Eucalyptol type sesquiterpenoids in bighead atractylodes rhizome as well as extraction method and application of eudesmol type sesquiterpenoids

Abstract

The invention discloses cineole type sesquiterpenoids in bighead atractylodes rhizome as well as an extraction method and application thereof, belongs to the field of traditional Chinese medicine extraction, and particularly relates to cineole type sesquiterpenoids which are separated from the bighead atractylodes rhizome and are shown in general formulas (I)-(V) and an extraction method of the cineole type sesquiterpenoids. The invention also discloses an application of the compound or an isomer of the compound or a pharmaceutically acceptable salt of the compound or a pharmaceutical composition containing the compound in preparation of anti-inflammatory drugs. The invention further enriches the structural diversity of active substances in bighead atractylodes rhizome, provides an active lead compound for new drug development, and also provides a theoretical basis for deep research and development of the traditional Chinese medicinal material.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of traditional Chinese medicine extraction, and particularly relates to a eudesmane-type sesquiterpene compound in Atractylodes macrocephala Koidz and an extraction method thereof and application of the eudesmane-type sesquiterpene compound in preparation of anti-inflammatory drugs. BACKGROUND

[0002] The chemical components in Atractylodes macrocephala Koidz mainly include sesquiterpenes, polyacetylenes and polysaccharides (Chen T, Zheng X, Ouyang L, et al. Six polyacetylenes from Atractylodes macrocephala Koidz and their anti-colon cancer activity [J], Fitoterapia, 2023, 167, 105490.), and it is found through experiments that Atractylodes macrocephala Koidz has anti-inflammatory activity (Yao CM, Yang XW. Bioactivity-guided isolation of polyacetylenes with inhibitory activity against NO production in LPS-activated RAW264.7 macrophages from the rhizomes of Atractylodes macrocephala [J], Journal of Ethnopharmacology, 2014, 151(2), 791-799.), gastrointestinal protective effect (Zhang H, Lin C, Yin L, et al. Bioactive constituents from the rhizomes of Atractylodes macrocephala [J], Fitoterapia, 2023, 165, 105431.). In order to further explore the eudesmane-type sesquiterpene chemical components in Atractylodes macrocephala Koidz and the pharmacological activity thereof, the dried rhizomes of Atractylodes macrocephala Koidz are subjected to systematic component research, and a new eudesmane-type sesquiterpene compound is extracted, the structure of the compound is confirmed by means of nuclear magnetic resonance, infrared and mass spectrometry, and the inhibitory effect of the extracted compound on NO production in LPS-induced RAW264.7 cells is detected. SUMMARY

[0003] The primary object of the present application is to provide a eudesmane-type sesquiterpene compound or an isomer of the compound or a pharmaceutically acceptable salt of the compound.

[0004] The second object of the present application is to provide an extraction method of a eudesmane-type sesquiterpene compound.

[0005] A third object of the present application is to provide a pharmaceutical composition containing the eudesmane-type sesquiterpenoid compound.

[0006] A fourth object of the present application is to provide an application of the eudesmane-type sesquiterpenoid compound or an isomer of the compound or a pharmaceutically acceptable salt of the compound or a pharmaceutical composition containing the compound in the preparation of an anti-inflammatory drug.

[0007] The technical solution of the present application comprises the following: The eudesmane-type sesquiterpenoid compound or an isomer of the compound or a pharmaceutically acceptable salt of the compound as shown in general formula (I)~(V).

[0008]

[0009] wherein: R1, R2, R3, R4 are each independently H or hydroxyl; the parent nucleus skeleton is at Δ 7（11） Optionally has a carbon-carbon double bond, when the double bond is not present, the carbon atom at this position is a saturated carbon atom, respectively connected with the corresponding hydrogen atom or substituent.

[0010]

[0011] wherein: R1, R3 are each independently H or hydroxyl; R2 is H, hydroxyl or acetoxy; the parent nucleus skeleton is at Δ 7（8） Optionally has a carbon-carbon double bond, when the double bond is not present, the carbon atom at this position is a saturated carbon atom, respectively connected with the corresponding hydrogen atom or substituent.

[0012]

[0013] wherein: R1, R4 are each independently H or acetoxy; R2 is hydroxyl, or when Δ 7（11） forms a carbon-carbon double bond, R2 is not present; R3 is H or carbonyl; the parent nucleus skeleton is at Δ 7（11） or Δ 11 Optionally has a carbon-carbon double bond, when the carbon-carbon double bond is present, the substituent R2 is not present, when the carbon-carbon double bond is not present, R2 is hydroxyl.

[0014]

[0015] wherein: R1 is H or hydroxyl.

[0016]

[0017] wherein: R1 is H or hydroxyl.

[0018] Further, the eudesmane-type sesquiterpenes are any one of the compounds shown in the following structural formulae 1-12 or any one of isomers or pharmaceutically acceptable salts of the compounds.

