Linderane-type sesquiterpene dimer and preparation method and use thereof

Abstract

The present invention belongs to the field of phytochemistry and medicine, and relates to a linderane-type sesquiterpene dimer, a preparation method and use thereof, and also relates to a medicine or a pharmaceutical composition containing the linderane-type sesquiterpene dimer. The linderane-type sesquiterpene dimers chlorahololides H, J, L and chlorajaponilide E of the present invention have significant NLRP3 inflammasome inhibitory activity and can be used to treat chronic inflammatory diseases mediated by NLRP3 inflammasome.

Description

Technical Field

[0001] The present invention belongs to the field of plant chemistry and pharmaceutical technology, and in particular, relates to chlorahololides H, J, L and chlorajaponilide E, a medicine or a pharmaceutical composition containing the chlorahololides H, J, L and chlorajaponilide E, and also relates to a preparation method of the chlorahololides H, J, L and chlorajaponilide E and an application of the chlorahololides H, J, L and chlorajaponilide E in preparing a medicine or a pharmaceutical composition for treating chronic inflammatory diseases. Background Art

[0002] Inflammasomes are a class of protein complexes that play an important role in immune regulation and inflammatory processes. As an intracellular signaling platform, inflammasomes can sense and respond to intracellular and extracellular stimuli, including microbial infection, cell damage, and environmental stress. Among them, the NLRP3 inflammasome is a member of the NOD-like receptor (NLR) family, and members of this family play an important role in sensing and regulating endogenous and exogenous signals in host cells. The NLRP3 inflammasome is composed of multiple components, including NLRP3 protein, apoptosis-associated speck-like protein containing a CARD (ASC), and pro-cysteinyl aspartate specific proteinase-1 (pro-caspase-1). The NLRP3 inflammasome is activated when it is infected by microorganisms, cell damage, or other stress stimuli. The signals that activate the NLRP3 inflammasome usually involve disorders in the intracellular and extracellular environment, such as intracellular K + Ion loss, Ca 2+ Signaling, accumulation of reactive oxygen species (ROS), mitochondrial dysfunction and lysosomal rupture, etc. After activation, NLRP3 inflammasome cleaves pro-caspase-1 to generate biologically active caspase-1, which further promotes the maturation and release of pro-inflammatory cytokines such as IL-1β (interleukin-1β) and IL-18 (interleukin-18), thereby inducing inflammatory effects.

[0003] Abnormal activation of NLRP3 inflammasomes is associated with the occurrence and development of a variety of inflammatory-related diseases, including rheumatoid arthritis, inflammatory bowel disease, type II diabetes, etc. Therefore, regulation of NLRP3 inflammasomes has become a potential therapeutic strategy for the treatment of these chronic inflammatory diseases. The development of inhibitors of NLRP3 inflammasome activation assembly provides a new treatment for related diseases. MCC950 has been widely studied as a specific small molecule inhibitor of NLRP3 inflammasomes. Its potential application in the treatment of inflammatory diseases such as rheumatoid arthritis has attracted widespread attention and even entered the Phase II clinical trial stage. However, due to the problem of liver toxicity, it ultimately failed to pass the clinical trial.

[0004] As an important source for the development of drug lead compounds, natural products play an indispensable role in drug research and development. It is of great significance to find NLRP3 inflammasome small molecule inhibitors with both low toxicity and high activity from natural products. Many plants in the genus Chloranthus are called "four tiles". They are commonly used in folk medicine to treat trauma, snake bites, and rheumatic joint pain. They are Chinese herbal medicines with important clinical value. There is great potential in finding natural products with therapeutic effects on chronic inflammatory diseases. Summary of the invention

[0005] In view of this, the object of the present invention is to provide chlorahololide-type sesquiterpene dimers with NLRP3 inflammasome inhibitory activity, namely compounds chlorahololides H, J, L and chlorajaponilide E; drugs or pharmaceutical compositions containing these compounds; the use of these compounds in the preparation of drugs or pharmaceutical compositions for the treatment of chronic inflammatory diseases, and methods for preparing these compounds, thereby providing new options for the development of NLRP3 inflammasome inhibitors.

[0006] The purpose of the present invention can be achieved through the following technical solutions.

[0007] In the first aspect, the present invention provides a linderane-type sesquiterpene dimer or a pharmaceutically acceptable derivative thereof represented by the following structural formulas I, II, III and IV:

[0008]

[0009] In a second aspect, the present invention provides a drug or a pharmaceutical composition for treating chronic inflammatory diseases, comprising at least one of the linderane-type sesquiterpene dimers shown in Formulas I, II, III and IV or pharmaceutically acceptable derivatives thereof.

[0010] In a third aspect, the present invention relates to the use of chlorahololides H, J, L and chlorajaponilide E or pharmaceutically acceptable derivatives thereof in the preparation of a medicament or pharmaceutical composition for treating chronic inflammatory diseases.

[0011] In an embodiment of the present invention, the chronic inflammatory disease is a chronic inflammatory disease mediated by NLRP3 inflammasome, including but not limited to arthritis, such as rheumatoid arthritis, rheumatoid arthritis, gouty arthritis; type II diabetes; non-alcoholic fatty liver disease; inflammatory bowel disease; neurological diseases and brain damage, including multiple sclerosis, Alzheimer's disease, etc.

[0012] In a fourth aspect, the present invention provides a method for preparing chlorahololides H, J, L and chlorajaponilide E, comprising the following steps:

[0013] 1) drying and crushing a Chloranthus plant material, extracting with an organic solvent, and then desolventizing to obtain a Chloranthus plant material extract; and

[0014] 2) The extract of the genus Chrysanthemi is subjected to column chromatography to obtain chlorahololides H, J, L and chlorajaponilide E, which are linderane-type sesquiterpene dimers.

[0015] In an embodiment of the method of the present invention, the organic solvent used in step 1) can be at least one of petroleum ether, chloroform, dichloromethane, ethyl acetate, acetone, ethanol, methanol, n-butanol and acetonitrile; preferably, the organic solvent is ethyl acetate, methanol, 95% ethanol or a mixture thereof.

[0016] Compared with the prior art, the present invention has obvious beneficial effects. Specifically, the chlorahololides H, J, L and chlorajaponilide E of the present invention have significant NLRP3 inflammasome inhibitory activity and can be used to treat chronic inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The chemical structural formulas are the chlorahololides H, J, L and chlorajaponilide E of the linderane-type sesquiterpene dimers of the present invention.

