Guaiarene-type sesquiterpene derivatives prepared from Daphne koreana and their application

Abstract

The guaiacyl sesquiterpene derivatives prepared from Daphne koreana and their applications belong to the field of medical technology, and specifically relate to 40 guaiacyl sesquiterpene derivatives extracted and separated from Daphne koreana of the Thymelaeaceae family by chromatographic methods such as silica gel, ODS column chromatography, HP20 column chromatography, and HPLC. The guaiacyl sesquiterpene derivatives were evaluated for anti-neuroinflammatory activity, and the effect was obvious, among which compounds 3, 8, 35, 36, 39 and 40 had better inhibitory effects than the positive control dexamethasone. Therefore, the derivatives disclosed in the present invention can be used in the preparation of anti-neuroinflammatory drugs. The preparation method of the present invention is simple, reproducible, and the obtained compound has high purity. The obtained compound shows good anti-neuroinflammatory activity on the LPS-induced BV-2 cell line.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to 40 guaiacyl sesquiterpene derivatives prepared by guided separation of sesquiterpene components in Daphne koreana using LC-MS / MS molecular network technology, and their application in the preparation of anti-neuroinflammatory drugs. Background Art

[0002] Neuroinflammation is a disease observed in the central nervous system (CNS) under the stimulation of infection, toxic metabolites, trauma or autoimmunity; its main characteristics are symmetrical sensory motor and autonomic dysfunction. At present, many common diseases have been found to be associated with neuroinflammation, including Alzheimer's disease (AD), cerebral hemorrhage and traumatic brain injury. The main methods for treating neuroinflammation include supplementing vitamin B 12 , give some neuroprotective drugs, etc.

[0003] In recent years, molecular network-guided technology based on LC-MS / MS has been widely used in the study of natural products from microorganisms, marine organisms, fungi and plants. By uploading the secondary mass spectrometry data of the sample to the Global Natural Products Society Molecular Network (GNPS) data analysis platform (http: / / gnps.ucsd.edu), a visual molecular network (MN) can be constructed to identify the components in the sample and build the relationship between these components, thereby achieving accurate positioning and rapid separation of the target compound.

[0004] In the previous study of the chemical components of Daphne penicillata Rehd., it was found that guaiacyl sesquiterpene derivatives showed good anti-neuroinflammatory activity. With further in-depth research on the plant, 40 guaiacyl sesquiterpene derivatives with anti-neuroinflammatory properties were found. The sesquiterpene compounds involved in the present invention are obtained by rapid separation of sesquiterpene components in Daphne penicillata Rehd. by LC-MS / MS molecular network technology, and the compounds have good anti-neuroinflammatory activity. The new compounds involved in the present invention and their configurations have been determined, and there has been no patent or literature report so far. Summary of the invention

[0005] The present invention provides 40 sesquiterpenoid compounds and preparation methods thereof;

[0006] The present invention also provides the use of the sesquiterpenoid compound in preparing an anti-neuroinflammatory drug;

[0007] The 40 sesquiterpenoid compounds extracted and separated from Daphne koreana of the Thymelaeaceae family have the following structures:

[0008]

[0009] The preparation technology scheme of the present invention comprises the following steps:

[0010] (1) Dried Daphne koreana was extracted 2-3 times with 70-80% industrial ethanol under reflux, and the extracts were combined and concentrated to obtain an extract. The extract was extracted with ethyl acetate and n-butanol in turn, and the ethyl acetate and n-butanol fractions were subjected to silica gel column chromatography and gradient eluted with a dichloromethane-methanol system to collect a total of 4 fractions Fr A to Fr D;

[0011] (2) Fraction Fr.A was subjected to polyamide column chromatography and eluted with ethanol and water gradient to collect two fractions Fr.A1 to Fr.A2;

[0012] (3) Fraction Fr.A1 was separated by HP20 column chromatography and eluted with ethanol and water gradient to obtain two fractions Fr.A1-1 to Fr.A1-2;

[0013] (4) Fractions Fr.A1-1 and Fr.A1-2 were separated by ODS column and eluted with ethanol-water gradient to obtain 5 fractions Fr.1 to Fr.5;

[0014] (5) The five fractions were subjected to secondary mass spectrometry testing using an HPLC-QTOF-MS mass spectrometer. By analyzing the structural type and distribution of the fraction nodes, three target fractions were finally selected, namely Fr.3, Fr.4 and Fr.5;

[0015] (6) The above three target fractions Fr.3, Fr.4 and Fr.5 were passed through a silica gel column and eluted with a petroleum ether-ethyl acetate gradient. Then, they were subjected to preparative HPLC and eluted with methanol-water to obtain fractions Fr.3.1.1 to Fr.3.1.4, Fr.3.2.1 to Fr.3.2.7, Fr.3.3.1 to Fr.3.3.3, Fr.4.1.1 to Fr.4.1.4 and Fr.5.1.1 to Fr.5.1.4. Fr.3.1.1 was then subjected to semi-preparative HPLC chromatography and eluted with acetonitrile-water to separate and purify compounds 1, 2, 5 and 6; Fr.3.1.3 was subjected to semi-preparative HPLC chromatography and eluted with acetonitrile-water to separate and purify compounds 3, 4, 7 and 9; Fr.3.2.4 was subjected to semi-preparative HPLC chromatography and eluted with acetonitrile-water to separate and purify compounds 11, 14, 16, 17 and 20; Fr.3.2.5 was subjected to semi-preparative HPLC chromatography and eluted with acetonitrile-water to separate and purify compounds 8, 10, 12, 13 and 15; Fr.3 .3.1 and Fr.3.3.3 were separated and purified by semi-preparative HPLC chromatography with acetonitrile-water as eluent to obtain compounds 18, 19, 21, 22, 24 and 25; Fr.4.1.2 and Fr.4.1.3 were separated and purified by semi-preparative HPLC chromatography with acetonitrile-water as eluent to obtain compounds 23, 26, 27, 29, 33 and 34; Fr.5.1.2 and Fr.5.1.3 were separated and purified by semi-preparative HPLC chromatography with acetonitrile-water as eluent to obtain compounds 28, 30, 31, 32, 35, 36, 37, 38, 39 and 40.

[0016] The above preparation method, wherein

[0017] The gradient of dichloromethane-methanol in step (1) is: 100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1.

[0018] The concentration of ethanol in the ethanol water in step (2) is: 20%, 70%.

[0019] The concentration of ethanol in the ethanol water in step (3) is: 20%, 50%, 90%.

[0020] The concentration of ethanol in the ethanol water in step (4) is: 40%, 50%, 60%, 70%, 80%, 90%.

[0021] The specific operation in step (5) is to upload the secondary mass spectrometry data of the sample to the Global Natural Product Society Molecular Network (GNPS) data analysis platform, and construct a visual molecular network (Molecular Networking, MN) through the cytoscape software.

[0022] In step (6), the volume ratio of petroleum ether to ethyl acetate is 100:1, 50:1, 30:1, 20:1, 10:1, 5:1, the volume ratio of methanol to water for the preparative liquid phase is 40:60, and the volume ratio of acetonitrile to water for the semi-preparative liquid phase is 20:80.

[0023] like Figure 1 As shown, a molecular network was established by analyzing the fractions from Daphne koreana L. by HPLC-MS / MS.

[0024] The obtained compounds were systematically identified, and the results are as follows: Figure 2-18 , as shown in 22-26:

[0025] Compound 1 is a colorless oil (methanol), which is easily soluble in solvents such as methanol and chloroform. HRESIMS gives a quasi-molecular ion peak of 309.1471 [M+Na] + (calcd for C 18 H 22 O 2 Na, 309.1461), the molecular weight of the compound was determined to be 286, and the molecular formula was C 18 H 22 O 3 , the calculated unsaturation is 8. 1 H NMR (600 MHz, CDCl 3 ) spectrum shows two double bond olefinic hydrogen proton signals δ H 6.04 (1H, d, J = 15.6 Hz), 7.56 (1H, d, J = 15.7 Hz), and 2 terminal double bond olefin proton signals δ H 4.75 (1H, dd, J = 1.7, 0.9 Hz), 4.76 (1H, t, J = 1.5 Hz). In addition, there are 2 methyl signals δ H 1.77 (3H, s), 1.85 (3H, s), a methoxy signal δ H 3.78(3H,s). 13 C NMR (150 MHz, CDCl 3 ) spectrum showed 18 carbon signals. Combined with HSQC spectrum analysis, it was inferred that the compound contained 10 sp 2 Hybridized carbon signal (δ C 203.0,143.4,166.2,148.9,110.1,118.4,143.9,167.7,143.3,133.5), 1 methine carbon signal (δ C 42.8), 4 methylene carbon signals (δ C 39.7, 32.8, 31.8, 25.6) and three methyl carbon signals (δ C9.1, 20.8, 51.9). In the HMBC spectrum, H-7 is correlated with C-8 and C-6, H-6 is correlated with C-8, C-7, C-4, C-1 and C-5, H-14 is correlated with C-10 and C-1, H-9 is correlated with C-10 and C-1, and H-6 is correlated with H-7, H-7 with H-8, and H-8 with H-9. 1 H- 1 H COSY correlation indicates the existence of a seven-membered ring fragment formed by C-1, C-5, C-6, C-7, C-8, C-9, and C-10. 3 -17 is related to C-4, C-5 and C-3, H-2 is related to C-10, C-1, C-4, C-5 and C-3, and the existence of a five-membered ring fragment composed of C-1, C-2, C-3, C-4 and C-5 is confirmed, and it is connected to the seven-membered ring fragment through C-1 and C-5, among which CH 3 -17 is connected to C-4. Therefore, it is confirmed that compound 1 contains a guaiacyl structure skeleton. 3 -13 is related to C-7, C-12 and C-11, H 2 -12 and C-7, C-11 and CH 3 -13 is related, indicating the existence of a group of terminal double bond fragments. 3 -16 is correlated with C-16, H-15 is correlated with C-16, C-10 is correlated with H-14, and C-16 is correlated with C-16, indicating the presence of a side chain at C-10, in which the methoxy group is connected to C-16, thus determining the planar structure of the compound. Therefore, the general structure of 1 was determined, which contains an additional acetic acid connected to C-14 and can be biosynthesized by intermolecular Aldol reactions (such as Fig. 20 ).

[0026] The absolute configuration of the compound was determined by comparing the calculated ECD with the measured ECD. First, the conformation search was performed using Spartan software, and the conformations with Boltzmann distribution greater than 1% were selected for optimization at the B3LYP / 6-31G(d) basis set level. The excited state calculation was further performed at the B3LYP / 6-311+G(d,p) basis set level, and the ECD spectrum was finally fitted using SpecDis software. The final absolute configuration of the molecule was determined to be 7R. Therefore, the absolute configuration of compound 1 was determined and named Daphnenicillata A.

[0027] Compounds 2-5 have the same molecular formula, and HRESIMS gives a quasi-molecular ion peak of 273.1472 [M+H] + (calcdfor C 15 H 22 O3 Na,273.1461), combined 1 H, 13 C NMR data confirmed that its molecular formula is C 15 H 22 O 3 , and its unsaturation degree is calculated to be 5. Comprehensive spectral analysis shows that compounds 2-4 are known compounds. However, although compound 5 is consistent with the data of the known compound Daphnepapytone E, further quantum chemical calculations (nuclear magnetic resonance and electron capture spectroscopy) and single crystal X-ray diffraction analysis determined the absolute configuration of 5. Therefore, the configuration of compound Daphnepapytone E is wrong and should be corrected to 1R, 7R, 10S (such as Fig.21 ).

[0028] Compound 6 is a colorless oil (methanol). HRESIMS gives a quasi-molecular ion peak of 289.1411 [M+H] + (calcdfor C 15 H 22 O 4 Na, 289.1410), the molecular weight of the compound is determined to be 266, and the molecular formula is C 15 H 22 O 4 , the calculated unsaturation degree is 5. By comparing the 1 H and 13 C NMR data showed that the nuclear magnetic data of the three were very similar. 1 H- 1 The existence of C6-C7-C8-C9 spin system in H COSY spectrum and the existence of H-2 with C-1, C-3, C-5 and C-10, H-6 with C-1, C-4, C-8 and C-11, H-9 with C-1, C-7, H-14, H-2 in HMBC spectrum 2 The key correlation signals of C-14 and C-10, C-9 and C-1 indicate that the difference between this compound and compound 3 is that there is a hydroxyl substitution at the C-14 position. Through the HSQC spectrum, the compound was assigned to the carbon-hydrogen direct correlation and its planar structure was determined. It is speculated that this compound is a typical guaiacyl sesquiterpene. In the NOESY spectrum, H-9 and H 2 -14 has NOESY correlation, thus confirming H 2 -14 and H-9 are on the same side, and the other chiral centers cannot be accurately determined by NOESY spectrum correlation. Therefore, quantum chemical ECD and NMR calculations were performed for all possible configurations of the structure. The measured NMR data were compared with the calculated NMR data, and a variety of statistical analysis methods (R 2, DP4+ probability analysis), comparing the correlation coefficient and confidence probability between compound 6 and the four possible configurations, it was found that the compound had the best agreement with the calculated results of 1R*, 7R*, and 10R* configurations. The DP4+ probability analysis of hydrocarbon data was 100%, and there was a small calculation deviation in the linear correlation analysis (R 2 =0.9981). The absolute configuration of the compound was determined by comparing the calculated ECD with the measured ECD. The calculated 1R, 7R, 10R configuration of compound 6 was consistent with that of compound 6, so its absolute configuration was determined to be 1R, 7R, 10R.