[0019] The pharmaceutically acceptable salt in the present application includes sodium salt, potassium salt, ammonia salt, hydrochloride and sulfate formed by the eudesmane-type sesquiterpenes and inorganic acid / base or organic acid / base.

[0020] The isomer in the present application includes optical isomer, cis-trans isomer, racemate and mixture of the eudesmane-type sesquiterpenes.

[0021] The present application also provides an extraction method of eudesmane-type sesquiterpenes, which comprises the following steps: (1) taking dry rhizome of Atractylodes macrocephala as raw material, adding 8-15 mass times of 70%-95% volume concentration ethanol aqueous solution, refluxing and extracting 2-5 times, each time for 2-4 hours, combining to obtain an extract, recovering solvent under reduced pressure, and concentrating to obtain total extract; (2) dispersing the total extract into 1.5-3 mass times of water, extracting with equal mass times of ethyl acetate, recovering solvent, and obtaining ethyl acetate extraction concentrated solution and water phase; (3) subjecting the ethyl acetate extraction concentrated solution to silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate in a volume ratio of 100:0-0:1 as eluent, collecting fraction E2 in a volume ratio of 80:1 and fraction E4 in a volume ratio of 10:1; (4) concentrating fraction E2, subjecting the concentrated solution to silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate in a volume ratio of 100:1-0:1 as eluent, and further purifying to obtain eudesmane-type sesquiterpenes 7 and 8; concentrating fraction E4, subjecting the concentrated solution to silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate in a volume ratio of 100:1-0:1 as eluent, and further purifying to obtain eudesmane-type sesquiterpenes 1-6 and 9-12.

[0022] In the above extraction method, the term "mass times" refers to volume-mass ratio of solvent to material, in L / kg; for example, "8-15 mass times" specifically refers to volume-mass ratio of added ethanol aqueous solution to raw material, in L / kg, as (8-15):1; "1.5-3 mass times" refers to volume-mass ratio of added water and ethyl acetate to total extract, in L / kg, as (1.5-3):1. The similar "mass times" expressions appearing elsewhere in the present application have the same meaning as defined above, i.e. corresponding volume-mass ratio of solvent to material, in L / kg.

[0023] In the above extraction method, the specific separation and purification process of flow fraction E2 and E4 in step (4) is as follows: The flow fraction E2 was concentrated and separated by silica gel column chromatography, gradient elution was performed with petroleum ether-ethyl acetate (100:1 to 0:1, by volume ratio) as eluent, the flow fraction with petroleum ether-ethyl acetate (80:1, by volume ratio) was collected and recorded as E22; the flow fraction with petroleum ether-ethyl acetate (15:1, by volume ratio) was collected and recorded as E25.

[0024] The flow fraction E22 was concentrated and separated by ODS column chromatography, gradient elution was performed with methanol-water (60:40 to 100:0, by volume ratio) as eluent, the flow fraction with methanol-water (80:20, by volume ratio) was collected and recorded as E221; the flow fraction E25 was concentrated and separated by ODS column chromatography, gradient elution was performed with methanol-water (60:40 to 100:0, by volume ratio) as eluent, the flow fraction with methanol-water (70:30, by volume ratio) was collected and recorded as E252.

[0025] The flow fraction E221 was concentrated and purified by semi-preparative HPLC chromatography with methanol-water (60:40 to 80:20, by volume ratio) as mobile phase to obtain compound 7; the flow fraction E252 was concentrated and purified by semi-preparative HPLC chromatography with methanol-water (70:30 to 90:10, by volume ratio) as mobile phase to obtain compound 8.

[0026] The flow fraction E4 was concentrated and separated by silica gel column chromatography, gradient elution was performed with petroleum ether-ethyl acetate (100:1 to 0:1, by volume ratio) as eluent, the flow fraction with petroleum ether-ethyl acetate (50:1, by volume ratio) was collected and recorded as E42; the flow fraction with petroleum ether-ethyl acetate (30:1, by volume ratio) was collected and recorded as E43; the flow fraction with petroleum ether-ethyl acetate (20:1, by volume ratio) was collected and recorded as E45.

[0027] The flow fraction E42 was concentrated and separated by ODS column chromatography, gradient elution was performed with methanol-water (50:50 to 100:0, by volume ratio) as eluent, the flow fraction with methanol-water (70:30, by volume ratio) was collected and recorded as E423; the flow fraction E43 was concentrated and separated by MCI column chromatography, gradient elution was performed with methanol-water (80:20 to 100:0, by volume ratio) as eluent, the flow fraction with methanol-water (80:20, by volume ratio) was collected and recorded as E431; the flow fraction E45 was concentrated and separated by MCI column chromatography, gradient elution was performed with methanol-water (80:20 to 100:0, by volume ratio) as eluent, the flow fraction with methanol-water (80:20, by volume ratio) was collected and recorded as E451.

