CN116514757A - Sesquiterpene coumarin compound in ferula sinkiangensis as well as preparation method and application thereof - Google Patents
Sesquiterpene coumarin compound in ferula sinkiangensis as well as preparation method and application thereof Download PDFInfo
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- CN116514757A CN116514757A CN202310152090.5A CN202310152090A CN116514757A CN 116514757 A CN116514757 A CN 116514757A CN 202310152090 A CN202310152090 A CN 202310152090A CN 116514757 A CN116514757 A CN 116514757A
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- 229930004725 sesquiterpene Natural products 0.000 title claims abstract description 51
- -1 Sesquiterpene coumarin compound Chemical class 0.000 title claims abstract description 42
- 241000134109 Ferula sinkiangensis Species 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 title abstract description 64
- 235000001671 coumarin Nutrition 0.000 title abstract description 52
- 229960000956 coumarin Drugs 0.000 title abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 17
- 239000003814 drug Substances 0.000 claims abstract description 9
- 230000004770 neurodegeneration Effects 0.000 claims abstract description 6
- 208000015122 neurodegenerative disease Diseases 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 186
- 150000001875 compounds Chemical class 0.000 claims description 84
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 78
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 67
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 65
- 239000012046 mixed solvent Substances 0.000 claims description 60
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 58
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 57
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 44
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 44
- 239000012071 phase Substances 0.000 claims description 41
- 239000003208 petroleum Substances 0.000 claims description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 24
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 24
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 20
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 20
- 239000000287 crude extract Substances 0.000 claims description 14
- 241000510609 Ferula Species 0.000 claims description 12
- 239000000284 extract Substances 0.000 claims description 12
- 239000012074 organic phase Substances 0.000 claims description 12
- 239000003960 organic solvent Substances 0.000 claims description 12
- 238000010992 reflux Methods 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 10
- 238000010898 silica gel chromatography Methods 0.000 claims description 9
- 150000003839 salts Chemical class 0.000 claims description 8
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 5
- 239000008194 pharmaceutical composition Substances 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 3
- 238000002481 ethanol extraction Methods 0.000 claims description 3
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000003808 methanol extraction Methods 0.000 claims description 3
- 238000002137 ultrasound extraction Methods 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000002265 prevention Effects 0.000 claims 1
- 229940079593 drug Drugs 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 3
- 230000004112 neuroprotection Effects 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 229910052799 carbon Inorganic materials 0.000 description 70
- 229910052739 hydrogen Inorganic materials 0.000 description 47
- 239000001257 hydrogen Substances 0.000 description 47
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 31
- 210000004027 cell Anatomy 0.000 description 25
- 125000004084 sesquiterpene group Chemical group 0.000 description 25
- 238000013375 chromatographic separation Methods 0.000 description 24
- 238000001514 detection method Methods 0.000 description 23
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 16
- 150000004354 sesquiterpene derivatives Chemical class 0.000 description 13
- 238000001228 spectrum Methods 0.000 description 13
- 229940126214 compound 3 Drugs 0.000 description 12
- 150000004775 coumarins Chemical class 0.000 description 12
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 12
- 229940125904 compound 1 Drugs 0.000 description 11
- 229940125782 compound 2 Drugs 0.000 description 10
- 229940125898 compound 5 Drugs 0.000 description 10
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 10
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- 150000002500 ions Chemical class 0.000 description 10
- 230000002025 microglial effect Effects 0.000 description 10
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 125000000332 coumarinyl group Chemical group O1C(=O)C(=CC2=CC=CC=C12)* 0.000 description 8
- CJIJXIFQYOPWTF-UHFFFAOYSA-N 7-hydroxycoumarin Natural products O1C(=O)C=CC2=CC(O)=CC=C21 CJIJXIFQYOPWTF-UHFFFAOYSA-N 0.000 description 7
- 244000228957 Ferula foetida Species 0.000 description 7
- ORHBXUUXSCNDEV-UHFFFAOYSA-N umbelliferone Chemical compound C1=CC(=O)OC2=CC(O)=CC=C21 ORHBXUUXSCNDEV-UHFFFAOYSA-N 0.000 description 7
- 238000005160 1H NMR spectroscopy Methods 0.000 description 6
- WSNKEJIFARPOSQ-UHFFFAOYSA-N 3-[4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy-N-(1-benzothiophen-2-ylmethyl)benzamide Chemical compound NCC1=CC(=NC(=C1)C(F)(F)F)OC=1C=C(C(=O)NCC2=CC3=C(S2)C=CC=C3)C=CC=1 WSNKEJIFARPOSQ-UHFFFAOYSA-N 0.000 description 6
- 235000007162 Ferula assa foetida Nutrition 0.000 description 6
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- 229940125773 compound 10 Drugs 0.000 description 6
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- 238000006467 substitution reaction Methods 0.000 description 6
- 238000005481 NMR spectroscopy Methods 0.000 description 5
- OBOXTJCIIVUZEN-UHFFFAOYSA-N [C].[O] Chemical group [C].[O] OBOXTJCIIVUZEN-UHFFFAOYSA-N 0.000 description 5
- 125000003668 acetyloxy group Chemical group [H]C([H])([H])C(=O)O[*] 0.000 description 5
- 238000004611 spectroscopical analysis Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 239000002024 ethyl acetate extract Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 125000004430 oxygen atom Chemical group O* 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 230000003215 anti-neuroinflammatory effect Effects 0.000 description 3
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 3
- VILAVOFMIJHSJA-UHFFFAOYSA-N dicarbon monoxide Chemical group [C]=C=O VILAVOFMIJHSJA-UHFFFAOYSA-N 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 229930185068 farnesiferol Natural products 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000401 methanolic extract Substances 0.000 description 3
- 210000000274 microglia Anatomy 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- OCHZHKVSLMBEJP-YPXWKZOUSA-N 7-[(e)-3-methyl-5-[(1r,4s)-2,2,4-trimethyl-7-oxabicyclo[2.2.1]heptan-3-yl]pent-2-enoxy]chromen-2-one Chemical compound C1=CC(=O)OC2=CC(OC\C=C(CCC3C([C@H]4CC[C@]3(C)O4)(C)C)/C)=CC=C21 OCHZHKVSLMBEJP-YPXWKZOUSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 125000002619 bicyclic group Chemical group 0.000 description 2
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- 238000011161 development Methods 0.000 description 2
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- 239000000469 ethanolic extract Substances 0.000 description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
- 125000002950 monocyclic group Chemical group 0.000 description 2
- 238000000238 one-dimensional nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
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- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- MOMFXATYAINJML-UHFFFAOYSA-N 2-Acetylthiazole Chemical group CC(=O)C1=NC=CS1 MOMFXATYAINJML-UHFFFAOYSA-N 0.000 description 1
- GOLORTLGFDVFDW-UHFFFAOYSA-N 3-(1h-benzimidazol-2-yl)-7-(diethylamino)chromen-2-one Chemical compound C1=CC=C2NC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 GOLORTLGFDVFDW-UHFFFAOYSA-N 0.000 description 1
- 241000208173 Apiaceae Species 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 238000001061 Dunnett's test Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
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- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 102000004889 Interleukin-6 Human genes 0.000 description 1
- 208000036110 Neuroinflammatory disease Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 102100029469 WD repeat and HMG-box DNA-binding protein 1 Human genes 0.000 description 1
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- 125000003118 aryl group Chemical group 0.000 description 1
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- 239000002026 chloroform extract Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
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- 238000005138 cryopreservation Methods 0.000 description 1
- 239000002027 dichloromethane extract Substances 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
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- 229940088598 enzyme Drugs 0.000 description 1
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- 150000004676 glycans Chemical class 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical group 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 125000003253 isopropoxy group Chemical group [H]C([H])([H])C([H])(O*)C([H])([H])[H] 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
- A61K31/37—Coumarins, e.g. psoralen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
The invention belongs to the technical field of medicines, and particularly relates to a sesquiterpene coumarin compound in ferula sinkiangensis root, and a preparation method and application thereof.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a sesquiterpene coumarin compound in ferula sinkiangensis root, and a preparation method and application thereof.
Background
Sinkiang Ferula asafetida (Ferula sinkiangensis K.M. shen.) is a plant of the genus Ferula (Ferula L.) of the family Umbelliferae. Over 150 plants of Ferula are distributed worldwide, mainly in the southern Europe, mediterranean and North Africa, iran, affori, well-known and Siberian, india, pakistan etc. There are 26 varieties of Ferula plants in China.
Xinjiang asafetida is herb with one or more results of multiple years, is mainly produced in Xinjiang province in China, and few kinds are distributed in Gansu, ningxia, shanxi, inner Mongolia, liaoning, hei Longjiang, hebei, henan, shandong, jiangsu, anhui, yunnan, tibet and other provinces. Xinjiang asafetida is a traditional Chinese medicine in China, has long medicinal history, is recorded in Tang Ben Cao, and is used for treating digestive system diseases in China. Modern pharmacological researches show that the Xinjiang asafetida has wide pharmacological actions of bacteriostasis, disinsection, antiallergic, anti-tumor and the like, and mainly contains various chemical components such as sesquiterpene coumarin, sulfur-containing compounds, polysaccharide, aromatic and the like.
Disclosure of Invention
The invention provides sesquiterpene coumarin compounds and pharmaceutically acceptable salts thereof in ferula sinkiangensis having the following structures, wherein the compounds are extracted from ferula sinkiangensis (Ferula sinkiangensis K.M.Shen.).
