Andrographis paniculata diterpene lactone compound, Anti-inflammatory agent, as well as extraction method and application thereof

Abstract

To provide an andrographis paniculata diterpene lactone compound as well as extraction method and application thereof.SOLUTION: The andrographis paniculata diterpene lactone compound provided by the invention has a structure as shown in any one of formulas 1-7 and is novel in structure. The andrographis paniculata diterpene lactone compound extracted and separated from andrographis paniculata has a good anti-inflammatory effect, and has a good down-regulation effect on secretion of NO, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in a lipopolysaccharide (LPS) induced mouse mononuclear macrophage (RAW264.7 cell) inflammation model; the compound also has a good inhibition effect on expression of inducible nitric oxide synthase (INOS) and cyclooxygenase-2 (COX-2) proteins, and can be applied to preparation of anti-inflammatory drugs.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and specifically relates to andrographolide diterpene lactone compounds, and an extraction method and application thereof.

Background Art

[0002] Inflammation is a defensive response of the human body to damaged stimuli, which can regulate the body's immunity, remove pathogens to reduce cell damage, start tissue repair functions, and autonomously repair damaged tissues. When macrophages are invaded by foreign pathogenic microorganisms, they can release pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) family cytokines, NO, and start the secretion of related proteins that regulate inflammation, causing inflammation and immune responses.

[0003] NO is a small molecule discovered in vascular endothelium, and the occurrence of both acute and chronic inflammation is related to it. NO can inhibit the proliferation of T cells and B cells and regulate cytokines of multiple inflammatory reactions, thereby causing inflammation. Multiple anti-inflammatory effects of andrographis paniculata are all related to the regulatory effect on NO. When immune cells are stimulated by microbial endotoxins, inflammatory mediators, etc., they generate a large amount of inducible NO synthase (iNOS) to produce NO for immune responses. Therefore, inhibiting the production of NO is a direct indicator for calculating the anti-inflammatory activity of compounds. Dexamethasone is a common anti-inflammatory drug in this field. Dexamethasone can reduce and prevent the reaction of tissues to inflammation, thereby reducing the expression of inflammation. However, with the development of the times, the anti-inflammatory effect of dexamethasone is difficult to meet the needs of patients.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide andrographolide diterpene lactone compounds having a good anti-inflammatory effect and applicable to the preparation of anti-inflammatory drugs, an extraction method thereof, and an application thereof.

Means for Solving the Problems

[0006] In order to achieve the above object of the present invention, the present invention provides the following technical solutions. The present invention provides andrographolide diterpene lactone compounds having a structure shown in any one of Formulas 1 to 7 (Chemical Formulas 1 to 7 below):

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

[0007] The present invention provides an extraction method of andrographolide diterpene lactone compounds described in the above technical solution. The extraction method of andrographolide diterpene lactone compounds having a structure shown in Formulas 1 to 6 is Adopt the first solvent to extract Andrographis paniculata, obtain an extract, remove the solvent in the extract to obtain a first extract, and the first solvent includes an aqueous methanol solution or acetone. Suspend the first extract and water, extract with petroleum ether, collect the water extract, remove the solvent in the water extract to obtain a second extract. After mixing the second extract and methanol, sequentially perform separation by first silica gel column chromatography and separation by second silica gel column chromatography to obtain a first mixture and a second mixture respectively. The mobile phases used for the separation by first silica gel column chromatography and the separation by second silica gel column chromatography are independently dichloromethane and methanol. Perform separation by first reverse-phase chromatography, separation by second reverse-phase chromatography, and separation by third reverse-phase chromatography on the first mixture sequentially to obtain a third mixture. The mobile phases used for the separation by first reverse-phase chromatography, the separation by second reverse-phase chromatography, and the separation by third reverse-phase chromatography are independently mobile phase A and mobile phase B. Mobile phase A is methanol, and mobile phase B is water. Perform separation by first high-performance liquid chromatography on the third mixture to obtain andrographis diterpenelactone compounds having the structures shown in Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, and Formula 6 respectively. The mobile phase used for the separation by first high-performance liquid chromatography includes mobile phase A and mobile phase B. Mobile phase A is acetonitrile, and mobile phase B is water. The extraction method of andrographis diterpenelactone compounds having the structure shown in Formula 7 is as follows. Recrystallize the second mixture to obtain a fourth mixture. Perform separation by second high-performance liquid chromatography on the fourth mixture to obtain andrographis diterpenelactone compounds having the structure shown in Formula 7. The mobile phase used for the separation by second high-performance liquid chromatography is a mixture of acetonitrile and water.

[0008] Preferably, the first solvent contains an aqueous methanol solution or acetone, the number of extraction times is 2 to 4 times, the extraction temperature each time is independently 20 to 30 °C, the extraction time each time is independently 20 to 30 h, and the solid-liquid ratio extracted each time is independently 1 to 1.5 kg: 3 to 4.5 L.

[0009] Preferably, the separation by the first silica gel column chromatography is gradient elution, and the volume ratio of dichloromethane to methanol used for the separation by the first silica gel column chromatography is 50 to 1:1 (50:1 to 1:1).

[0010] Preferably, the volume ratio of dichloromethane to methanol used for the separation by the first silica gel column chromatography is sequentially 50:1, 25:1, and 20:1. After the separation by the first silica gel column chromatography, the eluent when the volume ratio of dichloromethane to methanol is 20:1 is collected.

[0011] Preferably, the separation by the second silica gel column chromatography is gradient elution, and the volume ratio of dichloromethane to methanol used for the separation by the second silica gel column chromatography is 20 to 1:1 (20:1 to 1:1).

[0012] Preferably, the volume ratio of dichloromethane to methanol used for the separation by the second silica gel column chromatography is sequentially 20:1, 15:1, and 10:1. After the separation by the second silica gel column chromatography, the eluents when the volume ratios of dichloromethane to methanol are 15:1 and 10:1 are collected.