[0028] The stream E423 was concentrated and purified by semi-preparative HPLC chromatography with methanol-water (70:30 to 90:10 by volume ratio) as the mobile phase to obtain E4232, which was further purified by semi-preparative HPLC chromatography with acetonitrile-water (50:50 to 70:30 by volume ratio) as the mobile phase to obtain compound 4.

[0029] The stream E431 was separated by ODS column chromatography with methanol-water (30:70 to 100:0 by volume ratio) as the eluent for gradient elution, and the stream with a methanol-water volume ratio of 70:30 was collected and recorded as E4314, and the stream with a methanol-water volume ratio of 80:20 was collected and recorded as E4315; the stream E4314 was purified by semi-preparative HPLC chromatography with methanol-water (50:50 to 70:30 by volume ratio) as the mobile phase to obtain E43144; the stream E4315 was purified by semi-preparative HPLC chromatography with acetonitrile-water (20:80 to 50:50 by volume ratio) as the mobile phase to obtain compound 1; the stream E4314 was purified by semi-preparative HPLC chromatography with methanol-water (60:40 to 80:20 by volume ratio) as the mobile phase to obtain E43153; the stream E43153 was purified by semi-preparative HPLC chromatography with methanol-water (40:60 to 60:40 by volume ratio) as the mobile phase to obtain E431533 and E431534; the stream E431533 was further purified by semi-preparative HPLC chromatography with acetonitrile-water (30:70 to 50:50 by volume ratio) as the mobile phase to obtain compound 2, compound 3, compound 9 and compound 11; the stream E431534 was further purified by semi-preparative HPLC chromatography with acetonitrile-water (30:70 to 50:50 by volume ratio) as the mobile phase to obtain compound 10 and compound 12.

[0030] The stream E451 was concentrated and separated by ODS column chromatography with methanol-water (30:70 to 100:0 by volume ratio) as the eluent for gradient elution, and the stream with a methanol-water volume ratio of 50:50 was collected and recorded as E4513.

[0031] The stream E4513 was concentrated and purified by semi-preparative HPLC chromatography with methanol-water (60:40 to 80:20 by volume ratio) as the mobile phase to obtain compound 5 and compound 6.

[0032] The eudesmane-type sesquiterpene compound according to the present application is from a dry rhizome part extract of Atractylodes macrocephala Koidz.

[0033] The present application also provides an application of the dry rhizome part extract of Atractylodes macrocephala Koidz. in preparing anti-inflammatory drugs.

[0034] A pharmaceutical composition comprising the eudesmane sesquiterpenoid compound or one or more of isomers or pharmaceutically acceptable salts of the compound, and further comprising one or more of pharmaceutically acceptable carriers, excipients, diluents, or combinations thereof. The pharmaceutical composition is administered orally or by injection, and the dosage forms include tablets, capsules, powders, syrups, and injections.

[0035] The present application also provides the use of the eudesmane sesquiterpenoid compound or one or more of isomers or pharmaceutically acceptable salts of the compound, or the pharmaceutical composition comprising the compound, in the preparation of an anti-inflammatory drug.

[0036] Advantages of the present application: The eudesmane sesquiterpenoid compound or one or more of isomers or pharmaceutically acceptable salts of the compound, or the pharmaceutical composition comprising the compound, of the present application has an inhibitory effect on the production of NO in LPS-induced RAW264.7 cells, and is used in the preparation of an anti-inflammatory drug. The method of the present application further enriches the structural diversity of active substances in Atractylodes lancea, and lays a foundation for subsequent biological activity tests of monomeric compounds, provides active lead compounds for new drug development, and provides a theoretical basis for the in-depth research and development of Atractylodes lancea dried rhizome. DETAILED DESCRIPTION

[0037] The technical solutions of the present application will be further described below in combination with specific examples. The raw materials and reagents used in the present application can be obtained through commercial channels unless otherwise specified.

[0038] Example 1

[0039] The extraction method of the eudesmane sesquiterpenoid compound in Atractylodes lancea includes the following steps: (1) Take 20 kg of Atractylodes lancea dried rhizome as raw material, add 8 times the mass of 90% volume concentration of ethanol aqueous solution (160 L) to the raw material, reflux extract 3 times, each time for 3 hours, combine to obtain the extract, recover the solvent under reduced pressure, and concentrate to obtain the total extract (3.2 kg); (2) Disperse the total extract obtained into about 1.5 times the mass of water (5 L), extract with an equal mass of ethyl acetate, concentrate the extract to recover the solvent, and obtain the ethyl acetate extraction concentrate (390 g) and the water phase; (3) The ethyl acetate extraction concentrate is separated by silica gel column chromatography, and eluted with petroleum ether-ethyl acetate with a volume ratio of 100:0, 80:1, 50:1, 10:1, 4:1 and 0:1 as eluent gradient, and collect the fraction E2 with a volume ratio of 80:1 and the fraction E4 with a volume ratio of 10:1; (4) The concentrated liquid of stream E2 was separated by silica gel column chromatography, and compounds 7 and 8 were further purified. The concentrated liquid of stream E4 was separated by silica gel column chromatography, and compounds 1-6 and 9-12 were further purified.

