Method for extracting and separating phthalide dimer compound by taking angelica sinensis as raw material and application of obtained compound

Abstract

The invention discloses a method for extracting and separating a phthalide dimer compound by taking angelica sinensis as a raw material and application of the obtained compound, and the method comprises the following steps: 1) carrying out normal phase chromatography separation by adopting an angelica sinensis extract Fr.C silica gel column, carrying out gradient elution by taking petroleum ether-ethyl acetate as an eluent, merging the same eluted fractions according to a TLC detection result, and concentrating the eluted fractions to obtain a filtrate; a component FrC-1, a component FrC-2 and a component FrC-3 are obtained; (2) carrying out segmented treatment on the component FrC-2 by adopting a silica gel column, carrying out gradient elution by taking dichloromethane / methanol as an eluent, carrying out TLC (Thin Layer Chromatography) detection on all elution fractions, and combining the same fractions to obtain a component FrC-2-1, a component FrC-2-2 and a component FrC-2-3; and (3) eluting the component FrC-2-2 through Sephadex LH-20 column chromatography, carrying out isocratic elution with a methanol solution, carrying out TLC detection on elution fractions, and merging the same fractions to obtain a component FrC-2-2-1 and a component FrC-2-2-2. According to the method, 10 kinds of phthalide dimers with novel structures can be separated from angelica sinensis, and the compounds all show remarkable anti-inflammatory activity and have wide application prospects.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a method for extracting and separating phthalide dimer compounds using angelica as a raw material and the application of the obtained compounds. Background Art

[0002] Inflammation is a complex biological response of animal tissues to harmful stimuli. Increased NO may lead to cytotoxicity and pro-inflammatory effects. These phenomena indicate that inhibiting NO release is an important therapeutic target for treating inflammatory diseases. Jian-Bo Yang, Ai-Guo Wang, Qian Wei, Jin Ren, Shuang-Cheng Ma, Ya-Lun Su. New dimeric phthalides from Ligusticum sinense Oliv cv. Chaxiong. [J]. Journal of Asian Natural Products Research, 2014, 16 (7): 747-52. DOI: 10.1080 / 10286020.2014.914501. discloses four phthalide dimers extracted from Angelica sinensis that have the effect of inhibiting NO expression. The extraction method of the compound is as follows: the dried Angelica sinensis rhizome is crushed, extracted with 95% ethanol by reflux method, and then evaporated under reduced pressure to obtain the residue, which is suspended in distilled water, and purified with CH2Cl2 and EtOAc in turn to obtain CH2Cl2, EtOAc and H2O soluble parts. Among them, the CH2Cl2 soluble part was further dissolved with petroleum ether and partitioned with 90% MeOH to remove low polar fatty oil, and the obtained 90% MeOH part was separated by flash column chromatography (CC) and gradient elution, and finally purified by HPLC to obtain the target compound. However, this method is only briefly described and lacks practical feasibility.

[0003] In addition, Lu Huang, Cheng Peng et al. published "Six pairs of enantiomeric phthalide dimers from the rhizomes of Ligusticum chuanxiong and their absolute configurations and anti-inflammatory activities" on 2022-06-16 in Bioorganic Chemistry (IF4.5), which disclosed six phthalide dimers extracted from Chuanxiong with the effect of inhibiting NO expression. The extraction method of phthalide dimers is as follows: the rhizome of Ligusticum chuanxiong is extracted with 95% ethanol under reflux conditions for three times, each time for 2.5 hours, the extract is evaporated under reduced pressure, the residue is suspended in water, and then distributed with ethyl acetate and n-butanol, the ethyl acetate extract is separated on a silica gel column, and gradient elution is performed using petroleum ether-ethyl acetate and ethyl acetate-methanol, and finally 16 components are obtained. Among them, compound 4 crystallized from the 12th component, and the 11th component was further purified by reversed-phase medium pressure liquid chromatography and semi-preparative reversed-phase high-performance liquid chromatography to obtain compounds 2 and 3, and compound 6 and other compounds were obtained by further separation and purification steps.

[0004] Phthalide dimers are mainly used in the treatment of cardiovascular and cerebrovascular diseases, anti-cerebral ischemia and nerve repair, anti-tumor, and vasodilation. Phthalide dimers have complex multi-ring skeletons and multi-chiral centers, and have become a hot topic of research due to their rich chemical structure and important pharmacological activities.

[0005] However, the sources of existing phthalide dimers are mainly extracted from natural drugs or synthesized by biological or chemical methods through artificial means, among which the natural drug sources are Chuanxiong, Angelica sinensis and celery seeds.

[0006] Angelica sinensis is the dried root of Angelica sinensis (Oliv.) Diels, a plant of the Umbelliferae family. The chemical composition of Angelica sinensis is complex and rich, including volatile oils, polysaccharides, ferulic acid, phthalide compounds, etc. These components together give Angelica sinensis its unique pharmacological activity. In terms of pharmacological effects, Angelica sinensis has been proven to have multiple biological activities such as antioxidant, anti-inflammatory, anti-tumor, and anti-thrombotic, and has a good regulatory effect on multiple systems such as the cardiovascular system, immune system, and nervous system.

[0007] Among them, phthalide compounds are considered to be one of the main biologically active components of Angelica sinensis, which mainly include phthalide monomers and phthalide dimers. Angelica sinensis is mainly used to extract phthalide compounds.

[0008] However, due to the complex components of Angelica sinensis, substances such as polysaccharides will form a coating on phthalide compounds, affecting the extraction efficiency and yield of phthalide compounds.

[0009] For example, CN201610131491.2 discloses an extraction method and use of phthalide dimer compounds. This method uses supercritical CO2 to extract angelica slices, and then performs multiple silica gel column chromatography on the extract to obtain three structural compounds with anti-tumor effects. However, due to the complex structure of phthalide dimers, there are also many other phthalide dimers with special effects that need further research and development for their utilization.

[0010] The information disclosed in the background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0011] In response to the above technical problems, the present application provides a method for extracting and separating phthalide dimer compounds using Angelica sinensis as a raw material and the application of the obtained compounds. The method can achieve the effective extraction of phthalide dimer compounds in Angelica sinensis, and 10 compounds with novel structures are obtained. The obtained compounds all have the effect of inhibiting NO expression and have anti-inflammatory effects, thereby increasing the types of phthalide dimer compounds with anti-inflammatory effects.