[0029] Compounds 7 and 8 have the same planar structure, and HRESIMS gives a quasi-molecular ion peak of 287.1609 [M+H] + (calcd for C 16 H 24 O 3 Na,287.1618), combined 1 H, 13 C NMR data confirmed that its molecular formula is C 16 H 24 O 3 The unsaturation degree is calculated to be 5. 1 H and 13 Comparison of C NMR data revealed that the NMR data of compounds 7, 8 and guaiacyl sesquiterpene 5 were very similar, with the same parent nucleus and the same substituent connection position. The difference was that the hydroxyl group at the C-1 position in compounds 7 and 8 was replaced by a methoxy group. The HMBC spectra showed that the hydroxyl group at the C-1 position of compounds 7 and 8 was replaced by a methoxy group. 3 The HMBC cross peaks from H-14 to C-1, C-9 and C-10 indicate that the methoxy group is substituted at C-1. In addition, other related and combined 1 H- 1 The existence of the C6-C7-C8-C9 spin system in the H COSY spectrum further confirmed the skeleton of the guaiacyl sesquiterpene. The compound was assigned to carbon and hydrogen directly through the HSQC spectrum to determine the planar structure. The relative configuration of compound 7 was determined by observing the relevant signals of the NOESY spectrum. 1-OCH 3 Correlation with H-7 and H-2α indicates that 1-OCH 3 and H-7 are both α-configuration. 3 -14 is correlated with H-2β, thus the relative configuration of compound 7 was determined. In the NOESY spectrum of compound 8, H 2 -12 and 1-OCH 3 Related to H-8β, CH 3-14 is related to H-8β, and the relative configuration of compound 8 can be determined. By comparing the calculated ECD spectra with the measured ECD spectra, the absolute configurations of compounds 7-8 are 1S, 7R, 10R and 1R, 7R, 10R, respectively.

[0030] Compound 9 is a colorless oil (methanol). HRESIMS gives a quasi-molecular ion peak of 303.1570 [M+Na] + (calcd for C 16 H 24 O 4 Na, 303.1567), the molecular weight of the compound was determined to be 280, and the molecular formula was C 16 H 24 O 4 , the calculated unsaturation is 5. 1 H NMR (600 MHz, CDCl 3 ) spectrum shows two terminal double bond olefinic hydrogen proton signals δ H 4.78 (1H, d, J = 1.5 Hz), 4.75 (1H, d, J = 1.5 Hz), 2 methyl signals δ H 1.79(3H,s),1.75(3H,s). 13 C NMR (150 MHz, CDCl 3 ) spectrum showed 15 carbon signals. Combined with HSQC spectrum analysis, it was inferred that the compound contained 5 sp 2 Hybrid carbon signal, 5 methylene carbon signals, 1 methine carbon signal, 2 quaternary carbon signals, 2 methyl carbon signals and 1 methoxy carbon signal, it is speculated that the compound is a typical guaiacyl sesquiterpene. 3 -13 is related to C-7, C-12 and C-11, H 2 -12 and C-7, CH 3 -13 is related to C-11, indicating the presence of a terminal double bond fragment, in which CH 3 -13 is connected to C-11. H-7 is related to C-8 and C-6, H-6 is related to C-8, C-7, C-4, C-1 and C-5, H-9 is related to C-10, H-11 is related to C-12, H-13 is related to C-13, H-11 is related to C-13, H-13 is related to C-11, H-11 is related to C-12, H-13 is 2 -14 is correlated with C-1, and H-2 is correlated with C-10, C-1, C-4, C-5 and C-3, indicating that the compound is similar in structure to compound 6. 3 -1 and C-1 have a key HMBC correlation, proving the presence of a methoxy substitution at the C-1 position, thus confirming the planar structure of the compound.

[0031] OCH can be observed in the NOESY spectrum 3-14 is associated with H-2α and H-7, and H-2β is associated with H 2 -14 is correlated, thus determining the relative configuration of compound 9. The absolute configuration of the compound was determined by comparing the calculated ECD with the measured ECD. The calculated 1S, 7R, 9S configuration of compound 9 is consistent with the calculated one, so its absolute configuration is determined to be 1S, 7R, 9S.

[0032] Compound 10 is a colorless block crystal (methanol). HRESIMS gives a quasi-molecular ion peak of 259.1307 [M+Na] + (calcd for C 14 H 20 O 3 Na, 259.1305), the molecular weight of the compound was determined to be 236, and the molecular formula was C 14 H 20 O 3 , the calculated unsaturation is 5. Analysis of 1D NMR data showed that compound 10 is a structural analog of compound 22, with the obvious difference that there is no methyl group at C-10. This inference led to the 1 H- 1 The H COSY correlation and HMBC of H-10 to C-1 and C-8 confirmed the absolute configuration of 10 to be 1S, 7R, 10S by single crystal X-ray diffraction analysis and calculated ECD.

[0033] Compound 11 is a colorless oil (methanol). HRESIMS gives a quasi-molecular ion peak of 257.1182 [M+Na] + (calcd for C 14 H 20 O 3 Na, 257.1148), the molecular weight of the compound is determined to be 234, and the molecular formula is C 14 H 18 O 3 , the calculated unsaturation degree is 6. By comparing compound 11 with compound 10 1 H and 13 C NMR data show that the nuclear magnetic data of the two are very similar. Combined with the CH 3 -13 is related to C-7, C-12 and C-11, H 2 -12 and C-7, C-11 and CH 3-13, H-7 is related to C-8 and C-6, H-6 is related to C-8, C-7, C-4, C-1 and C-5, and H-2 is related to C-1, C-3, C-5 and C-10, indicating that the difference between this compound and 10 is that the hydroxyl group at the C-10 position is oxidized to a keto carbonyl group, indicating that this compound is a C-10-decarbonized guaiacyl-type sesquiterpenoid. Through the HSQC spectrum, the compound was assigned to a direct carbon-hydrogen correlation and the planar structure was determined. Through NMR calculation, the relative configuration of compound 11 was determined to be 1R*, 7R*. The absolute configuration of compound 11 was verified by comparing the calculated ECD spectrum with the measured ECD spectrum. The compound 11 has a good agreement with the calculated 1R, 7R configuration, so its absolute configuration is determined to be 1R, 7R.