[0040] The specific separation and purification process of streams E2 and E4 is as follows: The concentrated liquid of stream E2 was separated by silica gel column chromatography, and compounds 7 and 8 were further purified. The concentrated liquid of stream E4 was separated by silica gel column chromatography, and compounds 1-6 and 9-12 were further purified.

[0041] The concentrated liquid of stream E2 was separated by silica gel column chromatography, and compounds 7 and 8 were further purified. The concentrated liquid of stream E4 was separated by silica gel column chromatography, and compounds 1-6 and 9-12 were further purified.

[0042] The concentrated liquid of stream E2 was separated by silica gel column chromatography, and compounds 7 and 8 were further purified. The concentrated liquid of stream E4 was separated by silica gel column chromatography, and compounds 1-6 and 9-12 were further purified.

[0043] The concentrated liquid of stream E2 was separated by silica gel column chromatography, and compounds 7 and 8 were further purified. The concentrated liquid of stream E4 was separated by silica gel column chromatography, and compounds 1-6 and 9-12 were further purified.

[0044] Stream E42 was concentrated and separated by ODS column chromatography, eluted with methanol-water (50:50 to 100:0, by volume) as eluent gradient, the stream of methanol-water (70:30, by volume) was collected and recorded as E423; Stream E43 was concentrated and separated by MCI column chromatography, eluted with methanol-water (80:20 to 100:0, by volume) as eluent gradient, the stream of methanol-water (80:20, by volume) was collected and recorded as E431; Stream E45 was concentrated and separated by MCI column chromatography, eluted with methanol-water (80:20 to 100:0, by volume) as eluent gradient, the stream of methanol-water (80:20, by volume) was collected and recorded as E451.

[0045] Stream E423 was concentrated and purified by semi-preparative HPLC chromatography with methanol-water (80:20, by volume) as mobile phase, to obtain E4232, which was further purified by semi-preparative HPLC chromatography with acetonitrile-water (60:40, by volume) as mobile phase, to obtain 12.6 mg of compound 4.

[0046] Stream E431 was separated by ODS column chromatography, eluted with methanol-water (30:70 to 100:0, by volume) as eluent gradient, the stream of methanol-water (70:30, by volume) was collected and recorded as E4314, the stream of methanol-water (80:20, by volume) was collected and recorded as E4315; Stream E4314 was purified by semi-preparative HPLC chromatography with methanol-water (60:40, by volume) as mobile phase, to obtain E43144; which was further purified by semi-preparative HPLC chromatography with acetonitrile-water (40:60, by volume) as mobile phase, to obtain 4.2 mg of compound 1. Stream E4315 was purified by semi-preparative HPLC chromatography with methanol-water (70:30, by volume) as mobile phase, to obtain E43153; E43153 was purified by semi-preparative HPLC chromatography with methanol-water (50:50, by volume) as mobile phase, to obtain E431533 and E431534; E431533 was further purified by semi-preparative HPLC chromatography with acetonitrile-water (40:60, by volume) as mobile phase, to obtain 18.6 mg of compound 2, 11.3 mg of compound 3, 4.9 mg of compound 9, 4.0 mg of compound 11; E431534 was further purified by semi-preparative HPLC chromatography with acetonitrile-water (37:63, by volume) as mobile phase, to obtain 14.1 mg of compound 10, 7.2 mg of compound 12.

[0047] Stream E451 was concentrated and separated by ODS column chromatography, eluted with methanol-water (30:70 to 100:0, by volume) as eluent gradient, the stream of methanol-water (50:50, by volume) was collected and recorded as E4513.

[0048] Stream E4513 was concentrated and purified by semi-preparative HPLC chromatography with methanol-water (70:30, v / v) as mobile phase to give 7.2 mg of compound 5 and 8.3 mg of compound 6.

[0049] The extracted eudesmane-type sesquiterpenes 1-12 were identified by structure, and the specific physical and chemical data are as follows: Compound 1: white amorphous powder (CH3OH). HR-ESI-MS 271.1309 [M + Na] m / z 271.1309 [M + Na] + (calcd. for C 15 H 20 O3Na + , 271.1310), molecular formula C 15 H 20 O3. : 243.0 ( c = 0.10, CH3OH). 1 H-NMR (600MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are shown in Table 1.

