CN113717105A - Diterpene alkaloid type compound and extraction method and application thereof - Google Patents
Diterpene alkaloid type compound and extraction method and application thereof Download PDFInfo
- Publication number
- CN113717105A CN113717105A CN202110907232.5A CN202110907232A CN113717105A CN 113717105 A CN113717105 A CN 113717105A CN 202110907232 A CN202110907232 A CN 202110907232A CN 113717105 A CN113717105 A CN 113717105A
- Authority
- CN
- China
- Prior art keywords
- compound
- fraction
- volume ratio
- concentrating
- eluent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 150000001875 compounds Chemical class 0.000 title claims abstract description 114
- 229930002995 diterpene alkaloid Natural products 0.000 title claims abstract description 34
- 150000003800 diterpene alkaloid derivatives Chemical class 0.000 title claims abstract description 27
- 238000000605 extraction Methods 0.000 title claims abstract description 14
- 241000202296 Delphinium Species 0.000 claims abstract description 18
- 150000003839 salts Chemical class 0.000 claims abstract description 13
- 241001503951 Phoma Species 0.000 claims abstract description 11
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 11
- -1 diterpene alkaloid compound Chemical class 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 6
- 239000002260 anti-inflammatory agent Substances 0.000 claims abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 48
- 238000010828 elution Methods 0.000 claims description 34
- 239000003208 petroleum Substances 0.000 claims description 31
- 239000000243 solution Substances 0.000 claims description 30
- 239000003480 eluent Substances 0.000 claims description 29
- 238000010898 silica gel chromatography Methods 0.000 claims description 23
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 18
- 238000002953 preparative HPLC Methods 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 238000004587 chromatography analysis Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- 239000002994 raw material Substances 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 11
- 229940125904 compound 1 Drugs 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 241000196324 Embryophyta Species 0.000 claims description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 229940125782 compound 2 Drugs 0.000 claims description 9
- 229940125898 compound 5 Drugs 0.000 claims description 8
- 229910052739 hydrogen Inorganic materials 0.000 claims description 8
- 239000001257 hydrogen Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 230000002829 reductive effect Effects 0.000 claims description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 5
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000004480 active ingredient Substances 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000002791 soaking Methods 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 201000001320 Atherosclerosis Diseases 0.000 claims description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 2
- 239000002775 capsule Substances 0.000 claims description 2
- 239000003085 diluting agent Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 2
- 239000006188 syrup Substances 0.000 claims description 2
- 235000020357 syrup Nutrition 0.000 claims description 2
- 239000003826 tablet Substances 0.000 claims description 2
- 238000011282 treatment Methods 0.000 claims description 2
- 229940121363 anti-inflammatory agent Drugs 0.000 claims 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims 1
- 238000011321 prophylaxis Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 12
- 230000005764 inhibitory process Effects 0.000 abstract description 7
- 229940124599 anti-inflammatory drug Drugs 0.000 abstract description 6
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 48
- 210000004027 cell Anatomy 0.000 description 29
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 12
- 238000005160 1H NMR spectroscopy Methods 0.000 description 12
- 101000588302 Homo sapiens Nuclear factor erythroid 2-related factor 2 Proteins 0.000 description 11
- 102100031701 Nuclear factor erythroid 2-related factor 2 Human genes 0.000 description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 229940126214 compound 3 Drugs 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- 230000003110 anti-inflammatory effect Effects 0.000 description 7
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 102000043136 MAP kinase family Human genes 0.000 description 5
- 108091054455 MAP kinase family Proteins 0.000 description 5
- 230000005937 nuclear translocation Effects 0.000 description 5
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 description 4
- 101710089543 Nitric oxide synthase, inducible Proteins 0.000 description 4
- 102100038280 Prostaglandin G/H synthase 2 Human genes 0.000 description 4
- 108050003267 Prostaglandin G/H synthase 2 Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 210000001700 mitochondrial membrane Anatomy 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 102000003777 Interleukin-1 beta Human genes 0.000 description 3
- 108090000193 Interleukin-1 beta Proteins 0.000 description 3
- 102000004889 Interleukin-6 Human genes 0.000 description 3
- 108090001005 Interleukin-6 Proteins 0.000 description 3
- 102000003945 NF-kappa B Human genes 0.000 description 3
- 108010057466 NF-kappa B Proteins 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 102000005747 Transcription Factor RelA Human genes 0.000 description 3
- 108010031154 Transcription Factor RelA Proteins 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 229930013930 alkaloid Natural products 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 238000013375 chromatographic separation Methods 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 241000227129 Aconitum Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241001254651 Brachycarpus Species 0.000 description 2
- 241000743774 Brachypodium Species 0.000 description 2
- 108091006146 Channels Proteins 0.000 description 2
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 2
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 241001148062 Photorhabdus Species 0.000 description 2
- 241000218201 Ranunculaceae Species 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 150000004141 diterpene derivatives Chemical class 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- LOGFVTREOLYCPF-KXNHARMFSA-N (2s,3r)-2-[[(2r)-1-[(2s)-2,6-diaminohexanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxybutanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-KXNHARMFSA-N 0.000 description 1
- 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
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical class N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 241000446816 Consolida hispanica Species 0.000 description 1
- 241001586342 Delphinium forrestii var. viride Species 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 102000018745 NF-KappaB Inhibitor alpha Human genes 0.000 description 1
- 108010052419 NF-KappaB Inhibitor alpha Proteins 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241001674048 Phthiraptera Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000000956 alloy Substances 0.000 description 1
- 229910045601 alloy Inorganic materials 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000005956 cytoplasmic translocation Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000006303 immediate early viral mRNA transcription Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 229940100601 interleukin-6 Drugs 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- GRVDJDISBSALJP-UHFFFAOYSA-N methyloxidanyl Chemical group [O]C GRVDJDISBSALJP-UHFFFAOYSA-N 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000005311 nuclear magnetism Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 208000004371 toothache Diseases 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- 230000006433 tumor necrosis factor production Effects 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/22—Bridged ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Rheumatology (AREA)
- Heart & Thoracic Surgery (AREA)
- Alternative & Traditional Medicine (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Mycology (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Pain & Pain Management (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Physical Education & Sports Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the field of traditional Chinese medicine extraction, and particularly relates to a diterpene alkaloid type compound separated from delphinium glabrum, an extraction method thereof and application thereof in preparing an anti-inflammatory drug. The compound is a compound shown as general formulas (I) and (II) or an isomer of the compound, or a pharmaceutically acceptable salt of the compound; the substituents are as described in the specification. The diterpene alkaloid compound separated from the delphinium phoma, the isomer of the compound, the pharmaceutically acceptable salt of the compound or the pharmaceutical composition containing the compound have the inhibition effect on NO generation in RAW264.7 cells induced by LPS, and can be used for preparing anti-inflammatory drugs.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicine extraction, and particularly relates to a diterpene alkaloid type compound separated from delphinium glabrum, an extraction method thereof and application thereof in preparing an anti-inflammatory drug.