[0034] Compound 12 is a light yellow powder (methanol). HRESIMS gives a quasi-molecular ion peak of 255.1351 [M+Na] + (calcd for C 15 H 20 O 2 Na, 255.1356), the molecular weight of the compound is determined to be 232, and the molecular formula is C 15 H 20 O 2 , the calculated unsaturation degree is 6. By comparing compound 12 with compound 11 1 H and 13 C NMR data show that the nuclear magnetic data of the two are very similar. Combined with the HMBC spectrum, H-2 is related to C-1, C-3, C-5 and C-10, H-6 is related to C-1, C-4, C-8 and C-11, and H 2 -14 is related to C-1, C-9 and C-10, and H-9 is related to C-1, C-10, H 2 The presence of C-14 and C-7 related signals indicates that the difference between this compound and compound 11 is that the keto carbonyl at C-10 becomes an exocyclic double bond. The compound was assigned to carbon and hydrogen directly through HSQC spectrum to determine the planar structure. The relative configuration of compound 12 was determined to be 1S*, 7R* through NMR calculation. The absolute configuration of compound 12 was verified by comparing the calculated ECD spectrum with the measured ECD spectrum, thus determining its absolute configuration to be 1S, 7R.

[0035] Compound 13 is a colorless oil, which is easily soluble in solvents such as methanol and chloroform. HRESIMS gives a quasi-molecular ion peak of 271.1295 [M+Na] + (calcd for C 15 H 20 O 3 Na, 273.1305), the molecular weight of the compound was determined to be 248, and the molecular formula was C15 H 20 O 3 , the calculated unsaturation degree is 6. 3 -13 is related to C-7, C-12 and C-11, H 2 -12 and C-7, CH 3 -13 is related to C-11, indicating the presence of a terminal double bond fragment, in which CH 3 -13 is connected to C-11. H-7 is related to C-8 and C-6, H-6 is related to C-8, C-7, C-4, C-1 and C-5, H-9 is related to C-10, CH 3 -14 is related to C-1, H-2 is related to C-10, C-1, C-4, C-5 and C-3, and the basic skeleton of the five-membered ring and the seven-membered ring is determined. Combined with the six unsaturated degrees in the structure, δ C 65.7(C-9) and δ C 63.7 (C-10) two carbon characteristic chemical shifts and CH 3 -14 has a key HMBC correlation with C-10, C-9 and C-1, indicating that C-9 and C-10 form a three-membered oxygen ring structure fragment, thereby determining the planar structure of the compound. By comparing the 1 H and 13 C NMR data show that the two NMR data are very similar. 1 H- 1 The existence of C6-C7-C8-C9 spin system in H COSY spectrum and the existence of H-2 with C-1, C-3, C-5 and C-10, H-6 with C-1, C-4, C-8 and C-11, H-9 with C-1, C-7, H-14, CH 3 -14 and the key related signals of C-10, C-9 and C-1, indicating that compounds 14 and 15 have the same planar structure as compound 13, but different configurations. Their relative configurations were determined by NMR calculation. The absolute configurations of compounds 13, 14 and 15 were verified by comparing the calculated ECD spectra with the measured ECD spectra, thus determining that the absolute configurations of compounds 13-15 were 1R, 7S, 9R, 10R, 1R, 7S, 9S, 10S, 1S, 7S, 9R, 10R, respectively.

[0036] Compound 16 is a light yellow oil (methanol). HRESIMS gives a quasi-molecular ion peak of 271.1294 [M+Na] + (calcd for C 15 H 20 O 3Na, 273.1305), the molecular weight of the compound was determined to be 248, and the molecular formula was C 15 H 20 O 3 , the calculated unsaturation degree is 6. 3 -13 is related to C-7, C-12 and C-11, H 2 -12 and C-7, CH 3 -13 is related to C-11, indicating the presence of a terminal double bond fragment, in which CH 3 -13 is connected to C-11. H-7 is related to C-8 and C-6, H-6 is related to C-8, C-7, C-4, C-1 and C-5, H-9 is related to C-10, CH 3 -14 is correlated with C-1, and H-2 is correlated with C-10, C-1, C-4, C-5 and C-3, indicating that the compound is similar in structure to compound 13. C 70.4(C-1) and δ C 67.7 (C-10) two carbon characteristic chemical shifts and CH 3 -14 has a key HMBC correlation with C-10, C-9 and C-1, indicating that a three-membered oxygen ring structure fragment is formed at C-1 and C-10, thereby determining the planar structure of the compound. In the NOESY spectrum, it can be observed that H-9 and CH 3 -14 and H-8β have NOESY correlation, and H-8α and H-7 have correlation, thus determining the relative configuration of compound 16. The absolute configuration of the compound was determined to be 1R, 7S, 9S, 10S by comparing the calculated ECD with the measured ECD.

[0037] Compound 17 is a light yellow oil (methanol). HRESIMS gives a quasi-molecular ion peak of 255.1359 [M+H] + (calcd for C 15 H 20 O 2 Na,255.1356), combined 1 H, 13 C NMR data confirmed that its molecular formula is C 15 H 20 O 2 , and its unsaturation is calculated to be 6. In the HMBC spectrum, H-2 is related to C-1, C-3, C-5 and C-10, H-6 is related to C-1, C-4, C-8 and C-11, H-9 is related to C-1, C-7, H-14, and CH 3 -13 is related to C-7, C-11 and C-12, H 2 -14 is related to C-1, C-9 and C-10, CH3 The presence of -15 related to C-3, C-4 and C-5 indicates that the structure of compound 17 is very similar to that of compound 13, a guaiacyl sesquiterpenoid. The difference between them is that there is a hydroxyl group substituted at the C-14 position in compound 17. 1 H- 1 The existence of C6-C7-C8-C9-C10-C14 spin system observed in H COSY spectrum further confirmed the planar structure. Then, the accurate configuration of compound 17 was determined to be 1R, 7S, 9S, 10S by NMR and ECD calculation.

[0038] Compound 18 is a white powder (methanol). HRESIMS gives a quasi-molecular ion peak of 235.1692 [M+H] + (calcdfor C 15 H 23 O 2 ,235.1693), the molecular weight of the compound was determined to be 234 and the molecular formula was C 15 H 22 O 2 , the calculated unsaturation degree is 5. Comparison of H-C spectra revealed that compound 18 and compound 22 have the same general structure, except that there is a hydroxymethyl substitution on C-10, which gives the key HMBC formula of H-2 / H-1 / H-10 / H-14 and 1 H- 1 Confirmation of the HCOSY correlation. Therefore, the exact configuration of compound 18 was determined to be 1S, 7R, 10S by NMR and ECD calculations.

[0039] Compound 23 is a colorless oil (methanol). HRESIMS gives a quasi-molecular ion peak of 235.1697 [M+H] + (calcd for C 15 H 23 O 2 ,235.1693), combined 1 H, 13 C NMR data confirmed that its molecular formula is C 15 H 22 O 2 , and its unsaturation is calculated to be 5. In the HMBC spectrum, H-1 is related to C-3, C-4 and C-14, H-2 is related to C-1, C-3, C-5 and C-10, H-6 is related to C-1, C-4, C-8 and C-11, H-9 is related to C-1, C-7, H-14, CH 3 -13 is related to C-7, C-11 and C-12, CH 3 -14 is related to C-1, C-9 and C-10, CH 3The presence of -15 associated with C-3, C-4, and C-5 confirmed that the structure of compound 23 was a typical guaiacyl-type sesquiterpenoid. 1 H- 1 The presence of correlation observed in the H COSY spectrum further confirms the planar structure. 1 H NMR spectra and 13 The C NMR spectra of compound 24 and compound 23 show similar signals, which proves that they have the same planar structure but different configurations. Since H-1 and H-7 are related in the structure of compound 23, H-6 and H-8β and CH 3 -13 is related, H-8β is related to CH 3 -14 is related, thus determining the relative configuration of compound 23. Since H-1 in the structure of compound 24 is related to H-6 and H-7, CH 3 -14 and CH 3 -13 is correlated, thus determining the relative configuration of compound 24. Finally, by comparing the calculated and experimental ECD curves, the absolute configurations of compound 23 and compound 24 were determined to be 1S, 6S, 7S, 10S and 1S, 6R, 7S, 10S.

[0040] Compound 25 is a colorless oil (methanol). HRESIMS gives a quasi-molecular ion peak of 237.1846 [M+H] + (calcd for C 15 H 25 O 2 ,237.1849), in the HMBC spectrum, H-2 is related to C-1, C-3, C-5 and C-10, H-6 is related to C-1, C-4, C-8 and C-11, H-1 is related to C-4, C-5, C-10 and H-14, combined with 1 H- 1 The spin system observed in the H COSY spectrum further confirmed the planar structure of the compound. 1 H NMR spectra and 13 The C NMR spectra showed similar signals to those of compound 25, proving that they have the same planar structure, but with different configurations. Based on the NOESY correlations of H-1 / H-7 and H-9α, CH3-14 / H-9β in H-1, H-7, and H-10 of compounds 25 and 26 were classified as α-oriented. However, due to the lack of conclusive evidence, the relative configuration of C-11 of compounds 25 and 26 remains unknown. To determine the configuration of C-11, the NMR of two possible isomers was calculated and the R 2 , CP3, DP4+ and MAE ΔΔδThey were analyzed and their absolute configurations were determined to be 1S, 7R, 10S, 11R and 1S, 7R, 10S, 11S according to the calculated ECD spectra.

[0041] Compound 27 is a colorless needle-shaped crystal (methanol). HRESIMS gives a quasi-molecular ion peak of 273.1462 [M+Na] + (calcd for C 15 H 22 O 3 Na,273.1461), combined 1 H, 13 C NMR data confirmed that its molecular formula is C 15 H 22 O 3 , and its unsaturation is calculated to be 5. In the HMBC spectrum, H-4 is related to C-1, C-2, C-5 and C-6, H-6 is related to C-1, C-4, C-8 and C-11, and H 2 -12 is related to C-7, C-11 and C-13, suggesting that compound 27 is a typical guaiacyl sesquiterpenoid compound. 1 H- 1 The spin system observed in the HCOSY spectrum further confirmed the planar structure of the compound. In the NOESY spectrum, H-8, H-6β and CH 3 -15 has a signal, H-6β and CH 3 -15 has a signal, so the relative configuration of compound 27 can be determined. Fortunately, a suitable single crystal of compound 27 was also obtained in methanol solvent, and the configuration of the compound was determined to be 4S, 7S, 8S, 10R.

[0042] Compound 28 is a colorless crystal. Its molecular formula was determined to be C based on HRESIMS and NMR data. 15 H 22 O 2 The 1D NMR data of 28 showed many similarities to (-)-rotundone. The only difference was the addition of a hydroxyl group at C-3 of 28, which was indicated by the chemical shift of C-3 and the shift of H-3 to C-1 and C-5. 3 The structure of 28 was further confirmed by X-ray single crystal analysis.

[0043] Compound 29 is a colorless block crystal (methanol). HRESIMS gives a quasi-molecular ion peak of 273.1466 [M+Na] + (calcd for C 15 H 22 O 3Na, 273.1461), in the HMBC spectrum, H-2 is correlated with C-1, C-3, C-5 and C-10, H-6 with C-1, C-4, C-8 and C-11, H-9 with C-1, C-7, H-14, which confirms that the compound is a guaiacyl sesquiterpene skeleton. 3 -13 and C-7, C-11 and C-12, CH 3 -14 and C-1, C-9 and C-10, CH 3 -15 is associated with C-3, C-4 and C-5, confirming the position of the methyl group. 1 H- 1 H COSY correlation shows that compound 29 has a typical basic skeleton of guaiacine, thus determining the planar structure of the compound. In the NOESY spectrum, due to severe signal overlap, it is impossible to accurately determine the relative configuration through the NOESY spectrum related signals. However, a suitable single crystal of compound 29 was obtained in methanol solvent, thus determining the configuration of the compound to be 4R, 5R, 7R, 10S.

[0044] Compound 30 is a colorless oil (methanol). HRESIMS gives a quasi-molecular ion peak of 255.1353 [M+H] + (calcd for C 15 H 20 O 2 Na, 255.1356), in the HMBC spectrum, H-2 is related to C-1, C-3, C-5 and C-10, H-6 is related to C-1, C-4, C-8 and C-11, and H-9 is related to C-1, C-7, H-14, which further confirms the planar structure of the compound. Compound 31 1 H NMR spectra and 13 The similarity of C NMR spectrum with the signal of compound 30 proves that they have the same planar structure, but different configurations. In the NOESY spectrum of compound 30, it can be observed that H-7 is correlated with H-8α, and H-9 is correlated with H-8β, thus determining the relative configuration of the compound. In the NOESY spectrum of compound 31, it can be observed that H-7 is correlated with H-9, thus determining the relative configuration of the compound. Finally, their absolute configurations were determined by NMR calculation / ECD calculation.

[0045] Compound 33 is a colorless oil (methanol). HRESIMS gives a quasi-molecular ion peak of 247.1697 [M+H] + (calcd for C 16 H 23 O 2 Na,247.1693), combined 1 H, 13C NMR data confirmed that its molecular formula is C 16 H 22 O 3 , compared with compound 30, the difference is that the C-14 position in this structure is methyl, and the H 2 -14 is related to C-1, C-9 and C-10, H-9 is related to C-1, C-7 and H 2 -14 related existence, combined 1 H- 1 The existence of C6-C7-C8-C9 spin system was observed in the H COSY spectrum, which further confirmed the planar structure of the compound. In the NOESY spectrum, H-7 and H-8α and CH 3 -9 is related, CH 3 -9 is correlated with H-8α, thus determining the relative configuration of the compound. The absolute configuration of the compound was determined by comparing the calculated ECD with the measured ECD. Compound 33 has a good agreement with the calculated 7S, 9R configuration, so its absolute configuration was determined to be 7S, 9R.

[0046] Compound 34 is a colorless oil (methanol). HRESIMS gives a quasi-molecular ion peak of 285.1469 [M+Na] + (calcd for C 16 H 22 O 3 Na,285.1461), combined 1 H, 13 C NMR data confirmed that its molecular formula is C 16 H 22 O 3 In the HMBC spectrum, H-2 is related to C-1, C-3, C-5 and C-10, H-6 is related to C-1, C-4, C-8 and C-11, H-9 is related to C-1, C-7 and H-14, and CH 3 The presence of -9 related to C-9 suggests that compound 34 is a typical guaiacyl sesquiterpenoid compound with a methoxy substitution at C-9. 1 H- 1 The presence of a C6-C7-C8-C9 spin system was observed in the H COSY spectrum, further confirming the planar structure of the compound. 1 H NMR spectra and 13 The C NMR spectra were similar to those of compound 34, indicating that they had the same planar structure but different configurations. Their relative configurations were determined based on NOESY correlation, and their absolute configurations were further determined by comparing experimental and calculated ECD data.

[0047] Compound 36 was a colored oil (methanol). HRESIMS gave a quasi-molecular ion peak of 305.1722 [M+Na] + (calcdfor C 16 H 26 O 4 Na,305.1723), combined 1 H, 13 C NMR data confirmed that its molecular formula is C 16 H 26 O 4 , and its unsaturation degree is calculated to be 4. Combined with the HMBC spectrum, H-4 is related to C-1, C-2, C-5 and C-6, H-6 is related to C-1, C-4, C-8 and C-11, and H 2 -12 is related to C-7, C-11 and C-13, and the planar structure of the compound is determined. 1 H NMR spectra and 13 The similarity of C NMR spectra with compound 37 indicated that they had the same planar structure but different configurations. Their relative configurations were determined by NOESY correlation, and finally, their absolute configurations were further determined to be 2R, 4R, 5S, 7S, 8S and 2S, 4R, 5S, 7S, 8S by comparing experimental and calculated ECD data.

[0048] Compound 38 is colorless needle-shaped crystals (methanol). HRESIMS gives a quasi-molecular ion peak of 289.1743 [M+Na] + (calcd for C 16 H 26 O 3 Na,289.1774), combined 1 H, 13 C NMR data confirmed that its molecular formula is C 16 H 26 O 3 , and its unsaturation is calculated to be 4. 1 H NMR spectra and 13 The C NMR spectrum showed signals similar to those of compound 42, giving 4 olefinic carbon signals, 4 methylene carbon signals, 5 methine carbon signals, 2 methyl carbon signals and 1 methoxy carbon signal. In the HMBC spectrum, H-4 correlated with C-1, C-2, C-5 and C-6, H-6 correlated with C-1, C-4, C-8 and C-11, H-7 correlated with C-6, C-9, C-11, C-12 and C-13, OCH 3 The presence of -2 associated with C-2 further confirmed the planar structure. In the NOESY spectrum, H-3β and OCH 3 -2 and CH 3-15, H-5 is related to H-3α and H-7, H-13 is related to H-6β and CH 3 -15 related, H 2 The presence of -12 related to H-8 can determine the relative configuration of compound 38. The absolute configuration of the compound was further verified by comparing the calculated ECD with the measured ECD. The calculated configuration of compound 38 was in good agreement with that of 2R, 4S, 5S, 7R, 8S, so the absolute configuration was determined to be 2R, 4S, 5S, 7R, 8S.

[0049] Compound 39 was a colored oil (methanol). HRESIMS gave a quasi-molecular ion peak of 301.1420 [M+Na] + (calcdfor C 16 H 22 O 4 Na,301.1410), combined 1 H, 13 C NMR data confirmed that its molecular formula is C 16 H 22 O 4 , and its unsaturation is calculated to be 6. In the HMBC spectrum, H-5 is related to C-2, C-3, C-7 and C-10, H-6 is related to C-1, C-4, C-8 and C-11, and H 2 -12 is related to C-7, C-11 and C-13, suggesting that compound 39 is a typical guaiacyl sesquiterpenoid compound. 1 H- 1 The spin system observed in the HCOSY spectrum further confirmed the planar structure of the compound. 1 H NMR spectra and 13 The similarity of C NMR spectra with compound 39 indicated that they had the same planar structure but different configurations. Their relative configurations were determined by NOESY correlation, and finally, their absolute configurations were further determined to be 4S, 5S, 7R, 10R and 4S, 5S, 7R, 10S, respectively, by comparing the experimental and calculated ECD data.

[0050] Compounds 2, 3, 4, 19, 20, 21, 22, 31, and 32 are known compounds, and their corresponding configurations were determined by comparing NMR data from literature and single crystal data.

[0051] A pharmaceutical composition comprises any one of the 40 guaiacane sesquiterpene derivatives in Daphne koreana or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0052] The invention discloses an application of the guaiacane sesquiterpene derivative in Daphne koreana or the pharmaceutical composition in the preparation of an anti-neuroinflammatory drug.

[0053] The anti-neuroinflammatory activity of 40 guaiacyl sesquiterpene derivatives of the present invention was evaluated. The results showed that compounds 3, 8, 35, 36, 39 and 40 had potential inhibitory effects, which were better than the positive control dexamethasone. Therefore, the guaiacyl sesquiterpene derivatives of the present invention have the potential to be clinically used as anti-neuroinflammatory drugs.

[0054] The advantages of the present invention are that the compounds are relatively large in number, most of which are new compounds with novel structures, and are all optically pure compounds with defined stereo configurations. They also have good anti-neuroinflammatory activity and are worth further development. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The molecular network was established by analyzing the fractions of Daphne koreana L. by HPLC-MS / MS.

[0056] Figure 2 Experimental and Calculation of Compounds 2 and 3 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0057] Figure 3 Experimental and Calculation of Compounds 4 and 5 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0058] Figure 4 Experimental and Calculation of Compound 6 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0059] Figure 5 Experimental and Calculation of Compound 11 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0060] Figure 6 Experimental and Calculation of Compound 12 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0061] Figure 7 Experimental and Calculation of Compound 13 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0062] Figure 8 Experimental and Calculation of Compound 14 13 CP3 calculation and MAE of C NMR chemical shiftsΔΔδ parameter;

[0063] Fig. 9 Experimental and Calculation of Compound 15 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0064] Fig.10 Experimental and Calculation of Compound 17 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0065] Fig.11 Experimental and Calculation of Compound 19 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0066] Fig.12 Experimental and Calculation of Compound 20 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0067] Fig.13 Experiments and calculations for compounds 15-26 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0068] Fig.14 Experimental and Calculation of Compounds 30-31 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0069] Fig.15 Experimental and Calculation of Compound 33 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0070] Fig.16 Experimental and Calculation of Compounds 34-35 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0071] Fig.17 Experimental and Calculation of Compounds 39-40 13 CP3 calculation and MAE of C NMR chemical shifts ΔΔδ parameter;

[0072] Fig.18 Calculated ECD of model compounds (M1A-M1D / M2A-M2D) and measured ECD of compounds 21 and 22;

[0073] Fig.19 Protective activity of compound 1-40 against LPS-induced neuroinflammatory injury in BV-2 mouse microglia; all data are expressed as means±SD (three independent experiments); Dex is a positive drug;

[0074] Fig. 20 The synthetic route for compound 1 was synthesized via intermolecular Aldol reaction;

[0075] Fig.21 Structural revisions of compounds 2-5 and Daphnepapytone E;

[0076] Fig. 22 The key NOESY correlations of compounds 7-9, 13-18, 23-28, 30-31, and 33-40;

[0077] Fig.23 Single crystal diffraction patterns of some compounds X;

[0078] Fig.24 ECD results were calculated for compound 1 and compounds 5-15;

[0079] Fig.25 ECD results were calculated for compounds 16-21 and compounds 23-28;

[0080] Fig.26 ECD results were calculated for compounds 29-31 and compounds 33-40. DETAILED DESCRIPTION

[0081] The following examples are intended to help those skilled in the art to better understand the present invention, but are not intended to limit the present invention in any way.

[0082] Example 1

[0083] The preparation of the guaiacane-type sesquiterpene derivatives 1-40 from Daphne koreana is specifically performed as follows:

[0084] (1) Take the whole plant of dried Daphne koreana (48 kg) and extract it with 75% industrial ethanol for 3 times, each time for 2 hours, and combine the extracts and concentrate to obtain an extract. The obtained extract is extracted with ethyl acetate and n-butanol successively. The ethyl acetate (600 g) part and the n-butanol (1.2 kg) part are subjected to vacuum silica gel column chromatography, and gradient elution is performed with dichloromethane-methanol system (100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1), and a total of 4 fractions Fr A to Fr D are collected.

[0085] (2) Fraction Fr.A (200 g) was subjected to polyamide column chromatography with an ethanol-water system for gradient elution (20%, 70%) to collect two fractions, Fr.A1 to Fr.A2.

[0086] (3) Fraction Fr.A1 (90 g) was subjected to HP20 column chromatography with a gradient elution of ethanol and water (20%, 50%, 90%) to collect two fractions, Fr.A1-1 to Fr.A1-2.

[0087] (4) Fractions Fr.A1-1 (35 g) and Fr.A1-2 (45 g) were subjected to ODS column chromatography and eluted with ethanol and water gradient (40%, 50%, 60%, 70%, 80%, 90%). The fractions were collected to obtain five fractions: Fr.1, Fr.2, Fr.3, Fr.4, and Fr.5.

[0088] (5) The above five fractions were subjected to secondary mass spectrometry testing using HPLC-QTOF-MS mass spectrometer. Based on the secondary mass spectrometry database of sesquiterpenes, flavonoids, etc. constructed by GNPS and the applicant's research group in the early stage, the target fractions Fr.3, Fr.4, and Fr.5 were selected for further experiments by analyzing the structural type and distribution of the fraction nodes.

[0089] (6) Fractions Fr.3 (15 g), Fr.4 (11 g) and Fr.5 (8 g) were passed through a silica gel column and eluted with petroleum ether-ethyl acetate (100:1, 50:1, 30:1, 20:1, 10:1, 5:1) gradient. Preparative HPLC was performed and eluted with methanol-water (40:60) to obtain fractions Fr.3.1.1 to Fr.3.1.4, Fr.3.2.1 to Fr.3.2.7, Fr.3.3.1 to Fr.3.3.3, Fr.4.1.1 to Fr.4.1.4, Fr.5.1.1 to Fr.5.1.4. Fr.3.1.1 was subjected to semi-preparative HPLC chromatography and eluted with acetonitrile-water (20:80) to separate and purify compounds 1 (5.0 mg), 2 (3.0 mg), 5 (3.0 mg) and 6 (10.0 mg); Fr.3.1.3 was subjected to semi-preparative HPLC chromatography and eluted with acetonitrile-water (20:80) to separate and purify compounds 3 (2.0 mg), 4 (5.0 mg), 7 (7.0 mg) and 9 (1.5 mg); Fr.3.2.4 was subjected to semi-preparative HPLC chromatography and eluted with acetonitrile-water (20:80) to separate and purify compounds Spectrum, eluted with acetonitrile-water (20:80), separated and purified to obtain compounds 11 (2.5 mg), 14 (3.5 mg), 16 (2.5 mg), 17 (2.5 mg) and 20 (6.7 mg); Fr.3.2.5 was subjected to semi-preparative HPLC chromatography, eluted with acetonitrile-water (20:80), separated and purified to obtain compounds 8 (6.5 mg), 10 (3.0 mg), 12 (4.0 mg), 13 (4.5 mg) and 15 (5.5 mg); Fr.3.3.1 and Fr.3. 3.3 was separated and purified by semi-preparative HPLC chromatography with acetonitrile-water (20:80) to give compounds 18 (3.5 mg), 19 (4.0 mg), 21 (4.5 mg), 22 (2.5 mg), 24 (1.5 mg) and 25 (1.5 mg); Fr.4.1.2 and Fr.4.1.3 were separated and purified by semi-preparative HPLC chromatography with acetonitrile-water (20:80) to give compounds 23 (3.0 mg), 26 (3.0 mg), 27 (1.5 mg), 29 (2.0 mg), 33 (4.5 mg) and 34 (5.0 mg); Fr.5.1.2 and Fr.5.1.3 were separated and purified by semi-preparative HPLC chromatography with acetonitrile-water (20:80) to give compounds 28 (1.5 mg), 30 (2.0 mg), 31 (3.5 mg), 32 (2.0 mg), 35 (5.5 mg), 36 (1.5 mg), 37 (2.5 mg), 38 (3.5 mg), 39 (2.5 mg) and 40 (1.5 mg).

[0090] Example 2

[0091] Anti-neuroinflammatory activity of guaiacane sesquiterpenoids 1-40 from Daphne koreana

[0092] BV2 mouse microglia were cultured in DMEM medium containing 10% FBS and placed in a 37°C, 5% CO 2 Culture in a saturated humidity incubator. Subculture when the cell confluence reaches 80%.

[0093] The anti-neuroinflammatory activity of guaiac sesquiterpenoids 1-40 from Daphne koreana was investigated by NO content detection. BV2 microglia were seeded in 96-well plates at a density of 6×10 4 Each well was filled with 100 μL of culture medium. Three replicate wells were set up and placed at 37°C and 5% CO. 2 The cells were cultured in a saturated humidity incubator. After 12 hours, the drug (10 μM) was added. After 1 hour, 10 μg / ml LPS was added to stimulate BV2 microglia. After 24 hours, the culture supernatant was collected and the nitric oxide content in the supernatant was measured using a nitric oxide assay kit.

[0094] First, Griess Reagent I and Reagent II were restored to room temperature, and 50 μL of culture medium supernatant was added to each well of a 96-well plate. Griess Reagent I and Reagent II were then added in sequence, and the absorbance was measured at 540 nm using an ELISA reader. All experiments were performed in parallel and repeated three times, with blank and positive control groups set up, and analyzed using GraphPad Prism 6 software. The results are shown in the figure. Fig.19 shown.

Claims

1. A guaiacyl sesquiterpene derivative prepared from Daphne koreana, characterized in that: It is any one of the compounds shown below:

2. A method for preparing guaiacyl sesquiterpene derivatives from Daphne koreana, characterized in that: The guaiacyl sesquiterpene derivative is any one of the following compounds: The steps include: (1) Dried Daphne koreana was extracted with 70-80% industrial ethanol for 2-3 times by refluxing, and the extracts were combined and concentrated to obtain an extract. The extract was extracted with ethyl acetate and n-butanol in turn, and the ethyl acetate part and the n-butanol part were subjected to silica gel column chromatography and gradient eluted with a dichloromethane-methanol system. The gradient of dichloromethane-methanol was 100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1, and a total of 4 fractions Fr A to Fr D were collected; (2) Fraction Fr.A was subjected to polyamide column chromatography and eluted with ethanol and water gradient to collect two fractions Fr.A1 to Fr.A2; (3) Fraction Fr.A1 was separated by HP20 column chromatography and eluted with ethanol and water gradient to obtain two fractions Fr.A1-1 to Fr.A1-2; (4) Fractions Fr.A1-1 and Fr.A1-2 were separated by ODS column and eluted with ethanol-water gradient to obtain 5 fractions Fr.1 to Fr.5; (5) The five fractions were subjected to secondary mass spectrometry testing using an HPLC-QTOF-MS mass spectrometer. By analyzing the structural type and distribution of the fraction nodes, three target fractions were finally selected, namely Fr.3, Fr.4 and Fr.5; (6) The three target fractions Fr.3, Fr.4 and Fr.5 were passed through a silica gel column and eluted with a petroleum ether-ethyl acetate gradient, with a petroleum ether-ethyl acetate volume ratio of 100:1, 50:1, 30:1, 20:1, 10:1, and 5:

1. The fractions Fr.3.1.1 to Fr.3.1.4, Fr.3.2.1 to Fr.3.2.7, and Fr.3.3.1 to Fr.3.3 were obtained by preparative HPLC with a methanol-water volume ratio of 40:

60. .3, Fr.4.1.1~Fr.4.1.4, Fr.5.1.1~Fr.5.1.4; Fr.3.1.1 was subjected to semi-preparative HPLC chromatography, eluted with acetonitrile-water volume ratio of 20:80, and separated and purified to obtain compounds 1, 2, 5 and 6; Fr.3.1.3 was subjected to semi-preparative HPLC chromatography, eluted with acetonitrile-water volume ratio of 20:80, and separated and purified to obtain compounds 3, 4, 7 and 9; Fr.3.2.4 was subjected to semi-preparative HPLC chromatography The semi-preparative HPLC chromatography of Fr.3.2.5 was eluted with acetonitrile-water volume ratio of 20:80 to separate and purify compounds 11, 14, 16, 17 and 20; the semi-preparative HPLC chromatography of Fr.3.2.5 was eluted with acetonitrile-water volume ratio of 20:80 to separate and purify compounds 8, 10, 12, 13 and 15; the semi-preparative HPLC chromatography of Fr.3.3.1 and Fr.3.3.3 was eluted with acetonitrile-water volume ratio of 20:80 to separate and purify compounds 18, 19, 21, 22, 24 and 25; Fr.4.1.2 and Fr.4.1.3 were separated and purified by semi-preparative HPLC chromatography with acetonitrile-water volume ratio of 20:80 to obtain compounds 23, 26, 27, 29, 33 and 34; Fr.5.1.2 and Fr.5.1.3 were separated and purified by semi-preparative HPLC chromatography with acetonitrile-water volume ratio of 20:80 to obtain compounds 28, 30, 31, 32, 35, 36, 37, 38, 39 and 40.

3. The method for preparing the guaiacane-type sesquiterpene derivatives from Daphne koreana according to claim 2, characterized in that: The concentration of ethanol in the ethanol water in step (2) is 20% and 70%.

4. The method for preparing the guaiacane-type sesquiterpene derivatives from Daphne koreana according to claim 2, characterized in that: The concentration of ethanol in the ethanol water in step (3) is: 20%, 50%, 90%.

5. The method for preparing the guaiacane-type sesquiterpene derivatives from Daphne koreana according to claim 2, characterized in that: The concentration of ethanol in the ethanol water in step (4) is: 40%, 50%, 60%, 70%, 80%, 90%.

6. A pharmaceutical composition, characterized in that It comprises any one of the guaiacane-type sesquiterpene derivatives prepared from the Daphne koreana of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

7. Use of the guaiacyl sesquiterpene derivative prepared from Daphne koreana of claim 1 in the preparation of anti-neuroinflammatory drugs.

8. Use of the pharmaceutical composition according to claim 6 in the preparation of anti-neuroinflammatory drugs.

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