10⁻⁶. 5 Each well was seeded with 100 μL of 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene at concentrations of 200, 100, 50, 25, and 12.5 μM, respectively, and incubated for 24 h in a constant temperature incubator. In the experimental group, each well was supplemented with 10 μL of 100, 50, 25, and 12.5 μM of 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene, respectively. The blank group was given an equal volume of DMEM complete medium. Six replicates were considered as one group, and the wells were incubated for another 24 h in a constant temperature incubator. Under light-protected conditions, 10 μL of MTT solution (5 mg / mL) was added to each well, and the wells were incubated at 37 °C in a 5% CO2 incubator. After 4 h, the supernatant was discarded, and 100 μL of DMSO was added. After incubation at 37 °C for 10 min, the absorbance was measured (490 nm).

[0095] (2) Measurement of inflammatory factors (NO)

[0096] RAW264.7 cells in logarithmic growth phase were stored at a cell density of 1×10⁻⁶. 6 Cells were seeded per well in 24-well plates, with a blank control group, a model LPS group, and an experimental group. The LPS group was given LPS at a final concentration of 1 μg / mL. In the experimental groups, 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM of 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene were added to DMEM complete medium, respectively. After 1 h, LPS was added to a concentration of 1 μg / mL, and the plates were cultured for 24 h. The supernatant was collected and centrifuged. NO production was detected using a NO kit. The experiment was repeated three times.

[0097] IC 50 = (Model - Dosage) / (Model - Blank) * 100

[0098] (3) Measurement of inflammatory factors (TNF-α and IL-6)

[0099] Cells were treated using the same method described above, with a blank group, a model LPS group, and an experimental group. The LPS group was given LPS at a final concentration of 1 μg / mL. In the experimental groups, 100 μM, 50 μM, and 25 μM of 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene were added to DMEM complete medium, respectively. After 1 h, LPS was added to a concentration of 1 μg / mL, and the cells were cultured for 24 h. The supernatant was collected and centrifuged. The cell supernatant was collected. The levels of inflammatory factors IL-6 and TNF-α in the cell supernatant were detected according to the instructions of the Mouse TNF-α ELISA kit and the Mouse IL-6 ELISA kit.

[0100] (4) RT-PCR method to examine the effect of inflammatory factor mRNA expression

[0101] RAW264.7 cells were grown at a density of 1×10⁻⁶ cells. 7 Cells were seeded per well in a 6-well plate, with 2 mL of the solution in each well. The cells were incubated in a 37°C, 5% CO2 incubator for 24 h. The supernatant was discarded, and the blank group, LPS group and experimental group were set up in the same grouping method as in (2). After incubation in the incubator for 24 h, the supernatant was discarded, and the cells were washed twice with PBS.

[0102] 1) Total RNA extraction was performed as follows: First, add 1 mL of Trizol reagent to each well, pipette the mixture, and transfer it to an enzyme-free EP tube. Lyse the mixture on ice for 5 min. Then, add 200 μL of chloroform to each tube, mix well, and incubate on ice for 3 min. Centrifuge at 12000 rpm for 15 min at 4°C. Transfer an appropriate amount of the RNA-containing supernatant to a new enzyme-free EP tube, add 250 μL of isopropanol, mix well, and incubate on ice for 10 min. Centrifuge at 12000 rpm for 15 min at 4°C, discard the supernatant, and retain the RNA precipitate at the bottom of the tube. Wash the precipitate with 1 mL of 75% ethanol to remove residual isopropanol. Centrifuge at 12000 rpm for 5 min at 4°C, discard the supernatant, and dry at room temperature for 5 min. Add 40 μL of enzyme-free water, mix well, and store at -80°C. Finally, RNA purity and concentration were tested using Nanodrop 2000, and enzyme-free water was added to bring the RNA concentration to approximately 500 ng / μL.

[0103] 2) Removal of gDNA: X (RNA volume) = 1000 ng / RNA concentration (ng / μL). The mixed reaction system is shown in Table 1.