Background
Delphinium gordonii (Delphinium forrestii var. viride) is a plant of the genus Delphinium of the family ranunculaceae, and is widely distributed and mainly produced in tibetan, Sichuan, Yunnan, etc. In 1979, the Chinese plant journal records that the plant has the folk action of medicine and is mainly used for treating traumatic injury, rheumatism, toothache, enteritis and the like, and is also used as a pesticide for killing lice, mosquitoes and fly larvae. In recent years, researchers at home and abroad have successively reported chemical components of diterpene alkaloids in plants of genera such as Delphinium and Aconitum of Ranunculaceae and corresponding pharmacological actions thereof. At present, most diterpene alkaloid compounds separated from delphinium are substituted by groups with simpler structures such as hydroxyl, methoxyl, acetyl and the like. The compounds isolated from delphinium with a short phoma part contain groups with a more complex structure, such as 2- (2-methyl-4-oxoquinazolin-3 (4H) -yl) benzoyloxy. Known Diterpene Alkaloids can be generally considered to have one or more of the following effects, for example Anti-Inflammatory effects (V.N.Yu, T.N.Povetieva, N.I.Suslov, G.N.Zyuz' Kov, A.V.Krapivin, Anti-Inflammatory Activity of Diperepen Alkaloids from Aconitum baikalense, Bulletin of Experimental Biology and Medicinal 2014,156(5) 665. 668.), analgesic effects (Wang, D.P., Lou, H.Y., Huang, Hao, X.J., Liang, G.Y., Yang, A. vary of Experimental peptides, G.2012, 4444. C.J., C.F.J., C.J., C.F.C., C.J., C.F.C.J., C.C.C.C.C.C.a; K.Wada; takahashi; T.Mori; kawahara, I.Kashiwakura, dominant effects of novel derivative analogs on molecular growth, Investigational New Drugs 2009,27(2) 111-.
A.H.
Ulubelen, H.K.Desai, S.W.Pelletier, Norterpendant and dipendant alloys from Turkish Consolida orientalis, Journal of Natural Products 2001,64(6) 787-. At present, the research on the anti-inflammatory activity of diterpenoid alkaloids only stays at the action of diterpenoid alkaloids, and no further discussion on the anti-inflammatory mechanism is carried out. In order to maximize the medicinal value of delphinium photorhabdus, further research on the systematic component of the dried whole plant of delphinium photorhabdus is now required.
Disclosure of Invention
The invention aims at providing a diterpene alkaloid type compound.
The second purpose of the invention is to provide a method for extracting diterpene alkaloid type compounds.
It is a third object of the present invention to provide a pharmaceutical composition comprising a diterpene alkaloid type compound.
The fourth purpose of the invention is to provide the application of the diterpene alkaloid type compound or the isomer of the compound, the pharmaceutically acceptable salt of the compound or the pharmaceutical composition containing the compound in the preparation of anti-inflammatory drugs.
To achieve the above object, the technical solution of the present invention is summarized as follows:
a diterpene alkaloid type compound which is a compound shown as general formulas (I) and (II) or an isomer of the compound, or a pharmaceutically acceptable salt of the compound;
wherein: r1，R2，R3,R4,R5Is hydrogen, hydroxy, methoxy, acetoxy, anthranilic acid group or 2- (2-methyl-4-oxoquinazolin-3 (4H) -yl) benzoyloxy.
The compound is shown in the following structural formula, isomers of the compound and pharmaceutically acceptable salts of the compound;
the pharmaceutically acceptable salt comprises sodium salt, potassium salt, ammonia salt, hydrochloride and sulfate.
The isomers include: optical isomers, cis-trans isomers, racemates and mixtures thereof.
The extraction method of diterpene alkaloid type compounds comprises the steps of extracting the whole plant of the short-pedicel delphinium which is taken as a raw material by ethanol to obtain the diterpene alkaloid type compounds.
The method specifically comprises the following steps:
(1) taking the dried whole plant of the delphinium glabrum with short stalk as a raw material, crushing, adding ethanol with the volume fraction of 70% -98% which is 0.1-1 mass time of the raw material, soaking for 1-5 times at room temperature for 5-10 days each time, and concentrating the extracting solution under reduced pressure to obtain an extract;
(2) dispersing the total extract into water 2-6 times of the total extract by mass, adjusting the pH of the suspension to 2-3 with hydrochloric acid solution, sequentially extracting with petroleum ether and ethyl acetate for 2-5 times, respectively, adjusting the pH of the suspension to 9-11 with ammonia water, and extracting the suspension with dichloromethane for 2-5 times to obtain dichloromethane layer extract;
(3) separating the dichloromethane layer extraction concentrated solution by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent in a volume ratio of 100:1: 0.1-0: 1:0.1, and collecting fractions in volume ratios of 50:1:0.1, 15:1:0.1 and 10:1:0.1, which are sequentially marked as D2, D3 and D5;
(4) fractions D2, D3 and D5 were concentrated and further purified to give compounds 1-6.