[0013] Preferably, the chromatography columns used for the separation by the first reverse-phase chromatography, the separation by the second reverse-phase chromatography, and the separation by the third reverse-phase chromatography are ODS chromatography columns, and the elution method for the separation by the first reverse-phase chromatography, the separation by the second reverse-phase chromatography, and the separation by the third reverse-phase chromatography is gradient elution. The gradient elution procedure for the separation by the first reverse-phase chromatography is as follows: From 0.00 to 2.00 h, maintain the volume fraction of mobile phase A at 40%. From 2.00 to 2.01 h, linearly increase the volume fraction of mobile phase A from 40% to 60%. From 2.01 to 4.00 h, maintain the volume fraction of mobile phase A at 60%. From 4.00 to 4.01 h, linearly increase the volume fraction of mobile phase A from 60% to 80%. From 4.01 to 5.50 h, set the volume fraction of mobile phase A to 80%. From 5.50 to 5.51 h, linearly increase the volume fraction of mobile phase A from 80% to 100%. From 5.51 to 6.00 h, maintain the volume fraction of mobile phase A at 100%. The gradient elution procedures for the separation by the second reverse-phase chromatography and the separation by the third reverse-phase chromatography are as follows: From 0.00 to 2.00 h, maintain the volume fraction of mobile phase A at 0%. From 2.00 to 2.01 h, linearly increase the volume fraction of mobile phase A from 0% to 60%. From 2.01 to 4.00 h, maintain the volume fraction of mobile phase A at 60%. From 4.00 to 4.01 h, linearly increase the volume fraction of mobile phase A from 60% to 80%. From 4.01 to 5.50 h, set the volume fraction of mobile phase A to 80%. From 5.50 to 5.51 h, linearly increase the volume fraction of mobile phase A from 80% to 100%. From 5.51 to 6.00 h, maintain the volume fraction of mobile phase A at 100%.

[0014] Preferably, the chromatography columns used for the separation by the first high performance liquid chromatography and the separation by the second high performance liquid chromatography are reverse phase C18 silica gel matrix chromatography columns. The volume fraction of acetonitrile in the mobile phase used for the separation by the first high performance liquid chromatography is 25 - 40%, and the volume fraction of acetonitrile in the mobile phase used for the separation by the second high performance liquid chromatography is 35 - 40%. The elution mode for the separation by the first high performance liquid chromatography and the separation by the second high performance liquid chromatography is isocratic elution.

[0015] The present invention provides the application of the andrographolide diterpene lactone compounds described in the above technical solution means in the preparation of anti-inflammatory drugs.

Effects of the Invention

[0016] The andrographolide diterpene lactone compounds provided by the present invention have the structure shown in any one of Formulas 1 - 7, and the structure is novel. The andrographolide diterpene lactone compounds described in the present invention have good anti-inflammatory effects, and also have good down-regulation effects on the secretion of NO, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in a bacterial lipopolysaccharide (LPS)-induced mouse monocyte macrophage leukemia cell (RAW264.7 cell) inflammation model. They also have good inhibitory effects on the expression of inducible nitric oxide synthase (INOS) and cyclooxygenase-2 (COX-2) proteins, and have a concentration effect, and can be used in the preparation of anti-inflammatory drugs. The examples show that the andrographolide diterpene lactone compounds provided by the present invention have good in vitro anti-inflammatory activities and are superior to the positive control drugs dexamethasone and andrographolide.

[0017] Furthermore, the present invention provides a method for extracting andrographolide compounds. By adopting the method provided by the present invention, seven andrographolide compounds with different structures can be extracted only once. The present invention uses the medicinal plant resource of Andrographis paniculata, obtains andrographolide compounds by extraction, not only broadens the types of anti-inflammatory drugs, but also provides a reference for the research of other natural products, and has good practical value.

[0018] Furthermore, the extraction method provided by the present invention is simple, has high purification degree, high operability and reproducibility. The andrographolide compounds obtained by extraction thereby have excellent anti-inflammatory effects. Since the present invention has also clarified the anti-inflammatory mechanism of androlata E, the andrographolide compounds provided by the present invention can be used as raw materials for synthesizing other compounds, for pilot studies, new drug development and pharmacological activity research, and can also be used for the preparation of anti-inflammatory drugs suitable for various inflammatory phenotypes, and have good economic benefits and availability.

[0019] To more clearly explain the technical solutions in the embodiments of the present invention or in the prior art, the drawings that need to be used in the following examples will be briefly described. Obviously, the drawings in the following description are only some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.

Brief Description of the Drawings

[0020]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Mode for Carrying Out the Invention

[0021] The present invention provides andrographolide diterpene lactone compounds having a structure shown in any one of Formulas 1 to 7. JPEG2025093266000009.jpg191144

[0022] The present invention provides a method for extracting andrographolide diterpene lactone compounds described in the above technical solution. The method for extracting andrographolide diterpene lactone compounds having a structure shown in Formulas 1 to 6 is as follows: Using a first solvent to extract andrographis paniculata to obtain an extract, removing the solvent in the extract to obtain a first extract, and the first solvent includes a step of containing an aqueous methanol solution or acetone; Suspending the first extract and water, extracting with petroleum ether, collecting the water extract, and removing the solvent in the water extract to obtain a second extract; After mixing the second extract and methanol, successively performing separation by a first silica gel column chromatography and separation by a second silica gel column chromatography to obtain a first mixture and a second mixture respectively. The mobile phases used for the separation by the first silica gel column chromatography and the separation by the second silica gel column chromatography are independently dichloromethane and methanol; Successively perform separation by first reverse-phase chromatography, separation by second reverse-phase chromatography, and separation by third reverse-phase chromatography on the first mixture to obtain a third mixture. The mobile phases used for the separation by first reverse-phase chromatography, the separation by second reverse-phase chromatography, and the separation by third reverse-phase chromatography independently contain mobile phase A and mobile phase B. Mobile phase A is methanol and mobile phase B is water. Perform separation by first high-performance liquid chromatography on the third mixture to obtain andrographolide compounds having the structures shown in Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, and Formula 6 respectively. The mobile phase used for the separation by first high-performance liquid chromatography contains mobile phase A and mobile phase B. Mobile phase A is acetonitrile and mobile phase B is water. The extraction method of andrographolide compounds having the structure shown in Formula 7 is Recrystallize the second mixture to obtain a fourth mixture. Perform separation by second high-performance liquid chromatography on the fourth mixture to obtain andrographolide compounds having the structure shown in Formula 7. The mobile phase used for the separation by second high-performance liquid chromatography is a mixed solution of acetonitrile and water.

[0023] In the present invention, unless otherwise specifically described, all raw materials used are commercially available products well known to those skilled in the art or those obtained by methods well known to those skilled in the art.