[0104] Table 1. Removal of gDNA

[0105]

[0106] 3) Preparation of cDNA: The mixed reaction system is shown in Table 2.

[0107] Table 2 Reverse Transcription System

[0108]

[0109] 4) PCR amplification

[0110] The primer concentration was 100 μM, and the primer sequences for the relevant genes are shown in Table 3 below.

[0111] Table 3 Primer sequences

[0112]

[0113] The PCR reaction system is shown in Table 4 below:

[0114] Table 4 PCR amplification system

[0115]

[0116] After thoroughly mixing the PCR reaction mixture as described above, transfer it to 8-tube strips (10 μL / tube). Place the strips in a 7500 Real-Time PCR System for PCR amplification. Relative expression levels are determined according to 2... -△△Ct The relative expression intensity of the primers being tested is calculated using the following formula.

[0117] Expression intensity of the primer being tested = optical density of the primer being tested / optical density of GAPDH

[0118] (5) Western blot method to detect the expression of related proteins

[0119] 1) RAW264.7 cells were cultured at a cell density of 6 × 10⁻⁶. 5 Cells were seeded per well in a 6-well plate, with 2 mL of the solution in each well. The cells were incubated in a 37°C, 5% CO2 incubator for 24 h. The supernatant was discarded, and the blank group, LPS group, and experimental group were set up in the same grouping method as in (2). After incubation for 24 h, the cells were collected and washed twice with PBS.

[0120] 2) Extraction of total cellular protein:

[0121] Add 200 μL of total protein lysis buffer (RIPA cell lysis buffer: PMSF protease inhibitor: protease phosphatase inhibitor = 100:1:1) to the cells, mix well, and incubate on ice for 10 min. Centrifuge for 10 min (4℃, 12000 rpm), collect the supernatant, and transfer the total protein to an EP tube. Add 1 / 4 volume of protein loading buffer and mix well. Incubate at 100℃ for 5 min to denature the protein, then store at -20℃.

[0122] 3) Determination of protein concentration:

[0123] Dilute the protein sample (2 μL / tube) in the 96-well plate 10-fold with PBS, with 3 replicates per sample. Add 200 μL of working solution (BCA:copper reagent = 50:1) to each well, mix well, incubate at 37°C for 30 min, and then measure the absorbance (562 nm) using a microplate reader to calculate the protein concentration. Calculate the loading volume of protein, with a loading amount of 30 μg per protein sample.

[0124] 4) SDS-PAGE:

[0125] Table 5. Proportioning System for Separating Gel and Stacking Gel

[0126]

[0127]

[0128] Prepare the separating gel according to the table above. Add isopropanol solution to remove air bubbles, and allow it to solidify at 37°C. Pour off the isopropanol, rinse with distilled water, and blot dry. Prepare the stacking gel, insert a comb, and allow it to solidify at a constant temperature. Pour 1× protein electrophoresis buffer into the electrophoresis tank, and add protein sample (30 μg) and marker (2 μL) to the sample wells. After sample addition, electrophoresis for 30 min (80 V) until the bands appear at the boundary between the stacking and separating gels, and then electrophoresis for another 60 min (120 V).

[0129] The 5× protein electrophoresis buffer was prepared by dissolving 15.15g Tris-base, 93.85g glycine, and 5g sodium dodecyl sulfate (SDS) in 900ml of deionized water and bringing the volume to 1L; the 1× protein electrophoresis buffer was prepared by mixing 400ml of deionized water with 100ml of 10× protein electrophoresis buffer and bringing the volume to 1L.

[0130] 5) Immunoblot development

[0131] ① Transfer: After electrophoresis, fix the electrodes in the transfer tank in the following order: positive electrode—sponge pad—filter paper—PVDF membrane—gel—filter paper—sponge pad—negative electrode. Add 1× transfer buffer for transfer. Voltage 72V, time 60min.

[0132] 10× transfer buffer is prepared by dissolving 30.3g Tris-base and 142.6g glycine in 900mL of deionized water and bringing the volume to 1L; 1× transfer buffer is prepared by mixing 350mL of deionized water with 50mL of 10× transfer buffer and bringing the volume to 100mL of methanol.

[0133] ② Seal: Seal in 5% skim milk powder for 2 hours.

[0134] ③ Incubation with primary antibody: According to the marker instructions, determine the location of the target protein and cut it. Then incubate the band with the corresponding primary antibody at 4°C overnight.

[0135] ④ Washing: After incubation with primary antibodies (iNOS and COX-2 protein, P38, ERK, JNK protein and its phosphorylated protein), wash the protein bands 5 times in TBST solution for 5 minutes each time.

[0136] ⑤ Incubate with secondary antibody: Based on the source of the primary antibody, administer the corresponding secondary antibody and incubate at room temperature for 1-2 hours.

[0137] ⑥ Washing: Same as ④, i.e., wash the protein bands 5 times in TBST solution, 5 minutes each time.

[0138] ⑦ Development Analysis: ECL luminescence solution was used. The developer was prepared according to the instructions (prepare fresh each time, and avoid light). Excess TBST solution was blotted off the bands with filter paper, and the developer was evenly spread onto the bands. Development and analysis were performed using a multicolor fluorescence, chemiluminescence, and visible light imager. The band grayscale was quantitatively analyzed using Image software, with β-actin as an internal control.

[0139] IV. Statistical Processing

[0140] The experimental results were statistically analyzed using GraphPad Prism 7 software. Experimental data are expressed as mean ± SD. One-way ANOVA was used for comparisons among multiple groups, with P < 0.05 considered statistically significant.

[0141] V. Experimental Results

[0142] (1) Effects of the compound on the viability of RAW264.7 cells

[0143] The effect of 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene on the viability of RAW 264.7 macrophages in the concentration range of 12.5–200 μM was detected by the MTT assay. The results are as follows: Figure 3As shown, within the concentration range of 12.5–100 μM, 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene had no significant effect on cell viability. Therefore, the concentration range of 6.25–100 μM for 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene was selected for the next step of the experiment.

[0144] (2) Effects of the compound on NO secretion in RAW264.7 cells

[0145] Figure 4 The effects of the compound on NO secretion in RAW264.7 cells stimulated by LPS were presented. Figure 4 As shown, the NO release from the blank group was low, while LPS stimulation increased NO release (P<0.001). Compared to the LPS group, 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene reduced NO release from the cells (P<0.001, P<0.01). The figure shows that 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene exhibited good activity. Based on the NO content in the cell supernatant, the IC50 of 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene was calculated using SPSS software. 50 The value is 52.50 μM.

[0146] (3) Effects of the compound on LPS-induced secretion of pro-inflammatory factors in RAW264.7 cells

[0147] To further evaluate the anti-inflammatory effects of 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene, ELISA and RT-PCR were used to detect the compound's influence on the secretion of inflammatory factors by inflammatory cells. Figure 5As shown, compared with the control group, the LPS group significantly promoted the secretion of TNF-α and IL-6 by RAW264.7 cells (P<0.001, P<0.001), and the expression levels of TNF-α, IL-6 and IL-1β mRNA were significantly increased (P<0.001, P<0.001, P<0.001). Compared with the LPS group, after administration of 100 μM of 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene, the release of TNF-α and IL-6 was significantly reduced (P<0.001, P<0.001), and the expression levels of TNF-α, IL-6 and IL-1β mRNA were significantly decreased at the 100 μM dose (P<0.01, P<0.001, P<0.001). The above results suggest that this compound may exert its anti-inflammatory effect by inhibiting the expression of TNF-α, IL-6 and IL-1β mRNA in LPS-induced RAW264.7 cells, thereby affecting the release of TNF-α and IL-6.

[0148] (4) Effects of the compound on LPS-induced expression of iNOS and COX-2 proteins in RAW264.7 cells

[0149] The anti-inflammatory activity of 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene was assessed by Western blot analysis of iNOS and COX-2 protein expression in cells. Figure 6 As shown, compared with the control group, the expression levels of iNOS and COX-2 proteins were significantly increased after LPS stimulation (P<0.001, P<0.001). Treatment with 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene at concentrations of 100, 50, and 25 μM resulted in varying degrees of decrease in the expression levels of iNOS and COX-2 proteins, all of which showed highly significant differences after quantitative calculation (P<0.001, P<0.001). This indicates that the compound can exert an anti-inflammatory effect by inhibiting the expression of iNOS and COX-2 proteins induced by LPS in RAW264.7 cells.

[0150] (5) Effects of compounds on the MAPK signaling pathway

[0151] like Figure 7As shown, compared with the blank group, LPS stimulation significantly increased the phosphorylation levels of P38, ERK, and JNK in the MAPK signaling pathway, and the quantitative analysis also showed significant differences (P<0.001, P<0.001, P<0.001). After administration of different concentrations of 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene, compared with the model LPS group, it was found that the compound could inhibit the phosphorylation level of P38 at lower concentrations. At a dosage of 100 μM, the phosphorylation levels of P38, ERK, and JNK were significantly reduced. This indicates that 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene exerts its anti-inflammatory effect through the MAPK pathway.

[0152] In summary, this invention experimentally demonstrates that the compound 1-carbonyl-2,8-dihydroxy-11-oxabicyclo[4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene, extracted from Cornus officinalis, can inhibit the secretion of NO in LPS-stimulated RAW264.7 cells, inhibit the secretion of TNF-α and IL-6 and their mRNA and IL-1β mRNA expression levels, and inhibit the phosphorylation levels of P38, ERK, and JNK in RAW264.7 cells. This indicates that its anti-inflammatory effect is achieved by inhibiting the MAPK signaling pathway, and that it can inhibit the overactivation of the MAPK signaling pathway to exert its anti-inflammatory effect.

Claims

1. A method for preparing an extract of Cornus officinalis, characterized by, Comprising the following steps: 1) taking dried fruit pulp of Cornus officinalis as raw material, extracting with ethanol aqueous solution to obtain an extract solution, recovering the solvent under reduced pressure to obtain an extract infusion; dissolving the extract infusion with methanol, loading onto a macroporous resin column, and then eluting with 20% ethanol, 40% ethanol, 60% ethanol and 95% ethanol in sequence, and collecting the 60% ethanol elution part; 2) loading the 60% ethanol elution part onto a column after mixing with silica gel, gradient eluting with dichloromethane-methanol in a volume ratio of 30:1~2:1, detecting by thin layer chromatography, combining the same components, and recovering the solvent under reduced pressure to obtain 17 components; according to the polarity of the obtained components from small to large, they are marked as Fr.1-1 to Fr.1-17 in sequence; 3) mixing Fr.1-1 with silica gel and loading onto a Flash column, gradient eluting with petroleum ether-ethyl acetate in a volume ratio of 15:1~1:1, detecting by thin layer chromatography, combining the same components, and recovering the solvent under reduced pressure to obtain Fr.1-1-1; dissolving Fr.1-1-1 and purifying with an LH-20 gel chromatography column, eluting with methanol, detecting by thin layer chromatography, combining the same components, and recovering the solvent under reduced pressure to obtain a Cornus officinalis extract; The Cornus officinalis extract is 1-carbonyl-2,8-dihydroxy-11-oxabicyclo [4,4,1]germacra-2(3),4(5),6(7),8(9)-tetraene, and its structural formula is as shown below: ； In step 1), the volume fraction of the ethanol aqueous solution is 70-80%, and each time is 6-8 days. In step 2), gradient elution is performed with dichloromethane-methanol in a volume ratio of 30:1, 20:1, 10:1, 5:1, 3:1 and 2:1; and in step 3), gradient elution is performed with petroleum ether-ethyl acetate in a volume ratio of 15:1, 12:1, 10:1, 5:1 and 1:

1.

2. The method of claim 1, wherein the extract of Cornus officinalis is prepared by the steps of: In step 1), the ethanol aqueous solution is extracted at room temperature for 2-4 times.

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