The specific separation and purification processes of the fractions D2, D3 and D5 in the step (4) are as follows:
concentrating the fraction D2, separating by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at a volume ratio of 100:1: 0.1-0: 1:0.1, and collecting fractions at a volume ratio of 10:1:0.1 and 8:1:0.1, which are respectively marked as D26 and D27;
concentrating the fraction D26, separating by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at a volume ratio of 50:1: 0.1-0: 1:0.1, and collecting a fraction at a volume ratio of 5:1, and recording the fraction as D264;
concentrating fraction D264, performing preparative HPLC chromatography, and purifying with methanol-water as mobile phase at volume ratio of 75:25 to obtain compounds 3 and 4;
concentrating the fraction D27, separating by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at a volume ratio of 20:1: 0.1-0: 1:0.1, and collecting a fraction at a volume ratio of 5:1:0.1, and marking the fraction as D274;
concentrating fraction D274, subjecting to preparative HPLC chromatography with methanol-water at a volume ratio of 75:25 as mobile phase, and purifying to obtain compound 2;
concentrating the fraction D3, separating by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at a volume ratio of 100:1: 0.1-0: 1:0.1, and collecting fractions at a volume ratio of 5:1:0.1 and 3:1:0.1, which are respectively marked as D37 and D39;
concentrating the fraction D37, separating by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at a volume ratio of 20:1: 0.1-0: 1:0.1, and collecting a fraction at a volume ratio of 5:1:0.1, wherein the fraction is marked as D374;
concentrating fraction D374, performing preparative HPLC chromatography, and purifying with methanol-water as mobile phase at volume ratio of 80:20 to obtain compound 6;
concentrating the fraction D39, separating by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at a volume ratio of 20:1: 0.1-0: 1:0.1, and collecting a fraction at a volume ratio of 5:1:0.1, wherein the fraction is marked as D394;
concentrating fraction D394, separating by silica gel column chromatography, performing gradient elution with petroleum ether-acetone-diethylamine as an eluent at a volume ratio of 10:1: 0.1-0: 1:0.1, and collecting fraction D3944 at a volume ratio of 5:1: 0.1;
concentrating fraction D3944, performing preparative HPLC chromatography, and purifying with methanol-water as mobile phase at volume ratio of 75:25 to obtain compound 1;
concentrating fraction D5, eluting with 40-90% methanol-water as eluent by reversed-phase ODS column chromatography, and collecting 60% fraction D52;
fraction D52 was concentrated and purified by preparative HPLC chromatography on methanol-water as the mobile phase at a volume ratio of 75:25 to give compound 5.
A pharmaceutical composition comprising one or more of said diterpene alkaloid type compound, an isomer of said compound, a pharmaceutically acceptable salt of said compound.
A pharmaceutical preparation comprises active ingredient and one or combination of pharmaceutically acceptable carrier, excipient, and diluent; wherein the active ingredient is the compound or the composition.
The administration route of the preparation is oral administration or injection administration, and the preparation form is as follows: tablet, capsule, powder, syrup or injection.
The application of diterpene alkaloid type compounds or pharmaceutical compositions or pharmaceutical preparations, and the application of the diterpene alkaloid type compounds, the compositions and the pharmaceutical preparations in the preparation of anti-inflammatory drugs.
The diterpene alkaloid type compound, the isomer of the compound, the pharmaceutically acceptable salt of the compound or the pharmaceutical composition has the inhibiting effect on NO generation in RAW264.7 cells induced by LPS, and can be applied to the preparation of anti-inflammatory drugs.
The diterpene alkaloid type compound, the composition and the pharmaceutical preparation are applied to the medicines for preventing and treating rheumatoid arthritis, inflammatory bowel disease or atherosclerosis.
The invention has the advantages that:
the diterpene alkaloid type compound obtained by the invention is extracted from the phoma brachycarpus delphinium, and the structure of the compound is confirmed by means of nuclear magnetism, mass spectrum and the like, so that part of diterpene alkaloids separated from the phoma brachycarpus delphinium contain groups with more complex structures such as 2- (2-methyl-4-oxo quinazoline-3 (4H) -yl) benzoyloxy and the like; meanwhile, the obtained diterpene alkaloid type compound or isomer thereof or pharmaceutically acceptable salt or pharmaceutical composition thereof has an inhibition effect on NO generation in RAW264.7 cells induced by LPS, and plays an anti-inflammatory effect by inhibiting the generation of ROS and regulating NF-kappa B, MAPK and Nrf2 signal pathways, so that the diterpene alkaloid type compound or isomer thereof or pharmaceutically acceptable salt or pharmaceutical composition thereof is applied to the preparation of medicines for treating inflammation. The invention further enriches the structural diversity of the phoma short-distance delphinium active substance, lays a foundation for the relevant biological activity test of the subsequently obtained monomer compound, provides an active lead compound for the development of new drugs, and simultaneously provides a theoretical basis for the deep research and development of the phoma short-distance delphinium medicinal material.
Drawings
FIG. 1 is a graph of the effect of varying concentrations of Compound 6 on the viability of RAW264.7 cells in the presence or absence of LPS.
FIG. 2 is a graph showing the effect of Compound 6 on the expression of inflammatory cytokines secreted by LPS-induced macrophage RAW 264.7. (a) (b) (c) schematic ELISA kit for detecting the expression of inflammatory factors IL-1 beta, TNF-alpha and IL-6 in cell supernatant.###p < 0.001vs control group<0.001vs LPS group.
FIG. 3 is a graph showing the effect of Compound 6 on the expression of iNOS, COX-2, NF- κ B p65, MAPKs, and Nrf2 signaling pathway-related proteins. (a) (b) (c) is a graph showing the expression of the inflammatory factors iNOS and COX-2 protein levels in the western-blot detection and corresponding gray level statistical graphs. (d) The expression of p-p65, p65, p-I κ B α and I κ B α detected by western-blot is shown in the figure, and the grayscale analysis of the figure (e) shows the relative expression levels of p-p65 and p-I κ B α proteins. (g) The illustrated western-blot assays for p-p38, p38, p-ERK, N-terminal-,Expression of ERK, p-JNK, (f) Gray scale analysis of graph (g), showing the relative expression levels of p-p38, p-ERK, and p-JNK proteins. (h) (i) graphical representation of western-blot detection of protein expression of keap-1, Nrf2, HO-1 and corresponding grey scale statistical plots.###p < 0.001vs control group<0.01 vs. LPS panel,. SP<0.001vs LPS group.
FIG. 4 is a graph showing the effect of Compound 6 on nuclear translocation of NF-. kappa. B p65 and Nrf 2. (a) Illustrating the inhibitory effect of compound 6 on LPS-induced nuclear translocation of NF- κ B p65, blue fluorescence is for DAPI-labeled nuclei and red fluorescence is for NF- κ B p 65. (b) Illustrating the promoting effect of compound 6 on LPS-induced nuclear translocation of Nfr2, blue fluorescence is DAPI-labeled nucleus and red fluorescence is Nrf 2.
