A new moroxydine trimer with anti-inflammatory activity and its preparation method and application

Abstract

The application discloses a new cinnabarinum acetyl dopamine trimer with anti-inflammatory activity. The application is based on the systematic and in-depth research on chemical components of cinnabaris, and the new cinnabarinum acetyl dopamine trimer compound is separated and purified by using a silica gel column, a reverse ODS column and a high performance liquid chromatograph in combination, and identified by nuclear magnetic resonance and mass spectrometry data. The compound separated by the application can reduce the levels of NO, IL-6, TNF-alpha and PGE2 in LPS-induced RAW264.7 macrophages to different degrees, indicating that the new compound has obvious anti-inflammatory activity.

Description

TECHNICAL FIELD

[0001] The present application relates to a new compound isolated from the exuviae, in particular to a new exuviae acetyl dopamine trimer with anti-inflammatory activity isolated from the exuviae, and a preparation method and use thereof, belonging to the technical field of medicine. BACKGROUND

[0002] The exuviae is the exuviae of Cryptotympana pustulata Fabricius, which is an important animal medicinal material in China. The exuviae is rich in acetyl dopamine, acetyl dopamine dimer, acetyl dopamine trimer, acetyl dopamine tetramer and other oligomers. The structure of the compounds is that the amino group on the dopamine side chain is acetylated to form a compound as a basic mother nucleus structure, and then the double hydroxyl on the benzene ring is polymerized with the carbon atom on the side chain of another mother nucleus to form N-acetyl dopamine polymers with different polymerization degrees, which have anti-tumor, anti-oxidation and other activities, and are important active substances in the exuviae. Exploring acetyl dopamine oligomers with strong activity from exuviae resources has positive significance and broad application prospect for the discovery of drug precursors. SUMMARY

[0003] The purpose of the present application is to further study the chemical composition of the exuviae, and to isolate and purify a new exuviae acetyl dopamine trimer with anti-inflammatory activity. Another purpose of the present application is to provide a preparation method and anti-inflammatory use of the new exuviae acetyl dopamine trimer.

[0004] Technical scheme: In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0005] A new exuviae acetyl dopamine trimer with anti-inflammatory activity, the structural formula of which is as follows:

[0006] .

[0007] As a preferred scheme, the new exuviae acetyl dopamine trimer with anti-inflammatory activity is prepared into tablets, capsules, injections, granules, drop pills, mixtures, oral liquids and the like by mixing acetyl dopamine trimer and a pharmaceutically acceptable carrier.

[0008] When the acetyl dopamine trimer provided by the present application is prepared into tablets, the acetyl dopamine trimer and lactose or corn starch are mixed uniformly, a lubricant magnesium stearate is added if necessary, the mixture is granulated, and then the tablets are pressed to prepare the tablets.

[0009] When the acetyl dopamine trimer provided by the present application is prepared into capsules, the acetyl dopamine trimer and the carrier lactose or corn starch are mixed uniformly, granulated, and then the capsules are filled to prepare the capsules.

[0010] The acetyldopamine trimer is mixed with diluent lactose or corn starch, granulated, dried, and formed into granules.

[0011] The acetyldopamine trimer is mixed with diluent lactose or corn starch, granulated, dried, and formed into granules.

[0012] The preparation method of the new acetyldopamine trimer with anti-inflammatory activity provided by the application comprises the following steps:

[0013] (1) Take a proper amount of periostracum cicadae medicinal materials, place them in a round-bottom flask, add ethanol, reflux extract, filter, combine the filtrates, place them in a water bath after being concentrated under reduced pressure, and freeze-dry after the ethanol is volatilized to obtain an extract;

[0014] (2) Dissolve the extract in step (1) in ethanol, mix with silica gel, place it in a water bath to volatilize the solvent, and perform silica gel column chromatography, gradient elution with different volume ratios of chloroform and methanol, collect the effluent, reduce pressure and concentrate, monitor with thin layer chromatography, and combine the same fractions to obtain a total of 47 fractions, namely Fr.1-Fr.47;