[0024] The present invention extracts Andrographis paniculata using a first solvent to obtain an extract, removes the solvent in the extract to obtain a first extract. Before the extraction, the present invention preferably pulverizes Andrographis paniculata to obtain Andrographis paniculata powder. The present invention does not particularly limit the particle size of the Andrographis paniculata powder, as long as the subsequent operations are facilitated. After obtaining the Andrographis paniculata powder, the present invention preferably employs a first solvent to extract the Andrographis paniculata powder to obtain an extract. In the present invention, the first solvent preferably contains an aqueous methanol solution or acetone, more preferably an aqueous methanol solution, and the volume fraction of the aqueous methanol solution is preferably 70-90%, more preferably 80%. In the present invention, the number of extraction times is preferably 2-4 times, more preferably 3 times. The extraction temperature for each time is independently preferably 20-30°C, more preferably independently 25°C. The extraction time for each time is independently preferably 20-30 h, more preferably 24 h. The solid-liquid ratio extracted each time is independently preferably 1-1.5 kg: 3-4.5 L, more preferably 1 kg: 3 L. In the present invention, the method for removing the solvent in the extract preferably includes concentrating the extract. The concentration described in the present invention is preferably vacuum concentration. The present invention does not particularly limit the specific method and conditions of the concentration, and a concentration method well known to those skilled in the art may be used.

[0025] After obtaining the first extract, the present invention suspends the first extract and water, extracts using petroleum ether, collects the water extract, removes the solvent in the water extract to obtain a second extract. In the present invention, the number of extraction times is preferably 2-4 times, more preferably 3 times. The collection method of the water extract of the present invention is preferably liquid separation. In the present invention, the method for removing the solvent in the water extract preferably includes concentrating the water extract. In the present invention, the concentration is preferably vacuum concentration. The present invention does not particularly limit the specific method and conditions of the concentration, and a concentration method well known to those skilled in the art may be used.

[0026] After obtaining the second extract, the present invention mixes the second extract with methanol, and then sequentially performs separation by first silica gel column chromatography and separation by second silica gel column chromatography to obtain a first mixture and a second mixture, respectively. Preferably, the present invention dissolves the second extract in methanol to obtain a first solution, mixes the first solution with silica gel, then adds a sample, and performs separation by first silica gel column chromatography. In the present invention, the methanol is preferably methanol and chloroform, more preferably methanol. In the present invention, the particle size of the silica gel is preferably 200 - 300 mesh, more preferably 250 - 300 mesh, and the mass ratio of the first solution to the silica gel is preferably 1:2, more preferably 1:1. The mobile phase used for the separation by the first silica gel column chromatography described in the present invention is preferably dichloromethane and methanol. In the present invention, the separation by the first silica gel column chromatography is preferably gradient elution, and the volume ratio of dichloromethane to methanol used for the separation by the first silica gel column chromatography is preferably 50 - 1:1, more preferably sequentially 50:1, 25:1, and 20:1. After the separation by the first silica gel column chromatography, the present invention preferably employs TLC thin layer chromatography to combine the same elution parts, collect the eluent eluted when the volume ratio of dichloromethane to methanol is 20:1, and dissolve the obtained elution material in a second solvent to obtain a second solution. After mixing the second solution with silica gel, loading is performed, and separation by second silica gel column chromatography is carried out. In the examples of the present invention, after the separation by the first silica gel column chromatography, TLC thin layer chromatography is employed to combine the same elution parts, and then six components named component A, component B, component C, component D, component E, and component F are obtained. Further, separation by second silica gel column chromatography is performed on component D. In the present invention, the second solvent is preferably at least one of methanol and chloroform, more preferably methanol.In the present invention, the particle size of the silica gel is preferably 200 - 300 mesh, more preferably 250 - 300 mesh, and the mass ratio of the second eluent to the silica gel is preferably 1:2, more preferably 1:1. The mobile phase used for the separation by the second silica gel column chromatography according to the present invention is preferably dichloromethane and methanol. In the present invention, the separation by the second silica gel column chromatography is preferably gradient elution, and the volume ratio of dichloromethane to methanol used for the separation by the second silica gel column chromatography is preferably 20 - 1:1, more preferably sequentially 20:1, 15:1, and 10:1. After the separation by the second silica gel column chromatography, the present invention preferably employs TLC thin layer chromatography to combine the same elution parts, and collect the eluents eluted when the volume ratios of dichloromethane to methanol are 15:1 and 10:1 respectively, to obtain a first mixture and a second mixture respectively. In the examples of the present invention, after the separation by the second silica gel column chromatography, TLC thin layer chromatography is employed to combine the same elution parts, and then five components named component D1, component D2, component D3, component D4, and component D5 are obtained, and further separation of component D1 by the first reverse phase chromatography is performed.

[0027] After obtaining the first mixture, the present invention performs separation by the first reverse phase chromatography, separation by the second reverse phase chromatography, and separation by the third reverse phase chromatography on the first mixture sequentially to obtain a third mixture. In the present invention, the chromatography columns used for the separation by the first reverse phase chromatography, the separation by the second reverse phase chromatography, and the separation by the third reverse phase chromatography are preferably ODS chromatography columns. In the present invention, the mobile phases used for the separation by the first reverse phase chromatography, the separation by the second reverse phase chromatography, and the separation by the third reverse phase chromatography independently include mobile phase A and mobile phase B, the mobile phase A is methanol, and the mobile phase B is water.

[0028] In the present invention, the elution methods for the separation by the first reversed-phase chromatography, the separation by the second reversed-phase chromatography, and the separation by the third reversed-phase chromatography are preferably gradient elution.

[0029] The gradient elution procedure for the separation by the first reversed-phase chromatography according to the present invention is preferably from 0.00 to 2.00 h, maintaining the volume fraction of mobile phase A at 40%, from 2.00 to 2.01 h, increasing the volume fraction of mobile phase A from 40% to 60% at a constant rate, from 2.01 to 4.00 h, maintaining the volume fraction of mobile phase A at 60%, from 4.00 to 4.01 h, increasing the volume fraction of mobile phase A from 60% to 80% at a constant rate, from 4.01 to 5.50 h, setting the volume fraction of mobile phase A to 80%, from 5.50 to 5.51 h, increasing the volume fraction of mobile phase A from 80% to 100% at a constant rate, from 5.51 to 6.00 h, maintaining the volume fraction of mobile phase A at 100%.

[0030] The gradient elution procedures for the separation by the second reversed-phase chromatography and the separation by the third reversed-phase chromatography according to the present invention are preferably from 0.00 to 2.00 h, maintaining the volume fraction of mobile phase A at 0%, from 2.00 to 2.01 h, increasing the volume fraction of mobile phase A from 0% to 60% at a constant rate, from 2.01 to 4.00 h, maintaining the volume fraction of mobile phase A at 60%, from 4.00 to 4.01 h, increasing the volume fraction of mobile phase A from 60% to 80% at a constant rate, from 4.01 to 5.50 h, setting the volume fraction of mobile phase A to 80%, from 5.50 to 5.51 h, increasing the volume fraction of mobile phase A from 80% to 100% at a constant rate, from 5.51 to 6.00 h, maintaining the volume fraction of mobile phase A at 100%.

[0031] In the present invention, after the separation by the first reverse-phase chromatography, the separation by the second reverse-phase chromatography, and the separation by the third reverse-phase chromatography, the present invention preferably all adopts TLC thin-layer chromatography to combine the same elution parts to obtain a third mixture. In the examples of the present invention, after the separation by the first reverse-phase chromatography, TLC chromatography is adopted to combine the same elution parts to obtain seven components named component D11, component D12, component D13, component D14, component D15, component D16, and component D17 respectively. Next, the separation by the second reverse-phase chromatography is performed on component D17. After the separation by the second reverse-phase chromatography, TLC chromatography is adopted to combine the same elution parts to obtain six components named component D151, component D152, component D153, component D154, component D155, and component D156 respectively. Next, the separation by the third reverse-phase chromatography is performed on component D155. After the separation by the third reverse-phase chromatography, TLC chromatography is adopted to combine the same elution parts to obtain three components named component D1551, component D1552, and component D1553 respectively. Next, the separation by the first high-performance liquid chromatography is performed on component D1552. In the present invention, component D155 has a purple spot with the characteristics of diterpenoids in TLC thin-layer chromatography, and the HPLC chromatography peak in component D155 has strong absorption at about 210 nm, which facilitates distinguishing it from other components.

[0032] After obtaining the third mixture, the present invention performs separation of the third mixture by first high performance liquid chromatography to obtain andrographolide compounds having the structures shown in Formula 1, Formula 2, Formula 3, Formula 4, Formula 5 and Formula 6 respectively. In the present invention, the chromatography column used for the separation by the first high performance liquid chromatography is preferably a reverse phase C18 silica gel matrix chromatography column. In the present invention, the mobile phase used for the separation by the first high performance liquid chromatography preferably includes mobile phase A and mobile phase B, the mobile phase A is preferably acetonitrile, the mobile phase B is preferably water, and the volume fraction of acetonitrile in the mobile phase used for the separation by the first high performance liquid chromatography is preferably 25-40%, more preferably 30-35%. In the present invention, the elution method for the separation by the first high performance liquid chromatography is preferably isocratic elution. The time for the separation by the first efficient liquid (high performance liquid) chromatography described in the present invention is preferably 60 min, and the flow rate of the mobile phase used for the separation by the first high performance liquid chromatography is preferably 7-9 mL / min, more preferably 8 mL / min. In the present invention, the separation order of the andrographolide compounds having the structures shown in Formula 1, Formula 2, Formula 3, Formula 4, Formula 5 and Formula 6 is preferably, in order, the andrographolide compounds having the structure shown in Formula 5, the andrographolide compounds having the structure shown in Formula 6, the andrographolide compounds having the structure shown in Formula 1, the andrographolide compounds having the structure shown in Formula 3, the andrographolide compounds having the structure shown in Formula 4 and the andrographolide compounds having the structure shown in Formula 2.

[0033] After obtaining the second mixture, the present invention recrystallizes the second mixture to obtain a fourth mixture. Before the recrystallization, the present invention preferably allows the second mixture to stand. In the present invention, the time for the standing is preferably 300-800 min, more preferably 600 min. The present invention does not particularly limit the recrystallization method and conditions, and a recrystallization method well known to those skilled in the art may be used.

[0034] After obtaining the fourth mixture, the present invention performs separation of the fourth mixture by second high performance liquid chromatography to obtain an andrographolide compound having the structure shown in Formula 7. In the present invention, the chromatography column used for the separation by second high performance liquid chromatography is preferably a reverse phase C18 silica gel matrix chromatography column. In the present invention, the mobile phase used for the separation by the second high performance liquid chromatography is preferably a mixed solution of acetonitrile and water, and the volume fraction of acetonitrile in the mobile phase used for the separation by the second high performance liquid chromatography is preferably 35 to 40%, more preferably 37%. In the present invention, the elution method for the separation by the second high performance liquid chromatography is preferably isocratic elution. The time for the separation by the second efficient liquid phase (high performance liquid) chromatography described in the present invention is preferably 50 min, and the flow rate of the mobile phase used for the separation by the second high performance liquid chromatography is preferably 7 to 9 mL / min, more preferably 8 mL / min.

[0035] The present invention further provides the application of the andrographis diterpenelactone compounds described in the above technical solution in the preparation of anti-inflammatory drugs. In the present invention, the anti-inflammatory drug preferably contains the andrographis diterpenelactone compounds described in the above technical solution and pharmaceutically acceptable pharmaceutical additives. In the present invention, the pharmaceutically acceptable pharmaceutical additives preferably include at least one of polyethylene glycol 4000, polyethylene glycol 6000, glycerin gelatin, cyclodextrin, microcrystalline cellulose, cross-linked carboxymethyl starch sodium and cross-linked povidone, and more preferably polyethylene glycol 4000, polyethylene glycol 6000 or cyclodextrin. In the present invention, the mass ratio of the andrographis diterpenelactone compounds to the pharmaceutically acceptable pharmaceutical additives is preferably 1-2:3-8. The dosage form of the anti-inflammatory drug described in the present invention preferably includes at least one of tablets, capsules, granules and dripping pills, more preferably tablets or capsules, the dosing method is preferably oral administration, and the dosing dose is preferably 0.1-0.2 g.

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the examples in the present invention. Obviously, the described examples are only some examples of the present invention, not all examples. Based on the examples in the present invention, all other examples obtained by those skilled in the art without creative efforts shall fall within the protection scope of the present invention.

[0037] The andrographis used in the examples of the present invention is the dried aerial part of the plant Andrographis paniculata, which is adopted in Linquan, Fuyang City, Anhui Province, and is identified as Andrographis paniculata (Burm.f.) Nees, a plant of the genus Andrographis in the family Acanthaceae, by Associate Professor Yang Qingshan of Anhui University of Chinese Medicine. The crude drug specimen (specimen number: 202104) is stored in Warehouse 400 of the School of Pharmacy, Anhui University of Chinese Medicine.

[0038] The names and sources of some of the devices, materials, and reagents used in the test examples of the present invention are as follows. Agilent 1260 LC-MS from the United States, Bruker AV-500 MHz nuclear magnetic resonance apparatus (internal standard is TMS, solvent is deuterated acetone or deuterated methanol), Mouse monocyte macrophage RAW264.7 (Shanghai Cell Bank, Chinese Academy of Sciences), Low serum medium (DMEM medium) and fetal bovine serum (both from BI Co., Ltd.), Griess Reagent, LPS (bacterial lipopolysaccharide), and the control drug dexamethasone (all from Sigma-Aldrich Co., LLC).

[0039] Example 1 Preparation of the first extract: After pulverizing 20 kg of Andrographis paniculata, it was placed in 60 L of an aqueous methanol solution (the volume fraction of the aqueous methanol solution was 80%) and extracted at 25 °C for 24 h each time, repeating 3 times. The extracts were combined, and the solvent in the extract was recovered by vacuum concentration to obtain 1350 g of the first extract. Preparation of the second extract: The first extract was suspended with 13 L of water and extracted with 9 L of petroleum ether, separated, and the extraction was repeated 3 times. The aqueous extract in the lower layer was collected, and the solvent in the aqueous extract was recovered by vacuum concentration to obtain 550 g of the second extract. Separation by the first silica gel column chromatography: The second extract was dissolved in methanol to obtain the first dissolved solution. The first dissolved solution and silica gel with a particle size of 200 - 300 mesh were mixed at a mass ratio of 1:1, then loaded onto a column, and gradient elution was performed using dichloromethane and methanol as the mobile phase (the volume ratio of dichloromethane to methanol was successively 50:1, 25:1, and 20:1). After separation by the first silica gel column chromatography, TLC thin layer chromatography was used to combine the same elution parts, and six components named component A (5 g), component B (10 g), component C (52 g), component D (108 g), component E (172 g), and component F (58 g) were obtained. Separation by the second silica gel column chromatography: Component D was dissolved in methanol to obtain a second solution. After mixing the second solution with silica gel having a particle size of 200 to 300 mesh at a mass ratio of 1:1, it was loaded onto a column, and dichloromethane and methanol were used as mobile phases for gradient elution (the volume ratio of dichloromethane to methanol was sequentially 20:1, 15:1, and 10:1). After separation by the second silica gel column chromatography, TLC thin layer chromatography was used to combine the same elution parts, and five components named component D1 (2.3 g), component D2 (13.3 g), component D3 (17.2 g), component D4 (12.5 g), and component D5 (3.6 g) were obtained. Separation by the first reverse-phase chromatography: Component D1 (2.3 g) was loaded onto an ODS column, and methanol was used as mobile phase A and water as mobile phase B for gradient elution (the elution procedure was that from 0.00 to 2.00 h, the volume fraction of mobile phase A was maintained at 40%, from 2.00 to 2.01 h, the volume fraction of mobile phase A increased linearly from 40% to 60%, from 2.01 to 4.00 h, the volume fraction of mobile phase A was maintained at 60%, from 4.00 to 4.01 h, the volume fraction of mobile phase A increased linearly from 60% to 80%, from 4.01 to 5.50 h, the volume fraction of mobile phase A was 80%, from 5.50 to 5.51 h, the volume fraction of mobile phase A increased linearly from 80% to 100%, and from 5.51 to 6.00 h, the volume fraction of mobile phase A was maintained at 100%). TLC chromatography was used to combine the same elution parts, and seven components named component D11, component D12, component D13, component D14, component D15, component D16, and component D17 were obtained. Separation by second reverse-phase chromatography: Component D15 (0.5271 g) was loaded onto an ODS column, and methanol was used as mobile phase A and water as mobile phase B for gradient elution. (The elution procedure was as follows: from 0.00 to 2.00 h, the volume fraction of mobile phase A was maintained at 0%; from 2.00 to 2.01 h, the volume fraction of mobile phase A increased linearly from 0% to 60%; from 2.01 to 4.00 h, the volume fraction of mobile phase A was maintained at 60%; from 4.00 to 4.01 h, the volume fraction of mobile phase A increased linearly from 60% to 80%; from 4.01 to 5.50 h, the volume fraction of mobile phase A was set at 80%; from 5.50 to 5.51 h, the volume fraction of mobile phase A increased linearly from 80% to 100%; from 5.51 to 6.00 h, the volume fraction of mobile phase A was maintained at 100%). TLC chromatography was used to combine the same eluted portions, and six components named component D151, component D152, component D153, component D154, component D155, and component D156 were obtained respectively. Separation by third reverse-phase chromatography: Component D155 (0.2452 g) was loaded onto an ODS column, and methanol was used as mobile phase A and water as mobile phase B for gradient elution. (The elution procedure was as follows: from 0.00 to 2.00 h, the volume fraction of mobile phase A was maintained at 0%; from 2.00 to 2.01 h, the volume fraction of mobile phase A increased linearly from 0% to 60%; from 2.01 to 4.00 h, the volume fraction of mobile phase A was maintained at 60%; from 4.00 to 4.01 h, the volume fraction of mobile phase A increased linearly from 60% to 80%; from 4.01 to 5.50 h, the volume fraction of mobile phase A was set at 80%; from 5.50 to 5.51 h, the volume fraction of mobile phase A increased linearly from 80% to 100%; from 5.51 to 6.00 h, the volume fraction of mobile phase A was maintained at 100%). TLC chromatography was used to combine the same eluted portions, and three components named component D1551, component D1552, and component D1553 were obtained respectively. Separation by the First High-Performance Liquid Chromatography: Component D1552 was loaded onto a reverse-phase C18 silicon matrix chromatography column. Acetonitrile was used as mobile phase A and water as mobile phase B for isocratic elution (the volume fraction of acetonitrile in the mobile phase was 35%). The flow rate of the mobile phase was 8 mL / min, the elution time was 60 min, and six components named compound 5 (9.5 mg, 26.9 min), compound 6 (12.3 mg, 27.9 min), compound 1 (14.2 mg, 28.3 min), compound 3 (10.5 mg, 28.7 min), compound 4 (5.3 mg, 29.0 min), and compound 2 (9.6 mg, 32.4 min) were obtained successively. Recrystallization and Separation by the Second High-Performance Liquid Chromatography: After standing component D2 for 600 min, needle-like crystals precipitated, and then recrystallization was carried out to obtain one component named component D2a. Component D2a was loaded onto a reverse-phase C18 silicon matrix chromatography column. A mixed solution of acetonitrile and water was used as the mobile phase for isocratic elution (the volume fraction of acetonitrile in the mobile phase was 37%). The flow rate of the mobile phase was 8 mL / min, the elution time was 50 min, and one component named compound 7 (5.2 mg, 22.1 min) was obtained.

[0040] Test Example 1 Structure Identification of Compounds 1 - 7 Instrument and Materials: All one-dimensional nuclear magnetic resonance spectra and two-dimensional nuclear magnetic resonance spectra (1D / 2D NMR) were measured by a Bruker AV-500 MHz nuclear magnetic resonance spectrometer (the internal standard was TMS, and the solvent was deuterated acetone or deuterated methanol). The high-resolution electrospray ionization mass spectrum (HR-ESI-MS) was measured by an Agilent 1260 LC-MS in the United States. Take the compounds 1 - 7 prepared in Example 1 and identify their structures by 1D / 2D NMR and HR-ESI-MS respectively. The measurement results are as follows. The 1D / 2D NMR data of compounds 1 - 7 are shown in Tables 1 - 4.

[0041] Table 1 Hydrogen spectrum (500 MHz) and carbon spectrum (125 MHz) data of Compounds 1, 3 and 4 [Table 1]

[0042] Table 2 Hydrogen spectrum (500 MHz) and carbon spectrum (125 MHz) data of Compound 2 [Table 2]

[0043] Table 3 Hydrogen spectrum (500 MHz) and carbon spectrum (125 MHz) data of Compounds 5 and 6 [Table 3]

[0044] Table 4 Hydrogen spectrum (500 MHz) and carbon spectrum (125 MHz) data of Compound 7 JPEG2025093266000013.jpg212135 Note: Sample a was placed in a deuterated acetone solution and measured, and sample b was placed in a deuterated methanol solution and measured.

[0045] The physicochemical data of Compounds 1 - 7 are as follows: Compound 1: androlata A, molecular formula is C 24 H 32 O7, it is a yellow jelly-like substance, easily soluble in methanol and acetone, [α] 25 D -5.0 (c 1.8, CH3OH); UV (CH3OH) λ max (log ε): 249 (3.77) nm, HR-ESI-MS m / z: 455.2041 [M+Na] + (calculated value: 455.2040). Compound 2: androlata B, molecular formula is C 28 H 36 O 10It is a yellow jelly-like substance, easily soluble in methanol and acetone, [α] 25 D -41.8 (c 1.2, CH3OH); UV (CH3OH) λ max (log ε): 249 (3.79) nm, HR-ESI-MS m / z: 555.2201 [M+Na] + (calculated value: 555.2201). Compound 3: androlata C, molecular formula is C 24 H 32 O7, it is a yellow jelly-like substance, easily soluble in methanol and acetone, [α] 20 D -13.7 (c 2.4, CH3OH); UV (CH3OH) λ max (log ε): 248 (3.82) nm, HR-ESI-MS m / z: 455.2040 [M+Na] + (calculated value: 455.2040). Compound 4: androlata D, molecular formula is C 25 H 34 O7, it is a yellow jelly-like substance, easily soluble in methanol and acetone, [α] 20 D -3.3 (c 2.0, CH3OH); UV (CH3OH) λ max (log ε): 249 (3.81) nm, HR-ESI-MS m / z: 469.2198 [M+Na] + (calculated value: 469.2197). Compound 5: androlata E, molecular formula is C 25 H 36 O8, it is a yellow jelly-like substance, easily soluble in methanol and acetone, [α] 20 D -35.6 (c 0.5, CH3OH); UV (CH3OH) λ max (log ε): 245 (3.24) and 265 (3.11) nm, HR-ESI-MS m / z: 487.2306 [M+Na] + (calculated value: 487.2302). Compound 6: androlata F, molecular formula is C 24 H 34It is O7, a yellow jelly-like substance, easily soluble in methanol and acetone, [α] 25 D -27.4 (c1.4, CH3OH); UV (CH3OH) λ max (logε): 244 (3.29) nm, HR-ESI-MS m / z: 457.2195 [M+Na] + (calculated value: 457.2197). Compound 7: androlataG, 12-methoxy-14-deoxyandrographside, the molecular formula is C 27 H 42 O 10 It is a white powder, [α] 25 D -7.8 (c1.1, CH3OH); UV (CH3OH) λ max (logε): 246 (3.39) nm, easily soluble in DMSO, HR-ESI-MS m / z: 549.2671 [M+Na] + (calculated value: 549.2670).

[0046] The structural formulas of Compounds 1 to 7 are shown below respectively. JPEG2025093266000014.jpg57170JPEG2025093266000015.jpg57170

[0047] The hydrogen spectrum, carbon spectrum, heteronuclear single quantum correlation spectrum (HSQC spectrum), heteronuclear single quantum coherent spectrum ( 1 H- 1 H COSY spectrum), heteronuclear multiple bond correlation spectrum (HMBC spectrum), nuclear Overhauser effect spectroscopy (ROESY spectrum), high-resolution mass spectrometry spectrum, ultraviolet spectrum and circular dichroism spectrum (CD spectrum) of Compound 5 are shown in Figures 1 to 9 respectively.

[0048] Test Example 2 Anti-inflammatory activity screening of Compounds 1 to 7 Materials and Reagents: Mouse monocyte macrophage RAW264.7, low serum medium (DMEM medium), fetal bovine serum, Griess Reagent, LPS, and dexamethasone as a control drug. Principle: LPS is one of the main components of the outer membrane of Gram-negative bacteria and is widely used to induce the RAW264.7 cell inflammation model for screening potential anti-inflammatory active natural products. When cells are stimulated by LPS, they can release inflammatory neurotransmitters such as TNF-α, IL-6, and NO, thereby initiating the function of resisting the inflammatory response. NO is a multifunctional free radical messenger molecule, mainly catalyzed and generated by nitric oxide synthases (NOS). However, the excessive production of NO and inflammatory mediators is related to the inflammatory process. The activation of inducible nitric oxide synthase (iNOS) causes various inflammatory diseases, and the down-regulated expression of iNOS is regarded as a target for treating inflammation. Usually, cyclooxygenase-2 (COX-2) has very low activity in normal cells. After being activated by external factors, its expression level rapidly increases, which is an important symbol enzyme for the inflammatory response.

[0049] (1) Inhibitory rate of compounds 1-7 on the production of NO RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco), and the culture conditions were a temperature of 37°C, a humidity of 95%, and a CO2 concentration of 5%. RAW264.7 macrophages were seeded at a density of 5×10 4Cells were inoculated into a 96-well plate at 150 μL per well at a density of [X] cells / mL and cultured in a cell incubator for 24 h. After the culture was completed, the culture medium was aspirated and removed, and a two-fold gradient treatment was performed on test compounds 1-7 at their corresponding concentrations with a culture medium containing 1 μg / mL LPS. A group without drug and a dexamethasone positive drug group were set as controls. After culturing the cells for 24 h, the medium was taken to detect the production of NO, and the absorbance value was measured at the 570 nm site. The CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was added to the remaining medium to detect cell viability and eliminate the effect of the compound on cell toxicity. NO production inhibition rate (%) = (OD of non-drug treatment group 570nm - OD of sample group 570nm ) / (OD of non-drug treatment group 570nm - OD of blank group 570nm ) × 100%. Half inhibitory concentration (IC 50 , 50% concentration of inhibition) was calculated and fitted with Prism 8, and the IC 50 standard deviation of the three groups was calculated by Excel, and the specific results are shown in Table 5.

[0050] Table 5 In vitro anti-inflammatory activity IC 50 values JPEG2025093266000016.jpg140147

[0051] The data in Table 5 show that the anti-inflammatory activity of compound 5 (androlata E) is superior to that of the positive control drug and andrographolide, and is the highest.

[0052] (2) Effect of compound 5 on the expression levels of RAW264.7 inflammatory factors (IL-6, IL-1β, TNF-α) According to the method in (1), RAW264.7 cells were seeded at a density of 1×10 5 Please note that the value of [X] in the translation of is not provided in the original text, so it remains as [X] in the translation. Also, the specific content of 570nm , 570nm , etc. are not clear in terms of their specific meaning in this context, so they are just translated as is while preserving the tags.Inoculated into a 6-well plate at 2 mL per well at a density of [X] cells / mL (the culture conditions were the same as (1)). The experiment was divided into a blank group, an LPS group, and an LPS + gradient concentration drug group. The LPS group was treated with 1 μg / mL LPS, and the drug groups were simultaneously treated with 1 μg / mL LPS and 2.5 μM, 5 μM, and 10 μM of compound 5, respectively. After co-culturing for 24 h, the operation was carried out according to the instruction manual of the enzyme-linked immunosorbent assay kit (ELISA kit), and the levels of inflammatory factors IL-6, IL-1β, and TNF-α were measured. The measurement results are shown in A - C of Figure 10 (here, A of Figure 10 is the result diagram of detecting IL-6 by the Elisa method, B of Figure 10 is the result diagram of detecting IL-1β by the Elisa method, and C of Figure 10 is the result diagram of detecting TNF-α level by the Elisa method). Combined with A - C of Figure 10, it can be seen that compound 5 (androlata E) significantly reduces the secretion of TNF-α, IL-6, and IL-1β induced by LPS, and similarly, it has concentration dependence.

[0053] (3) Effect of compound 5 on the expression of COX-2 and iNOS proteins in RAW264.7 cells Compound 5 has stronger anti-inflammatory activity. Protein immunoblotting (Western blot) was used to detect the expression levels of COX-2 and iNOS proteins in RAW264.7 cell inflammation to further investigate its anti-inflammatory mechanism of action. According to the method of (1), RAW 264.7 was adjusted to a density of 1×10 5Inoculated into a 6-well plate at 2 mL per well at a density of / mL (the culture conditions were the same as (1)). The experiment was divided into a blank group, an LPS group, and an LPS + gradient concentration drug group. After continuous culture for 24 h, the cells were collected. According to the method in the kit's instruction manual, the cells were lysed to extract total protein, and the bicinchoninic acid (BCA) protein assay was used to measure the protein concentration. Western Blot was used to detect the intracellular protein expression levels of COX-2 and iNOS, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference to normalize the protein strips. The measurement results are shown in D - F of Figure 10 (here, D in Figure 10 is the result diagram of detecting the iNOS and COX-2 protein levels in cells by Western blot, E in Figure 10 is the result diagram of the COX-2 protein expression level, F in Figure 10 is the result diagram of the iNOS protein expression level, where n = 3, x ± s, vs blank group, #P < 0.05, ##P < 0.01, P < 0.001, vs LPS group, *P < 0.05, **P < 0.01, ***P < 0.001). As can be seen from D - F of Figure 10, compound 5 (androlata E) can significantly reduce the production of NO in inflammatory cells and decrease the protein expression levels of iNOS and COX-2.

[0054] (4) Docking of compound 5 with COX-2, iNOS, and IL-6, IL-1β, and TNF-α molecules Method: For compound 5 with COX-2, IL-1β, IL-6, and TNF-α, molecular docking of IL-1β and IL-6 was performed using AutoDockTools and AutoDock Vina. The docking method is as follows. (1) For the diterpene compound structure file (mol2 format), ChemOffice software was used to convert it into a 3D structure to minimize the structural energy. Then, AutoDockTools software was used to hydrogenate the three-dimensional structure and calculate and save it as a pdbqt file. (2) Obtain the target crystal structure from the RCSB Protein Data Bank (https: / / www.pdb.org / ), select the crystal structure with the PDB code 6Y8I as the docking crystal structure of IL-1β, select the crystal structure with the PDB code 1ALU as the docking crystal structure of IL-6, select the crystal structure with the PDB code 5F19 as the docking crystal structure of COX-2, select the crystal structure with the PDB code 1A8M as the docking crystal structure of TNF-a, import it into PyMOL, dehydrate it, hydrogenate it, and use it for ligand separation. Then, in AutoDockTools, construct a docking grid box at the active site of each target protein and save it in the pdbqt format. (3) Use AutoDock Vina 4.2 to perform molecular docking on the hypothetical target and the active compound and evaluate the free binding energy. (4) Use PyMOL to visualize the active compound and analyze its interactions.

[0055] Figure 11 is the molecular docking diagram of compound 5 and each inflammatory factor protein molecule (where A in Figure 11 is the docking diagram of compound 5 and COX-2 protein, B in Figure 11 is the docking diagram of compound 5 and IL-1β protein, C in Figure 11 is the docking diagram of compound 5 and IL-6 protein, D in Figure 11 is the docking diagram of compound 5 and TNF-α, and E in Figure 11 is the docking diagram of compound 5 and iNOS protein).

[0056] As can be seen with reference to FIG. 11, from the molecular docking simulation, the proton at C-3’ in compound 5 forms a hydrogen bond with the GLY-574 amino residue in the COX-2 protein, the protons at C-3’ and 16 form hydrogen bonds with the GLN-246 and GLY-243 amino residues in the IL-1β protein, the protons at C-1’ and 3’ form a hydrogen bond with the LYS-46 amino residue in the IL-6 protein, the protons at C-3’ and C-1’ / 3 / 16 form hydrogen bonds with the ALA-33 and ARG-32 amino residues in the TNF-α protein, and the protons at C-3 / 16 and C-16 form hydrogen bonds with the LYS-191 and LEU-209 amino residues in the iNOS protein.

[0057] The above examples have described the present invention in detail, but they are only a part of the examples of the present invention, not all examples. People can also obtain other examples based on these examples without being creative, and all examples belong to the protection scope of the present invention.

Claims

1. A compound which is a diterpene lactone compound having any one of the structures shown in the following formulas 1 to 7.

2. An anti-inflammatory drug comprising the diterpene lactone compound of claim 1.

3. A method for extracting a diterpene lactone compound having a structure represented by any one of formulas 1 to 6 according to claim 1, comprising: Using a first solvent to extract Lianthus chinensis to obtain an extract, and removing the solvent in the extract to obtain a first extract, the first solvent comprising methanol aqueous solution or acetone; suspending the first extract with water, extracting with petroleum ether, collecting the aqueous extract, and removing the solvent in the aqueous extract to obtain a second extract; Mixing the second extract with methanol, and then sequentially separating the first mixture and the second mixture by a first silica gel column chromatography and a second silica gel column chromatography, respectively, and the mobile phases used in the first silica gel column chromatography and the second silica gel column chromatography are independently dichloromethane and methanol; performing a first reverse phase chromatographic separation, a second reverse phase chromatographic separation, and a third reverse phase chromatographic separation on the first mixture in order to obtain a third mixture, wherein the mobile phases used in the first reverse phase chromatographic separation, the second reverse phase chromatographic separation, and the third reverse phase chromatographic separation independently comprise a mobile phase A and a mobile phase B, wherein the mobile phase A is methanol and the mobile phase B is water; separating the third mixture by a first high performance liquid chromatography to obtain a diterpene lactone compound having a structure represented by any one of formulas 1, 2, 3, 4, 5, and 6, wherein the mobile phase used for the separation by the first high performance liquid chromatography includes mobile phase A and mobile phase B, the mobile phase A being acetonitrile and the mobile phase B being water; The method for extracting the diterpene lactone compound of Formula 7 according to claim 1 comprises: recrystallizing the second mixture to obtain a fourth mixture; A method for extracting Acanthus persicae diterpene lactone compounds, comprising: separating the fourth mixture by a second high performance liquid chromatography to obtain Acanthus persicae diterpene lactone compounds having the structure shown in formula 7; and a step in which the mobile phase used for the separation by the second high performance liquid chromatography is a mixture of acetonitrile and water.

4. The extraction method according to claim 3, characterized in that the extraction is performed 2 to 4 times, the extraction temperature for each extraction is independently 20 to 30°C, the extraction time for each extraction is independently 20 to 30 hours, and the solid-liquid ratio extracted for each extraction is independently 1 to 1.5 kg: 3 to 4.5 L.

5. The extraction method according to claim 3, wherein the separation by the first silica gel column chromatography is gradient elution, and the volume ratio of dichloromethane and methanol used in the separation by the first silica gel column chromatography is 50 to 1:

1.

6. 6. The method of claim 5, wherein the volume ratio of dichloromethane to methanol used in the first silica gel column chromatography separation is 50:1, 25:1, and 20:1, respectively, and after the first silica gel column chromatography separation, an eluent when the volume ratio of dichloromethane to methanol is 20:1 is collected.

7. The extraction method according to claim 3, wherein the separation by the second silica gel column chromatography is gradient elution, and the volume ratio of dichloromethane and methanol used in the separation by the second silica gel column chromatography is 20 to 1:

1.

8. The extraction method according to claim 7, wherein the volume ratio of dichloromethane to methanol used in the separation by the second silica gel column chromatography is 20:1, 15:1 and 10:1, respectively, and after the separation by the second silica gel column chromatography, the eluents when the volume ratio of dichloromethane to methanol is 15:1 and 10:1 are collected.

9. the chromatography columns used in the first, second and third reversed phase chromatographic separations are ODS chromatography columns, and the elution methods in the first, second and third reversed phase chromatographic separations are gradient elution; The gradient elution procedure of the first reversed phase chromatographic separation comprises: From 0.00 to 2.00 h, the volume fraction of mobile phase A was kept at 40%; 2.00 to 2.01 h, the volume fraction of mobile phase A was increased from 40% to 60% at a constant rate; 2.01 to 4.00 h, the volume fraction of mobile phase A was kept at 60%, 4.00-4.01h, the volume fraction of mobile phase A is increased from 60% to 80% at a constant rate; 4.01 to 5.50 h, the volume fraction of mobile phase A is 80%, 5.50-5.51h, the volume fraction of mobile phase A was increased from 80% to 100% at a constant rate; 5.51 to 6.00 h, the volume fraction of mobile phase A is kept at 100%; The gradient elution procedure for the second and third reversed phase chromatographic separations comprises: From 0.00 to 2.00 h, the volume fraction of mobile phase A was kept at 0%; 2.00-2.01h: The volume fraction of mobile phase A was increased from 0% to 60% at a constant rate. 2.01 to 4.00 h, the volume fraction of mobile phase A was kept at 60%, 4.00 to 4.01 h, the volume fraction of mobile phase A was increased from 60% to 80% at a constant rate; 4.01 to 5.50 h, the volume fraction of mobile phase A is 80%, 5.50-5.51h, the volume fraction of mobile phase A was increased from 80% to 100% at a constant rate; The extraction method according to claim 3, characterized in that the volume fraction of mobile phase A is maintained at 100% for 5.51 to 6.00 h.

10. The extraction method according to claim 3, wherein the chromatography columns used in the first high performance liquid chromatography separation and the second high performance liquid chromatography separation are reverse-phase C18 silica gel matrix chromatography columns, the volume fraction of acetonitrile in the mobile phase used in the first high performance liquid chromatography separation is 25 to 40%, the volume fraction of acetonitrile in the mobile phase used in the second high performance liquid chromatography separation is 35 to 40%, and the elution method for the first high performance liquid chromatography separation and the second high performance liquid chromatography separation is isocratic elution.

11. A method for preparing an anti-inflammatory drug, comprising applying the C. persicae diterpene lactone compound described in claim 1.

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