FIG. 5 is a graph showing the effect of Compound 6 on the level of active oxygen and mitochondrial membrane potential. (a) In the figure, in addition to the control and LPS groups, cells were treated with different concentrations of Compound 6 for 3 hours before addition of LPS for 12 hours. Cells were collected and stained by DCFH-DA and images were obtained by fluorescence microscopy. (b) Cells were treated as described above and the fluorescence intensity of DCFH-DA was measured by flow cytometry. (c) DCFH-DA fluorescence intensity calculated by Flow jo software. (d) (e) graph by flow cytometry detection of mitochondrial membrane potential level, and calculation of JC-1 two states of cell proportion.###p < 0.001vs control group<0.01 vs. LPS panel,. SP<0.001vs LPS group.
Detailed Description
The technical solution of the present invention will be further described with reference to specific examples.
Example 1
The extraction method of diterpene alkaloid type compounds 1-6 in short-stalked delphinium comprises the following steps:
(1) the phoma brachypodium is used as a raw material, the total dry weight of the phoma brachypodium is 10.0kg, 95% ethanol reagent (4L) which is 0.4 times of the raw material by mass is added, and the raw material is soaked for 3 times at room temperature, and each time lasts for 7 days. Concentrating the extractive solution under reduced pressure to obtain extract (360 g).
(2) Dispersing the total extract into water (720mL) 2 times by mass, adjusting the pH of the suspension to 2 with hydrochloric acid solution, sequentially extracting with petroleum ether and ethyl acetate for 3 times, adjusting the pH of the suspension to 10 with ammonia water, and extracting with dichloromethane for 3 times to obtain dichloromethane layer extract (75 g).
(3) Carrying out dry sample mixing on the dichloromethane layer extraction concentrated solution, carrying out chromatographic separation on the dichloromethane layer extraction concentrated solution by using a 200-mesh 300-mesh silica gel column, carrying out gradient elution by using petroleum ether-acetone-diethylamine as an eluent in a volume ratio of 100:1: 0.1-0: 1:0.1 at an elution speed of 1.2mL/min, and collecting fractions in volume ratios of 50:1:0.1, 15:1:0.1 and 10:1:0.1, which are sequentially marked as D2, D3 and D5;
(4) fractions D2, D3 and D5 were concentrated under reduced pressure and further purified to give 27.2mg of compound 1, 55mg of compound 2, 235mg of compound 3, 53mg of compound 4, 32.9mg of compound 5 and 10.7mg of compound 6. The specific separation and purification process comprises the following steps:
concentrating the fraction D2 under reduced pressure to obtain 22.6g of concentrated solution, performing dry sample mixing, performing 200-mesh 300-mesh silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as eluent at the volume ratio of 50:1:0.1, 15:1:0.1, 10:1:0.1,8:1:0.1, 5:1:0.1 and 3:1:0.1, wherein the elution flow rate is 2.5mL/min, and collecting fractions at the volume ratio of 10:1:0.1 and 8:1:0.1, which are respectively marked as D26 and D27;
concentrating the fraction D26 under reduced pressure to obtain 2.6g of concentrated solution, mixing the sample with a dry method, separating by 200-mesh 300-mesh silica gel column chromatography, carrying out gradient elution by using petroleum ether-acetone-diethylamine as an eluent at the volume ratio of 50:1:0.1, 40:1:0.1, 30:1:0.1, 20:1:0.1, 15:1:0.1, 10:1:0.1, 5:1:0.1 and 2:1:0.1, wherein the elution flow rate is 2.5mL/min, and collecting the fraction with the volume ratio of 5:1:0.1, and marking as D264;
concentrating fraction D264 to obtain 1.7g, and purifying by preparative HPLC chromatography on SHIMADZU5 μm C1820 × 250mm with methanol-water as mobile phase at 75:25 volume ratio and flow rate of 8mL/min to obtain 235mg of compound 3 and 53mg of compound 4;
concentrating the fraction D27 to obtain 1.9g of concentrated solution, mixing the concentrated solution with a dry method, separating by 200-mesh 300-mesh silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at the volume ratio of 50:1:0.1, 40:1:0.1, 30:1:0.1, 20:1:0.1, 15:1:0.1, 10:1:0.1, 5:1:0.1 and 2:1:0.1, wherein the elution flow rate is 2.5mL/min, and collecting the fraction with the volume ratio of 5:1:0.1, which is marked as D274;
concentrating fraction D274 to obtain 335mg, and purifying by preparative HPLC chromatography on SHIMADZU5 μm C1820 × 250mm with methanol-water as mobile phase at volume ratio of 75:25 and flow rate of 8mL/min to obtain 55mg of Compound 2;
concentrating the fraction D3 to obtain 3g of concentrated solution, mixing the concentrated solution with a dry method, separating by silica gel column chromatography with 200-300 meshes, performing gradient elution by using petroleum ether-acetone-diethylamine as eluent at the volume ratio of 50:1:0.1, 30:1:0.1, 15:1:0.1, 10:1:0.1, 5:1:0.1 and 3:1:0.1, wherein the elution flow rate is 2.5mL/min, and collecting fractions with the volume ratio of 5:1:0.1 and 3:1:0.1 respectively, namely D37 and D39;
concentrating the fraction D37 to obtain 2.1g of concentrated solution, mixing the concentrated solution with a dry method, separating by 200-mesh 300-mesh silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at the volume ratio of 50:1:0.1, 40:1:0.1, 30:1:0.1, 20:1:0.1, 15:1:0.1, 10:1:0.1, 5:1:0.1 and 2:1:0.1, wherein the elution flow rate is 2.5mL/min, and collecting the fraction with the volume ratio of 5:1:0.1 and marking as D374;
concentrating fraction D374 to obtain 40mg, and purifying by preparative HPLC chromatography on SHIMADZU5 μm C1820 × 250mm with methanol-water as mobile phase at volume ratio of 75:25 and flow rate of 8mL/min to obtain 10.7mg of compound 6;
concentrating the fraction D39 to obtain 1.1g of concentrated solution, carrying out dry-method sample mixing, carrying out chromatographic separation by using a 200-mesh 300-mesh silica gel column, carrying out gradient elution by using petroleum ether-acetone-diethylamine as an eluent in a volume ratio of 20:1:0.1, 15:1:0.1, 10:1:0.11, 8:1:0.1, 5:1:0.1 and an elution flow rate of 2mL/min, and collecting the fraction in a volume ratio of 5:1:0.1, wherein the fraction is marked as D394;
concentrating the fraction D394 to obtain 1.1g of concentrated solution, mixing the concentrated solution with a dry method, performing chromatographic separation by using a 200-mesh 300-mesh silica gel column, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at the volume ratio of 15:1:0.1, 10:1:0.11, 8:1:0.1, 5:1:0.1 and 2:1:0.1, wherein the elution flow rate is 2mL/min, and collecting the fraction with the volume ratio of 5:1:0.1, which is recorded as D3944;
fraction D3944 was concentrated to give 320mg, which was subjected to preparative HPLC chromatography on a column of SHIMADZU 5. mu. m C1820X 250mm, purified with methanol-water as the mobile phase at a volume ratio of 75:25, at a flow rate of 8mL/min to give 27.2mg of Compound 1;
fraction D5 was concentrated to give 10.5g, which was eluted by reverse phase ODS column chromatography using 40%, 60%, 70%, 90% by volume methanol-water as eluent, and the fraction collected was 60% by volume and designated as D52.