[0015] (3) Dissolve the Fr.12 fraction in methanol, mix with silica gel, place it in a water bath to volatilize the solvent, perform silica gel column chromatography, and gradient elution with different volume ratios of dichloromethane and methanol, respectively, collect the effluent, reduce pressure and concentrate, monitor with thin layer chromatography, and combine the same fractions to obtain a total of 48 fractions, namely Fr.12-1-Fr.12-48;

[0016] (4) Dissolve the Fr.12-32 fraction in methanol, uniformly load it into an ODS column, and gradient elution with different volume ratios of acetonitrile and water, respectively, collect the effluent, reduce pressure and concentrate, monitor with thin layer chromatography, and combine the same fractions to obtain a total of 19 fractions, namely Fr.12-32-1-Fr.12-32-19;

[0017] (5) Split the Fr.12-32-10 fraction, use an APS10 analytical high-performance liquid chromatograph for separation and purification, use a C18 chromatographic column, and use a mobile phase of acetonitrile and water in a volume ratio of 22.5:77.5, and finally obtain the acetyldopamine trimer compound.

[0018] As a preferred scheme, the preparation method of the new acetyldopamine trimer with anti-inflammatory activity described above comprises the following steps:

[0019] (1) Take a proper amount of periostracum cicadae medicinal materials, place them in a round-bottom flask, add 10 times the amount of 95% ethanol, reflux extract twice, 3h each time, filter, combine the filtrates, place them in a water bath after being concentrated under reduced pressure, and freeze-dry after the ethanol is volatilized to obtain a dry extract;

[0020] (2) The dry extract of step (1) is dissolved with ethanol, 60-100 mesh silica gel is added and mixed, and then the solvent is evaporated on a water bath to obtain a sample for use, 100-200 mesh silica gel is soaked in chloroform and methanol at a volume ratio of 20:1 for 3-5 hours, and then the glass column is filled with the silica gel after ultrasonic degassing, the prepared sample is uniformly loaded into the glass column, and gradient elution is performed with chloroform and methanol at a volume ratio of 20:1, 15:1, 12:1, 10:1, 8:1, 5:1, 3:1 and 1:1, respectively, the effluent is collected, concentrated under reduced pressure, monitored by thin layer chromatography, and the same fractions are combined, and 47 fractions, i.e., Fr.1-Fr.47, are obtained;

[0021] (3) The Fr.12 fraction is dissolved with methanol, 60-100 mesh silica gel is added and mixed, and then the solvent is evaporated on a water bath to obtain a sample for use, 100-200 mesh silica gel is soaked in chloroform and methanol at a volume ratio of 20:1 for 3-5 hours, and then the glass column is filled with the silica gel after ultrasonic degassing, the prepared sample is uniformly loaded into the glass column, and gradient elution is performed with dichloromethane and methanol at a volume ratio of 20:1, 15:1, 10:1, 5:1 and 1:1, respectively, the effluent is collected, concentrated under reduced pressure, monitored by thin layer chromatography, and the same fractions are combined, and 48 fractions, i.e., Fr.12-1-Fr.12-48, are obtained;

[0022] (4) The Fr.12-32 fraction is dissolved with methanol and uniformly loaded into a 0.5m*0.1m ODS column, and gradient elution is performed with acetonitrile and water at a volume ratio of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 and 10:0, respectively, the effluent is collected, concentrated under reduced pressure, monitored by thin layer chromatography, and the same fractions are combined, and 19 fractions, i.e., Fr.12-32-1-Fr.12-32-19, are obtained;

[0023] (5) The Fr.12-32-10 fraction is separated and purified by using an APS10 analytical high performance liquid chromatograph, a HYPERSIL BDS C18 column with a size of 250mm*10mm, and a mobile phase of acetonitrile and water at a volume ratio of 22.5:77.5, and finally an acetyl dopamine trimer compound is obtained.

[0024] Advantages: The new cicada slough acetyl dopamine trimer with anti-inflammatory activity provided by the application has the following advantages compared with the prior art:

[0025] The application obtains the optimal preparation process by systematically and in-depth studying the chemical components of cicada slough, using silica gel column, reverse ODS column and high performance liquid chromatograph for systematic separation and purification, and identifies the new cicada slough acetyl dopamine trimer compound separated from the cicada slough by nuclear magnetic and mass spectrum data.

[0026] The compound separated by the application can reduce the levels of NO, IL-6, TNF-alpha and PGE2 in LPS-induced RAW264.7 macrophages to different degrees, indicating that the new compound has obvious anti-inflammatory activity. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The structural schematic diagram of the new cecropin acetyl dopamine trimer (T1) provided by the application is shown.

[0028] Figure 2 The ECD map is shown.

[0029] Figure 3 The acetyl dopamine trimer (T1) is shown.

[0030] Figure 4 The standard curve of the NO content is shown.

[0031] Figure 5 The acetyl dopamine trimer (T1) IC 50 Trend chart and influence on cell proliferation activity. Note: compared with the control group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0032] Figure 6 The acetyl dopamine trimer (T1) has an influence on the NO secretion amount of LPS-induced RAW264.7 cells. Note: compared with the model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0033] Figure 7 The acetyl dopamine trimer (T1) has an influence on the inflammatory factor secretion amount of LPS-induced RAW264.7 cells at a concentration of 300 mM. (a) IL-6 content, (b) TNF-alpha content, (c) PGE2 content. Note: compared with the model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0034] The application can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific material proportions, process conditions and their results described in the examples are only used to illustrate the application, and should not and will not limit the application described in detail in the claims.

[0035] Example 1

[0036] The application provides a preparation method of the new cecropin acetyl dopamine trimer with anti-inflammatory activity, which comprises the following steps:

[0037] (1) 10 kg of the crushed Periostracum Cicadae was taken, placed in a 5 L round bottom flask, and 10 times the amount of 95% ethanol was added. The mixture was extracted by reflux for 2 times, 3 hours each time. The filtrate was combined, concentrated under reduced pressure, and then placed in a water bath to heat. After the ethanol evaporated, the mixture was freeze-dried to obtain 277 g of dry extract.

[0038] (2) 270 g of the dry extract was dissolved in a small amount of ethanol, mixed with 270 g of 60-100 mesh silica gel, and then placed in a water bath to evaporate the solvent. 3 kg of 100-200 mesh silica gel was soaked in 5 L of chloroform:methanol (20:1) for 5 hours, and then loaded into a glass column (1 m x 0.15 m) after ultrasonic degassing. The mixed sample was then uniformly loaded into the silica gel column. The column was eluted with chloroform:methanol at a gradient of 20:1, 15:1, 12:1, 10:1, 8:1, 5:1, 3:1, and 1:1. The effluent was collected in 500 mL fractions, concentrated under reduced pressure, and monitored by thin layer chromatography. Fractions with the same elution time were combined. A total of 47 fractions were obtained, i.e., Fr. 1-Fr. 47.

[0039] (3) Fr. 12 (80 g) was dissolved in a small amount of methanol, mixed with 80 g of 60-100 mesh silica gel, and then placed in a water bath to evaporate the solvent. 800 g of 100-200 mesh silica gel was soaked in 1 L of chloroform:methanol (20:1) for 5 hours, and then loaded into a glass column (0.8 m x 0.15 m) after ultrasonic degassing. The dissolved sample was then uniformly loaded into the glass column. The column was eluted with dichloromethane:methanol at a gradient of 20:1, 15:1, 10:1, 5:1, and 1:1. The effluent was collected in 250 mL fractions, concentrated under reduced pressure, and monitored by thin layer chromatography. Fractions with the same elution time were combined. A total of 48 fractions were obtained, i.e., Fr. 12-1-Fr. 12-48.

[0040] (4) Fr. 12-32 (4 g) was dissolved in a small amount of methanol and uniformly loaded into a 0.5 m x 0.1 m ODS column. The column was eluted with acetonitrile:water at a gradient of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0. The effluent was collected in 20 mL fractions, concentrated under reduced pressure, and monitored by thin layer chromatography. Fractions with the same elution time were combined. A total of 19 fractions were obtained, i.e., Fr. 12-32-1-Fr. 12-32-19.

[0041] (5) The Fr. 12-32-10 fraction was separated using an APS10 analytical high-performance liquid chromatograph, a HYPERSIL BDS C18 (250 mm x 10 mm) column, and a mobile phase of acetonitrile:water = 22.5 / 77.5. Finally, an acetyl dopamine trimer compound was obtained, as shown in the structural formula Figure 1 .

[0042] Acetyldopamine trimer compound structure analysis:

[0043] The compound shows quasi-molecular ion peaks at positive ion mode [M+Na]+m / z 600.1970 and [M+H]+m / z 578.2145, and its elemental composition is determined as C 30 H 31 N3O9.

[0044] 1 The H-NMR spectrum contains three ABX type spin system signals in the aromatic region (δ6.72-6.99), three methyl proton signals at δ1.83 (s, 3H, H-2”-2), δ1.87 (s, 3H, H-3'-2) and δ1.88 (s, 3H, H-3-2), two methylene proton signals at δ2.69 (t, 2H, J = 7.2 Hz, H-1”) and δ3.34 (t, 2H, J = 7.2 Hz, H-2”), four methine signals at δ4.72 (d, 1H, J = 7.0 Hz, H-2), δ4.76 (d, 1H, J = 7.0 Hz, H-2’), δ5.66 (d, 1H, J = 7.2 Hz, H-3’) and δ5.71 (d, 1H, J = 7.2 Hz, H-3). 13 C-NMR and DEPT data show 30 signals, including 3 methyl groups, 2 methylene groups, 13 methine groups and 12 quaternary ammonium salts (3 carbonyl groups, 9 olefins), as shown in Table 1.

[0045] The above signals indicate that the compound is an acetyldopamine trimer structure. Combined with the 1 H- 1 The signals of the compound are assigned by analyzing the H-COSY spectrum, HMBC spectrum and HSQC spectrum, and the planar structure of the compound is determined. C-5' / H-5' (d, J = 8.2 Hz), C-8' / H-8' (d, J = 1.9 Hz) and C-6', C-7' indicate that the side chain is connected to C-7'. 1 H- 1 The HCOSY spectrum shows the correlation of H-2 / H-3, H-2' / H-3' and H1” / H-2”. The HMBC correlation indicates that the long-range coupling exists between the methane proton signal at δ2.69 (H-1”) and the carbon signals at δ123 (C-6') and δ118 (C-8'), further indicating that the side chain structure is connected to the position of C-7'. In addition, H-2 is related to C-2, C-6, H-2' is related to C-5 and C-7, indicating that C-2' is connected to C-6, and C-1”' is connected to C-2.

[0046] The coupling constant between H-2 and H-3, H-2' and H3' is 7.0 Hz, indicating a trans structure. In order to further clarify the absolute configuration of the compound, ECD spectrum was determined, as shown in Figure 2 After comparing the experimental CD data with the quantum mechanics calculation ECD data, the structure of the compound was finally confirmed as: N,N'-((2R,2'S,3S,3'R)-7-(2-acetamidoethyl)-2'-(3,4-dihydroxyphenyl)-2,2',3,3'-tetrahydro-[2,6'-bibenz o[b][1,4]dioxine]-3,3'-diyl)diacetamide. Named as acetyldopamine trimer.

[0047] Table 1 Anti-inflammatory experimental study of acetyldopamine trimer 13 C-NMR and 1 H-NMR data

[0048]

[0049]

[0050] Example 2 Anti-inflammatory experimental study of acetyldopamine trimer (hereinafter referred to as T1)

[0051] I. Drugs and reagents

[0052] (l) DMEM high-sugar culture medium: Jiangsu Keygen Biotech Co., Ltd.

[0053] (2) Fetal bovine serum: Zhejiang Tianhang Biotechnology Co., Ltd.

[0054] (3) Cell Counting Kit-8 (CCK-8): Jiangsu Keygen Biotech Co., Ltd.

[0055] (4) Dimethyl sulfoxide (DMSO): Beijing Solabio Technology Co., Ltd.

[0056] (5) Lipopolysaccharide LPS: Biosharp.

[0057] (6) ELISA kit: Shanghai Enzyme-linked Biotechnology Co., Ltd.

[0058] II. Cell lines and cell culture

[0059] Mouse RAW264.7 macrophage cells were provided by the Experimental Center of Jiangsu Province Institute of Traditional Chinese Medicine, and the cells were from ATCC (American National Cell Bank). The cells were cultured in DMEM high-sugar medium containing 10% fetal bovine serum at 37°C, saturated humidity, and 5% CO2 in a culture box. The cells were adherent growth, and the logarithmic growth phase cells were taken for experiments, and the cell concentration was adjusted to 1-5 x 10 5 / mL. Drug intervention was started after the cells were inoculated for 24 hours into the logarithmic growth phase.

[0060] III. Preparation of main solutions

[0061] (1) Griess reagent: A solution: 0.1% naphthyl ethylenediamine dihydrochloride (40 mg of drug powder + 40 mL of PBS mixed uniformly and stored at 4°C in the dark). B solution: 1.5% sulfanilamide + 5% phosphoric acid solution (400 mg of sulfanilamide + 2.352 mL of phosphoric acid + 37.65 mL of double distilled water mixed uniformly and stored at 4°C in the dark). Before use, mix equal amounts of A and B solutions uniformly, and use the same amount of liquid as the cell supernatant.

[0062] (2) PBS: Weigh 8 g of NaCl, 0.2 g of KCI, 1.44 g of Na2HPO4, and 0.24 g of K2PO4, and dissolve them in 800 mL of distilled water. Adjust the pH of the solution to 7.4 with HC1, and finally add distilled water to make up to 1 L. Sterilize by steam under high pressure (at least 20 min), and store at room temperature or in a 4°C refrigerator.

[0063] IV. Main instruments and equipment

[0064] (1) Centrifuge: Heraeus Multifuge X1R type, Germany.

[0065] (2) CO2 incubator: HERAcell 150 type, Germany.

[0066] (3) Clean bench: Class II A2 type of 1300 series, Thermo Fisher Scientific.

[0067] (4) Inverted microscope: MODEL CKX41SF type, Philips.

[0068] (5) Enzyme marker: INFINITE 200PRO, Australia.

[0069] II. Experimental methods

[0070] 1. Cell culture and grouping:

[0071] The cells were cultured in DMEM high-sugar medium containing 10% fetal bovine serum at 37°C, saturated humidity, and 5% CO2 in a culture box. The cells were adherent growth, and the logarithmic growth phase cells were taken for experiments, and the cell concentration was adjusted to 2.0 x 105 / mL. Drug intervention was started 24 h after inoculation of cells into the logarithmic growth phase.

[0072] 2. CCK8 method for detecting cell proliferation and IC 50

[0073] Logarithmic proliferation RAW264.7 macrophages were collected and adjusted to a concentration of 2.0 x 10 5 / mL cell suspension, 100 μL per well, 6 replicates, and a blank control. After 24 h of inoculation, the cells were observed under a microscope to confirm good cell adhesion, and the supernatant was discarded. The experimental group was added with the prepared drug solution of each concentration (cells + 10 μL of each concentration of drug + 90 μL of culture medium), the blank group (only culture medium without cells), and the normal control group (cells + 100 μL of culture medium). The cells were cultured at 37°C in a CO2 incubator for 24 h, then 10 μL of CCK-8 solution was added to each well, and the cells were further cultured at 37°C for 1.5 h. The optical density value (OD value) of each well at a wavelength of 490 nm was detected by an enzyme label instrument. The experiment was repeated three times. The proliferation rate was calculated according to the formula:

[0074] Proliferation rate = (OD value of drug group - OD value of blank group) / (OD value of control group - OD value of blank group) x 100%

[0075] 3. Griess method for detecting the content of nitric oxide (NO) in cells

[0076] RAW264.7 cells (2 x 10 5 Cells / well) were inoculated in a 24-well plate and cultured at 37°C in a 5% CO2 cell incubator for 24 hours. The culture medium was removed, 800 uL of fresh culture medium and 100 ul of drug (100-600 μM) were added to the cells for 2 h, then LPS 100 μL was supplemented to a final concentration of 1 μg / mL for further culture for 24 hours. Finally, the cell-free supernatant was collected and treated, and NO was quantified by mixing 100 μL of cell-free supernatant with 100 μL of Griess reagent. After 10 minutes of incubation, the absorbance at 540 nm was recorded, and the results were expressed as NO%.

[0077] 4. ELISA kit for measuring cytokines (IL-6, TNF-a) and PGE2

[0078] RAW264.7 cells (2 x 10 5Cells were seeded in 24-well plates at a density of 1 x 105cells / well and incubated at 37°C in a 5% CO2incubator for 24 h. The medium was removed and the cells were treated with 800 μL fresh medium and 100 μL drug (300 μM) for 2 h, then LPS 100 μL was added to a final concentration of 1 μg / ml and incubated for another 24 h. Finally, cell-free supernatants were collected and processed, and the IL-6, TNF-a and PGE2were quantified using ELISA kits according to the manufacturer's instructions.

[0079] III. Experimental results

[0080] 1. Cell proliferation and IC 50 Experimental results

[0081] CCK-8 method for detecting the proliferation toxicity of compound T1 on RAW264.7 cells Figure 3 Six drug concentrations (100, 200, 300, 400, 600, 800 μM) were set respectively, and the drugs were added to RAW264.7 cells for 24 h. Each concentration was compared with the normal control group. When the drug concentration was 100 and 200 μM, it had a promoting effect on cell proliferation. When the drug concentration was 300 μM, there was no statistical difference (p>0.05). When the drug concentration was 400 and 600 μM, there was a statistical difference (p<0.05). The results showed that when the concentration of T1 was ≤300 μM, it did not inhibit the growth of RAW264.7 cells and the cell growth state was good. The IC of T1 on RAW264.7 cells 50 was 814.2±5.38 μM.

[0082] 2. Effect of T1 on the secretion of inflammatory mediator NO in LPS-induced RAW264.7 cells.

[0083] NO standard curve

[0084] The standard solution concentration was taken as the abscissa (X), and the corresponding absorbance was taken as the ordinate (Y). The data of each point was fitted to draw a dot curve, and the regression equation Y=0.0052X+0.0538 was obtained. Therefore, by measuring the OD value of the sample, the NO content of the sample was calculated through the standard curve and the regression equation, as shown in Figure 4 The NO concentration showed good linearity in the range of 0-50 μM, and the correlation coefficient R 2 = 0.9991.

[0085] The NO secretion in the supernatant of the blank group cells was very low, and it could significantly increase the secretion of NO after LPS stimulation, as shown in Figure 5As shown, compared with the model group, the positive drug group dexamethasone and the new compound T1 can dose-dependently inhibit the secretion of NO of LPS-induced RAW264.7 macrophages. When the concentration of T1 is 200 μM, the amount of NO release is 70.98% of the model group, and when the concentration is 300 μM, the amount of NO release is 63.62% of the model group.

[0086] The release amount of each inflammatory factor is determined by ELISA method, and the results show that the contents of IL-6, TNF-α and PGE2 of the macrophages induced by LPS are significantly increased, and after the action of the acetyl dopamine trimer T1 with a concentration of 300 μM, the release amounts of IL-6, TNF-α and PGE2 are significantly reduced.

[0087] The anti-inflammatory activity of the new acetyl dopamine trimer T1 separated from cicada slough is studied by using the LPS-induced RAW264.7 macrophage in vitro anti-inflammatory model, and the contents of inflammatory mediators NO and pro-inflammatory cytokines IL-6, TNF-α and PGE2 in cells are used as indexes. The results show that the new compound can reduce the levels of NO, IL-6, TNF-α and PGE2 in LPS-induced RAW264.7 macrophages to different degrees at a lower cytotoxicity concentration, indicating that the new compound has obvious anti-inflammatory activity.