[0050] Compound 2: colorless needle-like crystal (CH3OH). HR-ESI-MS 271.1312 m / z [M + Na] + (calcd. for C 15 H 20 O3Na + , 271.1310), molecular formula C 15 H 20 O3. : −15.9 ( c = 0.05, CH3OH). 1 H-NMR (600MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are shown in Table 2.

[0051] Compound 3: colorless needle-like crystal (CH3OH). HR-ESI-MS 271.1312 m / z [M + Na] + (calcd. for C 15 H 20 O3Na + , 271.1310), molecular formula C 15 H 20 O3. : 201.0 ( c = 0.10, CH3OH). 1 H-NMR (600 MHz, CD3OD) and 13 C-NMR (150 MHz, CD3OD) data are listed in Table 5.

[0052] Compound 4: colorless oil (CH3OH). HR-ESI-MS m / z 301.1782 [M + Na] + (calcd. for C 17 H 26 O3Na + , 301.1780), molecular formula C 17 H 26 O3. : -53.3 ( c = 0.16, CH3OH). 1 H-NMR (600 MHz, CD3OD) and 13 C-NMR (150 MHz, CD3OD) data are listed in Table 5.

[0053] Compound 5: colorless oil (CH3OH). HR-ESI-MS m / z 259.1671 [M + Na] + (calcd. for C 15 H 24 O2Na + , 259.1674), molecular formula C 15 H 24 O2. : 35.9 ( c = 0.10, CH3OH). 1 H-NMR (600 MHz, CD3OD) and 13 C-NMR (150 MHz, CD3OD) data are listed in Table 5.

[0054] Compound 6: colorless oil (CH3OH). HR-ESI-MS m / z 277.1780 [M + Na] + (calcd. for C 15 H 26 O3Na + , 277.1781), molecular formula C 15 H 26 O3. : 75.0 ( c= 0.16, CH3OH). 1 H-NMR (600 MHz, CD3OD) and 13 C-NMR (150 MHz, CD3OD) data are listed in Table 6.

[0055] Compound 7: colorless oil (CH3OH). HR-ESI-MS m / z 257.1513 [M + Na] + (calcd. for C 15 H 22 O2Na + , 257.1518), molecular formula C 15 H 22 O2. : -8.7 ( c = 0.22, CH3OH). 1 H-NMR (600 MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are listed in Table 7.

[0056] Compound 8: colorless oil (CH3OH). HR-ESI-MS m / z 359.1833 [M + K] + (calcd. for C 19 H 28 O4K + , 359.1831), molecular formula C 19 H 28 O4. : 87.2 ( c = 0.10, CH3OH). 1 H-NMR (600 MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are listed in Table 8.

[0057] Compound 9: colorless irregular granules (CH3OH). HR-ESI-MS m / z 289.1419 [M + Na] + (calcd. for C 17 H 26 O3Na + , 289.1416), molecular formula C 17 H 26 O3. : 63.0 ( c = 0.12, CH3OH). 1H-NMR (600 MHz, CD3OD) and 13 C-NMR (150 MHz, CD3OD) data are listed in Table 9.

[0058] Compound 10: colorless oil (CH3OH). HR-ESI-MS m / z 243.1361 [M + Na] + (calcd. for C 14 H 20 O2Na + , 243.1361), molecular formula C 14 H 20 O2. : -101.9 ( c = 0.10, CH3OH). 1 H-NMR (600MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are listed in Table 10.

[0059] Compound 11: colorless prismatic crystals (CH3OH). HR-ESI-MS m / z 233.1155 [M + Na] + (calcd. for C 12 H 18 O3Na + , 233.1154), molecular formula C 12 H 18 O3. : 50.1 ( c = 0.10, CH3OH). 1 H-NMR (600MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are listed in Table 11.

[0060] Compound 12: colorless prismatic crystals (CH3OH). HR-ESI-MS m / z 233.1154 [M + Na] + (calcd. for C 12 H 18 O3Na + , 233.1154), molecular formula C 12 H 18 O3. : 57.8 ( c = 0.10, CH3OH). 1 H-NMR (600MHz, CDCl3) and13 C-NMR (150 MHz, CDCl3) data are shown in Table 12.

[0061] Table 1 Carbon and hydrogen spectral data of compound 1

[0062] Table 2 Carbon and hydrogen spectral data of compound 2

[0063] Table 3 Carbon and hydrogen spectral data of compound 3

[0064] Table 4 Carbon and hydrogen spectral data of compound 4

[0065] Table 5 Carbon and hydrogen spectral data of compound 5

[0066] Table 6 Carbon and hydrogen spectral data of compound 6

[0067] Table 7 Carbon and hydrogen spectral data of compound 7

[0068] Table 8 Carbon and hydrogen spectral data of compound 8

[0069] Table 9 Carbon and hydrogen spectral data of compound 9

[0070] Table 10 Carbon and hydrogen spectral data of compound 10

[0071] Table 11 Carbon and hydrogen spectral data of compound 11

[0072] Table 12 Carbon and hydrogen spectral data of compound 12

[0073] The structures of the above compounds were identified by physicochemical data and modern spectroscopic methods (HRESIMS and NMR), combined with relevant data in the published literature, and it was determined that compounds 1-12 are all new compounds that have not been reported in the literature, as shown below:

[0074] Example 2

[0075] The extraction method of eudesmane-type sesquiterpenes in Atractylodes macrocephala Koidz. comprises the following steps: (1) taking 16 kg of dried rhizome of Atractylodes macrocephala Koidz. as raw material, adding 8.7 times of volume concentration of 90% ethanol aqueous solution (140 L) to the raw material, refluxing and extracting for 3 times, each time for 3 hours, combining to obtain an extract, recovering the solvent under reduced pressure, and concentrating to obtain total extract (2.6 kg); (2) dispersing the obtained total extract into about 1.5 times of water (4 L), extracting with equal mass times of volume of ethyl acetate, concentrating the extract to recover the solvent, and obtaining ethyl acetate extraction concentrated liquid (300 g) and water phase; (3) gradient eluting the ethyl acetate extraction concentrated liquid with petroleum ether-ethyl acetate with a volume ratio of 100:0, 80:1, 50:1, 10:1, 4:1 and 0:1 as eluent, collecting the fraction E2 with a volume ratio of 80:1 and the fraction E4 with a volume ratio of 10:1; (4) concentrating the fraction E2 to obtain 65.0 g of concentrated liquid, further purifying the concentrated liquid by silica gel column chromatography to obtain 27.1 mg of compound 7 and 3.6 mg of compound 8. Concentrating the fraction E4 to obtain 62.0 g of concentrated liquid, further purifying the concentrated liquid by silica gel column chromatography to obtain 3.5 mg of compound 1, 16.0 mg of compound 2, 9.7 mg of compound 3, 10.5 mg of compound 4, 5.8 mg of compound 5, 6.2 mg of compound 6, 3.8 mg of compound 9, 11 mg of compound 10, 3 mg of compound 11 and 5.7 mg of compound 12. The specific separation and purification process is the same as that of Example 1.

[0076] Example 3

[0077] The extraction method of eudesmane-type sesquiterpenes in Atractylodes macrocephala Koidz. comprises the following steps: (1) taking 16 kg of dried rhizome of Atractylodes macrocephala Koidz. as raw material, adding 8.7 times of volume concentration of 90% ethanol aqueous solution (140 L) to the raw material, refluxing and extracting for 3 times, each time for 3 hours, combining to obtain an extract, recovering the solvent under reduced pressure, and concentrating to obtain total extract (2.6 kg); (2) dispersing the obtained total extract into about 1.5 times of water (4 L), extracting with equal mass times of volume of ethyl acetate, concentrating the extract to recover the solvent, and obtaining ethyl acetate extraction concentrated liquid (300 g) and water phase; (3) The ethyl acetate extract concentrate was separated by silica gel column chromatography, using petroleum ether-ethyl acetate with volume ratios of 100:0, 80:1, 50:1, 10:1, 4:1 and 0:1 as eluents for gradient elution, and the fraction E2 with a volume ratio of 80:1 and the fraction E4 with a volume ratio of 10:1 were collected. (4) After concentration of fraction E2, 40.0 g of concentrate was obtained. The concentrate was further purified by silica gel column chromatography to obtain 16.3 mg of compound 7 and 2.0 mg of compound 8. After concentration of fraction E4, 36.0 g of concentrate was obtained. The concentrate was further purified by silica gel column chromatography to obtain 2.0 mg of compound 1, 9.2 mg of compound 2, 5.6 mg of compound 3, 6.0 mg of compound 4, 3.2 mg of compound 5, 3.6 mg of compound 6, 2.5 mg of compound 9, 7.0 mg of compound 10, 1.8 mg of compound 11, and 3.0 mg of compound 12. The specific separation and purification process is the same as in Example 1.

[0078] Test Example 1

[0079] The eucalyptane-type sesquiterpenoid compounds 1-12 extracted above were tested for their inhibitory effect on LPS-induced NO production in RAW264.7 cells, as detailed below: (1) Cell culture Mouse mononuclear macrophages RAW264.7 were cultured in a solution containing 10% fetal bovine serum and 100 mg / L of sodium chloride. μ The cells were incubated and grown in DMEM medium containing 100 U / mL streptomycin and 100 U / mL penicillin sodium at 37°C and 5% CO2.

[0080] (2) The effect of compound 1-12 on inhibiting the release of nitric oxide (NO) from mouse RAW264.7 macrophages induced by lipopolysaccharide was detected by the Griess method.

[0081] 1. Principle: Excessive LPS induces the activation and expression of nitric oxide synthase (NOS) in macrophages, leading to the production of NO. NO then reacts with oxygen free radicals to rapidly form NO2. - NO2 formed - It can be quantitatively detected by Griess Reagent under acidic conditions. First, NO2 - It undergoes a diazotization reaction with sulfanilamide, followed by reaction with hydrochloric acid. N -1-Naphthyl-ethylenediamine ( NThe coupling reaction of NO with 1-naphthylethylene-diamine dihydrochloride generates a purple-red azo compound. The absorbance value of the azo compound at 540 nm is measured by a microplate reader, and the NO content in the sample is calculated by combining the standard curve.

[0082] 2. Method: Take the logarithmic phase growth of mouse monocyte macrophage RAW264.7, adjust the cell concentration to 3.5 × 10 4 cells / well, and inoculate in a 96-well plate. 100 μL of cell suspension is added to each well. In the experiment, a control group (RAW264.7 cells, DMSO), a model group (RAW264.7 cells, DMSO, 0.5 μg / mL of LPS), and a test drug group (RAW264.7 cells, each test compound (20 μM), 0.5 μg / mL of LPS) are set up at the same time. Incubate in a 5% CO2, 37°C constant temperature incubator for 24 h, then aspirate 40 μL of cell supernatant and place it in an enzyme-labeled plate. Add an equal volume of Griess reagent and measure the nitrite accumulation in the culture medium at 540 nm using a microplate reader. The results are shown in Table 13. Except for compounds 3, 6, and 10, the rest of the compounds have certain inhibitory activity on NO release.

[0083] Table 13 Effect of compounds 1-12 on the survival rate of LPS-induced RAW264.7 cells

[0084] The above examples are only used to help understand the method of the present application and its central idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the present application.

Claims

1. Eucalyptane-type sesquiterpenoids as shown in general formulas (I) to (V), or their pharmaceutically acceptable salts: in: R1, R2, R3, and R4 are each independently H or hydroxyl groups; the parent nucleus skeleton is in Δ 7（11） Optionally, it has a carbon-carbon double bond, and when the double bond is absent, the carbon atom at that position is a saturated carbon atom, which is connected to the corresponding hydrogen atom or substituent. Wherein: R1 and R3 are each independently H or hydroxyl; R2 is H, hydroxyl, or acetoxy; the parent nucleus skeleton is in Δ 7（8） Optionally, it has a carbon-carbon double bond, and when the double bond is absent, the carbon atom at that position is a saturated carbon atom, which is connected to the corresponding hydrogen atom or substituent. Wherein: R1 and R4 are each independently H or acetoxy; R2 is a hydroxyl group, or when Δ 7（11） When a carbon-carbon double bond is formed, R2 is absent; R3 is H or a carbonyl group; the parent skeleton is in Δ 7（11） or Δ 11 Optionally has a carbon-carbon double bond, where the carbon-carbon double bond is present and the substituent R2 is absent, and where the carbon-carbon double bond is absent and R2 is a hydroxyl group; Wherein: R1 is H or a hydroxyl group; Wherein: R1 is an α or β oriented hydroxyl group.

2. The eucalyptane-type sesquiterpene compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, It is any one of the following compounds or a pharmaceutically acceptable salt thereof; 。 3. A method for extracting eucalyptane-type sesquiterpenoids as described in claim 2, characterized in that, Includes the following steps: (1) Using the dried rhizome of Atractylodes macrocephala as raw material, add 8 to 15 times the mass of the raw material in a 70% to 95% ethanol aqueous solution, reflux extract 2 to 5 times, each extraction for 2 to 4 hours, combine the extracts, recover the solvent under reduced pressure, and concentrate to obtain the total extract. (2) Disperse the total extract into 1.5 to 3 times the mass of water, extract with an equal mass of ethyl acetate, recover the solvent, and obtain ethyl acetate extract concentrate and aqueous phase; (3) The ethyl acetate extract concentrate was separated by silica gel column chromatography, using petroleum ether-ethyl acetate with a volume ratio of 100:0~0:1 as the eluent gradient, and the fraction E2 with a volume ratio of 80:1 and the fraction E4 with a volume ratio of 10:1 were collected. (4) Concentrated fraction E2: The concentrate was separated by silica gel column chromatography and eluted with a gradient of petroleum ether-ethyl acetate at a volume ratio of 100:1 to 0:1 to obtain eucalyptane-type sesquiterpenoids 7 and 8. Concentrated fraction E4: The concentrate was separated by silica gel column chromatography and eluted with a gradient of petroleum ether-ethyl acetate at a volume ratio of 100:1 to 0:1 to obtain eucalyptane-type sesquiterpenoids 1-6 and 9-12.

4. The method for extracting eucalyptane-type sesquiterpenoids according to claim 3, characterized in that, The specific separation and purification process of fractions E2 and E4 in step (4) is as follows: After concentration, fraction E2 was separated by silica gel column chromatography, using a gradient elution of petroleum ether-ethyl acetate at a volume ratio of 100:1 to 0:

1. The fraction with a petroleum ether-ethyl acetate volume ratio of 80:1 was collected and designated as E22; the fraction with a petroleum ether-ethyl acetate volume ratio of 15:1 was collected and designated as E25. After concentration, fraction E22 was separated by ODS column chromatography, using a gradient elution of methanol-water at a volume ratio of 60:40 to 100:

0. The fraction with a methanol-water volume ratio of 80:20 was collected and designated as E221. After concentration, fraction E25 was separated by ODS column chromatography, using a gradient elution of methanol-water at a volume ratio of 60:40 to 100:

0. The fraction with a methanol-water volume ratio of 70:30 was collected and designated as E252. Fraction E221 was concentrated and purified by semi-preparative HPLC with a mobile phase of methanol-water at a volume ratio of 60:40 to 80:20 to obtain compound 7; Fraction E252 was concentrated and purified by semi-preparative HPLC with a mobile phase of methanol-water at a volume ratio of 70:30 to 90:10 to obtain compound 8. After concentration, fraction E4 was separated by silica gel column chromatography using a gradient elution of petroleum ether-ethyl acetate at a volume ratio of 100:1 to 0:

1. The fraction with a petroleum ether-ethyl acetate volume ratio of 50:1 was collected and designated as E42; the fraction with a petroleum ether-ethyl acetate volume ratio of 30:1 was collected and designated as E43; and the fraction with a petroleum ether-ethyl acetate volume ratio of 20:1 was collected and designated as E45. Fraction E42 was concentrated and separated by ODS column chromatography, using a methanol-water gradient elution with a volume ratio of 50:50 to 100:

0. The fraction with a methanol-water volume ratio of 70:30 was collected and designated as E423. Fraction E43 was concentrated and separated by MCI column chromatography, using a methanol-water gradient elution with a volume ratio of 80:20 to 100:

0. The fraction with a methanol-water volume ratio of 80:20 was collected and designated as E431. Fraction E45 was concentrated and separated by MCI column chromatography, using a methanol-water gradient elution with a volume ratio of 80:20 to 100:

0. The fraction with a methanol-water volume ratio of 80:20 was collected and designated as E451. After concentration, fraction E423 was purified by semi-preparative HPLC with methanol-water (v / v) at a mobile phase of 70:30 to 90:10 to obtain E4232. It was further purified by semi-preparative HPLC with acetonitrile-water (v / v) at a mobile phase of 50:50 to 70:30 to obtain compound 4. Fraction E431 was separated by ODS column chromatography using a methanol-water gradient elution at a volume ratio of 30:70 to 100:

0. The fraction with a methanol-water volume ratio of 70:30 was collected and designated E4314, and the fraction with a methanol-water volume ratio of 80:20 was collected and designated E4315. Fraction E4314 was purified by semi-preparative HPLC using a methanol-water mobile phase at a volume ratio of 50:50 to 70:30 to obtain E43144. Further purification was carried out by semi-preparative HPLC using acetonitrile-water at a volume ratio of 20:80 to 50:50 to obtain compound 1. Fraction E4315 was purified by semi-preparative HPLC using a methanol-water mobile phase at a volume ratio of 60: The mobile phase was 40-80:20 methanol-water to obtain E43153. E43153 was further purified by semi-preparative HPLC with a mobile phase of 40:60-60:40 methanol-water to obtain E431533 and E431534. E431533 was further purified by semi-preparative HPLC with a mobile phase of 30:70-50:50 acetonitrile-water to obtain compounds 2, 3, 9, and 11. E431534 was further purified by semi-preparative HPLC with a mobile phase of 30:70-50:50 acetonitrile-water to obtain compounds 10 and 12. After concentration, fraction E451 was separated by ODS column chromatography with a gradient elution of methanol-water at a volume ratio of 30:70 to 100:

0. The fraction with a methanol-water volume ratio of 50:50 was collected and designated as E4513. After concentration of fraction E4513, it was purified by semi-preparative HPLC with methanol-water as the mobile phase at a volume ratio of 60:40 to 80:20 to obtain compounds 5 and 6.

5. A pharmaceutical composition, characterized in that, It comprises one or more of the eucalyptane-type sesquiterpenoid compounds of claim 1 or 2 or their pharmaceutically acceptable salts, and one or a combination of pharmaceutically acceptable carriers, excipients, diluents, or the like; the pharmaceutical composition is administered orally or by injection, and the dosage forms include tablets, capsules, powders, syrups, and injections.

6. The use of the eucalyptane-type sesquiterpenoid compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, in the preparation of an anti-inflammatory drug.

7. The use of the pharmaceutical composition of claim 5 in the preparation of an anti-inflammatory drug.

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