Fraction D52 was concentrated to give a preparative HPLC chromatogram on a column at SHIMADZU5 μm C1820X 250mm, purified with methanol-water as the mobile phase at a volume ratio of 75:25 to give 32.9mg of Compound 5.
The physicochemical and constants of the compounds are as follows:
compound 1: white amorphous powder; HRESIMS M/z 670.3463[ M + H ]]+(calcd for C39H48N3O7670.3487), determination of the formula C for Compound 139H47N3O7；
-202.0(c 0.3,MeOH)；1H-NMR(600MHz,CDCl3) And13C-NMR(150MHz,CDCl3) The data are shown in Table 1.
Compound 2: white amorphous powder; HRESIMS M/z 684.3623[ M + H ]]+(calcd for C40H50N3O7684.3643), determination of the formula C for Compound 240H49N3O7；
-143.0(c 0.3,MeOH)；1H-NMR(600MHz,CDCl3) And13C-NMR(150MHz,CDCl3) The data are shown in Table 2.
Compound 3: white amorphous powder; HRESIMS M/z 698.3767[ M + H ]]+(calcd for C41H51N3O7698.3799), determining the formula of Compound 3 as C41H50N3O7；
-331.5(c 0.4,MeOH)；1H-NMR(600MHz,CDCl3) And13C-NMR(150MHz,CDCl3) The data are shown in Table 3.
Compound 4: white amorphous powder; HRESIMS M/z 569.3210[ M + H ]]+(calcd for C32H45N2O7569.3221), determination of the formula C for Compound 432H44N3O7；
-332.0(c 0.2,MeOH)；1H-NMR(600MHz,CDCl3) And13C-NMR(150MHz,CDCl3) The data are shown in Table 4.
Compound 5: white amorphous powder; HRESIMS M/z 436.3045[ M + H ]]+(calcd for C25H42NO5436.3058), determination of the formula C for Compound 525H41NO5；
-141.0(c 0.5,MeOH)；1H-NMR(600MHz,CDCl3) And13C-NMR(150MHz,CDCl3) The data are shown in Table 5.
Compound 6: white amorphous powder; HRESIMS M/z 316.2261[ M + H ]]+(calcd for C20H30NO2316.2271), determination of the formula C for Compound 620H29NO2；
-468.5(c 0.1,MeOH)；1H-NMR(600MHz,CDCl3) And13C-NMR(150MHz,CDCl3) The data are shown in Table 6.
TABLE 1 carbon and hydrogen spectra data for Compound 1
Note:1H-NMR，600MHz，CDCl3；13C-NMR，150MHz，CDCl3。
TABLE 2 carbon and hydrogen spectra data for Compound 2
Note:1H-NMR，600MHz，CDCl3；13C-NMR，150MHz，CDCl3。
TABLE 3 carbon and hydrogen spectra data for Compound 3
Note:1H-NMR，600MHz，CDCl3；13C-NMR，150MHz，CDCl3。
TABLE 4 carbon and hydrogen spectra data for Compound 4
Note:1H-NMR，600MHz，CDCl3；13C-NMR，150MHz，CDCl3。
TABLE 5 carbon and hydrogen spectra data for Compound 5
Note:1H-NMR，600 MHz，CDCl3；13C-NMR，150 MHz，CDCl3。
TABLE 6 carbon and hydrogen spectra data for Compound 6
Note:1H-NMR，600 MHz，CDCl3；13C-NMR，150 MHz，CDCl3。
its structure, compound 1, 2, 3,4, 5 and 6 structures, was identified by physicochemical constants and modern spectroscopic means (HRESIMS and NMR), in combination with literature-relevant data, as follows:
example 2
(1) Taking 15.0kg of phoma brachypus as a raw material, adding a 95% ethanol reagent (4.5L) which is 0.3 times of the raw material by mass, and soaking for 3 times at room temperature for 6 days each time. Concentrating the extractive solution under reduced pressure to obtain extract (400g),
(2) dispersing the total extract into 2 mass times of water (800mL), adjusting the pH of the suspension to 2.5 by using a hydrochloric acid solution, sequentially extracting for 3 times by using petroleum ether ethyl acetate, adjusting the pH of the suspension to 10.5 by using ammonia water, and extracting by using dichloromethane to obtain a dichloromethane layer extract (81 g).
(3) Separating the dichloromethane layer extraction concentrated solution by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent in a volume ratio of 100:1: 0.1-0: 1:0.1, and collecting fractions in volume ratios of 50:1:0.1, 15:1:0.1 and 10:1:0.1, which are sequentially marked as D2, D3 and D5;
(4) fractions D2, D3 and D5 were concentrated and further purified to yield 29.2mg of compound 1, 61mg of compound 2, 272mg of compound 3, 61mg of compound 4, 34.9mg of compound 5 and 10.8mg of compound 6.
Example 3
Taking 12.0kg of phoma brachypus as a raw material, adding a 95% ethanol reagent (2.4L) which is 0.2 times of the raw material by mass, and soaking for 7 days at room temperature for 3 times. Concentrating the extractive solution under reduced pressure to obtain extract (350 g).
(2) Dispersing the total extract into water (700mL) with the mass of 2 times, adjusting the pH of the suspension to 3 by using a hydrochloric acid solution, sequentially extracting for 3 times by using petroleum ether ethyl acetate, adjusting the pH of the suspension to 11 by using ammonia water, and extracting by using dichloromethane to obtain a dichloromethane layer extract (77 g).