[0088] Preparation of tablets in Example 3

[0089] The acetyl dopamine trimer prepared in Example 1 above, a suitable amount of starch, magnesium stearate and other pharmaceutical excipients are taken, uniformly mixed, and then compressed into tablets for oral use.

[0090] Preparation of capsules in Example 4

[0091] The acetyl dopamine trimer prepared in Example 1 above, a suitable amount of starch, magnesium stearate and other pharmaceutical excipients are taken, uniformly mixed, and then filled into capsules for oral use.

[0092] Preparation of granules in Example 5

[0093] The acetyl dopamine trimer prepared in Example 1 above, a suitable amount of starch, magnesium stearate and other pharmaceutical excipients are taken, uniformly mixed, and then filled into capsules for oral use.

[0094] The above implementation manners are only for illustrating the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and implement it, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made according to the spirit and essence of the present application should be covered within the protection scope of the present application.

Claims

1. A method for preparing a novel cicada molting acetyldopamine trimer with anti-inflammatory activity, characterized in that, Includes the following steps: (1) Take an appropriate amount of cicada molting medicinal material, place it in a round-bottom flask, add 10 times the amount of 95% ethanol, reflux extract twice, 3 hours each time, filter, combine the filtrates, concentrate under reduced pressure, place on a water bath and heat until the ethanol evaporates, freeze dry to obtain dry extract. (2) Dissolve the dry extract from step (1) in ethanol, add 60-100 mesh silica gel and mix well, then place it in a water bath to evaporate the solvent and set aside. Soak 100-200 mesh silica gel in chloroform and methanol at a volume ratio of 20:1 for 3-5 hours, remove bubbles by sonication and pack it into a glass column. Then pack the mixed sample evenly into the silica gel column and elute with a gradient of chloroform and methanol at volume ratios of 20:1, 15:1, 12:1, 10:1, 8:1, 5:1, 3:1 and 1:1 respectively. Collect the eluent, concentrate under reduced pressure, monitor by thin-layer chromatography, and combine the same fractions to obtain a total of 47 fractions, namely Fr.1 to Fr.

47. (3) Dissolve the Fr.12 fraction in methanol, add 60-100 mesh silica gel and mix well, then place it in a water bath to evaporate the solvent and set aside; soak 100-200 mesh silica gel in chloroform:methanol at a volume ratio of 20:1 for 3-5 hours, remove bubbles by sonication and then pack it into a glass column; pack the dissolved sample evenly into the glass column, and then elute with dichloromethane and methanol at volume ratios of 20:1, 15:1, 10:1, 5:1, and 1:1 respectively, collect the eluent, concentrate under reduced pressure, monitor by thin-layer chromatography, combine the same fractions, and obtain a total of 48 fractions, namely Fr.12-1 to Fr.12-48; (4) The Fr.12-32 fraction was dissolved in methanol and uniformly packed into a 0.5m×0.1m ODS column. It was then eluted with acetonitrile:water gradients at volume ratios of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0, respectively. The eluent was collected, concentrated under reduced pressure, and monitored by thin-layer chromatography. Fractions with the same characteristics were combined to obtain a total of 19 fractions, namely Fr.12-32-1 to Fr.12-32-19. (5) The Fr.12-32-10 fraction was separated and purified using an APS10 high-performance liquid chromatograph with a HYPERSIL BDSC18 column (250 mm × 10 mm) and a mobile phase of acetonitrile and water (volume ratio 22.5:77.5). The final product was an acetyl-dopamine trimer compound with the following structural formula:

2. The method for preparing the novel cicada molting acetyldopamine trimer with anti-inflammatory activity according to claim 1, characterized in that, The obtained novel cicada molting acetyldopamine trimer and a pharmaceutically acceptable carrier are used to prepare drugs in the form of tablets, capsules, granules, pellets, powders, and injections.

Images