The invention also provides a preparation method of the sesquiterpene coumarin compound and the pharmaceutically acceptable salt thereof in the ferula sinkiangensis, which is characterized by comprising the following steps of:
(1) Extracting root of Ferula sinkiana (Ferula sinkiangensis K.M.) with ethanol or methanol, and recovering extractive solution to obtain crude extract;
(2) Dissolving the ethanol crude extract obtained in the step (1) with water, sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol, and extracting each organic phase for 3 times to obtain extracts with different polarity positions;
(3) Separating the extract obtained in the step (2) by silica gel column chromatography, and gradient eluting with petroleum ether and ethyl acetate mixed solvent, or petroleum ether and acetone mixed solvent, or chloroform and acetone mixed solvent, or dichloromethane and acetone mixed solvent, or chloroform and methanol mixed solvent, or dichloromethane and methanol mixed solvent;
(4) Separating the fraction obtained in the step (3) by ODS column chromatography, and gradient eluting with methanol-water or acetonitrile-water mixed solvent as mobile phase;
(5) And (3) further separating the methanol-water or acetonitrile-water eluate obtained in the step (4) by HPLC, and performing gradient elution by using a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as a mobile phase to obtain the compounds 1-10.
Preferably, the extraction method in the step (1) is a heating reflux ethanol extraction, a heating reflux methanol extraction or a heating ultrasonic extraction for 2-5 times, wherein the volume concentration of ethanol is 70% -95%, the volume concentration of methanol is 60% -90%, and the feed-liquid ratio is 1:8-1:20 g/mL.
Preferably, the organic solvent extraction method in the step (2) uses petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate and n-butanol to extract for 3-5 times according to the volume ratio of the water phase to the organic phase of 1:1-1:5, and the organic solvent is recovered under reduced pressure.
Preferably, the volume ratio of petroleum ether to ethyl acetate, the volume ratio of petroleum ether to acetone mixed solvent in the step (3) is 100:10-1:1, the volume ratio of dichloromethane to acetone, the volume ratio of chloroform to acetone mixed solvent, dichloromethane to methanol mixed solvent, or chloroform to methanol mixed solvent is 100:1-1:1.
Preferably, in the step (4), the volume ratio of methanol to water in the mixed solvent of methanol and water is 1:9-9:1, and the volume ratio of acetonitrile to water in the mixed solvent of acetonitrile and water is 1:9-9:1.
Preferably, the volume ratio of methanol to water in the mixed solvent of methanol and water in the step (5) is 4:6-9:1, and the volume ratio of acetonitrile to water in the mixed solvent of acetonitrile and water is 4:6-9:1.
It is another object of the present invention to provide a pharmaceutical composition.
A pharmaceutical composition comprises sesquiterpene coumarin compound in Ferula sinkiana with the following structure, pharmaceutically acceptable salt and pharmaceutically acceptable carrier,
the invention also aims to provide the sesquiterpene coumarin compound in the ferula sinkiangensis and application of the sesquiterpene coumarin compound and pharmaceutically acceptable salt or the pharmaceutical composition in preparation of medicines for preventing or treating neurodegenerative diseases.
The beneficial effects of the invention are as follows: the invention provides a method for preparing and identifying sesquiterpene coumarin derivatives in 10 Xinjiang asafetida by taking Xinjiang asafetida roots as raw materials for the first time, and systematically evaluates the activity of the sesquiterpene coumarin derivatives in neuroprotection, thereby clarifying the application of the sesquiterpene coumarin derivatives in developing and treating neurodegenerative diseases.
Detailed Description
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way.
The test methods described in the following examples, unless otherwise specified, are all conventional; the reagents and materials, unless otherwise specified, are commercially available.
The invention aims to provide a series of sesquiterpene coumarin compounds in Xinjiang asafetida, a preparation method and novel medical application thereof.
The invention provides the following 10 specific compounds:
the invention also provides a preparation method of the sesquiterpene coumarin compound 1-10 in the ferula sinkiangensis, which comprises the following steps:
(1) Heating and reflux-extracting the root of the Ferula sinkiana (Ferula sinkiangensis K.M.) with 70-95% ethanol or 60-90% methanol, and recovering the extractive solution to obtain crude extract;
(2) Dissolving the crude extract obtained in the step (1) by water, and sequentially extracting for 3 times by using petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate and n-butanol according to the volume ratio of a water phase to an organic phase of 1:1-1:5 to obtain extracts with different polarities;
(3) Dissolving the extract obtained in the step (2) by an organic solvent, taking a silica gel, stirring and drying, separating by a silica gel column chromatography, and performing gradient elution by using a mixed solvent of petroleum ether and ethyl acetate of 100:10-1:1, or a mixed solvent of petroleum ether and acetone of 100:10-1:1, or a mixed solvent of dichloromethane and acetone of 100:1-1:1, or a mixed solvent of chloroform and methanol of 100:1-1:1, or a mixed solvent of dichloromethane and methanol of 100:1-1:1;
(4) Separating the fraction obtained in the step (3) by ODS column chromatography, and performing gradient elution by taking a mixed solvent of methanol-water 1:9-9:1 or acetonitrile-water 2:8-8:2 as a mobile phase;
(5) And (3) separating methanol and water, acetonitrile and water eluates obtained in the step (4) by HPLC, and performing gradient elution by taking a mixed solvent of methanol and water as a mobile phase or a mixed solvent of acetonitrile and water as a mobile phase of 4:6-9:1 or 3:7-8:2 to obtain the compounds 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.
The extraction method in the step (1) is heating reflux ethanol extraction, heating reflux methanol extraction or heating ultrasonic extraction for 2-5 times, and the solvent is 70-95% ethanol or 60-90% methanol, preferably 80-95% ethanol or 70-90% methanol. The feed liquid ratio is 1:8-1:20 g/mL, preferably 1:10-1:15 g/mL.
According to the preparation method of the sesquiterpene coumarin compound 1-10, in the step (2), the organic solvent extraction method is adopted, water is adopted to dissolve the crude extract, petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate and n-butanol are respectively used for extraction for 3 times, preferably 4 times according to the volume ratio of the water phase to the organic phase of 1:1-1:5, preferably 1:1-1:3, and the organic solvent is recovered under reduced pressure.
According to the preparation method of the sesquiterpene coumarin compound 1-10, the volume ratio of petroleum ether to ethyl acetate or the mixed solvent of petroleum ether and acetone in the step (3) is 100:5-1:1, preferably 100:10-1:1; the volume ratio of the mixed solvent of the dichloromethane to the acetone, the chloroform to the acetone, the dichloromethane to the methanol, or the chloroform to the methanol is 100:1-3:1, preferably 100:3-5:1.
According to the preparation method of the sesquiterpene coumarin compound 1-10, the volume ratio of the methanol and water mixed solvent in the step (4) is 1:9-9:1, preferably 4:6-9:1, and the volume ratio of the acetonitrile and water mixed solvent is 2:8-8:2, preferably 3:7-8:2.
According to the preparation method of the sesquiterpene coumarin compound 1-10, the volume ratio of the methanol and water mixed solvent in the step (5) is 4:6-9:1, preferably 5:5-9:1, and the volume ratio of the acetonitrile and water mixed solvent is 3:7-8:2, preferably 4:6-8:2.
The invention evaluates the anti-neuroinflammation of the preparation method of the prepared sesquiterpene coumarin compound 1-10 by using an LPS-induced BV-2 microglial cell overactivation model. The results show that the novel compounds 2,3,4,6,7, 10 are capable of inhibiting LPS-induced release of over-activated BV-2 microglial NO, exhibiting significant anti-neuroinflammatory activity. Therefore, the novel sesquiterpene coumarin compound prepared in the invention can be applied to the development of medicaments for treating neurodegenerative diseases.
Example 1
(1) Extracting 1000g of Ferula sinkiangensis root with 95% ethanol under reflux for 3 times (9L), and recovering extractive solution under reduced pressure to obtain crude extract;
(2) Dissolving the crude extract obtained in the step (1) by water, and sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol for 3 times according to the volume ratio of the water phase to the organic phase of 1:1 to obtain extracts with different polarities;
(3) Separating the dichloromethane extract obtained in the step (2) by silica gel column chromatography, and eluting with petroleum ether and ethyl acetate mixed solvents in sequence of 100:5, 10:1,8:1,5:1,3:1 and 1:1;
(4) Petroleum ether obtained in the above step (3): the ethyl acetate 8:1-1:1 fraction is subjected to ODS chromatography and is eluted by the gradient of mixed solvents of methanol-water 3:7,5:5,7:3 and 9:1;
(5) The methanol-water (5:5-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the flow rate is 3mL/min after 210nm detection, and the mobile phase is methanol: water=80:20, giving compound 7 (t R =14 min) (yield 0.0014%o) and compound 1 (t) R =16 min) (yield 0.00006%o), compound 2 (t R =22 min) (yield 0.00004%o).
(6) The methanol-water (5:5-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the flow rate is 3mL/min after 210nm detection, and the mobile phase is methanol: water=70:30, giving compound 9 (t R =15 min) (yield 0.00055%o) and compound 8 (t) R =20 min) (yield 0.00009%o).
(7) The methanol-water (5:5-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the flow rate is 3mL/min after 210nm detection, and the mobile phase is methanol: water=72:28, giving compound 5 (t R =35 min) (yield 0.0029%o) and compound 4 (t) R =30 min) (yield 0.00007%o).
(8) The methanol-water (5:5-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the flow rate is 3mL/min after 210nm detection, and the mobile phase is methanol: water=69:31 to give compound 6 (t R =70 min) (yield 0.0006%o).
(9) The methanol-water (5:5-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the flow rate is 3mL/min after 210nm detection, and the mobile phase is methanol: water=65:35, giving compound 10 (t R =38 min) (yield 0.0053%o) and compound 3 (t) R =40 min) (yield 0.0011%o).
The structure of the compounds 1 to 10 was identified based on their physicochemical properties and spectroscopic data.
The structural identification data for compound 1 are as follows:
white solid (methanol),-27.3(c 0.3,CH 3 OH). HR-ESI-MS gives an excimer ion peak M/z453.2642[ M+H ]] + (calcd.453.2641for C 28 H 37 O 5 ) Prompt molecular formula C 28 H 36 O 5 The unsaturation was 11. 1 H NMR(600MHz,CDCl 3 ) The low field region gives a characteristic hydrogen signal of the 7-hydroxycoumarin parent nucleus: delta H 7.63 (1 h, d, j=9.5 hz, h-4), 7.36 (1 h, d, j=8.3 hz, h-5), 6.80 (1 h, overlapped, h-8), 6.78 (1 h, overlapped, h-6), 6.24 (1 h, d, j=9.5 hz, h-3); the high field region sees sesquiterpene mother nuclear hydrogen signals: delta H 4.48 (1H, m, H-3 '), 4.30 (1H, dd, J=10.0, 6.0Hz, H-11' a), 4.02 (1H, dd, J=10.0, 6.0Hz, H-11' b) are the oxygen-carbon hydrogen signals, δ H 1.02(3H,s,CH 3 -15'),0.92(3H,s,CH 3 -13'),0.90(3H,s,CH 3 -14') are three sets of methyl hydrogen signals. In addition, a set of hydrogen signals from the isopropanoyl substrate segments were also observed: delta H 2.54(1H,hept,J＝7.0Hz,CH-isopropyl),1.18(3H,d,J＝7.0Hz,CH 3 -isopropyl),1.16(3H,d,J＝7.0Hz,CH 3 -isopropyl)。 13 C-NMR(150MHz,CDCl 3 ) The characteristic carbon signal (delta) of 9 7-hydroxycoumarin parent nuclei can be seen in the middle C 162.0,161.4,156.0,143.5,128.9,113.2,113.1,112.7,102.0), 15 sesquiterpene skeleton carbon signals (delta) C 146.7,111.6,80.2,68.2,56.7,46.8,38.2,37.7,34.6,32.4,28.5,24.1,22.9,22.3,17.0) and a set of isopropanoyl carbon signals (delta) C :176.9,34.6,19.3,19.1). In conclusion, the compound 1 is suggested to be sesquiterpene coumarin which is a characteristic component of ferula plants.
All hydrogen-carbon signals were assigned by HSQC (table 1, table 2), and the attachment position of methoxy groups was confirmed by HMBC spectroscopy. In HMBC, delta H 4.30 (H, dd, H-11 a), 4.02 (H, dd, H-11 b) and delta C 162.0 (C-7) there is a remote association suggesting that the sesquiterpene fragment is linked to the C-7 position of the coumarin parent nucleus through an oxygen atom; delta H 4.48 (1H, m, H-3') and delta C 176.9 (c=o) remote correlation suggests that the isopropoxy substitution is at the C-3' position; delta H 4.82 (1H, br.s, H-12 'a), 4.73 (1H, br.s, H-12' b) and delta C 56.7(C-9')A remote correlation of 32.4 (C-7 ') suggests that the double bond is attached at the C-8' position. Delta H 1.02(3H,s,CH 3 -15') and delta C 56.7 (C-9 '), 46.8 (C-5 '), and 34.6 (C-1 '), suggesting CH 3 -15 'is attached at the C-10' position. The compound structure was thus determined to be isobutyryl farnesiferol, and further the absolute configuration of compound 1 was determined to be 3'r,5's,9's,10' r by comparison of experimental and computational ECD data.
In summary, compound 1 was identified as 3'R,5' S,9'S,10' R-isobutyryl farnesiferol, which was searched for a new compound not reported in the literature.
The structural identification data for compound 2 are as follows:
white solid (methanol),3.5(c 0.43,CH 3 OH). HR-ESI-MS gives an excimer ion peak M/z458.2539[ M+NH ] 4 ] + (calcd.458.2543for C 26 H 36 NO 6 ) Prompt molecular formula C 28 H 36 O 5 The unsaturation was 11. 1 H NMR(600MHz,CDCl 3 ) The low field region gives a characteristic hydrogen signal of the 7-hydroxycoumarin parent nucleus: delta H 7.63 (1 h, d, j=9.5 hz, h-4), 7.36 (1 h, d, j=8.5 hz, h-5), 6.83 (1 h, dd, j=8.5, 2.4hz, h-6), 6.80 (1 h, d, j=2.4 hz, h-8), 6.25 (1 h, d, j=9.5 hz, h-3); the high field region sees sesquiterpene mother nuclear hydrogen signals: delta H 4.88 (1H, br.s, H-12 'a), 4.79 (1H, br.s, H-12' b) is a terminal double bond hydrogen signal, δ H 4.62 (1H, br.s, H-3 '), 4.37 (1H, dd, J=9.9, 5.0Hz, H-11' a), 4.15 (1H, dd, J=9.9, 6.8Hz, H-11' b) are the oxygen-carbon hydrogen signals, δ H 1.20(3H,s,CH 3 -13'),1.11(3H,s,CH 3 -14'),1.02(3H,s,CH 3 -15') is three sets of methyl hydrogen signals; in addition, delta H 2.07(3H,s,COCH 3 ) Is a set of acetoxy hydrogen signals. 13 C-NMR(150MHz,CDCl 3 ) The characteristic carbon signal (delta) of 9 7-hydroxycoumarin parent nuclei can be seen in the middle C 162.1,161.4,156.0,143.6,128.9,113.5,113.2,112.7,101.5), 15 sesquiterpene skeleton carbon signals (delta) C 144.0,112.9,80.4,70.2,67.8,56.5,47.9,44.2,38.1,37.3,31.6,30.6,23.6,23.0,22.3), 2 acetyl carbonsSignal (delta) C 170.6,21.4) which has similar nuclear magnetic data to Compound 1, except that the C-3' position in Compound 2 is an acetoxy substitution and one more oxygen-hydrogen signal delta H 3.97 (1 h, td, j=10.7, 5.0hz, h-6') indicating that the sesquiterpene fragment has a hydroxy substitution.
All hydrogen-carbon signals were assigned by HSQC (table 1, table 2), and the attachment position of methoxy groups was confirmed by HMBC spectroscopy. In HMBC, delta H 4.37 (H, dd, H-11 a), 4.15 (H, dd, H-11 b) and delta C 162.1 (C-7) there is a remote association suggesting that the sesquiterpene fragment is linked to the C-7 position of the coumarin parent nucleus through an oxygen atom; delta H 4.62 (1H, br.s, H-3') and delta C 170.6 (c=o) remote correlation suggests that the acetoxy substitution is at the C-3' position; delta H 4.88 (1H, br.s, H-12 'a), 4.79 (1H, br.s, H-12' b) and delta C 56.5 (C-9 '), and the remote correlation of 44.2 (C-7 ') suggests that the double bond is attached at the C-8' position. Delta H 3.97 (1H, td, J=10.7, 5.0Hz, H-6') and delta C 47.9 Remote correlation of (C-5 '), suggesting a substitution of a hydroxy group at the C-6' position. Thus, the structure of the compound was determined to be sinkianol C, and further, the absolute configuration of the compound 2 was determined to be 3' S,5' S,6' R,9' S,10' S by comparison of experimental and computational ECD data.
In conclusion, the compound 2 was identified as 3' S,5' S,6' R,9' S,10' S-sinkianol C, which was searched for as a novel compound not reported in the literature.
The structural identification data for compound 3 are as follows:
white solid (methanol),-56.0(c 0.5,CH 3 OH). HR-ESI-MS gives an excimer ion peak m/z 397.2015[ M+H ]] + :(calcd.for C 24 H 29 O 5 397.2015) of the formula C 24 H 28 O 5 The unsaturation was 11.
1 H NMR(600MHz,CDCl 3 ) The low field region gives a characteristic hydrogen signal of the 7-hydroxycoumarin parent nucleus: delta H 7.63(1H,d,J＝9.5Hz,H-4),7.37(1H,d,J＝8.4Hz,H-5),6.82(1H,dd,J＝8.4,2.4Hz,H-6),6.81(1H,d,J＝2.4Hz,H-7),6.26(1H,d,J＝9.5Hz,H-3)。1D NMR data (Table 2-3) suggested that Compound 3 and Compound 2 were relatively similar, except that Compound 3 lacked an acetoxy hydrogen carbon signal, but one more ketone carbonyl carbon signal, delta C 217.2, and the C-1', C-2', C-4' carbon signal shifts to the low field. It is therefore presumed that compound 3 is structurally similar to compound 2, and that the C-3' position may be substituted with a carbonyl group.
All hydrogen carbon signals were attributed using HSQC (table 1, table 2). Delta in HMBC H 4.31 (1H, dd, J=9.8, 5.8Hz, H-11 'a), 4.15 (1H, dd, J=9.8, 6.3Hz, H-11' b) and delta C 161.8 (C-7) remote correlation, suggesting that the sesquiterpene fragment is attached to the C-7 position of the coumarin by an oxygen atom; delta H 4.96 (1H, t, J=2.0 Hz, H-12 'a), 4.87 (1H, t, J=2.0 Hz, H-12' b) and delta C 54.8 (C-9 '), 42.9 (C-7 '), remote correlation, suggested that the double bond substitution was at the C-8' position of the sesquiterpene (Figure 2-27). Delta H 1.36(3H,s,CH 3 -13'),1.33(3H,s,CH 3 -14') are respectively associated with delta C 217.2 (C-3') remote correlation, delta H 2.08 (m, H-1 'alpha), 1.66 (m, H-1' beta) and delta, respectively C 217.2 (C-3 ') remote correlation, suggesting that the ketocarbonyl group is attached at the C-3' position. Thus, the structure of the compound was determined to be farnesiferone C, and further, the absolute configuration of the compound 3 was determined to be 5'S,6' R,9'S,10' S by comparison of experimental and computational ECD data.
In summary, compound 3 was identified as (5 'S,6' R,9'S,10' S) -farnesiferone C, and was searched for as a novel compound not reported in the literature.
The structural identification data for compound 4 are as follows:
white solid (methanol),-34.2(c 0.3,CH 3 OH). HR-ESI-MS gives an excimer ion peak M/z425.2337[ M+H ]] + :(calcd.for C 26 H 33 O 5 425.2328) of the formula C 26 H 32 O 5 The degree of unsaturation was 10.
1 H NMR(400MHz,CDCl 3 ) The low field region gives a characteristic hydrogen signal of the 7-hydroxycoumarin parent nucleus: delta H 7.63(1H,d,J＝9.5Hz,H-4),7.37(1H,d,J＝9.3HzH-5), 6.89 (1H, m, H-6), 6.88 (1H, m, H-8), 6.24 (1H, d, j=9.5 hz, H-3); the high field region sees sesquiterpene mother nuclear hydrogen signals: delta H 4.70(1H,br.s,H-3'),δ H 4.56 (1H, d, J=10.0 Hz, H-11 'a), 4.42 (1H, d, J=10.0 Hz, H-11' b) is a hydrocarbon signal on oxygen, delta H 1.72(3H,s,CH 3 -12'),1.06(3H,s,CH 3 -15'),0.94(3H,s,CH 3 -13'),0.90(3H,s,CH 3 -14') is four sets of methyl hydrogen signals; in addition, delta H 2.07(3H,s,COCH 3 ) Is an acetyl hydrogen signal. 13 C-NMR(100MHz,CDCl 3 ) (Figure 2-11) 9 7-hydroxycoumarin mother nuclei characteristic carbon signals (. Delta.) C 162.6,161.4,156.1,143.5,128.8,113.3,113.2,112.6,101.6), 15 sesquiterpene skeleton carbon signals (delta) C 136.0,135.4,77.7,64.7,45.8,37.9,36.9,33.7,30.2,23.3,27.8,21.8,20.8,19.7,18.4) and 2 acetoxy carbon signals (. Delta.) C :170.9,21.5). The compound was presumed to be sesquiterpene coumarin based on the above NMR data.
All hydrogen carbon signals were attributed using HSQC (table 1, table 2). Delta in HMBC H 4.56 (1H, d, J=10.0 Hz, H-11 'a), 4.42 (1H, d, J=10.0 Hz, H-11' b) and delta C 162.6 (C-7) remote association, suggesting that the sesquiterpene fragment is attached to the C-7 position of the coumarin parent nucleus via an oxygen atom; delta H 4.70 (1H, br.s, H-3') and delta C 170.9(CRemote correlation suggests that acetoxy is attached at the C-3' position (Figure 2-12). Delta H 1.72(3H,s,CH 3 -12') and delta C 135.4 (C-9 '), 33.4 (C-7') remote correlation prompt CH 3 -12 'is attached at the C-8' position; delta H 1.06(3H,s,CH 3 -15') and delta C 135.4 (C-9 '), 45.8 (C-5 '), 30.2 (C-1 ') remote correlation prompt CH 3 -15 'is attached to C-10'. The compound structure was thus determined to be farnesiferol acetate, and further the absolute configuration of compound 4 was determined to be 3' r,5' r,10's by comparison of experimental and computational ECD data.
In conclusion, compound 4 was identified as (3 ' r,5' r, 10's) -farnesiferol acetate, which was searched for as a novel compound not reported in the literature.
The structural identification data for compound 5 are as follows:
yellow oil (methanol). HR-ESI-MS gives an excimer ion peak m/z 405.2046[ M+Na ]] + (calcd.405.2036for C 24 H 30 O 4 Na), prompt molecular formula C 24 H 30 O 4 The degree of unsaturation was 10.
1 H-NMR(400MHz,CDCl 3 ) In the spectrum, the low field region gives a characteristic hydrogen signal of the 7-O-substituted coumarin parent nucleus: delta H 7.62 (1 h, d, j=9.5 hz, h-4), 7.35 (1 h, d, j=9.3 hz, h-5), 6.83 (1 h, overlap, h-6), 6.79 (1 h, overlap, h-8), 6.23 (1 h, d, j=9.5 hz, h-3); group 1 oxymethylene hydrogen signals: delta H 4.53(1H,d,J＝10.0Hz,H a -11′),4.38(1H,d,J＝10.0Hz,H b -11'); 1 oxygen-linked methine hydrogen signal: delta H 3.27 (1 h, dd, j=11.3, 4.5hz, h-3'). The high field gives 4 methyl hydrogen signals: delta H 1.68(3H,s,CH 3 -12′),1.03(3H,s,CH 3 -13′),1.02(3H,s,CH 3 -15′),0.82(3H,s,CH 3 -14′)。 13 C-NMR(100MHz,CDCl 3 ) The spectrum shows 24 carbon signals, 9 are 7-O-substituted coumarin parent nuclear characteristic carbon signals delta C 162.6 (C-7), 161.4 (C-2), 156.0 (C-9), 143.6 (C-4), 128.8 (C-5), 113.3 (C-6), 113.1 (C-3), 112.6 (C-10), 101.6 (C-8); 15 carbon signals which are sesquiterpene units, comprising 1 set of double bond carbon signals: delta C 136.0 (C-8 '), 135.2 (C-9'); 2 oxygen-carbon signals: delta C 64.7 (C-11 '), 78.8 (C-3'). Since the coumarin parent nucleus and 1 double bond provide 8 unsaturations, the sesquiterpene moiety of compound 5 is suggested to be a bicyclic structure.
All hydrogen carbon signals were attributed using HSQC (table 1, table 2). Delta in HMBC H 1.03(CH 3 -13'),0.82(CH 3 -14') and delta C 78.8 (C-3 '), 38.9 (C-4 '), 50.7 (C-5 '), and suggesting CH 3 -13' and CH 3 -14 'is attached at the C-4' position; delta H 1.68(CH 3 -12') and delta C 34.1 (C-7 '), 136.0 (C-8 '), 135.2 (C-9 '), and suggesting CH 3 -12 'is attached at the C-8' position; delta H 1.02(CH 3 -15') and delta C 34.7(C-1′),37.9(C-10 '), 135.2 (C-9 '), 50.7 (C-5 '), and suggesting CH 3 -15 'is attached at the C-10' position. Delta H 3.27 (H-3') and delta C 34.7 (C-1 '), 27.7 (C-2'), and 38.9 (C-4 ') are related, suggesting that the hydroxyl group is attached at the C-3' position. Comprehensive delta H 4.53(H a -11′),4.38(H b -11') and delta C 37.9 (C-10 '), 135.2 (C-9 '), 136.0 (C-8 '), delta H 2.15(H 2 -7') and delta C 18.7 (C-6 '), 50.7 (C-5'), and the planar structure of the sesquiterpene fragment was determined. Delta H 4.53(H a -11′),4.38(H b -11') and delta C 162.6 The remote-related hint sesquiterpene fragment of (C-7) is linked to the C-7 position of the coumarin parent nucleus via an ether linkage. Thus, the structure of this compound was determined to be ferrongensine I, and further, the absolute configuration of compound 5 was determined to be 3' r,5's,10' r by comparison of experimental and computational ECD data.
In summary, compound 5 was identified as (3 ' R,5' S,10' R) -ferrodinginine I, which was searched for as a novel compound not reported in the literature.
Table 1 hydrogen Spectrometry Nuclear magnetic data belonging to Compounds 1 to 5
* Overlapping signals
Table 2 carbon Spectrum Nuclear magnetic data attribution for Compounds 1 to 10
The structural identification data for compound 6 are as follows:
yellow oil (methanol). HR-ESI-MS gives an excimer ion peak m/z 443.2436[ M+H ]] + (calcd.443.2428for C 26 H 35 O 6 ) Supposedly the molecular formula is C 26 H 34 O 6， Indicating the presence of 10 unsaturations in the structure。
1 H-NMR(400MHz,CDCl 3 ) The low field region in the spectrum gives a characteristic hydrogen signal of the 7-O-substituted coumarin parent nucleus: delta H 7.63 (1 h, d, j=9.5 hz, h-4), 7.36 (1 h, d, j=8.8 hz, h-5), 6.83 (1 h, dd, j=8.8, 2.2hz, h-6), 6.88 (1 h, d, j=2.2 hz, h-8), 6.23 (1 h, d, j=9.5 hz, h-3); group 1 oxymethylene hydrogen signals: delta H 4.52(1H,dd,J＝10.0,1.9Hz,H a -11′),4.25(1H,dd,J＝10.0,5.0Hz,H b -11'); 1 oxygen-linked methine hydrogen signal: delta H 4.45 (1H, m, H-3'). The high field gives 5 methyl hydrogen signals: delta H 2.03(3H,s,CH 3 -2″),1.51(3H,s,CH 3 -12′),1.18(3H,s,CH 3 -15′),0.91(3H,s,CH 3 -13′),0.88(3H,s,CH 3 -14′)。 13 C-NMR(100MHz,CDCl 3 ) The spectrum shows 26 carbon signals, 9 are characteristic carbon signals of 7-O-substituted coumarin parent: delta C 162.0 (C-7), 161.4 (C-2), 156.1 (C-9), 143.6 (C-4), 128.9 (C-5), 113.2 (C-6), 113.1 (C-3), 112.7 (C-10), 102.0 (C-8); 15 carbon signals that are sesquiterpene units comprising 3 oxygen-linked carbon signals: delta C 80.7 (C-3 '), 72.3 (C-8 '), 67.0 (C-11 '); 2 are acetoxy carbon signals: delta C 171.0 (C-1 '), 21 (C-2'). Since the coumarin parent nucleus and 1 acetoxy group provide 8 unsaturations in total, the sesquiterpene part of the compound is suggested to be a bicyclic structure.
All hydrogen-carbon signals were assigned using HSQC (table 2, table 3). Delta in HMBC H 0.88(CH 3 -14'),0.91(CH 3 -13') and delta C 80.7 (C-3 '), 38.1 (C-4 '), 47.4 (C-5 '), and suggesting CH 3 -13' and CH 3 -14 'is attached at the C-4' position; delta H 1.51(CH 3 -12') and delta C 39.1 (C-7 '), 72.3 (C-8 '), 59.2 (C-9 '), and suggesting CH 3 -12 'is attached at the C-8' position; delta H 1.18(CH 3 -15') and delta C 35.1 (C-1 '), 37.7 (C-10'), 59.2 (C-9 '), 47.4 (C-5'), and suggesting CH 3 -15 'is attached at the C-10' position. Synthesis, delta H 4.45 (H-3') and delta C 23.9 (C-2 '), 38.1 (C-4'), delta H 4.52(H a -11′),4.25(H b -11') and delta C 37.7 (C-10 '), 59.2 (C-9 '), 72.3 (C-8 '), and the planar structure of the sesquiterpene fragment was determined. Delta H 4.45 (H-3') and delta C 171.0 (C-2 ') is related, suggesting that the acetoxy group is attached at the C-3' position; delta H 4.53(H a -11′),4.38(H b -11') and delta C 162.0 The remote-related hint sesquiterpene fragment of (C-7) is linked to the C-7 position of the coumarin parent nucleus via an ether linkage. Thus, the structure of this compound was determined to be ferrongensine J, and further the absolute configuration of compound 6 was determined to be 3' r,5's,8's,9's,10' r by comparison of experimental and computational ECD data.
In summary, compound 6 was identified as (3 ' R,5' S,8' S,9' S,10' R) -ferusingensine J, and was searched for as a novel compound not reported in the literature.
The structural identification data for compound 7 are as follows:
White powder (methanol). HR-ESI-MS gives an excimer ion peak m/z 453.2662[ M-H ]] - :(calcd.453.2646 for C 28 H 37 O 5 ) Supposedly the molecular formula is C 28 H 38 O 5 Indicating that there are 10 unsaturations in the structure.
1 H-NMR(600MHz,CDCl 3 ) The low field region in the spectrum gives a characteristic hydrogen signal of the 7-O-substituted coumarin parent nucleus: delta H 7.62 (1 h, d, j=9.5 hz, h-4), 7.34 (1 h, d, j=8.6 hz, h-5), 6.81 (1 h, dd, j=8.6, 2.3hz, h-6), 6.75 (1 h, d, j=2.3 hz, h-8), 6.23 (1 h, d, j=9.5 hz, h-3); group 2 oxymethylene hydrogen signals: delta H 4.06(2H,m,H-3′),3.88(1H,d,J＝8.3Hz,H a -11′),3.69(1H,d,J＝8.3Hz,H b -11'). The high field gives 6 methyl hydrogen signals: delta H 1.62(3H,d,J＝0.4Hz,CH 3 -14′),1.45(3H,d,J＝1.7Hz,CH 3 -13′),1.18(6H,d,J＝7.0Hz,CH 3 -3″,CH 3 -4″),1.11(3H,s,CH 3 -15′),0.90(3H,d,J＝7.0Hz,CH 3 -12′)。 13 C-NMR(150MHz,CDCl 3 ) The spectrum shows 28 carbon signals, 9 are 7-O-substituted coumarin parent characteristic carbon signals: delta C 163.1(C-7),161.4(C-2),156.1 (C-9), 143.6 (C-4), 128.7 (C-5), 113.3 (C-6), 112.9 (C-3), 112.4 (C-10), 101.3 (C-8); 15 carbon signals which are sesquiterpene units, comprising 1 set of double bond carbon signals: delta C 130.3 (C-5 '), 125.3 (C-4'); 2 oxygen-carbon signals: delta C 71.9 (C-11 '), 64.9 (C-3'). The remaining 4 signals are isobutyryloxy substrate segments: delta C 177.4 (C-1 '), 34 (C-2 '), 19 (C-3 '). S22 is similar to S21 except that the substituents at the C-3 'position are different, and the C-3' position of S22 is an isobutyryloxy substrate fragment, not propionyloxy.
All hydrogen-carbon signals were assigned using HSQC (table 2, table 3). Delta in HMBC H 2.55 (H-2') and delta C 177.4 (C-1') correlation, delta H 1.18(CH 3 -3″,CH 3 -4') and delta C 34.2 (C-2 '), 177.4 (C-1 '), further confirming the structure of the fragment of the substituent isobutyryloxy at the C-3' position. Delta H 4.06 (H-3') and delta C 177.4 (C-1 ') suggesting that the fragment is linked at the C-3' position of the sesquiterpene unit. Thus, the structure of this compound was determined to be ferrodinginine N, and further the absolute configuration of compound 7 was determined to be 8's,9's,10's by comparison of experimental and calculated ECD data.
In summary, compound 7 was identified as (8 ' S,9' S,10' S) -ferusingensine N, and was searched for as a novel compound not reported in the literature.
The structural identification data for compound 8 are as follows:
yellow oil (methanol). HR-ESI-MS gives an excimer ion peak m/z 405.2046[ M+Na ]] + :(calcd.405.2036for C 24 H 30 O 4 Na), supposedly of the molecular formula C 24 H 30 O 4 Indicating that there are 10 unsaturations in the structure.
1 H-NMR(600MHz,CDCl 3 ) The low field region in the spectrum gives a characteristic hydrogen signal of the 7-O-substituted coumarin parent nucleus: delta H 7.63 (1 h, d, j=9.4 hz, h-4), 7.35 (1 h, d, j=8.6 hz, h-5), 6.84 (1 h, dd, j=8.6, 2.3hz, h-6), 6.81 (1 h, d, j=2.3 hz, h-8), 6.23 (1 h, d, j=9.4 hz, h-3); 1 alkene hydrogen signal: delta H 5.46 (1 h, t, j=6.5 hz, h-9'); group 1 continuous oxymethyleneBase hydrogen signal: delta H 4.57(2H,d,J＝6.5Hz,H 2 -11'). The high field gives 4 methyl hydrogen signals: delta H 1.74(3H,s,CH 3 -12′),1.03(3H,d,J＝6.8Hz,CH 3 -15′),1.02(3H,d,J＝7.0Hz,CH 3 -13′),0.88(3H,s,CH 3 -14′)。 13 C-NMR(150MHz,CDCl 3 ) The spectrum shows 24 carbon signals, 9 are characteristic carbon signals of 7-O-substituted coumarin parent: delta C 162.2 (C-7), 161.4 (C-2), 156.0 (C-9), 143.6 (C-4), 128.8 (C-5), 113.3 (C-6), 113.2 (C-3), 112.6 (C-10), 101.7 (C-8); 15 carbon signals which are sesquiterpene units comprising 1 carbonyl carbon signal: delta C 214.9 (C-3'); group 1 double bond carbon signals: delta C 142.8 (C-8 '), 118.6 (C-9'); 1 oxygen-carbon signal: delta C 65.6 (C-11'). Since the coumarin parent nucleus, 1 carbonyl group and 1 double bond provide 9 unsaturations in total, the sesquiterpene moiety of the compound is presumed to be a monocyclic structure.
All hydrogen-carbon signals were assigned using HSQC (table 2, table 3). Delta in HMBC H 1.74(CH 3 -12') and delta C 32.8 (C-7 '), 142.8 (C-8 '), 118.6 (C-9 '), and suggesting CH 3 -12 'is attached at the C-8' position; delta H 1.02(CH 3 -13') and delta C 214.9 (C-3 '), 51.5 (C-4 '), 42.1 (C-5 '), and suggesting CH 3 -13 'is attached at the C-4' position; delta H 0.88(CH 3 -14') and delta C 51.5 (C-4 '), 42.1 (C-5 '), and 34.2 (C-6 '), suggesting CH 3 -14 'is attached at the C-5' position; delta H 1.03(CH 3 -15') and delta C 29.9 (C-1 '), 33.5 (C-10 '), 42.1 (C-5 '), and delta C 65.6 (C-11') uncorrelated, suggesting CH 3 -15 'is attached at the C-10' position and the B ring of the sesquiterpene unit is cleaved; delta H 1.89(H α -1'),1.63(H β -1') and delta C 214.9 (C-3') correlation, delta H 1.39(H 2 -6') and delta C 32.8 (C-7') correlation; delta H 4.57(H 2 -11') and delta C 142.9 (C-8 '), 119.3 (C-9'), suggesting that the double bond is located at the C-8'/C-9' position, and the above HMBC correlations together define the plane of the sesquiterpene unitStructure is as follows. In addition, delta H 4.57(H 2 -11') and delta C 162.2 The remote-related hint sesquiterpene fragment of (C-7) is linked to the C-7 position of the coumarin parent nucleus via an ether linkage. Thus, the structure of this compound was determined to be ferrongensine L, and further the absolute configuration of compound 8 was determined to be 4's,5's,10's by comparison of experimental and computational ECD data.
In summary, compound 8 was identified as (4 ' S,5' S,10' S) -ferusingensine L, and was searched for as a novel compound not reported in the literature.
The structural identification data for compound 9 are as follows: (4 ' R,5' S,10' S) -ferrodinginine M
Yellow oil (methanol). HR-ESI-MS gives an excimer ion peak m/z 819.4074[2M+Na ]] + :(calcd.819.4077for C 48 H 60 O 10 Na), supposedly of the molecular formula C 24 H 30 O 5， The presence of 9 unsaturations in the structure is suggested.
1 H-NMR(600MHz,CDCl 3 ) In the spectrum, the low field region gives a characteristic hydrogen signal of the 7-O-substituted coumarin parent nucleus: delta H 7.63 (1 h, d, j=9.5 hz, h-4), 7.36 (1 h, d, j=8.6 hz, h-5), 6.86 (1 h, dd, j=8.6, 2.4hz, h-6), 6.82 (1 h, d, j=2.4 hz, h-8), 6.24 (1 h, d, j=9.5 hz, h-3); 1 alkene hydrogen signal: delta H 5.48 (1 h, t, j=6.5 hz, h-9'); group 1 oxymethylene hydrogen signals: delta H 4.60 (2 h, d, j=6.5 hz, h-11'). The high field gives 4 methyl hydrogen signals: delta H 1.78(3H,s,CH 3 -12′),1.38(3H,s,CH 3 -13′),0.94(3H,d,J＝6.7Hz,CH 3 -15′),0.69(3H,s,CH 3 -14′)。 13 C-NMR(150MHz,CDCl 3 ) The spectrum shows 24 carbon signals, 9 are characteristic carbon signals of 7-O-substituted coumarin parent: delta C 162.3 (C-7), 161.4 (C-2), 156.0 (C-9), 143.6 (C-4), 128.8 (C-5), 113.4 (C-6), 113.1 (C-3), 112.6 (C-10), 101.8 (C-8); the remaining 15 are carbon signals of sesquiterpene units, containing 1 carbonyl carbon signal: delta C 215.0 (C-3'); group 1 double bond carbon signals: delta C 143.8 (C-8 '), 118.0 (C-9'); 2 oxygen-carbon signals: delta C 81.3 (C-4 '), 65.7 (C-11'). Due to coumarin parent (7 unsaturations) and1 carbonyl group provides 8 unsaturations in total, so the sesquiterpene moiety of the compound is presumed to be a monocyclic structure. In comparison with the 1D NMR of Compound 8, the main difference was found in that the chemical shift value of C-4 'was significantly increased, and it was presumed that the C-4' of 8 was hydroxylated. Thereafter, all hydrogen carbon data were attributed according to HSQC (table 2, table 3). In HMBC spectra, δ H 1.38(CH 3 -13') and delta C 215.0 (C-3 '), 81.3 (C-4 '), 47.3 (C-5 '), and delta H 0.69(CH 3 -14') and delta C 81.3 (C-4') correlation, and the above estimation was confirmed. Thus, the structure of this compound was determined to be ferrodinginine M, and further the absolute configuration of compound 9 was determined to be 4' r,5's,10's by comparison of experimental and computational ECD data.
In summary, compound 9 was identified as (4 ' R,5' S,10' S) -ferusingensine M, and was searched for as a novel compound not reported in the literature.
The structural identification data for compound 10 are as follows:
yellow oil (methanol). HR-ESI-MS gives an excimer ion peak m/z 399.2175[ M+H ]] + :(calcd.399.2166 for C 24 H 31 O 5 ) Supposedly the molecular formula is C 24 H 30 O 5 Indicating that there are 10 unsaturations in the structure.
1 H-NMR(600MHz,CDCl 3 ) The low field region in the spectrum gives a characteristic hydrogen signal of the 7-O-substituted coumarin parent nucleus: delta H 7.61 (1 h, d, j=9.5 hz, h-4), 7.34 (1 h, d, j=8.6 hz, h-5), 6.81 (1 h, dd, j=8.6, 2.3hz, h-6), 6.77 (1 h, d, j=2.3 hz, h-8), 6.21 (1 h, d, j=9.5 hz, h-3); 1 alkene hydrogen signal: delta H 5.44 (1 h, t, j=6.5 hz, h-2'); group 1 oxymethylene hydrogen signals: delta H 4.55 (2 h, d, j=6.5 hz, h-1'). The high field gives 4 groups of methyl hydrogen signals: delta H 1.74(3H,s,CH 3 -13'),1.05(3H,overlap,CH 3 -14'),1.04(3H,overlap,CH 3 -15'),1.04(3H,overlap,CH 3 -12')。 13 C-NMR(150MHz,CDCl 3 ) The spectrum shows 24 carbon signals, 9 being 7-O-substituted coumarin parent carbon signals: delta C 162.1(C-7),161.3(C-2),155.9(C-9),143.5(C-4),128.8(C-5),113.2 (C-6), 113.1 (C-3), 112.6 (C-10), 101.6 (C-8); 15 carbon signals which are sesquiterpene units, comprising 1 set of double bond carbon signals: delta C 141.1 (C-3 '), 118.9 (C-2'); 2 carbonyl carbon signals: delta C 214.6 (C-10 '), 213.2 (C-6'); 1 oxygen-carbon signal: delta C 65.4(C-1')。。
All hydrogen-carbon signals were assigned using HSQC (table 2, table 3). Delta in HMBC H 1.74(CH 3 -13') and delta C 118.9 (C-2 '), 141.1 (C-3 '), and 33.0 (C-4 '), suggesting CH 3 -13 'is attached at the C-3' position; delta H 1.05(CH 3 -14') and delta C 213.2 (C-6 '), 45.5 (C-7 '), 26.4 (C-8 '), and suggesting CH 3 -14 'is attached at the C-7' position; delta H 1.04(CH 3 -12′),1.04(CH 3 -15') and delta C 40.9 (C-11 '), 214.2 (C-10'), suggesting CH 3 -12' and CH 3 -15 'is attached at the C-11' position. Delta H 2.31(H 2 -4') and delta C 39.0 (C-5 '), 213.2 (C-6'), delta H 1.89(H a -8′),1.59(H b -8') and delta C 37.4 (C-9 '), 214.26 (C-10'), related, delta H 4.60(H 2 -1') and delta C 118.9 (C-2 '), 141.1 (C-3'), 162.3 (C-7), further define the planar structure of the sesquiterpene fragment, and the sesquiterpene units are linked to the C-7 position of the coumarin parent through ether linkages. Thus, the structure of the compound was determined to be ferrodinginine O, and further, the absolute configuration of the compound 10 was determined to be 7'S by comparison of experimental and computational ECD data.
In summary, compound 10 was identified as (7'S) -ferusingensinine O, and was searched for as a novel compound not reported in the literature.
TABLE 3 assignment of hydrogen Spectrometry Nuclear magnetic data for Compounds 6-10
* Overlapping signals
Example 2
(1) Extracting 500g of Ferula sinkiangensis root with 95% ethanol under reflux for 3 times (6L), and recovering extractive solution under reduced pressure to obtain crude extract;
(2) Extracting the ethanol extract obtained in the step (1) by using an organic solvent, sequentially extracting with petroleum ether, ethyl acetate and n-butanol according to the volume ratio of the water phase to the organic phase of 1:2, and respectively extracting for 3 times to obtain extracts of different polar parts;
(3) Separating the ethyl acetate extract obtained in the step (2) by silica gel column chromatography, and eluting with a mixed solvent of dichloromethane and acetone in sequence of 100:1, 100:3, 100:5, 10:1,8:1,5:1 and 3:1;
(4) Methylene dichloride obtained in the step (3) is prepared: the acetone 100:3-8:1 fraction is subjected to ODS chromatography and is eluted by the gradient of mixed solvents of methanol-water 3:7,5:5,7:3 and 9:1;
(5) The methanol-water (5:5-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the flow rate is 3mL/min after 210nm detection, and the mobile phase is methanol: water=78:22 to give compound 5 (t R =13 min) (yield 0.0031%o), compound 7 (t R =18 min) (yield 0.0012%o) and compound 1 (t) R =27 min) (yield 0.00006%o), compound 2 (t R =31 min) (yield 0.00003%o).
(6) The methanol-water (5:5-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the flow rate is 3mL/min after 210nm detection, and the mobile phase is methanol: water=70:30, giving compound 4 (t R =46 min) (yield 0.00011%o) and compound 6 (t) R =66 min) (yield 0.0008%o).
(7) The methanol-water (5:5-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the flow rate is 3mL/min after 210nm detection, and the mobile phase is methanol: water=65:35, giving compound 9 (t R =32 min) (yield 0.00041%o) and compound 8 (t) R =47 min) (yield 0.00012%o).
(8) The methanol-water (5:5-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the flow rate is 3mL/min after 210nm detection, and the mobile phase is methanol: water=63:37 to give compound 10 (t R =45 min) (yield 0.0053%o) and compound 3 (t) R ＝52min) (yield 0.0011%.
The structural identification method of the sesquiterpene coumarin compounds 1-10 is shown in an example 1.
Example 3
(1) Extracting 1000g of Ferula sinkiangensis root with 80% ethanol under reflux for 3 times (15L), and recovering extractive solution under reduced pressure to obtain crude extract;
(2) Extracting the ethanol extract obtained in the step (1) by using an organic solvent, sequentially extracting with cyclohexane, ethyl acetate and n-butanol according to the volume ratio of a water phase to an organic phase of 1:3, and respectively extracting for 3 times to obtain extracts of different polar parts;
(3) Separating the ethyl acetate extract obtained in the step (2) by silica gel column chromatography, and eluting with chloroform and methanol mixed solvents of 100:1, 100:3, 100:5, 10:1,8:1,5:1 and 3:1 in sequence;
(4) Chloroform obtained in the above step (3): the methanol 100:3-8:1 fraction is subjected to ODS chromatography and is eluted by the gradient of a mixed solvent of acetonitrile-water 3:7,5:5,7:3 and 9:1;
(5) The acetonitrile-water (3:7-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the detection is carried out at 210nm, the flow rate is 3mL/min, and the mobile phase is acetonitrile: water=75:25, giving compound 7 (t R =14 min) (yield 0.0017%o) and compound 1 (t) R =22 min) (yield 0.00003%o), compound 2 (t R =25 min) (yield 0.00005%o).
(6) The acetonitrile-water (3:7-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the detection is carried out at 210nm, the flow rate is 3mL/min, and the mobile phase is acetonitrile: water=65:35, giving compound 5 (t R =32 min) (yield 0.0032%o), compound 4 (t R =41 min) (yield 0.00016%o) and compound 6 (t) R =44 min) (yield 0.0009%o).
(7) The acetonitrile-water (3:7-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the detection is carried out at 210nm, the flow rate is 3mL/min, and the mobile phase is acetonitrile: water=60:40, giving compound 9 (t R =18 min) (yield 0.00061%o) and compound 8 (t) R =32 min) (yield 0.00010%o).
(8) Step (4) above) The acetonitrile-water (3:7-9:1) fraction obtained in the step (a) is prepared by HPLC-UV chromatographic separation, the detection is carried out at 210nm, the flow rate is 3mL/min, and the mobile phase is acetonitrile: water=55:45, giving compound 10 (t R =41 min) (yield 0.0050%o) and compound 3 (t) R =45 min) (yield 0.0018%o).
The structural identification method of the sesquiterpene coumarin compounds 1-10 is shown in an example 1.
Example 4
(1) 1500g of Xinjiang ferula root is heated and extracted by reflux with 80% methanol for 4 times (the dosage is 20L), and the extracting solution is recovered under reduced pressure to obtain a crude extract;
(2) Extracting the methanol extract obtained in the step (1) by using an organic solvent, sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol according to the volume ratio of water phase to organic phase of 1:3, and respectively extracting for 3 times to obtain extracts of different polar parts;
(3) Separating the ethyl acetate extract obtained in the step (2) by silica gel column chromatography, and eluting sequentially with chloroform and acetone mixed solvents of 100:1, 100:3, 100:5, 10:1,8:1,5:1 and 3:1;
(4) Chloroform obtained in the above step (3): the acetone 100:3-8:1 fraction was subjected to ODS chromatography, and eluted with a gradient of acetonitrile-water 2:8,4:6,6:4,8:2 mixed solvent:
(5) The acetonitrile-water (4:6-8:2) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the detection at 210nm is carried out, the flow rate is 3mL/min, and the mobile phase is acetonitrile: water=76:24 to give sesquiterpene coumarins 2 (t R =18 min) (yield 0.00014%o) and compound 1 (t) R =19 min) (yield 0.00023%o).
(6) The acetonitrile-water (4:6-8:2) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the detection at 210nm is carried out, the flow rate is 3mL/min, and the mobile phase is acetonitrile: water=72:28, giving sesquiterpene coumarins 7 (t R =11 min) (yield 0.00038%o) and compound 5 (t) R =21 min) (yield 0.00011%o).
(7) The acetonitrile-water (4:6-8:2) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the detection at 210nm is carried out, the flow rate is 3mL/min, and the mobile phase is acetonitrile: water=65:35 to obtain the sesquiterpene coumarin compound 9 (t) R =18 min) (yield 0.00017%o), compound 8 (t R =22 min) (yield 0.00070%o), compound 4 (t R =29 min) (yield 0.00071%o) and compound 6 (t) R =61 min) (yield 0.00011%o).
(8) The acetonitrile-water (4:6-8:2) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the detection at 210nm is carried out, the flow rate is 3mL/min, and the mobile phase is acetonitrile: water=62:38, giving sesquiterpene coumarins 10 (t R =54 min) (yield 0.0008%o) and compound 3 (t) R =61 min) (yield 0.00011%o).
The structural identification method of the sesquiterpene coumarin compounds 1-10 is shown in an example 1.
Example 5
(1) Extracting 2000g of Ferula sinkiangensis root with 80% methanol under reflux for 3 times (20L), and recovering extractive solution under reduced pressure to obtain crude extract;
(2) Extracting the methanol extract obtained in the step (1) by using an organic solvent, sequentially extracting with cyclohexane, chloroform and n-butanol according to the volume ratio of water phase to organic phase of 1:1, and respectively extracting for 3 times to obtain extracts of different polarity parts;
(3) Separating the chloroform extract obtained in the step (2) by silica gel column chromatography, and eluting with petroleum ether and acetone mixed solvents in sequence of 100:5, 10:1,8:1,5:1,3:1 and 1:1;
(4) Petroleum ether obtained in the step (3): the acetone 8:1-1:1 fraction is subjected to ODS chromatography and is eluted by the gradient of mixed solvents of methanol-water 3:7,5:5,7:3,8:2 and 9:1;
(5) The methanol-water (5:5-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the flow rate is 3mL/min after 210nm detection, and the mobile phase is methanol: water=81:19, giving sesquiterpene coumarins 7 (t R =13 min) (yield 0.00016%o), compound 1 (t) R =15 min) (yield 0.00006%o) and compound 2 (t) R =20 min) (yield 0.00006%o).
(6) The methanol-water (5:5-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, 210nm detection is carried out, the flow rate is 3mL/min,the mobile phase is methanol: water=71:29, giving sesquiterpene coumarins 9 (t R =14 min) (yield 0.00061%o), compound 8 (t R =20 min) (yield 0.00011%o), compound 5 (t R =36 min) (yield 0.00027%o) and compound 4 (t) R =42 min) (yield 0.00007%o).
(7) The methanol-water (5:5-9:1) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the flow rate is 3mL/min after 210nm detection, and the mobile phase is methanol: water=67:33, giving sesquiterpene coumarins 10 (t R =35 min) (yield 0.00057%o), compound 3 (t R =37 min) (yield 0.00011%o) and compound 6 (t) R =72 min) (yield 0.0007%o).
The structural identification method of the sesquiterpene coumarin compounds 1-10 is shown in an example 1.
Example 6
(1) 2500g of Xinjiang ferula root is heated and extracted with 90% methanol under reflux for 3 times (the dosage is 25L), and the extracting solution is recovered under reduced pressure to obtain a crude extract;
(2) Extracting the methanol extract obtained in the step (1) by using an organic solvent, sequentially extracting with petroleum ether, ethyl acetate and n-butanol according to the volume ratio of a water phase to an organic phase of 1:2, and respectively extracting for 3 times to obtain extracts of different polar parts;
(3) Separating the ethyl acetate extract obtained in the step (2) by silica gel column chromatography, and eluting with chloroform and methanol mixed solvents of 100:1, 100:3, 100:5, 10:1,8:1,5:1 and 3:1 in sequence;
(4) Chloroform obtained in the above step (3): the methanol 100:3-8:1 fraction is subjected to ODS chromatography and is eluted by the gradient of mixed solvents of acetonitrile-water 2:8,4:6,6:4,8:2 and 9:1;
(5) The acetonitrile-water (4:6-8:2) fraction obtained in the step (4) is prepared by HPLC-RID chromatographic separation, the flow rate is 3mL/min, and the mobile phase is acetonitrile: water=81:19 to give sesquiterpene coumarins 2 (t R =11 min) (yield 0.00013%o).
(6) The acetonitrile-water (4:6-8:2) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the flow rate is 3mL/min after 210nm detection, and the mobile phase is acetonitrile: water=77:23, giving sesquiterpene coumarins 7 (t R =9 min) (yield 0.00042%o) and compound 1 (t) R =14 min) (yield 0.00021%o).
(7) The acetonitrile-water (4:6-8:2) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the detection at 210nm is carried out, the flow rate is 3mL/min, and the mobile phase is acetonitrile: water=62:38, giving sesquiterpene coumarins 9 (t R =17 min) (yield 0.00019%o), compound 8 (t R =22 min) (yield 0.00072%o), compound 5 (t R =26 min) (yield 0.00079%o), compound 4 (t R =40 min) (yield 0.00016%o) and compound 6 (t) R =81 min) (yield 0.00005%o).
(8) The acetonitrile-water (4:6-8:2) fraction obtained in the step (4) is prepared by HPLC-UV chromatographic separation, the detection at 210nm is carried out, the flow rate is 3mL/min, and the mobile phase is acetonitrile: water=59:41 to give sesquiterpene coumarins 10 (t R =31 min) (yield 0.0009%o) and compound 3 (t) R =40 min) (yield 0.00005%o).
The structural identification method of the sesquiterpene coumarin compounds 1-10 is shown in an example 1.
The test results were as follows:
test of anti-neuroinflammatory Activity of sesquiterpene coumarin Compounds 1 to 10 prepared in examples 1 to 6.
(1) Experimental principle: the chronic inflammatory reaction mediated by the overactivation of microglia makes an important link in the occurrence and development of neurodegenerative diseases, so that the inhibition of overactivated microglia is likely to become a new target for drug discovery. LPS can activate microglial cells to release NO, TNF-alpha, IL-6, IL-1 beta and the like. The experiment evaluates the anti-neuroinflammatory activity of the novel sesquiterpene coumarin compounds 1-10 by establishing an in vitro LPS-induced abnormal activated BV-2 microglial cell screening model and taking the NO release amount as an index.
(2) The experimental method comprises the following steps:
(1) culture of the mouse microglial cell line BV-2
All glassware and metal instruments used in cell culture and model establishment (culture Bottles, pipettes, solution bottles, etc.), were autoclaved at 121 ℃ for 30min to thoroughly remove contaminating LPS. A cell culture solution containing 10% fetal bovine serum was prepared based on DMEM medium. Microglial cells were present at about 2.0X10 5 The concentration of cells/mL was 5% CO 2 Subculturing in culture flask at 37deg.C until the adherent cells occupy 70-80% of the bottom area of culture flask about the third day, digesting adherent cells with pancreatin, and subculturing to another culture flask. BV-2 after cryopreservation and recovery at the ultralow temperature of-80 ℃ is used as the first generation, and 3 rd generation to 8 th generation BV-2 cells are selected for experiments.
(2) Medicine preparation method
Test compounds were all dissolved in DMSO. A mother liquor (100 mM) was prepared and stored at-20 ℃. The cells were diluted with DMEM medium to give 100. Mu.M, 30. Mu.M, 10. Mu.M and 1. Mu.M in this order. The final DMSO concentration is < 1%.
(3) Griess assay for inhibition of LPS-activated microglia by compounds
Taking BV-2 microglial cells in logarithmic growth phase, adjusting cell density to 2.0X10 by fresh DMEM culture medium containing 10% fetal bovine serum 5 cells/mL, inoculated in 96-well plates, 100. Mu.L/well, at 37℃with 5% CO 2 Is cultured in an incubator. After 24 hours of cell wall-attached culture, the cells are replaced by fresh culture solution without serum, and meanwhile, the dosing treatment is carried out. The compound concentrations tested were 100. Mu.M, 30. Mu.M, 10. Mu.M, 1. Mu.M, co-acting with LPS. And a blank control is set. The final LPS concentration in each of the dosing groups was 100ng/mL. After the cells are continuously cultured for 24 hours after the drug addition, collecting supernatant, and detecting NO in the supernatant by using Griess colorimetric method 2- The content is as follows.
(4) MTT method for detecting influence of compound on microglial cell survival rate
Taking BV-2 microglial cells cultured in logarithmic growth phase, and adjusting cell density to 2.0X10 with fresh DMEM medium containing 10% fetal bovine serum 5 cells/mL, inoculated in 96-well plates, 100. Mu.L/well, at 37℃with 5% CO 2 Is cultured in an incubator. After 24 hours of cell wall-attached culture, the cells are changed into fresh culture solution, and meanwhile, the dosing treatment is carried out. The compound doses were 100. Mu.M, 30. Mu.M, 10. Mu.M, 1. Mu.M in combination with LPS. And a blank control is set. Each feedThe final concentration of LPS in the drug group was 100ng/mL. After the cells were dosed, the culture was continued for 24 hours, then MTT solution (10. Mu.L/well) was added to the cell solution, the cells were incubated with 0.25mg/mL MTT at 37℃for 3 hours, the culture solution was aspirated, and then 150. Mu.L of DMSO solution was added to determine the OD value of the optical density. And (3) data processing, namely performing data processing by using enzyme-labeled instrument software, calculating the average value of OD values of 3 holes of each sample, and calculating the cell viability (CV%) by using the average value according to the following formula.
Cell viability% = [ average of sample group OD values/average of blank group OD values ] ×100%
(5) Statistical method
All data were checked using the SPSS (19.0) statistical software package. Results were expressed as mean ± standard error, the difference in integrity was evaluated, the mean between groups was analyzed for homogeneity of variance using One-Way ANOVA analysis, and the comparison between groups was performed in combination with Dunnett's test analysis. The multisample variance homogeneity test uses a level test, when p >0.05, the variance is homogeneous, the difference in mean between the multiple groups is tested using Dunnett's double sided T, when p <0.05, the variance is heterogeneous, the difference in mean between the multiple groups is tested using Dunnett T3.
⑥IC 50 Is calculated by the method of (a)
Calculating IC by non-linear regression fitting of parameters such as each dose and inhibition rate 50 。
(3) Experimental results:
the experimental results are shown in Table 4.
TABLE 4 Effect of Compounds 1-10 on LPS-activated BV-2 microglia release of NO experimental results
Note that: * P (P)<0.05,**P<0.01,***P<0.001 compared to LPS-induced group; ### P<0.001 compared to the control group.
As a result, it was found that the novel sesquiterpene coumarin compounds 2 (10. Mu.M, 30. Mu.M, 100. Mu.M), 3 (1. Mu.M, 10. Mu.M, 30. Mu.M, 100. Mu.M), 4 (1. Mu.M, 10. Mu.M, 30. Mu.M, 100. Mu.M), 6 (30. Mu.M, 100. Mu.M), 7 (10. Mu.M, 30. Mu.M, 100. Mu.M), 10 (10. Mu.M, 30. Mu.M, 100. Mu.M) prepared in examples 1 to 6 were able to significantly inhibit LPS-induced release of over-activated BV-2 microglial NO.
Claims (9)
1. Sesquiterpene coumarin compounds and pharmaceutically acceptable salts thereof in Ferula sinkiana having the structure:
2. a process for the preparation of a compound as claimed in claim 1 and pharmaceutically acceptable salts thereof, which comprises the steps of:
(1) Extracting root of Ferula sinkiana (Ferula sinkiangensis K.M.) with ethanol or methanol, and recovering extractive solution to obtain crude extract;
(2) Dissolving the ethanol crude extract obtained in the step (1) with water, sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol, and extracting each organic phase for 3 times to obtain extracts with different polarity positions;
(3) Separating the extract obtained in the step (2) by silica gel column chromatography, and gradient eluting with petroleum ether and ethyl acetate mixed solvent, or petroleum ether and acetone mixed solvent, or chloroform and acetone mixed solvent, or dichloromethane and acetone mixed solvent, or chloroform and methanol mixed solvent, or dichloromethane and methanol mixed solvent;
(4) Separating the fraction obtained in the step (3) by ODS column chromatography, and gradient eluting with methanol-water or acetonitrile-water mixed solvent as mobile phase;
(5) And (3) further separating the methanol-water or acetonitrile-water eluate obtained in the step (4) by HPLC, and performing gradient elution by using a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as a mobile phase to obtain the compounds 1-10.
3. The method according to claim 2, characterized in that: the extraction method in the step (1) comprises the steps of heating reflux ethanol extraction, heating reflux methanol extraction or heating ultrasonic extraction for 2-5 times, wherein the volume concentration of ethanol is 70-95%, the volume concentration of methanol is 60-90%, and the feed-liquid ratio is 1:8-1:20 g/mL.
4. The method according to claim 2, characterized in that: and (3) extracting the organic solvent in the step (2) for 3-5 times by using petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate and n-butanol respectively according to the volume ratio of the water phase to the organic phase of 1:1-1:5, and recovering the organic solvent under reduced pressure.
5. A method according to claim 2, characterized in that: the volume ratio of petroleum ether to ethyl acetate in the step (3) is 100:10-1:1, and the volume ratio of the mixed solvent of dichloromethane to acetone, the mixed solvent of chloroform and acetone, the mixed solvent of dichloromethane and methanol, or the mixed solvent of chloroform and methanol is 100:1-1:1.
6. A method according to claim 2, characterized in that: in the step (4), the volume ratio of the methanol to the water in the mixed solvent of the methanol and the water is 1:9-9:1, and the volume ratio of the acetonitrile to the water in the mixed solvent of the acetonitrile and the water is 1:9-9:1.
7. A method according to claim 2, characterized in that: the volume ratio of the methanol to the water in the mixed solvent of the methanol and the water in the step (5) is 4:6-9:1, and the volume ratio of the acetonitrile to the water in the mixed solvent of the acetonitrile and the water is 4:6-9:1.
8. A pharmaceutical composition comprising a compound of claim 1 and pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier.
9. Use of a compound according to claim 1, a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 8 for the manufacture of a medicament for the prevention or treatment of neurodegenerative diseases.
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| CN104447655A (en) * | 2014-12-05 | 2015-03-25 | 沈阳药科大学 | Novel sesquiterpene coumarins in ferula sinkiangensis K.M.Shen as well as preparation methods and medical applications of novel sesquiterpene coumarins |
| CN111646965A (en) * | 2020-06-03 | 2020-09-11 | 中国医学科学院药用植物研究所 | Compound Sinkiangel E and application thereof in preparation of antitumor drugs |
| CN113754625A (en) * | 2020-06-01 | 2021-12-07 | 沈阳药科大学 | Sesquiterpene coumarin compound and preparation method and application thereof |
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| CN104447655A (en) * | 2014-12-05 | 2015-03-25 | 沈阳药科大学 | Novel sesquiterpene coumarins in ferula sinkiangensis K.M.Shen as well as preparation methods and medical applications of novel sesquiterpene coumarins |
| CN113754625A (en) * | 2020-06-01 | 2021-12-07 | 沈阳药科大学 | Sesquiterpene coumarin compound and preparation method and application thereof |
| CN111646965A (en) * | 2020-06-03 | 2020-09-11 | 中国医学科学院药用植物研究所 | Compound Sinkiangel E and application thereof in preparation of antitumor drugs |
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