(3) Separating the dichloromethane layer extraction concentrated solution by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent in a volume ratio of 100:1: 0.1-0: 1:0.1, and collecting fractions in volume ratios of 50:1:0.1, 15:1:0.1 and 10:1:0.1, which are sequentially marked as D2, D3 and D5;
(4) fractions D2, D3, and D5 were concentrated and further purified to give 28.2mg of compound 1, 60mg of compound 2, 265mg of compound 3, 60mg of compound 4, 34.1mg of compound 5, and 10.2mg of compound 6.
Example 4
Research on influence of product of the invention on NO generation of RAW264.7 cells
RAW264.7 cells were seeded in a 96-well plate, treated with 20. mu.M of the compound prepared in example 1 above for 3 hours, and then incubated with LPS (0.5. mu.g/mL) for 24 hours. DMSO with or without LPS was treated as vehicle control or model. Nitrite accumulation in the medium was measured using a Griess reagent at 540nm with a microplate reader. The inhibition rate (%) of the compound-treated group was calculated to evaluate NO inhibitory activity.
TABLE 1 table of% values of inhibition rate of compound on RAW264.7 cell NO production
| Compound (I) | Inhibition ratio (%) |
| |
12.44 |
| |
37.20 |
| Compound 3 | 10.30 |
| |
99.14 |
From the above table, it can be found that compound 6 has a strong inhibitory activity.
Further research on the anti-inflammatory effect and mechanism of the compound 6 in RAW264.7 cells
(1) CCK8 method for detecting influence of compound 6 on cell survival rate
RAW264.7 cells in logarithmic growth phase were seeded at 25000/well in 96-well plates and cultured for 12 h. Cells were treated with different concentrations of compound 6(100, 50, 25, 12.5, 6.25 and 3.125. mu. mol/L). Wells with the corresponding volume of DMSO added were used as blanks. After 24h, the culture medium is discarded, 100 mu L of culture medium containing 10% CCK8 is added into each well, and after 40min of culture, the OD value of each well at 450nm is detected by an enzyme-labeling instrument to calculate the cell survival rate of the experimental group (the cell survival rate of the blank control group is 100%). The results are shown in FIG. 1:
as can be seen from fig. 1, compound 6 had no significant effect on the survival of RAW264.7 cells.
(2) ELISA method for detecting inhibition of Interleukin 1 beta (IL-1 beta), Interleukin 6(IL-6) and TNF-alpha production by Compound 6
RAW264.7 cells in logarithmic growth phase are inoculated in a 96-well plate for 12 h. The experimental groups were treated with different concentrations of compound 6(5, 10, 20 and 40. mu. mol/L). After 3h, LPS was added to the model and experimental groups to a final concentration of 0.5. mu.g/mL, while an equal volume of DMEM medium was added to the blank group and incubation was continued for 24 h. Cell supernatants were collected from each well, and OD values of each well at 450nm were measured with an ELISA reader according to the ELISA kit operating manual, and the cytokine content of each group was calculated. The results are shown in FIG. 2:
as can be seen from fig. 2, compound 6 can inhibit the level of cytokine secreted by macrophage RAW264.7 induced by LPS, and its inhibitory effect is dose-dependent.
(3) Western Blot for detecting inhibition effect of compound 6 on inflammation-related protein, NF-kB, MAPK, Nrf2/HO-1 channel expression
RAW264.7 cells were plated in 96-well plates for 12 h. Difference in experimental groupConcentration Compound 6(5, 10 and 20. mu. mol. L)-1) And (6) processing. After 3h, LPS was added to the model and experimental groups to a final concentration of 0.5. mu.g/mL, while an equal volume of DMEM medium was added to the blank group and incubation was continued for 24 h. The culture medium is discarded, the cells are collected, the cells are cracked by RIPA, a BCA quantification Western Blot experiment is carried out, the expression conditions of iNOS, COX-2, NF-kB, MAPKs and Nrf2 signal channel related proteins after the treatment of different concentrations of compound 6 are detected, and the exposure bands are subjected to gray scale analysis by a Gel-Pro analyzer. The results are shown in FIG. 3.
As shown in FIG. 3, compound 6 can exert anti-inflammatory effects by decreasing the expression of iNOS, COX-2, NF-. kappa.B and MAPKs pathways and by up-regulating the Nrf2 signaling pathway and downstream related proteins.
(4) Immunofluorescence method for detecting influence of compound 6 on nuclear translocation of NF-KB p65 and Nrf2
RAW264.7 cells were seeded into 8X 10 wells4Cells were cultured in 24-well plates for 12h, then pretreated with DMSO or 6 (20. mu.M) for 2h, and stimulated with 0.5. mu.g/mL LPS for 12 h. Cells were fixed with freshly prepared 4% paraformaldehyde for 10min, washed 3 times with PBS, and then permeabilized with 0.2% Triton X-100 for 10 min. After blocking with 5% Bovine Serum Albumin (BSA) for 1h at room temperature, add 1: NF-. kappa. B p65 (Proteintetech, Cat #10745-1-AP) antibody or Nrf2 (Proteintetech, Cat #16396-1-AP) was diluted 400 and incubated at 4 ℃ overnight. After washing by PBS, the mixture was washed at room temperature and in the dark at a 1: a dilution of 400 was incubated for 1h with the addition of secondary antibody. Finally, staining with DAPI was performed at room temperature and in the dark for 5 min. Then, PBS was washed and an anti-fluorescence quenching mounting solution was added, and observed and photographed under an immunofluorescence microscope, and an image was obtained. The results are shown in FIG. 4.
As can be seen from FIG. 4, compound 6 (20. mu.M) can significantly inhibit cytoplasmic translocation of the p65 subunit of NF-. kappa.B from the nucleus to LPS-activated RAW264.7 cells, while compound 6 (20. mu.M) can promote LPS-induced nuclear translocation of Nrf 2.
(5) Effect of Compound 6 on reactive oxygen levels and mitochondrial Membrane potential levels
LPS-induced RAW264.7 cells were collected and centrifuged, after which the cells were washed three times with PBS. Then, according to the kit instructions, adding DCFH-DA or JC-1 fluorescent dye, at 37 ℃ for 30min, collecting the stained cells and using the flow cytometry or fluorescence microscope for detection analysis, the results are shown in figure 5.
As can be seen from fig. 5, compound 6 inhibited active oxygen and mitochondrial membrane potential levels dose-dependently.