[0018] Figure 2 Figure 2 shows the inhibition of pyroptosis in monocyte-macrophage J774A.1 cells by chlorajaponilide E.

[0019] Figure 3 Quantification of chlorajaponilide E inhibiting pyroptosis in monocyte-macrophage J774A.1 cells. DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the present invention, the present invention will be described more fully below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0022] According to the first aspect, the present invention provides chlorahololides H, J, L and chlorajaponilide E or pharmaceutically acceptable derivatives thereof having NLRP3 inflammasome inhibitory activity, such as pharmaceutically acceptable salts, esters or prodrugs, wherein the structural formulas of the chlorahololides H, J, L and chlorajaponilide E are shown in the following formulas I, II, III and IV, respectively:

[0023]

[0024] According to the second aspect of the present invention, the present invention provides a medicine or pharmaceutical composition for treating chronic inflammatory diseases, comprising at least one of the linderane-type sesquiterpene dimers chlorahololides H, J, L and chlorajaponilide E or their pharmaceutically acceptable derivatives as active pharmaceutical ingredients. It should be understood by those skilled in the art that the medicine or pharmaceutical composition for treating chronic inflammatory diseases invented herein may also include a pharmaceutically acceptable carrier, adjuvant or excipient. Therefore, in a more specific embodiment, the medicine or pharmaceutical composition for treating chronic inflammatory diseases comprises at least one of the linderane-type sesquiterpene dimers chlorahololides H, J, L and chlorajaponilide E or their pharmaceutically acceptable derivatives as active pharmaceutical ingredients and a pharmaceutically acceptable carrier, adjuvant or excipient.

[0025] In an embodiment of the medicine or pharmaceutical composition of the present invention, the linderane-type sesquiterpene dimer as an active ingredient may be any one of chlorahololides H, J, L and chlorajaponilide E or any combination thereof.

[0026] Those skilled in the art will appreciate that pharmaceutically acceptable derivatives of the compounds chlorahololides H, J, L or chlorajaponilide E of the present invention, such as pharmaceutically acceptable salts, esters, solvates or hydrates or prodrugs of these compounds, can also be used as active ingredients in the medicaments or pharmaceutical compositions of the present invention. Therefore, in another embodiment of the medicament or pharmaceutical composition of the present invention, the active ingredient can be a pharmaceutically acceptable derivative of chlorahololides H, J, L or chlorajaponilide E, such as pharmaceutically acceptable salts, esters, solvates or hydrates or prodrugs of these compounds.

[0027] It is well known to those skilled in the art that pharmaceutically acceptable salts may be, for example, pharmaceutically acceptable base addition salts. Pharmaceutically acceptable base addition salts include salts derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc. Salts derived from pharmaceutically acceptable non-toxic organic bases include salts of primary, secondary and tertiary amines, including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine and triethanolamine.

[0028] The pharmaceutically acceptable esters may be esters formed between the linderane-type sesquiterpene dimer of the present invention and pharmaceutically acceptable non-toxic organic acids, such as acetic acid, propionic acid, malic acid, citric acid, oxalic acid and palmitic acid.

[0029] Therefore, in the embodiment of the pharmaceutical composition of the present invention, the active pharmaceutical ingredient can be any one, a mixture of two, three or four of chlorahololides H, J, L or chlorajaponilide E, or their esters, solvates, hydrates and other derivatives.

[0030] Pharmaceutically acceptable carriers, adjuvants or excipients are well known to those skilled in the art. Pharmaceutical carriers or excipients are one or more solid, semisolid and liquid diluents, fillers and adjuvants for pharmaceutical products, including but not limited to fillers (diluents), lubricants (glidants or anti-adhesives), dispersants, wetting agents, adhesives, solubilizers, antioxidants, antibacterial agents, emulsifiers, disintegrants, etc. Binders include syrup, gum arabic, gelatin, sorbitol, tragacanth, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose or hydroxypropyl methylcellulose, etc.), gelatin slurry, syrup, starch slurry or polyvinyl pyrrolidone, etc.; fillers include lactose, powdered sugar, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salts (such as calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol or glycine, etc.; lubricants include micropowdered silica gel, magnesium stearate, talc, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, etc.; Disintegrants include starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinyl pyrrolidone or microcrystalline cellulose, etc.; wetting agents include sodium lauryl sulfate, water or alcohol, etc.; antioxidants include sodium sulfite, sodium bisulfite, sodium metabisulfite, dibutyl benzoic acid, etc.; antibacterial agents include 0.5% phenol, 0.3% cresol, 0.5% trichlorobutanol, etc.; emulsifiers include polysorbate-80, sorbitan monohydrate, lecithin, soybean lecithin, etc.; solubilizers include Tween-80, bile, glycerol, etc.

[0031] When the linderane-type sesquiterpene dimer of the present invention is used as a medicine, it can be directly administered or administered in the form of a pharmaceutical composition. In the pharmaceutical composition of the present invention, the pharmaceutical composition can contain 0.1-99% chlorahololides H, J, L or chlorajaponilide E, preferably 0.5-50%, based on the total weight of the pharmaceutical composition.

[0032] An "effective amount" of chlorahololides H, J, L or chlorajaponilide E or a pharmaceutically acceptable derivative thereof, such as an ester, refers to an amount sufficient to achieve the desired biological effect. It should be understood that the effective dose will depend on the age, sex, health status and weight of the recipient. Typically, the effective amount is determined by the person administering the treatment, such as a treating physician.

[0033] The pharmaceutical composition of the present invention can be administered in the form of a dosage per unit body weight. All pharmaceutical compositions with chlorahololides H, J, L or chlorajaponilide E or their pharmaceutically acceptable derivatives such as esters as active ingredients are prepared into various dosage forms using methods recognized in the pharmaceutical and food fields, such as liquid preparations (injections, suspensions, emulsions, solutions, syrups, etc.), solid preparations (tablets, capsules, granules, granules, etc.), sprays, aerosols, etc. The pharmaceutical composition of the present invention can be used for the treatment of inflammatory diseases through injection (intravenous injection, intravenous drip, intramuscular injection, intraperitoneal injection, subcutaneous injection) and oral, sublingual administration, mucosal dialysis and other administration routes.

[0034] According to a third aspect, the present invention provides use of chlorahololides H, J, L or chlorajaponilide E or pharmaceutically acceptable derivatives thereof in the preparation of a medicament or pharmaceutical composition for treating chronic inflammatory diseases.

[0035] In an embodiment of the present invention, the chronic inflammatory disease is a chronic inflammatory disease mediated by NLRP3 inflammasome, including but not limited to arthritis, such as rheumatoid arthritis, rheumatoid arthritis, gouty arthritis; type II diabetes; non-alcoholic fatty liver disease; inflammatory bowel disease; neurological diseases and brain damage, including multiple sclerosis, Alzheimer's disease, etc.

[0036] According to a fourth aspect, the present invention provides a method for preparing chlorahololides H, J, L and chlorajaponilide E, comprising the following steps:

[0037] 1) drying and crushing the Chrysanthemi plant material, extracting and desolventizing with an organic solvent to obtain a Chrysanthemi plant extract; and

[0038] 2) subjecting the extract of the genus Chrysanthemi to column chromatography to obtain chlorahololides H, J, L and chlorajaponilide E.

[0039] In an embodiment of the present invention, the Chrysanthemi plant material can be any part of the Chrysanthemi plant, such as roots, stems, branches and leaves or the whole plant. In an embodiment of the present invention, the Chrysanthemi plant is a plant in the Chrysanthemi genus, preferably C. holostegius, C. henryi or C. holostegius var. shimianensis.

[0040] In an embodiment of the present invention, in step 1), the organic solvent may be at least one of petroleum ether, chloroform, dichloromethane, ethyl acetate, acetone, ethanol, methanol, n-butanol and acetonitrile; preferably, the organic solvent is ethyl acetate, methanol, 95% ethanol or a mixture thereof.

[0041] In an embodiment of the present invention, in step 1), the extraction method can be organic solvent cold extraction, heating reflux extraction or organic solvent ultrasonic extraction.

[0042] In an embodiment of the present invention, the Chrysanthemi plant material is extracted with an organic solvent at least once, for example, 2, 3, 4 times, preferably 3 times.

[0043] In an embodiment of the present invention, the volume ratio of the organic solvent to the dried Chrysanthemum plant material can be 1:1 to 5:1, for example 1:1, 2:1, 3:1, 4:1, 5:1, and ratios between any two of the above ratios, for example 1.5:1, 2.5:1, 3.5:1, 4.5:1, etc., preferably 3:1.

[0044] In a specific embodiment of the present invention, in step 2), column chromatography includes normal phase silica gel column chromatography, reverse phase silica gel column chromatography (e.g. RP-18 column chromatography), resin column chromatography (e.g. D101 macroporous resin), gel column chromatography (e.g. Sephadex LH-20), medium pressure chromatography separation gel (e.g. MCI gel) and semi-preparative high performance liquid chromatography (hereinafter referred to as semi-preparative HPLC).

[0045] In a specific embodiment of the present invention, in step 2), the eluent used for column chromatography is at least one of petroleum ether, dichloromethane, chloroform, ethyl acetate, acetone, ethanol, methanol, n-butanol, acetonitrile, water and formic acid, for example, a combination of any two, any three, any four of the above organic solvents. In a more specific embodiment of the present invention, for normal phase silica gel column chromatography, petroleum ether and acetone can be used for gradient elution in a volume ratio of 1:0 to 0:1, for example, 1:0, 50:1, 20:1, 10:1, 5:1, 1:1, 1:5, 0:1. In a more specific embodiment of the present invention, for normal phase silica gel column chromatography, dichloromethane and methanol can also be used for gradient elution in a volume ratio of 1:0 to 1:1, for example, 1:0, 200:1, 100:1, 50:1, 20:1, 10:1, 1:1. In a more specific embodiment of the present invention, for reversed-phase silica gel column chromatography, such as reversed-phase RP-18 column chromatography, methanol and water can be used for gradient elution in a volume ratio of 2:8 to 10:0, for example, the volume ratio of methanol to water can be 2:8, 5:5, 8:2, 10:0. In a more specific embodiment of the present invention, for resin column chromatography, such as D101 macroporous resin column chromatography, methanol and water can be used for gradient elution in a volume ratio of 2:8 to 10:0, for example, the volume ratio of methanol to water can be 2:8, 5:5, 8:2, 10:0. In a more specific embodiment of the present invention, for gel column chromatography, such as Sephadex LH-20 gel column chromatography, methanol or chloroform-methanol (1:1, v:v) can be used for elution. In a more specific embodiment of the present invention, for medium pressure chromatography separation gel, such as MCI gel, ethanol and water can be used for gradient elution in a volume ratio of 2:8 to 10:0, for example, the volume ratio of ethanol to water can be 2:8, 5:5, 8:2, 10:0. In a more specific embodiment of the present invention, for semi-preparative HPLC, acetonitrile and water can be used for isocratic elution, for example, elution is carried out with 50% acetonitrile-water as the mobile phase. In a more specific embodiment of the present invention, for semi-preparative HPLC, acetonitrile and water gradient elution can also be used, for example, acetonitrile and water can be used for gradient elution in a volume ratio of 2:8 to 8:2.

[0046] The preferred embodiments of the present invention are described in detail below in conjunction with the examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention, and all of these modifications and substitutions fall within the scope of the claims of the present invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained through commercial channels.

[0048] In the following examples, the optical rotation was measured using an Autopol VI polarimeter, the electronic circular dichroism (ECD) was measured using an Applied Photophysics circular dichroism spectrometer, and the ultraviolet spectrum (UV) was measured using a Shimadzu UV2700 ultraviolet spectrometer. 1 H NMR, 13 C NMR and 2D NMR spectra were recorded using a Bruker AM-500 or AM-600 nuclear magnetic resonance instrument. High-resolution mass spectrometry (HR-ESI-MS) was measured using an Agilent 1290UPLC / 6540Q-TOF mass spectrometer. Semi-preparative HPLC used an Agilent 1260 liquid phase, and the chromatographic column was a Zorbax SB-C18 preparative column, with a flow rate of 3.0 mL / min. Column chromatography involved the use of silica gel (200-300 mesh, Qingdao Ocean Chemical Company, China), RP-18 (40-60 μm, Merk), D101 macroporous resin (Shanghai McLean Biochemical Technology Co., Ltd., China), MCI gel (75-150 μm, Mitsubishi Chemical Corporation, Japan) and Sephadex LH-20 (GE Healthcare, USA). Thin layer chromatography (TLC) was performed on a silica gel GF254 plate (Qingdao Ocean Chemical Company, China), and its spots were observed by a 10% sulfuric acid-ethanol solution. The absorbance value was measured using a Biotek ELX800 multifunctional microplate reader, and the fluorescence images were taken using an inverted microscope (Axio Observer3, Zeiss; Oberkochen, Germany).

[0049] Example 1: Preparation of compounds chlorahololides H, J, L and chlorajaponilide E

[0050] The whole plant of Asbestos variegata (20 kg) was dried and crushed, and 60 L of methanol (the volume ratio of organic solvent to plant material was 3:1) was added, and the mixture was heated at 65 ° C for 3 times, 3 h / time, and the extracts were combined and distilled under reduced pressure to obtain a methanol extract (1.8 kg). The methanol extract was passed through a D101 macroporous resin column and eluted with a methanol-water gradient (2:8, 5:5, 8:2, 10:0, v / v), with 4 to 6 column volumes for each gradient. The fraction eluted with a methanol-water volume ratio of 8:2 (162.4 g) was passed through a normal phase silica gel column chromatography and eluted with a dichloromethane-methanol gradient (1:0, 200:1, 100:1, 50:1, 20:1, 10:1, 1:1, v / v), with 4 to 6 column volumes for each gradient. According to thin layer chromatography, 10% sulfuric acid-ethanol solution was used for color development and then combined to obtain 7 components, namely Fr.1-Fr.7. Fr.5 (22.6 g) was chromatographed on a RP-18 reverse phase silica gel column using a gradient elution of methanol-water (2:8, 3:7, 5:5, 7:3, 8:2, 10:0, v / v), with 4 to 6 column volumes for each gradient, to obtain 6 components, namely Fr.5.1-Fr.5.6. Fr.5.3 (2.9 g) was chromatographed on a normal phase silica gel column using a gradient elution of petroleum ether-acetone (20:1, 10:1, 5:1, 1:1, 1:5, v / v), with 4 to 6 column volumes for each gradient, to obtain 5 components, namely Fr.5.3.1-Fr.5.3.5. Fr.5.3.2 (675.2 mg) was prepared by semi-preparative HPLC using an isocratic elution method with 50% acetonitrile-water as the mobile phase to obtain 7.8 mg chlorahololide H (yield: 0.000039%) and 4.0 mg chlorajaponilide E (yield: 0.00002%). Fr.5.4 (3.6 g) was prepared by normal phase silica gel column chromatography using dichloromethane-methanol (100:1, v / v) for isocratic elution to obtain 6 components, namely Fr.5.4.1-Fr.5.4.6. Fr.5.4.3 (573.3 mg) was prepared by semi-preparative HPLC using an isocratic elution method with 60% acetonitrile-water as the mobile phase to obtain 10.0 mg chlorahololide J (yield: 0.00005%) and 14.0 mg chlorahololide L (yield: 0.00007%).

[0051] Physical constants and spectral data of Chlorahololide H: White powder; (c0.14,MeOH); UV(MeOH)λ max (logε)223(3.18)nm; CD(MeOH)λ max(Δε)208(-14.1),251(+9.75),335(-1.33)nm; 1 HNMR(500MHz)δ1.59(1H,m,H-1),0.93(1H,td,J=7.6,4.5Hz,H-2α),0.34(1H,q,J=4.2Hz,H-2β),1.86(1H,m,H-3),3.88(1H,d,J=3.4Hz,H-6),2.69(1H,d,J=19.1Hz,H-9α),2.53(1H,m,H-9β),1.83(3H,s,H3-13),1.17(3H,s,H3-14),2.74(1H,dd,J=16.1,1.5Hz,H-15α),2.53(1H,m,H-15β),1.39(1H,td,J=8.4,3.9Hz,H-1′),0.71(1H,td,J=8.4,5.6Hz,H-2′α),1.78(1H,m,H-2′β),1.19(1H,m,H-3′),1.62(1H,m,H-5′),2.38(2H,m,H-6),1.92(1H,dd,J=5.9,1.8Hz,H-9′),4.35(2H,m,H-13′),0.62(3H,s,H3-14′),3.53(1H,dd,J=11.4,6.1Hz,H-15′a),3.57(1H,dd,J=11.4,5.0Hz,H-15′b),3.72(3H,s,MeO-12); 13CNMR(CDCl3,125MHz)δ28.4(C-1,d),16.2(C-2,t),24.9(C-3,d),138.8(C-4,s),134.1(C-5,s),41.2(C-6,d),137.3(C-7,s),204 .9(C-8,s),52.2(C-9,t),45.2(C-10,s),142.9(C-11,s),170.8(C-12,s),19.4(C-13,q),22.8(C-14,q),25.5(C-15,t),24.1(C- 1′,d),16.7(C-2′,t),21.3(C-3′,d),47.2(C-4′,d),57.8(C-5′,d),24.9(C-6′,t),169.3(C-7′,s),93.3(C-8′,s),54.4(C-9′,d ),44.0(C-10′,s),126.1(C-11′,s),172.8(C-12′,s),55.1(C-13′,t),24.4(C-14′,q),63.2(C-15′,t),52.8(MeO-12,q).HRESIMS m / z 543.2360[M+Na] + (calcd for C 31 H 36 NaO7 + ,543.2359).