[0012] The present application provides a method for extracting and separating phthalide dimer compounds using Angelica sinensis as a raw material, comprising the following steps:

[0013] 1) After separation by normal phase chromatography on a silica gel column, the Angelica sinensis extract Fr.C was gradient eluted using petroleum ether-ethyl acetate as the eluent, and the eluted fractions were combined according to the TLC detection results to obtain component FrC-1, component FrC-2, and component FrC-3;

[0014] 2) After fractionating component FrC-2 using a silica gel column, gradient elution was performed using dichloromethane / methanol as the eluent, and each elution fraction was detected by TLC, and the same fractions were combined to obtain component FrC-2-1, component FrC-2-2, and component FrC-2-3;

[0015] 3) eluting the component FrC-2-2 through Sephadex LH-20 column chromatography and isocratically eluting with a methanol solution, and combining the eluted fractions after TLC detection to obtain component FrC-2-2-1 and component FrC-2-2-2;

[0016] Component FrC-2-2-1 was purified by semi-preparative high performance liquid chromatography to obtain racemate (±)-1, i.e., compound 1, (±)-2, i.e., compound 2, and component FrC-2-2-1-1;

[0017] The component FrC-2-2-1-1 was further separated by silica gel column chromatography and gradient eluted with petroleum ether / ethyl acetate to obtain racemic (±)-3, i.e., compound 3, and (±)-4, i.e., compound 4;

[0018] The component FrC-2-2-2 was purified by semi-preparative high performance liquid chromatography to obtain racemate (±)-5, i.e., compound 5, (±)-6, i.e., compound 6, and the component FrC-2-2-2-1;

[0019] The component FrC-2-2-2-1 was further separated by silica gel column chromatography and gradient eluted with petroleum ether / ethyl acetate to obtain racemic (±)-7, i.e., compound 7, and (±)-8, i.e., compound 8.

[0020] Component FrC.2-3 was eluted by Sephadex LH-20 column chromatography with 100% methanol isocratically, the eluted fractions were detected by TLC, and the same fractions were combined to obtain two components, components FrC-2-3-1 to FrC-2-3-2;

[0021] Component FrC-2-3-1 was purified by semi-preparative HPLC to obtain racemates (±)-9, i.e., compound 9, and (±)-10, i.e., compound 10;

[0022] The obtained compounds 1 to 10 are:

[0023]

[0024] Preferably, when treating component FrC-2-2-1, the mobile phase used for semi-preparative HPLC purification is a mixture of acetonitrile and water in a volume ratio of 68:32.

[0025] Preferably, when treating component FrC-2-2-2, the mobile phase used for semi-preparative HPLC purification is a mixture of acetonitrile and water in a volume ratio of 63:37.

[0026] Preferably, when treating component FrC-2-3-1, the mobile phase used for semi-preparative HPLC purification is a mixture of acetonitrile and water in a volume ratio of 45:55.

[0027] Preferably, the petroleum ether-ethyl acetate eluent transitions from a petroleum ether:ethyl acetate volume ratio of 100:1 to a petroleum ether:ethyl acetate volume ratio of 1:1.

[0028] Preferably, the dichloromethane / methanol eluent is transitioned from a dichloromethane:methanol volume ratio of 100:1 to a dichloromethane:methanol volume ratio of 1:1.

[0029] Preferably, the preparation method of Angelica extract Fr.C is as follows: using dry Angelica powder as raw material, soaking it in 80% ethanol for 3 to 5 times, soaking for 1 to 2 days each time, combining the extracts obtained each time, concentrating under reduced pressure to obtain an extract, loading the water-soluble extract with D101 polystyrene macroporous resin, and gradient eluting with water, methanol and methanol as eluents in sequence, collecting the methanol elution part, concentrating under reduced pressure, and dissolving in water to obtain Angelica extract Fr.C.

[0030] Preferably, the method further comprises: performing chiral separation on the obtained compounds 1 to 10 to obtain the optical enantiomers corresponding to the compounds 1 to 10 respectively.

[0031] Preferably, the chiral separation conditions are: using a chiral column AD-H was analyzed by analytical HPLC with n-hexane:isopropanol as the mobile phase, the flow rate of the mobile phase was 1 mL / min, and the detection wavelength was set at 254 nm.

[0032] Preferably, the mobile phase used in the chiral resolution of compounds 1 and 8 is a mixture of n-hexane and isopropanol in a volume ratio of 96:4; the mobile phase used in the chiral resolution of compounds 2 and 3 is a mixture of n-hexane and isopropanol in a volume ratio of 93:7; the mobile phase used in the chiral resolution of compounds 4 to 6 and 9 is a mixture of n-hexane and isopropanol in a volume ratio of 92:8; the mobile phase used in the chiral resolution of compound 7 is a mixture of n-hexane and isopropanol in a volume ratio of 94:6; the mobile phase used in the chiral resolution of compound 10 is a mixture of n-hexane and isopropanol in a volume ratio of 90:10.

[0033] Another aspect of the present application provides an anti-inflammatory composition, comprising at least: compounds 1 to 10 obtained by the above method.

[0034] Preferably, it includes at least any one of: Compound 2, the optical antipode of Compound 1, Compound 9 and its optical antipode, Compound 7 and its optical antipode; more preferably, it includes at least Compound 7 and its optical antipode.

[0035] Preferably, the composition is in the form of at least one of ointment, emulsion, aqueous solution, tablet, injection, and capsule. The composition can be made into various dosage forms according to the needs of use, and the auxiliary materials required for each dosage form can be selected according to the commonly used formula in the field, which will not be repeated here.

[0036] Preferably, the amount of compounds 1 to 10 added is less than 6.25 μM. This amount can avoid the cytotoxicity produced and can exert an anti-inflammatory effect. Specifically, it can be 0.05 to 6.25 μM; the concentrations of compounds 1-1, 1-2, 2, 2-2, 3, 3-1, 3-2, 4, 5, 5-1, 6, 7, 7-1, and 7-2 that meet the cell survival rate approaching 100% are 3.13, 3.13, 1.56, 12.5, 6.25, 3.13, 1.56, 6.25, 3.13, 1.56, 6.25, 0.1, 0.05, and 0.2 μM, respectively. Each compound below this amount will not produce obvious toxicity to cells.

[0037] Preferably, it at least comprises: compound 7 and its enantiomers in an addition amount of 0.5 to 3.5 μM.

[0038] Preferably, the inhibition rate of compound 2, the optical antipode of compound 1, compound 9 and its optical antipode, compound 7 and its antipode on NO is greater than 90%; more preferably, the inhibition rate of compound 7 and its optical antipode on NO is greater than 97.0%.

[0039] Preferably, compound 7 and its optical enantiomer can reduce the secretion of TNF-α, IL-6 and IL-1β in RAW264.7 cells induced by LPS;

[0040] Preferably, compound 7 and its optical enantiomer can reduce the secretion of inflammatory cytokines TNF-α, IL-6, and IL-1β in RAW264.7 cells induced by LPS.

[0041] The beneficial effects of this application include:

[0042] 1) The present application provides a method for extracting and separating phthalide dimer compounds using angelica as a raw material. The method can effectively avoid the coating of phthalide dimers by impurities such as polysaccharides in the angelica raw material, and achieve the separation of 10 novel phthalide dimers from angelica. These compounds all show significant anti-inflammatory activity and have broad application prospects. At the same time, the method can effectively enrich phthalide dimer compounds with the same planar structure but different relative configurations, and the obtained compounds can be used for subsequent research on the structure of the compounds and their activity difference characteristics.

[0043] 2) The present application provides a method for extracting and separating phthalide dimer compounds using angelica as a raw material. The method has a simple preparation process, the solvent can be recycled, the extraction cost is reduced, and the sugar-containing part in the angelica raw material is effectively removed, thereby effectively separating a large number of small molecular substances, and realizing accurate identification and enrichment of phthalide dimer compounds, thereby obtaining up to 10 phthalide dimers with novel structures. The obtained phthalide dimer compounds can be used for the study of anti-inflammatory activity.

[0044] 3) The present application provides a method for extracting and separating phthalide dimer compounds using Angelica sinensis as a raw material. In terms of chemical structure, some of the compounds obtained by the method have the same planar structure, but different configurations of the chiral center and different cis-trans configurations of the butyl side chain. These 10 racemates have 5 different polymerization modes. This indicates that the method can achieve effective separation and purification of such compounds.

[0045] 4) The phthalide dimer provided in this application, which uses Angelica sinensis as a raw material, has been studied for its anti-inflammatory effects on these 10 racemates and their optical enantiomers, and it has been found that all of them have certain anti-inflammatory activity, and can inhibit the secretion of NO by RAW264.7 cells induced by LPS within the corresponding concentration range. Among them, the racemate of compound 7 has good anti-inflammatory activity, and further anti-inflammatory mechanism evaluation has been carried out on it. Compound 7 can significantly inhibit the expression levels of IL-6 and IL-1β mRNA in RAW264.7 cells induced by LPS. The above research results can well prove that phthalide dimer compounds with different polymerization modes, different relative configurations, different racemates, different optical enantiomers but the same planar structure have different anti-inflammatory effects, which has a good development and application prospect for anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a schematic diagram of the structural formula of compounds 1 to 10 obtained in Example 1 provided in this application;

[0047] Figure 2 Schematic diagram of the effect of the racemic mixture of compounds 1 to 10 obtained in Example 1 provided in the present application and their optical antipodes on the viability of RAW264.7 cells; wherein 1 is racemic body 1; 1-1 and 1-2 are a pair of optical antipodes obtained from racemic body 1; 2 is racemic body 2; 2-1 and 2-2 are a pair of optical antipodes of racemic body 2; 3 is racemic body 3; 3-1 and 3-2 are a pair of optical antipodes of racemic body 3; 4 is racemic body 4; 4-1 and 4-2 are racemic body 4 5 is a pair of optical enantiomers of racemate 5; 5-1 and 5-2 are a pair of optical enantiomers of racemate 5; 6 is racemate 6; 7 is racemate 7; 7-1 and 7-2 are a pair of optical enantiomers of racemate 7; 8 is racemate 8; 8-1 and 8-2 are a pair of optical enantiomers of racemate 8; 9 is racemate 9; 9-1 and 9-2 are a pair of optical enantiomers of racemate 9; 10 is racemate 10; 10-1 and 10-2 are a pair of optical enantiomers of racemate 10. Note: Compared with the control group, ****P<0.0001 (n=3). The meanings of the numbers in the following figures are the same and will not be repeated here.

[0048] Figure 3It is a line graph showing the results of the NO release generation rate of the racemic mixture of compounds 1 to 10 obtained in Example 1 provided in the present application and their optically pure form in RAW 264.7 cells induced by lipopolysaccharide;

[0049] Figure 4 The half-maximal inhibitory concentration value of the racemic mixture of compounds 1 to 10 obtained in Example 1 and their optically pure form on NO production in RAW 264.7 cells induced by lipopolysaccharide (LPS) provided in the present application.

[0050] Figure 5 It is the half-maximal inhibitory concentration value of compound 7 obtained in Example 1 provided in the present application on NO production in RAW 264.7 cells induced by lipopolysaccharide at an addition amount of 0.2 to 3.13 μM Log value.

[0051] Figure 6 The results of the effects of compound 7 obtained in Example 1 provided in this application on the secretion of TNF-α, IL-6, and IL-1β by RAW264.7 cells induced by LPS and their mRNA expression levels are shown in the figure; wherein a) is the result of compound 7 on the secretion of TNF-α by RAW264.7 cells induced by LPS; b) compound 7 on the secretion of IL-6 by RAW264.7 cells induced by LPS; c) compound 7 on the secretion of IL-1β by RAW264.7 cells induced by LPS; d) is the result of the inhibitory effect of compound 7 on the secretion of inflammatory cytokine TNF-α; e) is the result of the inhibitory effect of compound 7 on the secretion of inflammatory cytokine IL-6; f) is the result of the inhibitory effect of compound 7 on the secretion of inflammatory cytokine IL-1β. Note: Compared with the model group, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05 (n=3). DETAILED DESCRIPTION

[0052] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the protection scope of the present invention.

[0053] Example

[0054] Unless otherwise specified, the materials and instruments used in the following examples were obtained from commercial channels; the detection methods used were all existing methods unless otherwise specified.

[0055] Example 1 Preparation of phthalide dimer

[0056] 1. Extraction and separation

[0057] (1) taking dried powder of Angelica sinensis rhizomes, soaking it in 80% ethanol for 3-5 times, each time for 2 days, combining the extracts, and concentrating them under reduced pressure to obtain an extract concentrate (TE);

[0058] (2) dissolving the total extract obtained in step (1) with purified water, loading the column with D101 polystyrene macroporous resin, loading the sample by wet method, and gradient eluting with water, 60% methanol and 100% methanol as eluents in sequence, and concentrating the eluent under reduced pressure to remove the sugar-containing part and achieve the purpose of enriching small molecular substances. The water elution part is the sugar-containing part, and the 100% methanol elution part (without sugar) is mainly collected as the Angelica extract Fr.C;

[0059] (3) The 100% methanol elution portion (Fr.C) obtained in step (2) was separated by normal phase chromatography on a silica gel column, and gradient eluted with petroleum ether-ethyl acetate in a volume ratio of 100:1→1:1. The eluted fractions were detected by TLC, and the same fractions were combined to collect three components FrC-1 to FrC-3 respectively;

[0060] (4) Component FrC-2 was segmented using a silica gel column and eluted with a gradient of dichloromethane / methanol in a volume ratio of 100:1→1:1. The eluted fractions were detected by TLC and the same fractions were combined to obtain three components, namely, components FrC-2-1 to FrC-2-3;

[0061] The component FrC.2-2 was eluted by Sephadex LH-20 column chromatography with 100% methanol isocratically, the eluted fractions were detected by TLC, and the same fractions were combined to obtain two components FrC-2-2-1 to FrC-2-2-2;

[0062] The component FrC-2-2-1 was purified by semi-preparative high performance liquid chromatography (acetonitrile-water, 68:32, v / v) to obtain racemates (±)-1, (±)-2 and component FrC-2-2-1-1;

[0063] The component FrC-2-2-1-1 was further separated by silica gel column chromatography, and gradient elution was performed with petroleum ether / ethyl acetate to obtain racemates (±)-3 and (±)-4;

[0064] Component FrC-2-2-2 was purified by semi-preparative HPLC (acetonitrile-water, 63:37, v / v) to obtain racemates (±)-5, (±)-6 and component FrC-2-2-2-1;

[0065] The component FrC-2-2-2-1 was further separated by silica gel column chromatography and gradient eluted with petroleum ether / ethyl acetate to obtain racemic (±)-7 and (±)-8.

[0066] Component FrC.2-3 was eluted by Sephadex LH-20 column chromatography with 100% methanol isocratically, the eluted fractions were detected by TLC, and the same fractions were combined to obtain two components, components FrC-2-3-1 to FrC-2-3-2;

[0067] The component FrC-2-3-1 was purified by semi-preparative HPLC (acetonitrile-water, 45:55, v / v) to obtain racemates (±)-9 and (±)-10.

[0068] 2. Chiral separation

[0069] The racemate 1 was purified by chiral column AD-H, by analytical HPLC, with n-hexane:isopropanol (96 / 4, v / v, 1 mL / min) as the mobile phase and the detection wavelength set at 254 nm, a pair of optical enantiomers 1-1 and 1-2 were separated;

[0070] The racemate 2 was purified by chiral column AD-H, by analytical HPLC, with n-hexane:isopropanol (93 / 7, v / v, 1 mL / min) as the mobile phase and the detection wavelength set at 254 nm, a pair of optical enantiomers 2-1 and 2-2 were separated;

[0071] The racemate 3 was purified by chiral column AD-H, by analytical HPLC, with n-hexane:isopropanol (93 / 7, v / v, 1 mL / min) as the mobile phase and the detection wavelength set at 254 nm, a pair of optical enantiomers 3-1 and 3-2 were separated;

[0072] The racemate 4 was prepared by chiral column AD-H, by analytical HPLC, with n-hexane:isopropanol (92 / 8, v / v, 1 mL / min) as the mobile phase and the detection wavelength set at 254 nm, a pair of optical enantiomers 4-1 and 4-2 were separated;

[0073] The racemate 5 was purified by chiral column AD-H, by analytical HPLC, with n-hexane:isopropanol (92 / 8, v / v, 1 mL / min) as the mobile phase and the detection wavelength set at 254 nm, a pair of optical enantiomers 5-1 and 5-2 were separated;

[0074] The racemate 7 was purified by chiral column AD-H, by analytical HPLC, with n-hexane:isopropanol (94 / 6, v / v, 1 mL / min) as the mobile phase and the detection wavelength set at 254 nm, a pair of optical enantiomers 7-1 and 7-2 were separated;

[0075] The racemate 8 was prepared by chiral column AD-H, by analytical HPLC, with n-hexane:isopropanol (96 / 4, v / v, 1 mL / min) as the mobile phase and the detection wavelength set at 254 nm, a pair of optical enantiomers 8-1 and 8-2 were separated;

[0076] The racemate 9 was prepared by chiral column AD-H, by analytical HPLC, with n-hexane:isopropanol (92 / 8, v / v, 1 mL / min) as the mobile phase and the detection wavelength set at 254 nm, a pair of optical enantiomers 9-1 and 9-2 were separated;

[0077] The racemate 10 is prepared by using a chiral column AD-H, by analytical HPLC, with n-hexane:isopropanol (90 / 10, v / v, 1 mL / min) as the mobile phase and the detection wavelength set at 254 nm, a pair of optical enantiomers 10-1 and 10-2 were separated;

[0078] Structural identification

[0079] Compound 1: white powder, HRESIMS m / z 381.2065[M+H] + (calcd for C 24 H 28 O4+H,381.2060),UV(MeOH)λ max (logε)284(3.69)nm;

[0080] (+)-(6S,7R,8′R,3′S)-1: (c 0.09, MeOH); ECD(MeOH)λ max (Δε)208(-3.48),239(+3.99),291(+4.13)nm.(-)-(6R,7S,8′S,3′R)-1: (c 0.12, MeOH); ECD(MeOH)λ max (Δε)207(+5.33),239(-4.14),291(-2.38)nm, 1 H NMR (500 MHz, CDCl3) and 13 C NMR (125 MHz, CDCl3) data are shown in Table 1;

[0081] Compound 2: white powder, HRESIMS m / z 403.1879[M+Na] + (calcd for C 24 H28 O4+Na,403.1880),UV(MeOH)λ max (logε)196(4.43),280(4.25)nm; (+)-(6R,7S,8′R,3′S)-2: (c 0.15, MeOH); ECD(MeOH)λ max (Δε)208(-1.81),229(-6.03),294(+13.43)nm.(-)-(6S,7R,8′R,3′R)-2: (c 0.12, MeOH); ECD(MeOH)λ max (Δε)207(-1.56),228(+8.04),290(-12.90)nm. 1 H (500MHz,CDCl3) and 13 C NMR (125 MHz, CDCl3) data are shown in Table 1;

[0082] Compound 3: white powder, HRESIMS m / z 403.1880[M+Na] + (calcd for C 24 H 28 O4+Na,403.1880).UV(MeOH)λ max (logε)198(4.21),278(4.01)nm; (+)-(6R,7S,8′R,3′S)-3: (c 0.16, MeOH); ECD(MeOH)λ max (Δε)211(+22.36),233(+9.73),288(-5.87)nm.(-)-(6S,7R,8′S,3′R)-3: (c 0.09, MeOH); ECD(MeOH)λ max (Δε)211(+22.36),232(-7.65),289(+5.07)nm. 1 H(500MHz,CDCl3)and 13 C NMR (125MHz, CDCl3) data are shown in Table 1:

[0083] Compound 4: white powder, HRESIMS m / z 401.1728[M+Na] + (calcd for C 24 H 26 O4+Na,401.1723);UV(MeOH)λ max(logε)196(4.48),277(4.23)nm; (+)-(6R,7R,8′R,3′S)-4: (c 0.10, MeOH); ECD(MeOH)λ max (Δε)211(+9.98),234(-5.37),289(+4.97)nm.(-)-(6S,7S,8′S,3′R)-4: (c 0.13,MeOH); ECD(MeOH)λ max (Δε)210(-8.65),233(+5.15),289(-5.10)nm. 1 H (500MHz,CDCl3)and 13 C NMR (125 MHz, CDCl3) data are shown in Table 2;

[0084] Compound 5: white powder, HRESIMS m / z 401.1731[M+Na] + (calcd for C 24 H 26 O4+Na,401.1723).UV(MeOH)λ max (logε)281(4.11)nm;

[0085] (+)-(6R,7S,8′R,3′S)-5: (c 0.13,MeOH); ECD(MeOH)λ max (Δε)204(-1.57),229(-10.67),293(+21.21)nm.(-)-(6S,7R,8′S,3′R)-5: (c 0.10, MeOH); ECD(MeOH)λ max (Δε)201(+5.20),222(+10.87),296(-23.40)nm. 1 H(500MHz,CDCl3)and 13 CNMR (125 MHz, CDCl3) data are shown in Table 2;

[0086] Compound 6: white powder, HRESIMS m / z 401.1709[M+Na] + (calcd for C 24 H 26 O4+Na,401.1723).UV(MeOH)λ max (logε)281(4.11)nm. 1H(500MHz,CDCl3)and 13 C NMR (125 MHz, CDCl3) data are shown in Table 2;

[0087] Compound 7: light yellow oil, HRESIMS m / z 403.1884 [M+Na] + , (calcd for C 24 H 28 O4+Na403.1880), UV(MeOH)λ max (logε)196(4.15),282(3.44)nm; (+)-(3′R,8′S,3aR,6S)-7: (c 0.16, MeOH); ECD(MeOH)λ max (Δε)208(+23.22),240(+13.87),281(-4.38)nm.(-)-(3′S,8′R,3aS,6R)-7: (c0.15,MeOH);ECD(MeOH)λ max (Δε)209(-23.42),239(-19.29),282(+8.92)nm. 1 H(500MHz,CDCl3)and 13 C NMR (125 MHz, CDCl3) data are shown in Table 3;

[0088] (8) Compound 8: light yellow oil, HRESIMS m / z 435.1783 [M+Na] + (calcd for C 24 H 28 O6+Na,435.1778);UV(MeOH)λ max (logε)196(4.85),277(4.29)nm; (+)-(6R,7S,3′R)-8: (c 0.09, MeOH); ECD(MeOH)λ max (Δε)199(-6.83),219(+7.40),272(+1.74)nm.(-)-(6S,7R,3′S)-8: (c 0.10, MeOH); ECD(MeOH)λ max (Δε)197(+12.16),239(-5.47),271(-1.78)nm. 1 H(500MHz,CDCl3)and 13 C NMR (125 MHz, CDCl3) data are shown in Table 3;

[0089] (9) Compound 9: white powder, HRESIMS m / z 414.2271 [M+NH4] + (calcd for C 24 H 28 O5+NH4,414.2275),UV(MeOH)λ max (logε)274(4.40)nm; IR(KBr)ν max 2955,2928,1773,1695,1458,1244,1067,953,754,739cm -1 ;

[0090] (+)-(6R,3′aR,6′R,7R,9R)-9: (c 0.18,MeOH); ECD(MeOH)λ max (Δε)202(+24.84),238(-4.45),261(+2.72),292(-10.16)nm.

[0091] (-)-(6S,3′aS,6′S,7S,9S)-9: (c 0.13,MeOH); ECD(MeOH)λ max (Δε)202(-27.33),238(+6.66),261(-1.62),292(+12.66)nm. 1 H(500MHz,CDCl3)and 13 C NMR (125 MHz, CDCl3) data are shown in Table 3;

[0092] (10) Compound 10: HRESIMS m / z 397.2015 [M+H] + (calcd for C 24 H 28 O5+H,397.2010).UV(MeOH)λ max (logε)278(3.56)nm;

[0093] (+)-(6S,7R,8′R,3′S)-10: (c 0.08, MeOH); ECD(MeOH)λ max (Δε)195(-11.59),226(-0.59),291(+11.25)nm.(-)-(6R,7S,8′S,3′R)-10: (c 0.07, MeOH); ECD (MeOH) λmax (Δε)195(+15.25),219(+2.04),256(-8.30)nm. 1 H(500MHz,CDCl3)and 13 CNMR (125MHz, CDCl3) data are shown in Table 3.

[0094] Table 1 Compounds 1 to 3 in CDCl3 1 H and 13 C NMR data (unit: ppm)

[0095] No. <![CDATA[Compound 1 a > <![CDATA[Compound 2 a > <![CDATA[Compound 3 a > <![CDATA[δ C ]]> <![CDATA[δ H ]]> <![CDATA[δ C ]]> <![CDATA[δ H ]]> <![CDATA[δ C ]]> <![CDATA[δ H ]]> 1 167.5 168.9 169.2 3 148.3 148.5 148.9 3a 151.5 149.4 150.9 4 22.5 2.50,m 20.7 2.64,m 17.3 2.45,m 2.88,dt(17.5,4.0) 2.87,dd(18.5,6.0) 2.60,d(5.5) 5 20.8 1.79,m 21.4 1.61,m 20.9 1.58,m 2.09,m 1.98,m 1.96,m 6 26.6 2.80,m 31.4 2.38,m 31.8 2.41,m 7 37.9 3.82,d(9.5) 36.5 3.42,d(8) 36.5 3.41,d(8.0) 7a 126.5 126.5 123.8 8 116.4 5.70,t(8.5) 115.9 5.71,t(8.5) 112.0 5.25,t(8.0) 9 27.9 2.35,m 27.9 2.36,m 27.9 2.35,m 10 23.2 1.53,m 23.2 1.53,m 22.4 1.50,m 11 13.7 0.96,t(7.5) 13.8 0.98,t(7.5) 13.9 0.95,t(7.0) 1' 169.4 169.5 169.6 3' 92.3 89.5 89.5 3'a 156.0 159.2 159.3 4' 23.9 2.19,m 21.9 2.72,t(10.0) 21.9 2.72,t(10.0) 2.40,m 5' 22.6 2.24,m 22.4 2.48,m 22.3 2.49,m 6' 128.9 5.91,dt(9.5,4.0) 128.8 5.96,m 128.8 5.96,m 7' 117.8 6.20,d(9.5) 117.4 6.20,dt(9.5,2.0) 117.4 6.20,dt(9.5,2.0) 7'a 127.0 125.1 125.1 8' 46.8 3.33,q(8.5) 44.6 2.64,m 44.6 2.65,q(8.0) 9' 28.3 1.34,m 32.4 1.43,m 32.4 1.42,m 1.46,m 10' 21.1 1.16,m 20.9 1.16,m 20.8 1.15,m 1.24,m 1.27,m 1.26,m 11' 14.1 0.85,t(7.5) 14.2 0.85,m 14.1 0.84,t(7.5)

[0096] Table 2 Compounds 4-6 in CDCl3 1 H and 13 C NMR data (unit: ppm)

[0097]

[0098]

[0099] Table 3 Compounds 7-10 in CDCl3 1 H and 13 C NMR data (unit: ppm)

[0100]

[0101]

[0102] a express 1 H(500MHz) and 13 C (125 MHz) NMR data

[0103] It can be seen from the test results in Tables 1 to 3 that the method provided by the present application produces a new structure compound having a structure different from the existing known phthalide dimer compound.

[0104] Example 2 Anti-inflammatory Experiment

[0105] In this example, the racemic forms of phthalide dimer compounds 1 to 10 prepared in Example 1 and their optical enantiomers were used to conduct anti-inflammatory experiments.

[0106] 1. Cell recovery and culture

[0107] Take out the frozen cells to be tested, quickly place them in 37℃ warm water, and thaw them within 2 minutes. Transfer the thawed cell suspension to the clean bench and add an appropriate amount of complete culture medium. Centrifuge at 1200r / min for 5 minutes, discard the supernatant, add 2mL of complete culture medium, resuspend, transfer to the culture dish, shake crosswise, and place in a 37℃, 5% CO2 incubator for culture.

[0108] When the cell fusion reaches 70% to 80%, take the cells with good growth status in the logarithmic phase for subsequent experiments. The preparation method of the complete culture medium used is as follows: add 10 mL of Gibco fetal bovine serum and 1 mL of double antibody to 90 mL of DMEM culture medium, mix well, seal at 4°C for later use.

[0109] 2. Drug preparation

[0110] All 28 compounds are solid and dissolved in DMSO. Prepare the stock solution with a concentration of 20mM and store at -20°C. Dilute with DMEM culture medium before use, and dilute to 0.78μM, 1.56μM, 3.13μM, 6.25μM, 12.5μM, 25μM or less concentrations in sequence, following the principle of isotropic dilution. The final DMSO concentration is <1%.

[0111] 3. Griess method to detect the effects of racemic isomers of phthalide dimer compounds 1-10 and their optical enantiomers on NO release from RAW264.7 cells induced by LPS

[0112] RAW264.7 cells in the logarithmic growth phase were inoculated in a 96-well plate (1.0×106 cells / mL) and cultured in an incubator at 37°C and 5% CO2. A normal group, a model group, and an experimental group were set up, with 3 replicates in each group. After the cells adhered to the wall for 3 hours, the drugs were added for 24 hours. The model group was given DMEM containing 1μg / mL of LPS at a final concentration, and the experimental group was given a series of compounds with concentrations of 0.78μM, 1.56μM, 3.13μM, 6.25μM, 12.5μM, 25μM or less to act together with LPS for 24 hours (the final concentration of LPS in each drug group was 1μg / mL). The supernatant was collected, and the content of NO released in the supernatant was detected by Griess colorimetry to determine its NO generation rate.

[0113] 4. MTS method to determine the effect of racemic isomers of phthalide dimer compounds 1 to 10 and their optical enantiomers on RAW264.7 cell viability

[0114] Add 20 μL of MTS solution to the 96-well cell plate from which the supernatant was collected in step 3, incubate at 37°C for 80 min in the dark, measure the absorbance (490 nm), and record the results for data analysis. Each sample should be replicated in triplicate.

[0115] Cell viability % = average OD value of sample group / average OD value of blank control group × 100%.

[0116] 5. ELISA method was used to detect the effect of compound 7 on the secretion of inflammatory factors by RAW 264.7 cells induced by LPS

[0117] The cells were treated in the same manner as above, and the cell supernatant was collected. According to the instructions of the ELISA kit, the contents of inflammatory factors TNF-α, IL-6 and IL-1β in the cell supernatant were detected.

[0118] 6. RT-PCR was used to detect the effect of compound 7 on the expression of inflammatory factor mRNA in RAW 264.7 cells induced by LPS.

[0119] (1) RNA extraction

[0120] ① Cell treatment and collection: RAW264.7 cells in logarithmic phase growth were plated in a 24-well plate at a density of 1×106 cells / well. The group setting and cell treatment method were the same as “3”.

[0121] ② Extract total RNA: Add 250 μL Trizol reagent to each well, pipette thoroughly, collect into enzyme-free EP tube, and place on ice for 5 min.

[0122] ③ RNA separation: Add 200 μL chloroform, gently invert to mix, and place on ice for 3-5 minutes. Centrifuge at 4°C, 12000r / min for 15 minutes. At this time, the liquid is divided into 3 layers, from bottom to top, chloroform layer, protein layer and aqueous layer, and RNA is dissolved in the aqueous layer.

[0123] ④ Precipitate RNA: Aspirate about 200 μL of the aqueous phase and place it in a new enzyme-free EP tube. Add an equal volume of isopropanol, invert and mix, let stand for 10 minutes, and centrifuge at 4°C, 12,000 r / min for 15 minutes. Discard the supernatant, and the RNA will sink to the bottom of the tube.

[0124] ⑤ Wash RNA: Add 0.5 mL of 75% ethanol prepared with pre-cooled enzyme-free water to the RNA precipitate, gently invert it several times to wash away the residual isopropanol. Centrifuge at 4°C, 12000r / min for 5 minutes, and discard the supernatant. Then let the sample stand to evaporate the residual ethanol, but do not leave it for too long, otherwise it will cause RNA degradation.

[0125] ⑥Dissolve RNA: Add an appropriate amount of enzyme-free water and mix well to fully dissolve it.

[0126] (2) Reverse transcription reaction

[0127] ① Residual genomic DNA volume: Prepare the following mixture in an RNase free centrifuge tube and gently pipette to mix evenly. Incubate at 42℃ for 10min. The components and their usage are shown in Table 4 below:

[0128] Table 4

[0129] Components Usage Template RNA Total RNA: 1 μg 5×g DNA digestion mixture 3.0μL <![CDATA[RNase-free ddH2O]]> to 15.0μL

[0130] The required RNA mass is 1000 ng. The RNA volume is calculated according to the formula: X (RNA volume) = 1000 ng / RNA concentration (ng / μL).

[0131] ②Preparation of reverse transcription reaction system (20 μL): After removing gDNA according to the kit, prepare cDNA. The reaction system is shown in Table 5:

[0132] Table 5

[0133]

[0134] (3) Polymerase chain reaction

[0135] ① Primers: The primers required for PCR experiments were synthesized by Shanghai Bioengineering. The specific sequences of the relevant genes are shown in Experimental Reagent Table 1-3. The concentration used was 10 μmol / L.

[0136] ②PCR amplification: The PCR amplification mixed reaction system is shown in Table 1-7. Reaction conditions: 95℃ denaturation for 3min; 95℃ for 5s, 55℃ for 20s, 35 cycles, 95℃ for 15s, 60℃ for 1min, 95℃ for 15s, 60℃ for 15s.

[0137] ③Except for the cDNA solution, the other reagents were prepared in advance, mixed and transferred to the 8-tube strip, and the cDNA solution was added last. Amplification was performed using the QuantStudio 7 Real Time PCR instrument. -ΔΔCt The relative expression was calculated by the method, and β-actin was used as the internal reference.

[0138] Where ΔΔCT=(CT 实验组目的基因 -CT 实验组内参基因 )-(CT 对照组目的基因 -CT 对照组内参基因 ).

[0139] The PCR amplification reagents and reaction conditions used are shown in Table 6:

[0140] Table 6

[0141]

[0142] 7. Results Analysis

[0143] The experimental results were statistically analyzed using GraphPad Prism 8 software. The experimental data were expressed as mean ± standard error (X ± SD) to evaluate the overall difference. The mean between groups was analyzed by One-Way ANOVA analysis method for variance homogeneity, and compared between groups using Dunnett's test analysis method. P < 0.05 was considered statistically significant.

[0144] 8. IC 50 Calculation method

[0145] The IC was calculated by nonlinear regression fitting of each dose and inhibition rate. 50 .

[0146] 9. Experimental results

[0147] 9.1 Effects of the racemates of compounds 1 to 10 and their optical enantiomers on the viability of RAW264.7 cells

[0148] In this experiment, the MTS method was used to detect the effects of ZDG series compounds on the survival rate of RAW264.7 cells. Figure 2 As shown, compared with the control group, compounds 1-1, 1-2, 2, 2-2, 3, 3-1, 3-2, 4, 5, 5-1, 6, 7, 7-1, and 7-2 showed obvious cytotoxicity within a certain concentration range, while the remaining compounds showed no obvious cytotoxicity, indicating that the concentrations of these compounds need to be further reduced in order to evaluate their anti-inflammatory activity while ensuring that the cell survival rate approaches 100%.

[0149] according to Figure 2 It can be seen that the concentrations of compounds 1-1, 1-2, 2, 2-2, 3, 3-1, 3-2, 4, 5, 5-1, 6, 7, 7-1, and 7-2 that meet the cell survival rate approaching 100% are 3.13, 3.13, 1.56, 12.5, 6.25, 3.13, 1.56, 6.25, 3.13, 1.56, 6.25, 0.1, 0.05, and 0.2 μM, respectively. Determining the safe dosage for cells can avoid the influence of cytotoxicity on the results in subsequent anti-inflammatory effect experiments.

[0150] 9.2 Effects of the racemates of compounds 1 to 10 and their optical enantiomers on NO secretion induced by LPS in RAW264.7 cells

[0151] The results are as follows Figure 3 As shown, the racemates of compounds 1 to 10 and their optical antipodes can inhibit NO release within the corresponding concentration range, and compounds 1-1, 1-2, 2, 2-1, 2-2, 4, 7, 7-1, 7-2, 8, 8-1, 8-2, 9, 9-1, and 9-2 all have strong anti-inflammatory activity, and their IC50 Value Figure 4 As shown, detailed data are shown in Table 7;

[0152] Table 7: Half-inhibitory concentration values ​​of the racemic mixture of compounds 1 to 10 and their optical antipodes and L-arginine (L-NMMA) on NO production in RAW 264.7 cells induced by lipopolysaccharide:

[0153]

[0154]

[0155] a ND, not detected.

[0156] b L-NMMA, positive control.

[0157] From Table 7 and Figure 4 The results show that compound 7 and its optical antipode have the highest NO inhibition rate, and other compounds with inhibition rates higher than 90% include compound 2, the optical antipode of compound 1, and compound 9 and its optical antipode.

[0158] 9.3 Effects of compound 7 on LPS-induced inflammatory factors in RAW264.7 cells

[0159] After preliminary screening, compound 7 has potential anti-inflammatory activity. Since it has obvious cytotoxicity above 6.25 μM, the administration concentration was further reduced to exclude the possibility of reduced NO release due to cytotoxicity. The Log value of 0.2-3.13 μM compound 7 was set for further investigation. The results are as follows: Figure 5 : This indicates that compound 7 still has good anti-inflammatory activity at low concentrations, and IC 50 The value was 425.2 nM. When the addition amount of compound 7 was 3.13 μM, it had an inhibitory effect on NO release close to 100%.

[0160] 9.4 RT-PCR detection of the effect of compound 7 on the secretion of inflammatory factors by RAW264.7 cells and inflammatory cells induced by LPS

[0161] In order to further evaluate the anti-inflammatory mechanism of compound 7. This experiment used ELISA and RT-PCR to detect the effect of compound 7 on the secretion of inflammatory factors by inflammatory cells. Figure 6 As shown, ELISA experiments showed that compared with the normal group, the model group significantly promoted the expression levels of TNF-α, IL-6 and IL-1β in RAW264.7 cells (P<0.0001).

[0162] Compared with the model group, the secretion of TNF-α, IL-6 and IL-1β was significantly decreased after treatment with compound 7 at concentrations of 1 and 0.5 μM (P<0.0001, P<0.05).

[0163] RT-PCR experiments showed that treatment with compound 7 at concentrations of 1 and 0.5 μM could significantly inhibit the LPS-induced IL-6 and IL-1β mRNA expression levels in RAW264.7 (P<0.0001, P<0.001).

[0164] This indicates that compound 7 significantly inhibits the secretion of inflammatory factors by inflammatory cells, such as TNF-α, IL-6, and IL-1β, and can also inhibit the expression of IL-6 and IL-1β mRNA in RAW264.7 cells induced by LPS, indicating that the compound has a good anti-inflammatory effect.

[0165] In summary, compounds 1 to 10 of phthalide dimers were isolated from the rhizomes of the medicinal part of Angelica sinensis, and the anti-inflammatory activities of each compound were studied. The results showed that all compounds had anti-inflammatory activity, especially compound 7 and its optical enantiomers, which had higher anti-inflammatory activity. This further enriched the chemical component library of phthalide components, provided new lead compounds for the development of new anti-inflammatory drugs, and had good application prospects.

[0166] Example 3

[0167] The difference from Example 1 is that when preparing the Angelica extract Fr.C, the ethanol is soaked for 1 day each time.

[0168] Example 4 Aqueous anti-inflammatory composition

[0169] 3 μM of compound 7 was mixed evenly with a mixture consisting of 0.1 parts by weight of sodium hyaluronate, 0.5 parts by weight of D-panthenol, 0.3 parts by weight of Alcaligenes polysaccharide, 2 parts by weight of glycerol, 0.1 parts by weight of HA (30-50W), 0.05 parts by weight of EDTA-2Na, 3 parts by weight of methylpropanediol, 0.5 parts by weight of p-hydroxyacetophenone, 0.5 parts by weight of hexylene glycol, and 0.2 parts by weight, and then 500 mL of water was added and stirred to dissolve to obtain the product.

[0170] Example 5 Tablet anti-inflammatory composition

[0171] 3 μM of compound 7 was mixed with 10 parts by weight of dry starch and 1 part by weight of magnesium stearate, and the viscosity was adjusted to a tablet-compressible state using 10% mass concentration starch slurry and lactose aqueous solution, followed by tablet compression and drying to obtain tablets.

[0172] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting and separating phthalide dimer compounds using Angelica sinensis as a raw material, characterized in that: The following steps are involved: 1) After separation by normal phase chromatography on a silica gel column, the Angelica sinensis extract Fr.C was gradient eluted using petroleum ether-ethyl acetate as the eluent, and the eluted fractions were combined according to the TLC detection results to obtain component FrC-1, component FrC-2, and component FrC-3; 2) After fractionating component FrC-2 using a silica gel column, gradient elution was performed using dichloromethane / methanol as the eluent, and each elution fraction was detected by TLC, and the same fractions were combined to obtain component FrC-2-1, component FrC-2-2, and component FrC-2-3; 3) eluting the component FrC-2-2 through Sephadex LH-20 column chromatography and isocratically eluting with a methanol solution, and combining the eluted fractions after TLC detection to obtain component FrC-2-2-1 and component FrC-2-2-2; Component FrC-2-2-1 was purified by semi-preparative high performance liquid chromatography to obtain racemate (±)-1, i.e., compound 1, (±)-2, i.e., compound 2, and component FrC-2-2-1-1; The component FrC-2-2-1-1 was further separated by silica gel column chromatography and gradient eluted with petroleum ether / ethyl acetate to obtain racemic (±)-3, i.e., compound 3, and (±)-4, i.e., compound 4; The component FrC-2-2-2 was purified by semi-preparative high performance liquid chromatography to obtain racemate (±)-5, i.e., compound 5, (±)-6, i.e., compound 6, and the component FrC-2-2-2-1; The component FrC-2-2-2-1 was further separated by silica gel column chromatography and gradient eluted with petroleum ether / ethyl acetate to obtain racemic (±)-7, i.e., compound 7, and (±)-8, i.e., compound 8; Component FrC.2-3 was eluted by Sephadex LH-20 column chromatography with 100% methanol isocratically, the eluted fractions were detected by TLC, and the same fractions were combined to obtain two components, components FrC-2-3-1 to FrC-2-3-2; Component FrC-2-3-1 was purified by semi-preparative HPLC to obtain racemates (±)-9, i.e., compound 9, and (±)-10, i.e., compound 10; The obtained compounds 1 to 10 are:

2. The method according to claim 1, characterized in that When treating component FrC-2-2-1, the mobile phase used for semi-preparative HPLC purification was a mixture of acetonitrile and water in a volume ratio of 68:32; Preferably, when treating component FrC-2-2-2, the mobile phase used for semi-preparative HPLC purification is a mixture of acetonitrile and water in a volume ratio of 63:37; Preferably, when treating component FrC-2-3-1, the mobile phase used for semi-preparative HPLC purification is a mixture of acetonitrile and water in a volume ratio of 45:

55.

3. The method according to claim 1, characterized in that The eluent of petroleum ether-ethyl acetate was changed from a volume ratio of petroleum ether to ethyl acetate of 100:1 to a volume ratio of petroleum ether to ethyl acetate of 1:1; Preferably, the dichloromethane / methanol eluent is transitioned from a dichloromethane:methanol volume ratio of 100:1 to a dichloromethane:methanol volume ratio of 1:

1.

4. The method according to claim 1, characterized in that: The preparation method of Angelica sinensis extract Fr.C is as follows: using dry Angelica sinensis powder as raw material, soaking it in 80% ethanol for 3 to 5 times, soaking for 1 to 2 days each time, combining the extracts obtained each time, concentrating under reduced pressure to obtain an extract, loading the water-soluble extract with D101 polystyrene macroporous resin column, and gradient eluting with water, methanol and methanol as eluents in sequence, collecting the methanol elution part, concentrating under reduced pressure, and dissolving in water to obtain Angelica sinensis extract Fr.C.

5. The method according to claim 1, characterized in that Also includes: The obtained compounds 1-10 are subjected to chiral resolution to obtain the optical antipodes of compounds 1-10 respectively.

6. The method according to claim 5, characterized in that Chiral separation conditions: using chiral column AD-H was analyzed by analytical HPLC with n-hexane:isopropanol as the mobile phase, the flow rate of the mobile phase was 1 mL / min, and the detection wavelength was set at 254 nm.

7. The method according to claim 6, characterized in that The mobile phase used in the chiral separation of compounds 1 and 8 was a mixture of n-hexane:isopropanol in a volume ratio of 96:4; The mobile phase used in the chiral separation of compounds 2 and 3 was a mixture of n-hexane:isopropanol in a volume ratio of 93:7; The mobile phase used in the chiral separation of compounds 4-6 and 9 was a mixture of n-hexane:isopropanol in a volume ratio of 92:8; The mobile phase used in the chiral separation of compound 7 was a mixture of n-hexane:isopropanol in a volume ratio of 94:6; The mobile phase used in the chiral separation of compound 10 was a mixture of n-hexane and isopropanol in a volume ratio of 90:

10.

8. An anti-inflammatory composition, characterized in that At least: Compounds 1 to 10 obtained by the method according to any one of claims 1 to 7.

9. The composition according to claim 8, characterized in that At least: Any one of compound 2, the optical enantiomer of compound 1, compound 9 and its optical enantiomer, compound 7 and its optical enantiomer; More preferably, it comprises at least compound 7 and its optical antipodes; More preferably, the dosage form of the composition is at least one of ointment, emulsion, aqueous solution, tablet, injection, and capsule.

10. The composition according to claim 8, characterized in that The added amount of compounds 1 to 10 was less than 6.25 μM; Preferably, it comprises at least: compound 7 and its enantiomers added in an amount of 0.5 to 3.5 μM; More preferably, the inhibition rate of compound 2, the optical antipodes of compound 1, compound 9 and its optical antipodes, and compound 7 and its optical antipodes on NO is greater than 90%; More preferably, the inhibition rate of compound 7 and its optical antipodes on NO is greater than 97.0%; More preferably, compound 7 and its optical enantiomers can reduce the secretion of TNF-α, IL-6 and IL-1β in RAW264.7 cells induced by LPS; More preferably, compound 7 and its optical antipodes can reduce the secretion of inflammatory cytokines TNF-α, IL-6, and IL-1β in RAW264.7 cells induced by LPS.

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