Claims (10)
1. A diterpene alkaloid type compound, which is a compound shown in general formulas (I) and (II) or an isomer of the compound, or a pharmaceutically acceptable salt of the compound;
wherein: r1，R2，R3,R4,R5Is hydrogen, hydroxy, methoxy, acetoxy, anthranilic acid group or 2- (2-methyl-4-oxoquinazolin-3 (4H) -yl) benzoyloxy.
2. The diterpene alkaloid type compound according to claim 1, wherein the compound is represented by the following structural formula, and isomers of the compound, pharmaceutically acceptable salts of the compound;
3. the method for extracting a diterpene alkaloid compound according to claim 1, wherein the diterpene alkaloid compound according to claim 1 is obtained by extracting the whole plant of the phoma brachycanthum's dried herb with ethanol.
4. The method for extracting a diterpene alkaloid type compound according to claim 3,
(1) taking the dried whole plant of the delphinium glabrum with short stalk as a raw material, crushing, adding ethanol with the volume fraction of 70% -98% which is 0.1-1 mass time of the raw material, soaking for 1-5 times at room temperature for 5-10 days each time, and concentrating the extracting solution under reduced pressure to obtain an extract;
(2) dispersing the total extract into water 2-6 times of the total extract by mass, adjusting the pH of the suspension to 2-3 with hydrochloric acid solution, sequentially extracting with petroleum ether and ethyl acetate for 2-5 times, respectively, adjusting the pH of the suspension to 9-11 with ammonia water, and extracting the suspension with dichloromethane for 2-5 times to obtain dichloromethane layer extract;
(3) separating the dichloromethane layer extraction concentrated solution by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent in a volume ratio of 100:1: 0.1-0: 1:0.1, and collecting fractions in volume ratios of 50:1:0.1, 15:1:0.1 and 10:1:0.1, which are sequentially marked as D2, D3 and D5;
(4) fractions D2, D3 and D5 were concentrated and further purified to give compounds 1-6.
5. The method for extracting diterpene alkaloid type compounds according to claim 3, wherein the specific separation and purification processes of the fractions D2, D3 and D5 in the step (4) are as follows:
concentrating the fraction D2, separating by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at a volume ratio of 100:1: 0.1-0: 1:0.1, and collecting fractions at a volume ratio of 10:1:0.1 and 8:1:0.1, which are respectively marked as D26 and D27;
concentrating the fraction D26, separating by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at a volume ratio of 50:1: 0.1-0: 1:0.1, and collecting a fraction at a volume ratio of 5:1, and recording the fraction as D264;
concentrating fraction D264, performing preparative HPLC chromatography, and purifying with methanol-water as mobile phase at volume ratio of 75:25 to obtain compounds 3 and 4;
concentrating the fraction D27, separating by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at a volume ratio of 20:1: 0.1-0: 1:0.1, and collecting a fraction at a volume ratio of 5:1:0.1, and marking the fraction as D274;
concentrating fraction D274, subjecting to preparative HPLC chromatography with methanol-water at a volume ratio of 75:25 as mobile phase, and purifying to obtain compound 2;
concentrating the fraction D3, separating by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at a volume ratio of 100:1: 0.1-0: 1:0.1, and collecting fractions at a volume ratio of 5:1:0.1 and 3:1:0.1, which are respectively marked as D37 and D39;
concentrating the fraction D37, separating by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at a volume ratio of 20:1: 0.1-0: 1:0.1, and collecting a fraction at a volume ratio of 5:1:0.1, wherein the fraction is marked as D374;
concentrating fraction D374, performing preparative HPLC chromatography, and purifying with methanol-water as mobile phase at volume ratio of 80:20 to obtain compound 6;
concentrating the fraction D39, separating by silica gel column chromatography, performing gradient elution by using petroleum ether-acetone-diethylamine as an eluent at a volume ratio of 20:1: 0.1-0: 1:0.1, and collecting a fraction at a volume ratio of 5:1:0.1, wherein the fraction is marked as D394;
concentrating fraction D394, separating by silica gel column chromatography, performing gradient elution with petroleum ether-acetone-diethylamine as an eluent at a volume ratio of 10:1: 0.1-0: 1:0.1, and collecting fraction D3944 at a volume ratio of 5:1: 0.1;
concentrating fraction D3944, performing preparative HPLC chromatography, and purifying with methanol-water as mobile phase at volume ratio of 75:25 to obtain compound 1;
concentrating fraction D5, eluting with 40-90% methanol-water as eluent by reversed-phase ODS column chromatography, and collecting 60% fraction D52;
fraction D52 was concentrated and purified by preparative HPLC chromatography on methanol-water as the mobile phase at a volume ratio of 75:25 to give compound 5.
6. A pharmaceutical composition comprising one or more of the diterpene alkaloid type compound of claim 1, an isomer of the compound, and a pharmaceutically acceptable salt of the compound.
7. A pharmaceutical preparation is characterized in that the preparation is an active ingredient and one or the combination of a pharmaceutically acceptable carrier, an excipient and a diluent; wherein the active ingredient is a compound according to claim 1 or a composition according to claim 6.
8. The pharmaceutical formulation of claim 7, wherein: the administration route of the preparation is oral administration or injection administration, and the preparation form is as follows: tablet, capsule, powder, syrup or injection.
9. Use of a diterpene alkaloid type compound or a pharmaceutical composition or a pharmaceutical preparation according to claim 1, a composition according to claim 6 or a pharmaceutical preparation according to claim 7 for the preparation of an anti-inflammatory agent.