[0052] Physical constants and spectral data of Chlorahololide J: Yellow colloid; (c 0.14, MeOH); UV(MeOH)λ max (logε)219(3.42)nm; CD(MeOH)λ max (Δε)209(-21.9),253(+18.6),333(-3.02)nm; 1HNMR(CDCl3,600MHz)δ1.60(1H,o,H-1),0.95(1H,td,J=7.7,4.7Hz,H-2α),0.36(1H,q,J=4.1Hz,H-2β),1.84(1H,o,H-3),3.88(1H,d,J=3.7Hz,H-6),2.36(1H,d,J=18.5Hz,H-9α),2.56(1H,d,J=18.5Hz,H-9β),1.81(3H,s,H3-13),1.15(3H,s,H3-14),2.75(1H,d,J=16.6Hz,H-15α),2.55(1H,o,H-15β),1.60(1H,o,H-1′),0.71(1H,td,J=8.7,5.8Hz,H-2′α),1.31(1H,qJ=4.0Hz,H-2′β),1.39(1H,td,J=8.8,3.5Hz,H-3′),1.70(1H,dd,J=13.6,6.2Hz,H-5′),2.46(1H,dd,J=18.5,6.1Hz,H-6′α),2.73(1H,dd,J=18.5,13.6Hz,H-6′β),1.84(1H,m,H-9′),4.65(1H,d,J=12.4Hz,H-13′a),4.98(1H,d,J=12.5Hz,H-13′b),0.82(3H,s,H3-14′),3.69(1H,d,J=11.6Hz,H-15′a),4.53(1H,d,J=11.7,Hz,H-15′b),6.89(1H,m,H-3″),4.35(1H,m,H-4″a),4.42(1H,dd,J=15.7,4.8Hz,H-4″b),1.87(3H,s,H3-5″),2.12(3H,s,H-2″′),3.65(3H,s,MeO-12); 13C NMR(CDCl3,150MHz)δ28.6(C-1,d),16.3(C-2,t),24.8(C-3,d),138.5(C-4,s) ,134.7(C-5,s),41.2(C-6,d),136.5(C-7,s),201.0(C-8,s),52.2(C-9,t),45. 2(C-10,s),143.0(C-11,s),171.0(C-12,s),19.1(C-13,q),22.8(C-14,q),25. 3(C-15,t),25.3(C-1′,d),11.7(C-2′,t),27.9(C-3′,d),77.7(C-4′,s),61.2( C-5′,d),23.1(C-6′,t),173.3(C-7′,s),93.2(C-8′,s),55.6(C-9′,d),45.0( C-10′,s),123.0(C-11′,s),171.9(C-12′,s),54.7(C-13′,t),26.5(C-14′,q), 71.8(C-15′,t),168.1(C-1″,s),127.2(C-2″,d),142.5(C-3″,d),60.1(C-4″,t ),12.8(C-5″,q),171.4(C-1″′,s),20.7(Me-2″′,q),53.0(MeO-12,q).HRESIMS m / z699.2781[M+Na] + (calcd for C 38 H 44 NaO 11 + ,699.2781).

[0053] Physical constants and spectral data of Chlorahololide L: Yellow colloid; (c 0.11,MeOH); UV(MeOH)λ max (logε)220(3.29)nm; CD(MeOH)λ max (Δε)209(-21.5),259(+34.2),330(-6.69)nm; 1H NMR(CDCl3,600MHz)δ1.56(1H,o,H-1),0.92(1H,m,H-2α),1.33(1H,m,H-2β),1.87(1H,o,H-3),2.25(1H,d,J=17.8Hz,H-9α),2.55(1H,d,J=17.8Hz,H-9β),1.70(3H,s,H3-13),1.23(3H,s,H3-14),3.00(1H,dd,J=14.1,7.1Hz,H-15α),1.52(1H,o,H-15β),1.52(1H,o,H-1′),0.58(1H,td,J=8.9,5.6Hz,H-2′α),1.18(1H,m,H-2′β),1.59(1H,m,H-3′),1.44(1H,dd,J=13.0,6.7Hz,H-5′),2.31(1H,dd,J=17.5,6.5Hz,H-6′α),2.83(1H,dd,J=17.5,13.1Hz,H-6′β),2.57(1H,d,J=17.2Hz,H-9′),4.38(1H,d,J=13.8Hz,H-13′a),4.44(1H,d,J=13.6Hz,H-13′b),0.97(3H,s,H3-14′),3.89(1H,d,J=11.6Hz,H-15′a),4.25(1H,d,J=11.5,Hz,H-15′b),6.90(1H,m,H-3″),1.83(3H,d,J=7.2Hz,H-4″),1.86(3H,s,H3-5″),3.70(3H,s,MeO-12); 13C NMR(CDCl3,150MHz)δ28.7(C-1,d),8.4(C-2,t),29.4(C-3,d),90.4(C-4, s),160.7(C-5,s),126.6(C-6,s),136.5(C-7,s),201.4(C-8,s),48.6(C-9 ,t),44.5(C-10,s),135.2(C-11,s),169.6(C-12,s),20.0(C-13,q),22.1( C-14,q),37.1(C-15,t),27.2(C-1′,d),10.5(C-2′,t),29.4(C-3′,d),77. 8(C-4′,s),54.6(C-5′,d),21.7(C-6′,t),166.4(C-7′,s),87.6(C-8′,s), 51.8(C-9′,d),45.2(C-10′,s),127.8(C-11′,s),173.0(C-12′,s),55.4(C -13′,t),24.3(C-14′,q),70.8(C-15′,t),168.4(C-1″,s),128.4(C-2″,d) ,138.6(C-3″,d),14.7(C-4″,q),12.3(C-5″,q),52.7(MeO-12,q).HRESIMS m / z 673.2626[M+Na] + (calcd for C 36 H 42 NaO 11 + ,673.2625).

[0054] Physical constants and spectral data of Chlorajaponilide E: White powder; 1H NMR(500MHz,Chloroform-d)δ2.00(1H,m,H-1),1.23(1H,m,H-2α),0.95(1H,m,H-2β),1.85(1H,m,H-3),3.75(1H,s,H-9),1.78(3H,s,H3-13),1.02(3H,s,H3-14),3.07(1H,dd,J=14.3,7.1Hz,H-15α),1.60(1H,m,H-15β),1.58(1H,m,H-1′),1.18(1H,m,H-2′α),0.60(1H,td,J=9.0,5.5Hz,,H-2′β),1.60(1H,m,H-3′),1.60(1H,m,H-5′),2.88(1H,dd,J=17.5,13.1Hz,H-6′α),2.25(1H,dd,J=17.5,6.7Hz,H-6′β),2.59(1H,dd,J=10.0,7.1Hz,H-9′),4.43(1H,d,J=13.7Hz,H-13′a),4.36(1H,d,J=13.9Hz,H-13′b),0.98(3H,s,H3-14′),4.15(1H,d,J=11.4Hz,H-15′a),3.93(1H,d,J=11.5Hz,H-15′b),6.84(1H,m,H-3″),1.84(3H,s,H-4″),1.85(3H,s,H3-5″),3.78(3H,s,MeO-12); 13C NMR(125MHz,Chloroform-d)δ26.1(C-1,d),8.4(C-2,t),27.8(C-3,d),90.6(C-4,s),158.7(C-5,s),127.1(C-6,s),143.0(C-7,s),198.9(C-8,s),7 7.9(C-9,d),50.3(C-10,d),129.2(C-11,s),170.3(C-12,s),21.3(C-13,q ),15.4(C-14.q),36.8(C-15,t),27.4(C-1′,d),10.5(C-2′,t),29.3(C-3′ ,d),77.7(C-4′,s),54.8(C-5′,d),21.9(C-6′,t),166.5(C-7′,s),87.6(C -8′,s),52.5(C-9′,d),45.2(C-10′,s),128.5(C-11′,s),173.1(C-12′,s) ,55.1(C-13′,t),24.4(C-14′,q),70.6(C-15′,t),168.3(C-1″,s),128.2( C-2″,s), 139.0(C-3″,d), 14.7(C-4″,q), 12.3(C-5″,q), 52.9(MeO-12,q).

[0055] Example 2: Preparation Method 2 of Compounds Chlorahololides H, J, L and Chlorajaponilide E

[0056] The dried root (20 kg) of the whole-margined golden millet plant variety of asbestos was crushed, added with 100L ethyl acetate (the volume ratio of organic solvent to plant material was 5:1), and cold-extracted 4 times, 3 days / time, and the extracts were combined and distilled under reduced pressure to obtain a paste extract (1.1 kg). The paste extract was chromatographed on a normal phase silica gel column, eluted with a petroleum ether-acetone gradient (1:0, 50:1, 10:1, 1:1, 1:5, 0:1, v / v), 4 to 6 column volumes per gradient, and 6 components were obtained, namely Fr.1-Fr.6. Fr.4 (88.2 g) was chromatographed on an MCI gel column, eluted with an ethanol-water gradient (2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 10:0, v / v), 4 to 6 column volumes per gradient, and 8 components were obtained, namely Fr.4.1-Fr.4.8. Fr.4.3 (7.8 g) was chromatographed on a normal phase silica gel column using a chloroform-methanol gradient elution (200:1, 50:1, 20:1, 10:1, 1:1, v / v), with 4 to 6 column volumes for each gradient, to obtain five components, namely Fr.4.3.1–Fr.4.3.5. Fr.4.3.3 (267.9 mg) was prepared by semi-preparative HPLC using an isocratic elution method with 55% acetonitrile-water as the mobile phase to obtain 12.0 mg chlorahololide H (yield: 0.00006%) and 6.0 mg chlorajaponilide E (yield: 0.00003%). Fr.4.3.4 (437.2 mg) was prepared by semi-preparative HPLC using a gradient elution method (0-30 min, acetonitrile concentration from 30% → 80%, v / v) to obtain 16.0 mg chlorahololide J (yield: 0.00008%) and 4.0 mg chlorahololide L (yield: 0.00002%).

[0057] Example 3: Preparation of compounds chlorahololides H, J, L and chlorajaponilide E

[0058] The whole plant of Chrysanthemi Integrinaceae (15 kg) was dried and crushed, and then extracted twice with 45 L 95% ethanol (the volume ratio of organic solvent to plant material was 3:1) at 70 ° C under heating and reflux, 3 h / time, and the extracts were combined and distilled under reduced pressure to obtain an ethanol extract (1.4 kg). After the ethanol extract was suspended in 6 L of pure water, 6 L of ethyl acetate was added and extracted three times. After the extracts were combined, the solvent was recovered to obtain an ethyl acetate extract (763.5 g). The ethyl acetate extract was chromatographed on a medium MCI gel column and eluted with a methanol-water gradient (2:8, 3:7, 5:5, 7:3, 8:2, 10:0, v / v), with 4 to 6 column volumes for each gradient, to obtain 6 components, namely Fr.1-Fr.6. Fr.3 (146.7 g) was chromatographed on a normal phase silica gel column using dichloromethane-methanol as the eluent for gradient elution (200:1, 100:1, 50:1, 10:1, 1:1, v / v), with 4 to 6 column volumes for each gradient, to obtain 5 components, namely Fr.3.1-Fr.3.5. Fr.3.2 (24.3 g) was chromatographed on a Sephadex LH-20 gel column using methanol as the eluent, and then prepared by semi-preparative HPLC using acetonitrile-water as the mobile phase and an isocratic elution method (acetonitrile concentration 55%) to obtain 4.5 mg chlorahololide H (yield: 0.00003%) and 15.0 mg chlorajaponilide E (yield: 0.0001%). Fr.3.3 (16.4 g) was prepared by Sephadex LH-20 gel column chromatography with chloroform-methanol (1:1, v:v) as eluent, and then by semi-preparative HPLC with acetonitrile-water as mobile phase and isocratic elution method (acetonitrile concentration 60%) to obtain 18 mg chlorahololide J (yield: 0.00012%) and 13.5 mg chlorahololide L (yield: 0.00009%).

[0059] Example 4: Preparation of compounds chlorahololides H, J, L and chlorajaponilide E

[0060] The whole plant of broad-leaved golden millet (20 kg) was dried and crushed, and ultrasonically extracted three times at room temperature with 20 L of methanol (the volume ratio of organic solvent to plant material was 1:1). The extracts were combined and distilled under reduced pressure to obtain a paste extract (1.3 kg). The paste extract was chromatographed on a normal phase silica gel column and eluted with a dichloromethane-methanol gradient (1:0, 200:1, 100:1, 50:1, 20:1, 10:1, 1:1, v / v), with 4 to 6 column volumes for each gradient, to obtain 7 components, namely Fr.1-Fr.7. Fr.5 (127.4 g) was chromatographed on an RP-18 reverse phase silica gel column and eluted with a methanol-water gradient (2:8, 5:5, 8:2, 10:0, v / v), with 4 to 6 column volumes for each gradient, to obtain 4 components, namely Fr.5.1-Fr.5.4. Fr.5.3 (16.4 g) was chromatographed on a normal phase silica gel column using a petroleum ether-acetone gradient elution (10:1, 1:1, 1:5, 0:1, v / v), with 4 to 6 column volumes for each gradient, to obtain four components, namely Fr.5.3.1-Fr.5.3.4. Fr.5.3.2 (478.6 mg) was prepared by semi-preparative HPLC using an isocratic elution method with 50% acetonitrile-water as the mobile phase to obtain 10.0 mg chlorahololide H (yield: 0.00005%) and 16.0 mg chlorajaponilide E (yield: 0.00008%). Fr.5.4 (23.3 g) was chromatographed on a normal phase silica gel column with a gradient elution of petroleum ether-acetone (20:1, 10:1, 1:1, 1:5, 0:1, v / v), 4 to 6 column volumes for each gradient, to obtain 5 components, namely Fr.5.4.1-Fr.5.4.5. Fr.5.4.2 (898.4 mg) was prepared by semi-preparative HPLC using a gradient elution method (0 to 30 min, acetonitrile concentration from 30% → 70%, v / v) to obtain 8.0 mg chlorahololide J (yield: 0.00004%) and 12.0 mg chlorahololide L (yield: 0.00006%).

[0061] Example 5: NLRP3 inflammasome inhibitory activity of chlorahololides H, J, L and chlorajaponilide E

[0062] Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that exists in all cells and is quickly released into the cell culture fluid when the cell membrane is damaged. After activation, NLRP3 inflammasomes will eventually lead to cell pyroptosis, cell membrane rupture, and LDH release into the cell culture fluid. The level of intracellular NLRP3 inflammasome activation can be monitored by measuring the LDH content in the cell culture fluid. Therefore, in this embodiment, a NLRP3 inflammasome activation model was constructed using mouse mononuclear macrophages J774A.1, and NLRP3 inflammasome activation was induced by lipopolysaccharide (LPS) and nigericin. For specific methods, please refer to Xing, Jie Zhang, et al., 2021, Leojaponin inhibits NLRP3 inflammasome activation through restoration of autophagy via upregulating RAPTORphosphorylation, Journal of Ethnopharmacology 278 (2021) 114322.

[0063] The specific experiment is as follows: mouse mononuclear macrophage J774A.1 (purchased from Kunming Institute of Zoology, Chinese Academy of Sciences) was cultured at 2×10 5Cells were seeded at a density of 10 cells per well in a 24-well plate, each well containing 500 μL of Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 8% heat-inactivated fetal bovine serum (FBS, Gibco) and cultured overnight in a CO2 incubator (Thermo Scientific) containing 5% CO2 and a constant temperature of 37°C. After overnight culture, the culture medium was removed. Subsequently, 500 μL of 200 ng / mL LPS (Shanghai Biotech Co., Ltd.) dissolved in Opti-MEM medium (Thermo Scientific, Eugene, OR, USA) was added to each well to stimulate the cells for 3.5 hours. Then, the culture medium was aspirated, and 500 μL of 10 μM compounds chlorahololides H, J, L and chlorajaponilide E dissolved in Opti-MEM medium were added, 500 μL of 0.1 μM MCC950 (Shanghai Aladdin Biochemical Technology Co., Ltd.) dissolved in Opti-MEM medium was added as a positive control, and 500 μL of 1 μL / mL DMSO dissolved in Opti-MEM medium was added as a negative control, and the cells were incubated in a CO2 incubator for 30 min. Finally, the culture medium was aspirated, and 500 μL of 10 μM nigericin (Shanghai Aladdin Biochemical Technology Co., Ltd.) dissolved in Opti-MEM medium was added to each well to stimulate the cells for 1 hour. After inflammasome activation and compound treatment, the cell culture supernatant (120 μL) was collected in a 96-well plate, and 60 μL of LDH detection working solution (Shanghai Biyuntian Biotechnology Co., Ltd.) was added, mixed, and incubated at room temperature in the dark for 30 min. The absorbance was measured at 490 nm, and the LDH release inhibition rate was calculated according to the following formula: LDH release inhibition rate (%) = (OD of DMSO treated group) 490 nm – Sample Group OD 490 nm ) / DMSO treatment group OD 490 nm ×100%. Half maximal inhibitory concentration (IC) of compounds chlorahololides H, J, L and chlorajaponilide E on NLRP3 inflammasome activation 50 ) was tested as follows: different concentration gradients (10 μM, 5 μM, 2.5 μM, 1.25 μM and 0.625 μM) were set for each compound, and the LDH release inhibition rate of the compound at different concentrations was tested respectively, and then the IC of the compound was calculated by nonlinear regression analysis using GraphPad Prism 7.0 software.50 Value. The cell viability was detected by MTT method to exclude the toxic effects of compounds chlorahololides H, J, L and chlorajaponilide E on cells. For specific experimental methods, please refer to Xing, Jie Zhang 2021 (ibid.). All experiments were repeated at least 3 times, and the experimental data were analyzed and calculated using GraphPadPrism7.0.

[0064] The experimental results are shown in Tables 1 and 2. Chlorahololides H, J, L and chlorajaponilide E have significant NLRP3 inflammasome inhibitory activity at a concentration of 10 μM. The IC values ​​of these compounds were further tested. 50 values, were 8.73 μM, 6.15 μM, 3.03 μM and 2.99 μM, respectively.

[0065] Table 1. Chlorahololides H, J, L and chlorajaponilide E inhibition rate on NLRP3 inflammasome (%)

[0066]

[0067] a MCC950 is a positive control drug

[0068] Table 2. IC of Chlorahololides H, J, L and chlorajaponilide E on NLRP3 inflammasome inhibition 50 value

[0069]

[0070] a CC 50 Values ​​represent the concentration of compound that induces 50% cytotoxicity

[0071] Example 6: Chlorajaponilide E significantly reduces pyroptosis in J774A.1 macrophages

[0072] This example explores the effect of chlorajaponilide E, a sesquiterpene dimer of the linderane type, which has significant inhibitory activity on the NLRP3 inflammasome, on pyroptosis of J774A.1 macrophages. For specific methods, see Xing, Jie Zhang, et al., 2021, Leojaponin inhibits NLRP3 inflammasome activation through restoration of autophagy via upregulating RAPTOR phosphorylation, Journal of Ethnopharmacology 278 (2021) 114322.

[0073] The specific experiment is as follows: mouse mononuclear macrophage J774A.1 (purchased from Kunming Institute of Zoology, Chinese Academy of Sciences) was cultured at 4×10 4 Cells were seeded at a density of 10 cells per well in a 96-well plate, each well containing 200 μL of Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 8% heat-inactivated fetal bovine serum (FBS, Gibco) and cultured overnight in a CO2 incubator (Thermo Scientific) containing 5% CO2 and a constant temperature of 37°C. After overnight culture, three different experiments were set up. The LPS and nigericin treatment groups were operated as follows: the culture medium was removed, and 200 μL of 200 ng / mL LPS dissolved in Opti-MEM medium was added to each well to stimulate the cells for 3.5 hours, then the culture medium was aspirated, and 200 μL of the compound chlorajaponilide E (1 μM, 3 μM, and 9 μM) dissolved in Opti-MEM medium and DMSO (negative control, 1 μL / mL) were added to each well, and the cells were incubated in a CO2 incubator for 30 min. Finally, the medium was removed and 200 μL of 10 μM nigericin dissolved in Opti-MEM medium was added to each well to stimulate the cells for 1 hour. The untreated group was operated as follows: the medium was removed and 200 μL of Opti-MEM medium was added to each well. The group treated with chlorajaponilide E (9 μM) alone was operated as follows: the medium was removed and 200 μL of Opti-MEM medium was added to each well. After 3.5 hours, the medium was aspirated and 200 μL of 9 μM compound chlorajaponilide E dissolved in Opti-MEM medium was added to each well.

[0074] After the activation of NLRP3 inflammasomes in the LPS and nigericin treatment groups, the supernatant was removed and 100 μL of 3 μg / mL PBS-prepared propidium iodide (PI, Shanghai Bio-Tech Co., Ltd.) and 0.5 μg / mL Hoechst 33342 (Shanghai Bio-Tech Co., Ltd.) were added as dyes to stain macrophages J774A.1. The staining was carried out at room temperature for 15 min and photographed with a fluorescent inverted microscope (Axio Observer3, Zeiss; Oberkochen, Germany). PI dyed pyroptotic cells red, and Hoechst 33342 dyed cell nuclei blue. The same operation was performed on the other two groups of experimental cells. All experiments were repeated at least 3 times. The experimental data were analyzed and calculated using GraphPad Prism7.0. One-way ANOVA was used to analyze the differences between the groups. *P<0.05, **P<0.01, ***P<0.001. When P<0.05, it is statistically significant and there is a significant difference.

[0075] The results are as follows Figure 2 and 3 As shown. Figure 2 It can be seen that compared with the untreated group, the LPS and nigericin treatment groups can significantly increase the proportion of PI staining, indicating that the number of pyroptotic cells has increased significantly. However, there is no difference between the cells treated with chlorajaponilide E (9 μM) and the untreated group, indicating that the compound chlorajaponilide E does not cause pyroptosis in J774A.1 cells. In addition, in the LPS and nigericin treatment groups, compared with the negative control DMSO, chlorajaponilide E can reduce the proportion of J774A.1 cell pyroptosis in a concentration-dependent manner, indicating that the compound can effectively inhibit cell pyroptosis caused by NLRP3 inflammasome activation. Figure 3 This is a quantitative graph of chlorajaponilide E inhibiting pyroptosis of J774A.1 cells. In the LPS and nigericin treatment groups, compared with the negative control DMSO, the pyroptosis of J774A.1 cells was inhibited after the administration of chlorajaponilide E (1μM, 3μM and 9μM), P<0.001, indicating that chlorajaponilide E can significantly inhibit the pyroptosis of J774A.1 cells induced by LPS and nigericin.

Claims

1. A linderane-type sesquiterpene dimer or a pharmaceutically acceptable salt thereof, characterized in that The linderane-type sesquiterpene dimer has any one of the following structural formulas:

2. A drug or pharmaceutical composition for treating chronic inflammatory diseases, characterized in that: Contains at least one of the linderane-type sesquiterpene dimers or pharmaceutically acceptable salts thereof as described in claim 1.

3. Use of a linderane-type sesquiterpene dimer or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating chronic inflammatory diseases, wherein the linderane-type sesquiterpene dimer has any one of the following structural formulas I to IV:

4. A method for preparing the linderane-type sesquiterpene dimer according to claim 1, characterized in that: The following steps are involved: 1) drying and crushing a Chloranthus plant material, extracting with an organic solvent, and then desolventizing to obtain a Chloranthus plant material extract; and 2) subjecting the extract of the Chrysanthemi plant material to column chromatography to obtain chlorahololides H, J and L, wherein the organic solvent is at least one of ethyl acetate, ethanol and methanol, And the plant of the genus C. holostegius is C. holostegius, C. henryi or C. holostegius var. shimianensis.

5. The method according to claim 4, characterized in that The organic solvent is ethyl acetate, methanol, 95% ethanol or a mixture thereof.

6. The method according to claim 4, characterized in that The Chrysanthemi plant material is the root, stem, branch, leaf, whole plant or a mixture thereof.

7. The method according to claim 4, characterized in that The volume ratio of the organic solvent to the Chrysanthemi plant material is 1:1 to 5:

1.

8. The method according to claim 7, characterized in that The volume ratio of the organic solvent to the Chrysantha plant material is 3:

1.

9. The method according to claim 4, characterized in that In step 2), the column chromatography includes normal phase silica gel column chromatography, reverse phase silica gel column chromatography, resin column chromatography, gel column chromatography, medium pressure chromatography separation gel and semi-preparative HPLC.

10. The method according to claim 9, characterized in that In step 2), the eluent used for the column chromatography is at least one of petroleum ether, dichloromethane, chloroform, ethyl acetate, acetone, ethanol, methanol, n-butanol, acetonitrile, water and formic acid.

11. The method according to claim 10, characterized in that The normal phase silica gel column chromatography uses petroleum ether and acetone in a volume ratio of 1:0 to 0:1 for gradient elution, or the normal phase silica gel column chromatography uses dichloromethane and methanol in a volume ratio of 1:0 to 1:1 for gradient elution; the reverse phase silica gel column chromatography uses methanol and water in a volume ratio of 2:8 to 10:0 for gradient elution; the resin column chromatography uses methanol and water in a volume ratio of 2:8 to 10:0 for gradient elution; the gel column chromatography uses methanol or methanol and chloroform in a volume ratio of 1:1 for elution; the medium pressure chromatography separation gel uses methanol or ethanol and water in a volume ratio of 2:8 to 10:0 for gradient elution; the semi-preparative HPLC uses acetonitrile and water for isocratic elution, or the semi-preparative HPLC uses acetonitrile and water in a volume ratio of 2:8 to 8:2 for gradient elution.

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