10. Use according to claim 9, characterized in that: use of the diterpene alkaloid type compound according to claim 1, the composition according to claim 6, or the pharmaceutical preparation according to claim 7 as a medicament for the prophylaxis and treatment of rheumatoid arthritis, inflammatory bowel disease or atherosclerosis.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110907232.5A CN113717105B (en) | 2021-08-09 | 2021-08-09 | Diterpene alkaloid compound and extraction method and application thereof |
| CN202310695867.2A CN116730919A (en) | 2021-08-09 | 2021-08-09 | Diterpene alkaloid compound and extraction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110907232.5A CN113717105B (en) | 2021-08-09 | 2021-08-09 | Diterpene alkaloid compound and extraction method and application thereof |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310695867.2A Division CN116730919A (en) | 2021-08-09 | 2021-08-09 | Diterpene alkaloid compound and extraction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113717105A true CN113717105A (en) | 2021-11-30 |
| CN113717105B CN113717105B (en) | 2023-06-30 |
Family
ID=78675210
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110907232.5A Active CN113717105B (en) | 2021-08-09 | 2021-08-09 | Diterpene alkaloid compound and extraction method and application thereof |
| CN202310695867.2A Pending CN116730919A (en) | 2021-08-09 | 2021-08-09 | Diterpene alkaloid compound and extraction method and application thereof |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310695867.2A Pending CN116730919A (en) | 2021-08-09 | 2021-08-09 | Diterpene alkaloid compound and extraction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN113717105B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023160302A1 (en) * | 2022-02-25 | 2023-08-31 | 西南交通大学 | C19 diterpene alkaloid, preparation method therefor and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0386824A (en) * | 1989-08-30 | 1991-04-11 | Mitsubishi Kasei Corp | Diterpene compound and antiinflammoatory drug containing the same as active ingredient |
| CN1178219A (en) * | 1997-07-23 | 1998-04-08 | 中国科学院昆明植物研究所 | Diterpene salicylate alkaloid, preparation and use thereof |
| CN102127016A (en) * | 2010-12-30 | 2011-07-20 | 成都大学 | Hetisin type diterpenoid alkaloid and preparation method and application thereof |
| CN107383006A (en) * | 2017-09-06 | 2017-11-24 | 遵义医学院 | The novel C separated in the Dulong rhizome of Chinese monkshood19Diterpene alkaloid preparation method and its usage |
| CN108690023A (en) * | 2018-07-03 | 2018-10-23 | 中国科学院新疆理化技术研究所 | Diterpenoid Alkaloids compound and preparation method and purposes in a kind of delphinium of match Tacheng |
| CN113004297A (en) * | 2021-03-08 | 2021-06-22 | 沈阳药科大学 | Diterpene alkaloid compound and extraction method and application thereof |
-
2021
- 2021-08-09 CN CN202110907232.5A patent/CN113717105B/en active Active
- 2021-08-09 CN CN202310695867.2A patent/CN116730919A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0386824A (en) * | 1989-08-30 | 1991-04-11 | Mitsubishi Kasei Corp | Diterpene compound and antiinflammoatory drug containing the same as active ingredient |
| CN1178219A (en) * | 1997-07-23 | 1998-04-08 | 中国科学院昆明植物研究所 | Diterpene salicylate alkaloid, preparation and use thereof |
| CN102127016A (en) * | 2010-12-30 | 2011-07-20 | 成都大学 | Hetisin type diterpenoid alkaloid and preparation method and application thereof |
| CN107383006A (en) * | 2017-09-06 | 2017-11-24 | 遵义医学院 | The novel C separated in the Dulong rhizome of Chinese monkshood19Diterpene alkaloid preparation method and its usage |
| CN108690023A (en) * | 2018-07-03 | 2018-10-23 | 中国科学院新疆理化技术研究所 | Diterpenoid Alkaloids compound and preparation method and purposes in a kind of delphinium of match Tacheng |
| CN113004297A (en) * | 2021-03-08 | 2021-06-22 | 沈阳药科大学 | Diterpene alkaloid compound and extraction method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| CHEN YAN，等: "Spiramine derivatives induce apoptosis of Bax/Bak cell and cancer cells", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》 * |
| WENLIANG XU,等: "TWO NEW ATISINE-TYPE C20-DITERPENOID ALKALOIDS FROM ACONITUM LEUCOSTOMUM", 《HETEROCYCLES》 * |
| 南泽东，等: "翠雀中二萜生物碱类成分的分离与鉴定", 《中国实验方剂学杂志》 * |
| 陈泗英，等: "大理翠雀中的新二菇生物碱", 《云南植物研究》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023160302A1 (en) * | 2022-02-25 | 2023-08-31 | 西南交通大学 | C19 diterpene alkaloid, preparation method therefor and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116730919A (en) | 2023-09-12 |
| CN113717105B (en) | 2023-06-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113105388B (en) | Euphorbia lathyris diterpene alkyl compound and extraction method and application thereof | |
| AU2022352631B2 (en) | Azulene compound, and preparation method therefor and use thereof | |
| CN111704544B (en) | Labdane diterpenoid compound and separation method and application thereof | |
| CN115716790B (en) | Extraction and separation method of amide ester alkaloid in purslane and application of extraction and separation method | |
| WO2022257995A1 (en) | Cryptotanshinone derivative and preparation method therefor and application thereof in lowering lipid and resisting obesity | |
| CN108689851B (en) | Tiglic alkane type diterpene compound and preparation method and application thereof | |
| CN113717105B (en) | Diterpene alkaloid compound and extraction method and application thereof | |
| CN111377994A (en) | Seven withanolides compounds from cape gooseberry and preparation method and application thereof | |
| CN110903268B (en) | Vorcaptan diterpene derivative as well as preparation method and application thereof | |
| CN115521245B (en) | Alkaloid compound in purslane, and extraction and separation method and application thereof | |
| CN114989084B (en) | Extraction and separation method of tetrahydroisoquinoline alkaloid in purslane and application of tetrahydroisoquinoline alkaloid | |
| CN115109014A (en) | Diterpenoid compounds in euphorbia pekinensis as well as extraction method and application thereof | |
| CN112898357B (en) | Diterpene glycoside novel compound in trollius chinensis bunge and separation and purification method and application thereof | |
| CN115215881A (en) | Guaiane type sesquiterpenoids prepared from Thorellan odorata, and preparation method and application thereof | |
| CN111253352B (en) | Compound extracted and separated from traditional Chinese medicine cymbidium maculatum, and preparation method and application thereof | |
| CN108948040B (en) | Gilmaxane type sesquiterpene compound extracted from herba Centellae and application thereof | |
| CN114853712B (en) | Chromane or chromene type hetero-terpenoid, and extraction method and application thereof | |
| CN115772076B (en) | Diterpenoid compound with anti-inflammatory activity in euphorbia pekinensis, and extraction method and application thereof | |
| CN115710172B (en) | Diterpenoid compound in euphorbia pekinensis, and extraction method and application thereof | |
| CN111110687A (en) | Albizzia julibrissin new lignan compound for resisting lipid metabolism disorder | |
| CN112047887B (en) | Tinospora sinensis amide and preparation method and application thereof | |
| CN115703753B (en) | Benzofuran derivative and preparation method and application thereof | |
| CN114805465B (en) | Triterpene compound, preparation method and application thereof | |
| CN114436983B (en) | Oleraze and Oleraoxazine acid in purslane and extraction and separation method thereof | |
| CN115504950B (en) | Lignan compound and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |