CN114956965B - Stilbene compound separated from lindera reflexa and preparation method and application thereof - Google Patents
Stilbene compound separated from lindera reflexa and preparation method and application thereof Download PDFInfo
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- CN114956965B CN114956965B CN202210379901.0A CN202210379901A CN114956965B CN 114956965 B CN114956965 B CN 114956965B CN 202210379901 A CN202210379901 A CN 202210379901A CN 114956965 B CN114956965 B CN 114956965B
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- 241000012106 Lindera reflexa Species 0.000 title claims abstract description 84
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- PJANXHGTPQOBST-VAWYXSNFSA-N Stilbene Natural products C=1C=CC=CC=1/C=C/C1=CC=CC=C1 PJANXHGTPQOBST-VAWYXSNFSA-N 0.000 title claims description 31
- -1 Stilbene compound Chemical class 0.000 title claims description 25
- 235000021286 stilbenes Nutrition 0.000 title claims description 18
- 150000001875 compounds Chemical class 0.000 claims abstract description 96
- 239000000284 extract Substances 0.000 claims abstract description 27
- 241000546273 Lindera <angiosperm> Species 0.000 claims abstract description 17
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000000741 silica gel Substances 0.000 claims abstract description 17
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 17
- PJANXHGTPQOBST-UHFFFAOYSA-N stilbene Chemical class C=1C=CC=CC=1C=CC1=CC=CC=C1 PJANXHGTPQOBST-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229940124599 anti-inflammatory drug Drugs 0.000 claims abstract description 5
- 238000000605 extraction Methods 0.000 claims abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 153
- 239000003480 eluent Substances 0.000 claims description 72
- 239000011347 resin Substances 0.000 claims description 57
- 229920005989 resin Polymers 0.000 claims description 57
- 238000001514 detection method Methods 0.000 claims description 49
- 238000010828 elution Methods 0.000 claims description 46
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 40
- 238000001179 sorption measurement Methods 0.000 claims description 40
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 34
- 238000004809 thin layer chromatography Methods 0.000 claims description 33
- 238000011068 loading method Methods 0.000 claims description 32
- 239000003795 chemical substances by application Substances 0.000 claims description 29
- 229930003935 flavonoid Natural products 0.000 claims description 27
- 150000002215 flavonoids Chemical class 0.000 claims description 27
- 235000017173 flavonoids Nutrition 0.000 claims description 27
- 239000003208 petroleum Substances 0.000 claims description 26
- 238000004262 preparative liquid chromatography Methods 0.000 claims description 25
- 239000012046 mixed solvent Substances 0.000 claims description 24
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 23
- NPOMSUOUAZCMBL-UHFFFAOYSA-N dichloromethane;ethoxyethane Chemical compound ClCCl.CCOCC NPOMSUOUAZCMBL-UHFFFAOYSA-N 0.000 claims description 17
- 238000005406 washing Methods 0.000 claims description 17
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 15
- 238000010898 silica gel chromatography Methods 0.000 claims description 11
- 238000004458 analytical method Methods 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 9
- 239000012535 impurity Substances 0.000 claims description 9
- 238000004440 column chromatography Methods 0.000 claims description 8
- 238000011049 filling Methods 0.000 claims description 8
- 238000011010 flushing procedure Methods 0.000 claims description 8
- 239000000178 monomer Substances 0.000 claims description 8
- 238000012856 packing Methods 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 238000010298 pulverizing process Methods 0.000 claims description 8
- 238000002791 soaking Methods 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 238000002604 ultrasonography Methods 0.000 claims description 2
- 238000002137 ultrasound extraction Methods 0.000 claims description 2
- 238000004811 liquid chromatography Methods 0.000 claims 1
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 12
- BIYGTLDPTJMNET-CMDGGOBGSA-N (E)-3,5-Dimethoxystilbene Chemical compound COC1=CC(OC)=CC(\C=C\C=2C=CC=CC=2)=C1 BIYGTLDPTJMNET-CMDGGOBGSA-N 0.000 abstract description 9
- BIYGTLDPTJMNET-UHFFFAOYSA-N cis-pinosylvin dimethyl ether Natural products COC1=CC(OC)=CC(C=CC=2C=CC=CC=2)=C1 BIYGTLDPTJMNET-UHFFFAOYSA-N 0.000 abstract description 9
- 238000000926 separation method Methods 0.000 abstract description 3
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 abstract description 2
- 229930003944 flavone Natural products 0.000 abstract description 2
- 150000002212 flavone derivatives Chemical class 0.000 abstract description 2
- 235000011949 flavones Nutrition 0.000 abstract description 2
- 238000002955 isolation Methods 0.000 abstract description 2
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 abstract description 2
- 238000013375 chromatographic separation Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 50
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 21
- 229910052799 carbon Inorganic materials 0.000 description 21
- 238000010586 diagram Methods 0.000 description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 239000003814 drug Substances 0.000 description 17
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 16
- 238000005160 1H NMR spectroscopy Methods 0.000 description 16
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 16
- 238000001228 spectrum Methods 0.000 description 16
- 229940079593 drug Drugs 0.000 description 13
- 238000004896 high resolution mass spectrometry Methods 0.000 description 12
- 238000010521 absorption reaction Methods 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 11
- 229910052739 hydrogen Inorganic materials 0.000 description 11
- 239000001257 hydrogen Substances 0.000 description 11
- 239000002609 medium Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 9
- 108090001005 Interleukin-6 Proteins 0.000 description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 8
- 239000012634 fragment Substances 0.000 description 6
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 6
- 210000002540 macrophage Anatomy 0.000 description 6
- 229940041616 menthol Drugs 0.000 description 6
- YCVPRTHEGLPYPB-VOTSOKGWSA-N trans-pinosylvin Chemical compound OC1=CC(O)=CC(\C=C\C=2C=CC=CC=2)=C1 YCVPRTHEGLPYPB-VOTSOKGWSA-N 0.000 description 6
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 5
- 238000002329 infrared spectrum Methods 0.000 description 5
- 238000001819 mass spectrum Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000002211 ultraviolet spectrum Methods 0.000 description 5
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 4
- 238000005100 correlation spectroscopy Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000003921 oil Substances 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 3
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- NOOLISFMXDJSKH-UTLUCORTSA-N (+)-Neomenthol Chemical compound CC(C)[C@@H]1CC[C@@H](C)C[C@@H]1O NOOLISFMXDJSKH-UTLUCORTSA-N 0.000 description 2
- NOOLISFMXDJSKH-UHFFFAOYSA-N DL-menthol Natural products CC(C)C1CCC(C)CC1O NOOLISFMXDJSKH-UHFFFAOYSA-N 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 150000001555 benzenes Chemical group 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- LECSHJWIACEDPZ-UHFFFAOYSA-N ethane-1,2-diamine naphthalene hydrochloride Chemical compound C(CN)N.C1=CC=CC2=CC=CC=C12.Cl LECSHJWIACEDPZ-UHFFFAOYSA-N 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- YCVPRTHEGLPYPB-UHFFFAOYSA-N pinosylvine Natural products OC1=CC(O)=CC(C=CC=2C=CC=CC=2)=C1 YCVPRTHEGLPYPB-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 2
- 229940124530 sulfonamide Drugs 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000007882 Gastritis Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000218195 Lauraceae Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 208000023652 chronic gastritis Diseases 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 239000000469 ethanolic extract Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000012306 spectroscopic technique Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 150000001629 stilbenes Chemical class 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
- C07C39/23—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic, containing six-membered aromatic rings and other rings, with unsaturation outside the aromatic rings
-
- 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]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C43/00—Ethers; Compounds having groups, groups or groups
- C07C43/02—Ethers
- C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
- C07C43/215—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring having unsaturation outside the six-membered aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/14—The ring being saturated
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
-
- 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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Stilbene compounds isolated from lindera Reflexanbene D (3, 5-dihydroxy-4- [ (1 '' -S3 '' -S4 '' -S) p-menthyl ] -trans-stilbene), reflexanbene G (3, 5-dimethoxy-2- [ (3 '' -R4 '' -R) p-menthyl ] -trans-stilbene), reflexanbene J (3, 5-dihydroxy-2, 6-bis- [ (3 '' -R4 '' -R3 '' -R4 '' -S) -p-menthenyl ] -trans-stilbene), reflexanbene H (3, 5-dihydroxy-4- [ (3 '' -S4 '' -R) p-menthenyl ] -trans-stilbene) and Reflexanbene K (3, 5-dimethoxy-trans-stilbene), the preparation method comprising the steps of: (1) preparing lindera reflexa total flavone extract; (2) silica gel chromatographic separation; (3) directed isolation of the target compound. The invention has simple extraction and separation process, easy operation, obvious anti-inflammatory effect, can be used for preparing anti-inflammatory drugs, exploits the new medicinal application of lindera reflexa, has practical clinical significance and obvious economic and social benefits.
Description
Technical Field
The invention relates to medicine, in particular to a stilbene compound separated from lindera reflexa and a preparation method and application thereof.
Background
The dry root of Lindera reflexa hemsl (Lindera reflexa hemsl.) belonging to the genus Lindera of the family Lauraceae is mainly distributed in Dabie mountain areas in Henan, and is a common drug for treating chronic gastritis and gastric ulcer in folk. The chemical components of lindera reflexa Hemsl mainly comprise volatile oils, alkaloids, flavonoids, stilbenes, etc. The pinosylvin is one of the stilbene compounds with higher content in lindera reflexa, and researches show that the pinosylvin has good anti-inflammatory and antioxidant activities. With the continuous and deep research on chemical components of lindera frustuca, how to separate more new compounds from lindera frustuca, the application range of lindera frustuca is widened, the material basis for confirming the drug effect of lindera fruca is an important problem of the technical personnel, and the invention has no remarkable report to date on the anti-inflammatory effect of Reflexanbenne D (stilbene 3, 5-dihydroxy-4- [ (1 "S3" S4 ") p-menthyl ] -trans-stilbene), reflexanben G (3, 5-dimethoxy-2- [ (3" R4 ") p-menthenyl ] -trans-stilbene), reflexanbenne J (3, 5-dihydroxy-2, 6-di- [ (3" R4' "S) -p-menthenyl ] -trans-stilbene), reflexanbenne H (3, 5-dihydroxy-4- [ (3" S4 "R) p-menthenyl ] -trans-stilbene) and Reflexan (3, 5-dimethoxy-stilbene) from lindera fruca.
Disclosure of Invention
Aiming at the situation, the invention aims to overcome the defects of the prior art and provide the stilbene compound separated from lindera reflexa and the preparation method and application thereof, which can effectively solve the problems of preparing a new compound from lindera reflexa and preparing a new anti-inflammatory medicament and solving the problem of the new application of lindera reflexa for medicines.
The invention solves the technical scheme that the stilbene compound separated from mountain comprises a compound of Reflexanbene D (3, 5-dihydroxy-4- [ (1 "S3" S4 ") p-menthyl ] -trans-stilbene), reflexanbene G (3, 5-dimethoxy-2- [ (3" R4 ") p-menthenyl ] -trans-stilbene), reflexanbene J (3, 5-dihydroxy-2, 6-bis- [ (3" R4' "S) -p-menthenyl ] -trans-stilbene), reflexanbene H (3, 5-dihydroxy-4- [ (3" S4 ") p-menthenyl ] -trans-stilbene) and Reflexanbene K (3, 5-dimethoxy-trans-stilbene), and has the molecular structural formulas of respectively:
the preparation method comprises the following steps:
(1) Preparing lindera reflexa total flavone extract: extracting lindera reflexa with ethanol, loading on a macroporous adsorbent resin wet method, loading on a column, adsorbing the ethanol extract of lindera reflexa, washing with pure water to remove impurities, collecting ethanol eluent, and recovering ethanol under reduced pressure to obtain purified lindera reflexa total flavonoids;
(2) Separating by silica gel chromatography: weighing purified lindera reflexia total flavonoids, performing ultrasonic dissolution with methanol, eluting with petroleum ether-dichloromethane-methanol system by silica gel column chromatography, combining the same fractions by thin layer chromatography to obtain 7 components Fr.1-Fr.7;
(3) Directional isolation of the target compound: fractions fr.1 and fr.3 were subjected to silica gel column chromatography using a petroleum ether-dichloromethane system, followed by separation and purification of the semi-prepared liquid phase using MCI medium pressure preparative column, and recovery of the solvent under reduced pressure to give the compounds Reflexanbene D (3, 5-dihydroxy-4- [ (1 "S3" S4 "S) p-menthyl ] -trans-stilbene), reflexanbene G (3, 5-dimethoxy-2- [ (3" R4 "R) p-menthenyl ] -trans-stilbene), reflexanbene J (3, 5-dihydroxy-2, 6-bis- [ (3" R4 "R3'" R4 "S) -p-menthenyl ] -trans-stilbene), reflexanbene H (3, 5-dihydroxy-4- [ (3" S4 "R) p-menthenyl ] -trans-stilbene), and Reflexanbene K (3, 5-dimethoxy-trans-stilbene).
The application of the stilbene compounds Reflexanbene D, reflexanbene G, reflexanbene J, reflexanbene H and Reflexanbene K prepared by the method in the preparation of anti-inflammatory drugs.
The invention has simple extraction and separation process and easy operation, can effectively extract the stilbene compound Reflexanbene D (3, 5-dihydroxy-4- [ (1 "S3" S) p-menthol ] -trans-stilbene), reflexanbene G (3, 5-dimethoxy-2- [ (3 "R4") p-menthenyl ] -trans-stilbene), reflexanbene J (3, 5-dihydroxy-2, 6-bis- [ (3 "R4'" S) -p-menthenyl ] -trans-stilbene), reflexanbene H (3, 5-dihydroxy-4- [ (3 "S4") p-menthenyl ] -trans-stilbene and Reflexanbene K (3, 5-dimethoxy-trans-stilbene) from mountain, has remarkable anti-inflammatory effect, can be used for preparing anti-inflammatory medicaments, exploits the novel medicinal application of lindera reflexa, and has practical clinical significance and remarkable economic and social benefits.
Drawings
FIG. 1 is a diagram showing molecular structural formulas of Reflexanbene D, reflexanbene G, reflexanbene J, reflexanbene H and Reflexanbene K extracted by the invention.
FIG. 2 is a diagram of a Reflexanbene D of the present invention 1 H-NMR chart.
FIG. 3 is a diagram of a Reflexanbene D of the present invention 13 C-NMR chart.
FIG. 4 is a DEPT135 diagram of the Reflexanbene D of the present invention.
FIG. 5 is a HSQC diagram of Reflexan Bene D of the present invention.
Fig. 6 is an HMBC chart of the Reflexanbene D of the present invention.
FIG. 7 is a diagram of a Reflexanbene D of the invention 1 H- 1 H COSY pattern.
FIG. 8 is a NOESY diagram of the Reflexanbene D of the present invention.
FIG. 9 is a HR-ESI-MS plot of the present invention Reflexanbene D.
Fig. 10 is an ECD plot of the Reflexanbene D of the present invention.
FIG. 11 is a diagram of a Reflexanbene G of the invention 1 H-NMR chart.
FIG. 12 is a diagram of a Reflexanbene G of the invention 13 C-NMR chart.
FIG. 13 is a HSQC chart of Reflexanbene G of the present invention.
Fig. 14 is an HMBC chart of the Reflexanbene G of the present invention.
FIG. 15 is a graph of the present invention of Reflexanbene G 1 H- 1 H COSY pattern.
FIG. 16 is a NOESY diagram of the Reflexanbene G of the present invention.
FIG. 17 is a HR-ESI-MS plot of the present invention Reflexanbene G.
FIG. 18 is an ECD diagram of the Reflexanbene G of the present invention.
FIG. 19 is a diagram of a Reflexanbene of the present inventionJ, J 1 H-NMR chart.
FIG. 20 is a diagram of a Reflexanbene J of the invention 13 C-NMR chart.
FIG. 21 is a DEPT135 diagram of the Reflexanbene J of the present invention.
FIG. 22 is a HSQC chart of Reflexanbene J of the present invention.
Fig. 23 is an HMBC plot of Reflexanbene J of the present invention.
FIG. 24 is a diagram of a Reflexanbene J of the invention 1 H- 1 H COSY pattern.
FIG. 25 is a NOESY diagram of a Reflexanbene J of the present invention.
FIG. 26 is a HR-ESI-MS plot of the present invention Reflexanbene J.
FIG. 27 is an ECD diagram of the present invention Reflexanbene J.
FIG. 28 is a diagram of a Reflexanbene H of the invention 1 H-NMR chart.
FIG. 29 is a diagram of a Reflexanbene H of the invention 13 C-NMR chart.
FIG. 30 is a HSQC chart of Reflexanbene H of the present invention.
Fig. 31 is an HMBC chart of the Reflexanbene H of the present invention.
FIG. 32 is a NOESY diagram of the Reflexanbene H of the present invention.
FIG. 33 is a HR-ESI-MS plot of the present invention Reflexanbene H.
Fig. 34 is an ECD plot of the Reflexanbene H of the present invention.
FIG. 35 is a graph of the present invention of Reflexanbene K 1 H-NMR chart.
FIG. 36 is a graph of the present invention of Reflexanbene K 13 C-NMR chart.
FIG. 37 is a HR-ESI-MS plot of the present invention Reflexanbene K.
FIG. 38 is a graph showing the effect of drug on macrophage viability.
FIG. 39 is a graph showing the effect of drug on macrophage NO release.
FIG. 40 is a graph showing the effect of drug on macrophage IL-6 release.
Detailed Description
The following describes in detail the embodiments of the present invention with reference to the drawings and examples.
Example 1
The invention discloses a preparation method of a stilbene compound Reflexanbene D, reflexanbene G, reflexanbene J, reflexanbene H and Reflexanbene K separated from lindera reflexa, wherein the molecular structural formulas of the compound Reflexanbene D, the compound Reflexanbene G, the compound Reflexanbene J, the compound Reflexanbene H and the compound Reflexanbene K are respectively as follows:
the preparation method comprises the following steps:
(1) Preparing lindera reflexa extract solution: pulverizing lindera reflexa hemsl medicinal materials into powder, adding ethanol with the volume concentration of 70% for ultrasonic extraction for 3-5 times, wherein the amount of ethanol added with 70% for each time is 10-14 times of the weight of the lindera reflexa hemsl, and extracting for 0.8-1.2 h each time; mixing the extracting solutions, recovering ethanol under reduced pressure until the extracting solutions have no ethanol smell, obtaining concentrated solution, adding water into the concentrated solution to dilute the concentrated solution into lindera reflexa Hemsl extract solution with the mass concentration of 0.04-0.06 mg/mL (equivalent to 0.04-0.06 mg of crude drug in each 1 mL), and reserving;
(2) Resin treatment and column filling: soaking macroporous adsorption resin in 95% ethanol for 10-14 hr, washing with 95% ethanol until the ethanol eluate is mixed with water to form no white turbidity, washing with distilled water until no ethanol smell exists, and wet packing the macroporous adsorption resin in the ratio of resin column diameter to column height of 1:8;
(3) Preparation of purified lindera reflexa total flavonoids: taking the lindera reflexa extract solution prepared in the step (1), loading the sample with the loading amount which is 3-5 times of the weight of the macroporous adsorption resin, wherein the loading flow rate is 0.5-1.5 mL/min, standing for 1.9-2.1 h after loading is finished, flushing impurities with pure water with the weight which is 3-5 times of that of the macroporous adsorption resin, eluting with 70% ethanol with the volume concentration which is 3-5 times that of the macroporous adsorption resin, collecting eluent, recovering the ethanol under reduced pressure, and obtaining purified lindera reflexa total flavonoids;
(4) Preparing monomer components: separating 500g of lindera reflexa total flavonoids prepared in the step (3) by silica gel column chromatography, and sequentially carrying out gradient elution by using petroleum ether-dichloromethane mixed solvents with volume ratios of 100:0, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1 and 1:1, wherein the volume of eluent used by each gradient is 12.5L, 16.8L, 20L, 28L, 100L, 70.8L, 67.5L and 65.5L in sequence, and the flow rate is 18-22 mL/min; then carrying out gradient elution by using methylene dichloride-methanol mixed solvents with volume ratios of 100:0, 100:1, 50:1, 10:1, 5:1, 3:1, 1:1 and 0:100, wherein the volume of eluent used by each gradient is 46L, 48.5L, 97L, 70.5L, 71L, 69L, 38L and 49L in sequence, the flow rate is 90-110 mL/min, each 500mL is one fraction, 1740 fractions are collected in total, each fraction is separated by silica gel thin layer chromatography detection, GF254 thin layer plates are respectively used as developing agents with volume ratios of 1:3 petroleum ether-methylene dichloride, volume ratios of 50:1 methylene dichloride-methanol and volume ratios of 3:1, anisaldehyde-concentrated sulfuric acid are used as developing agents, and Fr 7-7 components are obtained by heating at 105 ℃ for 3-5min, and respectively combining fractions 190-278, fractions-324, fractions-325, fractions 767-810, fractions 811-887, fractions 926-983, fractions 6-1428 and Fr 7.7-7 components according to the detection result of thin layer chromatography;
Eluting the component Fr.1 by semi-preparative liquid chromatography with methanol-water with a volume ratio of 98:5 as eluent at a flow rate of 2-5 mL/min, detecting the wavelength of 297nm, and collecting the peak with an elution time of 22.30min to obtain a compound of Reflexanbene G (3, 5-dimethoxy-2- [ (3 'R4' R) p-menthenyl ] -trans-stilbene);
subjecting the component Fr.3 to medium pressure MCI column chromatography, sequentially performing gradient elution with methanol-water mixed solvents with volume ratios of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0, respectively combining fractions 69-80, 85-90 and 91-96 according to detection results of thin layer chromatography by using 4L eluent and flow rate of 12-17 mL/min, collecting 96 fractions each with 250mL as a fraction, performing detection analysis by silica gel thin layer chromatography, using GF254 thin layer plate, using petroleum ether-dichloromethane with volume ratio of 1:3 as developing agent, using anisaldehyde-concentrated sulfuric acid solution as developing agent, heating at 105 ℃ for 3-5min, obtaining 3 subfractions Fr.3-1, fr.3-2 and Fr.3-3, eluting the subfractions Fr.3-2 by semi-preparative liquid chromatography with methanol-water with a volume ratio of 98:2 as eluent, and collecting peaks with an elution time of 10.44min and 20.00min respectively at a detection wavelength of 297nm to obtain a compound Reflexanbene H (3, 5-dihydroxyl-4- [ (3 "S4" R) p-menthenyl ] -trans-stilbene) and a compound Reflexanbene J (3, 5-dihydroxyl-2, 6-di- [ (3 "R4" R3 '"R4'" S) -p-menthenyl ] -trans-stilbene); eluting the subcomponent Fr.3-3 by semi-preparative liquid chromatography with methanol-water with volume ratio of 95:5 as eluent at flow rate of 2-5 mL/min, detecting wavelength of 297nm, collecting peak with eluting time of 17.90min to obtain compound Reflexanbene D (3, 5-dihydroxy-4- [ (1S 3S 4) p-menthol ] -trans-stilbene); and (3) performing semi-preparative liquid chromatography on the subfractions Fr.3-3, eluting with methanol-water with a volume ratio of 85:15 as eluent at a flow rate of 2-5 mL/min, detecting the wavelength of 297nm, and collecting the peak with an elution time of 15.37min to obtain a compound Reflexanbene K (3, 5-dimethoxy-trans-stilbene).
Example 2
The invention discloses a preparation method of a stilbene compound Reflexanbene D, reflexanbene G, reflexanbene J, reflexanbene H and Reflexanbene K separated from lindera reflexa, which comprises the following steps:
(1) Preparing lindera reflexa extract solution: pulverizing lindera reflexa Hemsl into powder, adding 70% ethanol by volume concentration, extracting with ultrasound for 3 times for 1 hr, wherein the amount of 70% ethanol added is 12 times of lindera Hemsl by weight; combining the extracting solutions, recovering ethanol under reduced pressure until the extracting solutions have no alcohol smell, obtaining concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into lindera reflexa Hemsl extract solution with the mass concentration of 0.05mg/mL for later use;
(2) Resin treatment and column filling: soaking macroporous adsorption resin in 95% ethanol for 12h, washing with 95% ethanol until ethanol eluent is mixed with water to form no white turbidity, washing with distilled water until no alcohol smell exists, and packing macroporous adsorption resin in a wet method according to the ratio of resin column diameter to column height of 1:8;
(3) Preparation of purified lindera reflexa total flavonoids: taking lindera reflexa extract solution prepared in the step (1), loading the solution with the loading amount being 4 times of the weight of the macroporous adsorption resin, wherein the loading flow rate is 1mL/min, standing for 2 hours after loading is finished, flushing impurities with pure water with the weight being 4 times of the weight of the macroporous adsorption resin, eluting with 70% ethanol with the volume concentration being 4 times of the weight of the macroporous adsorption resin, collecting eluent, and recovering the ethanol under reduced pressure to obtain purified lindera reflexa total flavonoids;
(4) Preparing monomer components: separating 500g of lindera reflexia total flavonoids prepared in the step (3) by silica gel column chromatography, and sequentially carrying out gradient elution by using petroleum ether-dichloromethane mixed solvents with volume ratios of 100:0, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1 and 1:1, wherein the volume of eluent used by each gradient is 12.5L, 16.8L, 20L, 28L, 100L, 70.8L, 67.5L and 65.5L in sequence, and the flow rate is 20mL/min; then carrying out gradient elution by using methylene dichloride-methanol mixed solvents with volume ratios of 100:0, 100:1, 50:1, 10:1, 5:1, 3:1, 1:1 and 0:100, wherein the volume of eluent used by each gradient is 46L, 48.5L, 97L, 70.5L, 71L, 69L, 38L and 49L in sequence, the flow rate is 100mL/min, each 500mL is one fraction, 1740 fractions are collected in total, each fraction is separated by silica gel thin layer chromatography detection, a GF254 thin layer plate is used, petroleum ether-methylene dichloride with volume ratio of 1:3, methylene dichloride-methanol with volume ratio of 50:1 and methylene dichloride-methanol with volume ratio of 3:1 are respectively used as developing agents, anisaldehyde-concentrated sulfuric acid is heated at 105 ℃ for 3-5min, and according to the detection result of thin layer chromatography, the fractions 190-278, the fractions of fractions 279-324, the fractions 767-325, the fractions 811-887, the fractions 926-983 and the fractions of 131887.7-1428 are respectively combined to obtain Fr 7.7 fractions;
Eluting the component Fr.1 by semi-preparative liquid chromatography with methanol-water with volume ratio of 98:5 as eluent at flow rate of 3mL/min, detecting wavelength of 297nm, and collecting peak with eluting time of 22.30min to obtain compound Reflexanbene G;
subjecting a component Fr.3 to medium-pressure MCI column chromatography, sequentially performing gradient elution by using methanol-water mixed solvents with volume ratios of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0, performing semi-preparative liquid chromatography on each of the gradient fractions by using 4L eluent with a flow rate of 15mL/min and each of 250mL as a fraction, collecting 96 fractions, performing silica gel thin-layer chromatography detection analysis on each of the fraction, performing detection by using a GF254 thin-layer plate, using petroleum ether-dichloromethane with a volume ratio of 1:3 as a developing agent, using an anisaldehyde-concentrated sulfuric acid solution as a color-developing agent, heating at 105 ℃ for 3-5min, respectively combining fractions 69-80, 85-90 and 91-96 according to the detection result of the thin-layer chromatography to obtain 3 subfractions Fr.3-1, fr.3-2 and Fr.3-3, performing elution by using methanol-water with a volume ratio of 98:2 as eluent with a flow rate of 3mL/min, and respectively collecting the compounds with detection wavelength of 297nm and 20.00 peak and reflne, thereby obtaining the reflne; eluting the subcomponent Fr.3-3 with methanol-water with volume ratio of 95:5 as eluent at flow rate of 3mL/min, detecting wavelength of 297nm, and collecting peak with elution time of 17.90min to obtain compound Reflexanbene D; and (3) performing semi-preparative liquid chromatography on the subfractions Fr.3-3, eluting with methanol-water with a volume ratio of 85:15 as eluent at a flow rate of 3mL/min, detecting the wavelength of 297nm, and collecting the peak with an elution time of 15.37min to obtain a compound, namely Reflexanbene K.
Example 3
The invention discloses a preparation method of a stilbene compound Reflexanbene D, reflexanbene G, reflexanbene J, reflexanbene H and Reflexanbene K separated from lindera reflexa, which comprises the following steps:
(1) Preparing lindera reflexa extract solution: pulverizing lindera reflexa Hemsl into powder, adding 70% ethanol by volume concentration, ultrasonic extracting for 3 times, extracting for 0.8 hr each time, wherein the amount of 70% ethanol added each time is 10 times of the weight of lindera reflexa Hemsl; combining the extracting solutions, recovering ethanol under reduced pressure until the extracting solutions have no alcohol smell, obtaining concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into lindera reflexa Hemsl extract solution with the mass concentration of 0.04mg/mL for later use;
(2) Resin treatment and column filling: soaking macroporous adsorption resin in 95% ethanol for 10h, washing with 95% ethanol until the ethanol eluent is mixed with water to form no white turbidity, washing with distilled water until no alcohol smell exists, and packing the macroporous adsorption resin in a wet method according to the ratio of the diameter of the resin column to the height of the column of 1:8;
(3) Preparation of purified lindera reflexa total flavonoids: taking lindera reflexa extract solution prepared in the step (1), loading the solution with the loading amount which is 3 times of the weight of the macroporous adsorption resin, wherein the loading flow rate is 0.5mL/min, standing for 1.9h after loading is finished, flushing impurities with pure water with the weight 3 times of the weight of the macroporous adsorption resin, eluting with 70% ethanol with the volume concentration which is 3 times of the weight of the macroporous adsorption resin, collecting eluent, and recovering ethanol under reduced pressure to obtain purified lindera reflexa total flavonoids;
(4) Preparing monomer components: separating 500g of lindera reflexia total flavonoids prepared in the step (3) by silica gel column chromatography, and sequentially carrying out gradient elution by using petroleum ether-dichloromethane mixed solvents with volume ratios of 100:0, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1 and 1:1, wherein the volume of eluent used by each gradient is 12.5L, 16.8L, 20L, 28L, 100L, 70.8L, 67.5L and 65.5L in sequence, and the flow rate is 18mL/min; then carrying out gradient elution by using methylene dichloride-methanol mixed solvents with volume ratios of 100:0, 100:1, 50:1, 10:1, 5:1, 3:1, 1:1 and 0:100, wherein the volume of eluent used by each gradient is 46L, 48.5L, 97L, 70.5L, 71L, 69L, 38L and 49L in sequence, the flow rate is 90mL/min, each 500mL is one fraction, 1740 fractions are collected in total, each fraction is separated by silica gel thin layer chromatography detection, GF254 thin layer plates are used as developing agents, petroleum ether-methylene dichloride with volume ratio of 1:3, methylene dichloride-methanol with volume ratio of 50:1 and methylene dichloride-methanol with volume ratio of 3:1 are used as developing agents, anisaldehyde-concentrated sulfuric acid is heated at 105 ℃ for 3-5min, and according to the detection result of thin layer chromatography, fractions 190-278, fractions 279-324, fractions 767-454, fractions 811-887, fractions-983 and fractions 1317.7-1427 are combined respectively, so as to obtain Fr-7.7 fractions;
Eluting the component Fr.1 by semi-preparative liquid chromatography with methanol-water with volume ratio of 98:5 as eluent at flow rate of 2mL/min, detecting wavelength of 297nm, and collecting peak with eluting time of 22.30min to obtain compound Reflexanbene G;
subjecting a component Fr.3 to medium-pressure MCI column chromatography, sequentially performing gradient elution by using methanol-water mixed solvents with volume ratios of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0, performing semi-preparative liquid chromatography on each of the gradient fractions by using 4L eluent with flow rate of 12mL/min and each of the gradient fractions of 250mL, collecting 96 fractions, performing silica gel thin-layer chromatography detection analysis on each of the fraction fractions, performing detection by using a GF254 thin-layer plate, using petroleum ether-dichloromethane with volume ratio of 1:3 as a developing agent, using an anisaldehyde-concentrated sulfuric acid solution as a color-developing agent, heating at 105 ℃ for 3-5min, respectively combining fractions 69-80, 85-90 and fractions 91-96 according to the detection result of the thin-layer chromatography to obtain 3 subfractions Fr.3-1, fr.3-2 and Fr.3-3, performing elution by using methanol-water with volume ratio of 98:2 as eluent with flow rate of 2mL/min, and respectively collecting the compounds with detection wavelength of 297nm and 20.00 peak and reflne, thereby obtaining the reflne; eluting the subcomponent Fr.3-3 with methanol-water with volume ratio of 95:5 as eluent at flow rate of 2mL/min, detecting wavelength of 297nm, and collecting peak with elution time of 17.90min to obtain compound Reflexanbene D; and (3) performing semi-preparative liquid chromatography on the subfractions Fr.3-3, eluting with methanol-water with a volume ratio of 85:15 as eluent at a flow rate of 2mL/min, detecting the wavelength of 297nm, and collecting the peak with an elution time of 15.37min to obtain a compound, namely Reflexanbene K.
Example 4
The invention discloses a preparation method of a stilbene compound Reflexanbene D, reflexanbene G, reflexanbene J, reflexanbene H and Reflexanbene K separated from lindera reflexa, which comprises the following steps:
(1) Preparing lindera reflexa extract solution: pulverizing lindera reflexa Hemsl into powder, adding 70% ethanol by volume concentration, ultrasonic extracting for 5 times for 1.2 hr each time, wherein the amount of 70% ethanol added each time is 14 times of the weight of lindera reflexa Hemsl; combining the extracting solutions, recovering ethanol under reduced pressure until the extracting solutions have no alcohol smell, obtaining concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into lindera reflexa Hemsl extract solution with the mass concentration of 0.06mg/mL for later use;
(2) Resin treatment and column filling: soaking macroporous adsorption resin in 95% ethanol for 14h, washing with 95% ethanol until ethanol eluent is mixed with water to form no white turbidity, washing with distilled water until no alcohol smell exists, and packing macroporous adsorption resin in a wet method according to the ratio of resin column diameter to column height of 1:8;
(3) Preparation of purified lindera reflexa total flavonoids: taking the lindera reflexa extract solution prepared in the step (1), loading the sample with the loading amount which is 5 times of the weight of the macroporous adsorption resin, standing for 2.1h after loading, flushing impurities with pure water with the weight which is 5 times of that of the macroporous adsorption resin, eluting with 70% ethanol with the volume concentration which is 5 times of that of the macroporous adsorption resin, collecting eluent, and recovering ethanol under reduced pressure to obtain purified lindera reflexa total flavonoids;
(4) Preparing monomer components: separating 500g of lindera reflexia total flavonoids prepared in the step (3) by silica gel column chromatography, and sequentially carrying out gradient elution by using petroleum ether-dichloromethane mixed solvents with volume ratios of 100:0, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1 and 1:1, wherein the volume of eluent used by each gradient is 12.5L, 16.8L, 20L, 28L, 100L, 70.8L, 67.5L and 65.5L in sequence, and the flow rate is 22mL/min; then carrying out gradient elution by using methylene dichloride-methanol mixed solvents with volume ratios of 100:0, 100:1, 50:1, 10:1, 5:1, 3:1, 1:1 and 0:100, wherein the volume of eluent used by each gradient is 46L, 48.5L, 97L, 70.5L, 71L, 69L, 38L and 49L in sequence, the flow rate is 110mL/min, each 500mL is one fraction, 1740 fractions are collected in total, each fraction is separated by silica gel thin layer chromatography detection, GF254 thin layer plates are used as developing agents, petroleum ether-methylene dichloride with volume ratio of 1:3, methylene dichloride-methanol with volume ratio of 50:1 and methylene dichloride-methanol with volume ratio of 3:1 are used as developing agents, anisaldehyde-concentrated sulfuric acid is heated at 105 ℃ for 3-5min, and according to the detection result of thin layer chromatography, fractions 190-278, fractions 279-324, fractions 767-454, fractions 811-887, fractions-983 and fractions 1317.7-1427 are combined respectively, so as to obtain Fr-7.7 fractions;
Eluting the component Fr.1 by semi-preparative liquid chromatography with methanol-water with volume ratio of 98:5 as eluent at flow rate of 5mL/min, detecting wavelength of 297nm, and collecting peak with eluting time of 22.30min to obtain compound Reflexanbene G;
subjecting a component Fr.3 to medium-pressure MCI column chromatography, sequentially performing gradient elution by using methanol-water mixed solvents with volume ratios of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0, performing semi-preparative liquid chromatography on each of the gradient fractions by using 4L eluent with flow rate of 17mL/min and each of the gradient fractions of 250mL, collecting 96 fractions, performing silica gel thin-layer chromatography detection analysis on each of the fraction fractions, performing detection by using a GF254 thin-layer plate, using petroleum ether-dichloromethane with volume ratio of 1:3 as a developing agent, using an anisaldehyde-concentrated sulfuric acid solution as a color-developing agent, heating at 105 ℃ for 3-5min, respectively combining fractions 69-80, 85-90 and fractions 91-96 according to the detection result of the thin-layer chromatography to obtain 3 subfractions Fr.3-1, fr.3-2 and Fr.3-3, performing elution by using methanol-water with volume ratio of 98:2 as eluent with flow rate of 5mL/min, and respectively collecting the compounds with detection wavelength of 297nm and 20.00 peak and reflne, thereby obtaining the reflne; eluting the subcomponent Fr.3-3 with methanol-water with volume ratio of 95:5 as eluent at flow rate of 5mL/min, detecting wavelength of 297nm, and collecting peak with elution time of 17.90min to obtain compound Reflexanbene D; and (3) performing semi-preparative liquid chromatography on the subfractions Fr.3-3, eluting with methanol-water with a volume ratio of 85:15 as eluent, and collecting peaks with an elution time of 15.37min at a detection wavelength of 297nm at a flow rate of 5mL/min to obtain a compound, namely Reflexanbene K.
The structure of the stilbene compound Reflexanbene D (3, 5-dihydroxy-4- [ (1 "S3" S4 ") p-menthyl ] -trans-stilbene), reflexanbene G (3, 5-dimethoxy-2- [ (3" R4 "R) p-menthyl ] -trans-stilbene), reflexanbene J (3, 5-dihydroxy-2, 6-bis- [ (3" R4"R3 '" R4' "S) -p-menthenyl ] -trans-stilbene), reflexanbene H (3, 5-dihydroxy-4- [ (3" S4 "R) p-menthenyl ] -trans-stilbene) and Reflexanbene K (3, 5-dimethoxy-trans-stilbene) prepared by the method of any of the embodiments 1 to 4 of the invention are the same, the structure of the compound Reflexanbene G is the same, the structure of the Reflexanbene J is the same, the structure of the Reflexanbene G is the same, and the structure of the Reflexanbene K is the same, and the anti-inflammatory effect of the drug is the same, and the structure of the Reflexanbene is the same, and the anti-inflammatory effect is obtained by repeated tests:
1. structural identification of compounds
Through nuclear magnetic resonance spectrum 1 H-NMR、 13 C-NMR) and high resolution mass spectrometry (HR-ESI-MS) spectroscopic techniques, wherein:
the compound Reflexanbene D3, 5-dihydroxy-4- [ (1 "S3" S4 "S) p-menthol radical ]Trans-stilbene) as a tan oil, the excimer ion peak M/z367.2267[ M+H ] given by high resolution mass spectrum HR-ESI-MS] + (calculated for C 24 H 31 O 3 367.2268) and the molecular formula of the compound is presumed to be C 24 H 30 O 3 The degree of unsaturation was 10. Ultraviolet spectrum shows compound RMaximum absorption wavelength of eflexan-bene D in methanol (lambda max ) 212nm,237nm and 314nm. IR spectrum shows that there is a hydroxyl group (3431 cm -1 ) And benzene rings (1422, 1505,1579,1616 cm) -1 ) Is not limited to the absorption of (a).
Compounds of the formula Reflexanbene D 1 H-NMR(500MHz，CDCl 3 ) The spectrum shows a signal of 1 benzene ring monosubstituted: delta H 7.47 (2H, d, j=7.3 hz, H-3' and H-5 '), 7.35 (2H, t, j=7.5, 7.8hz, H-2' and H-6 '), 7.30 (1H, m, H-4 '); 1 hydrogen signal to trans double bond: delta H 7.03 (2h, dd, j=16.3, 16.3hz, H- α and H- β); hydrogen signal delta of 1,3,4, 5-position 4 substituted benzene ring H 6.62 (1 h, d, j=1.3 hz, h-6), 6.45 (1 h, d, j=1.4 hz, h-2); methyl hydrogen atom signal group 3: delta H 1.36(3H,s,H-7”)，1.10(3H,d,J＝6.5Hz,H-10”)，0.97(3H,d,J＝6.5Hz,H-9”)。 13 The C-NMR (125 MHz, CDCl 3) spectrum and DEPT-135 spectrum gave 24 carbon signals, 6 quaternary carbons, 3 secondary carbons, 3 primary carbons and 12 tertiary carbons, of which 14 are the ethylenic carbon signals: delta C 136.6 (C-1), 104.2 (C-2), 152.6 (C-3), 114.5 (C-4), 157.9 (C-5), 106.3 (C-6), 128.3 (C- α), 128.5 (C- β), 137.4 (C-1 '), 128.6 (C-2' and C-6 '), 126.5 (C-3' and C-5 '), 127.5 (C-4'); 3 are methyl signals: delta C 29.3 (C-7 '), 22.1 (C-9 '), 21.1 (C-10 '). From the above hydrocarbon nuclear magnetic data and references, it was determined that compound Reflexanbene D was a 3, 5-dihydroxy-trans-stilbene as a parent core with a 10 carbon substituent attached, wherein 3 carbons were methyl carbons.
The structure of the compound Reflexanbene D and the position of attachment of each group were determined by two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR). H-9 ' and H-10 ' are related to each other and to C-8 ' at the same time in the HMBC spectra, which is determined to be an isopropyl structural fragment, H-9 ', H-10 ', H-8 "is related to C-4", indicating that the isopropyl structural fragment is attached to C-4 "with the 3 carbons and delta of the isopropyl fragment removed from the 10 carbons of the substituent C 29.3 (C-7 ') and also 6 carbon atoms, wherein H-5 ' and C-4 ', C-6 ' and C-1 ', H-3 ' and C-2 ', C-4 ' and C-1 ', 1 H- 1 h COSY displayH-3 "is shown as having associated signals with H-2" and H-4", and H-5" is shown as having associated signals with H-4 "and H-6", indicating that the remaining six carbons are a six membered ring; h-7 "is associated with C-1", C-6 "and C-2", indicating delta C 29.3 The methyl carbon of (C-7 ') is linked to C-1' and the DEPT-135 spectrum shows delta C 74.7 (C-1 ") is a quaternary carbon whose chemical shift value shifts to the lower field, judging that it is attached to a hydroxyl group, and combining the references to determine that the 10 carbon substituents are structural fragments of menthol; the C-3' linkage of menthol to C-4 on the benzene ring was determined by analysis in combination with signals associated with C-4, H-2 and H-6 and C-alpha in the HMBC spectra. NOESY spectra show that H-3 'has NOE relation with both H-4' and H-7 ', H-3', H-4 ', H-7' are illustrated as being on the same side, the compound was found to be in the 1"S3" S4"S configuration by comparison of the theoretical and actual values of ECD. In summary, the structure of the compound was determined to be 3, 5-dihydroxy-4- [ (1 "S3" S4 "S) p-menthol]Trans-stilbene, which is identified as a new compound by the scibinder database search for a report that the structure is not identical, is named Reflexanbene D, with the molecular structural formula:
TABLE 1 Reflexanbene D compound 1 H-NMR 13 C-NMR data (in CDCl) 3 )
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The compound Reflexanbene G (3, 5-dimethoxy-2- [ (3 "R4" R) p-menthenyl]Trans-stilbene) as a pale yellow oil, readily soluble in chloroform, high resolution mass spectrum HR-ESI-MS gives an excimer ion peak m/z 377.2476[ M+H ] ] + (calculated for C 26 H 33 O 2 377.2475) is assumed to have the molecular formula C 26 H 32 O 2 . UV spectrum shows the maximum absorption wavelength (lambda) of compound Reflexanbene G in methanol solution max ) Is 214nm and 299nm. IR spectrum shows hydroxyl groups (3420 cm -1 ) And benzene ring (1459, 1576 cm) -1 ) Is not limited to the absorption of (a).
Compounds of the Reflexanbene G 1 H-NMR(CDCl 3 500 MHz) shows a pair of trans double bond hydrogen signals: delta H 7.84 (1 h, d, j=15.6 hz, h- α), 6.84 (1 h, d, j=16.2 hz, h- β); 5 hydrogen signals on 1 monosubstituted benzene ring: delta H 7.43 (2H, d, j=7.5 hz, H-2' and H-6 '), 7.35 (2H, t, j=7.5, 7.8hz, H-3' and H-5 '), 7.25 (1H, t, j=6.0, 7.3hz, H-4 '); two sets of methoxy hydrogen signals: delta H 3.85(3H,s,5-OCH 3 )，3.77(3H,s,3-OCH 3 ) The method comprises the steps of carrying out a first treatment on the surface of the Group 3 methyl hydrogen signals: delta H 1.75 (3H, s, H-7 "), 0.78 (6H, dd, j=6.7, 6.7hz, H-9", and H-10 "); 13 C-NMR(125MHz，CDCl 3 ) The spectrum shows 26 carbon signals, and delta is determined by combining HSQC and HMBC spectra C 128.1 (C-beta) and 127.5 (C-alpha) are carbon signals on the trans double bond; delta C 138.1 (C-1 '), 126.3 (C-2' and C-6 '), 128.7 (C-3' and C-5 ') and 127.3 (C-4') are carbon signals on the monosubstituted benzene ring; delta C 55.8(3-OCH 3 ) And 55.3 (5-OCH) 3 ) Is a carbon signal on methoxy; delta C 23.6 (C-7 "), 21.7 (C-9"), 16.2 (C-10 ") are three methyl carbon signals; wherein delta C 133.0 (C-1 "), 127.4 (C-2"), 36.2 (C-3 "), 45.3 (C-4"), 22.9 (C-5 "), 31.3 (C-6"), 3.6 (C-7 "), 27.9 (C-8"), 221.7 (C-9 "), 16.2 (C-10") are a typical set of p-menthenyl carbon signals; residual delta C 138.6 (C-1), 111.3 (C-2), 159.2 (C-3), 102.7 (C-4), 158.5 (C-5), 97.8 (C-6) are carbon signals on a set of benzene rings; HMBC spectra display, delta H 3.85 (3H, s) is remotely related to the presence of C-5, delta H 3.77 (3H, s) is related to C-3 in a remote manner, indicating that two methoxy groups are attached at C-5 and C-3 positions, respectively; the presence of a remote correlation signal for H-6 with C-5 and C-alpha, the absence of a remote correlation signal for H-4 with C-alpha, the presence of a remote correlation signal for H-2 'and H-4' with C-2, indicating that the p-menthenyl fragment is linked to C-2, NOESY spectra showed that H-3 "and H-4" did not have NOE relationship, indicating that both are on opposite sides, and the compound was found to be 3"R4" R configuration by comparison of ECD data with calculated values, which were integrated with the above dataIdentifying the structure as 3, 5-dimethoxy-2- [ (3 "R4" R) p-menthenyl]-trans-stilbene. By searching the scibinder database, the compound is found not to be reported, and is determined to be a novel compound, named as Reflexanbene G, and the molecular structural formula is as follows:
TABLE 2 Reflexanbene G 1 H-NMR 13 C-NMR data (in CDCl) 3 )
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The compound reflexenobene J (3, 5-dihydroxy-2, 6-bis- [ (3 "R4" R3 '"R4'" S) -p-menthenyl]Trans-stilbene) in the form of a reddish-brown oil, the excimer ion peaks M/z485.3403[ M+H ] given by high-resolution mass spectrum HR-ESI-MS ] + (calculated for C 34 H 45 O 2 485.3414) and the molecular formula of the compound is presumed to be C 34 H 44 O 2 The unsaturation was 13. The ultraviolet spectrum shows the maximum absorption wavelength (lambda) of compound Reflexanbene J in methanol max ) 203nm and 296nm. IR spectrum shows hydroxyl groups (3434 cm -1 ) And benzene ring (1446, 1599 cm) -1 ) Is not limited to the absorption of (a).
Compounds of the Reflexanbene J 1 H-NMR(500MHz，CDCl 3 ) The spectrum shows a signal of 1 benzene ring monosubstituted: delta H 7.45 (2H, d, j=7.4 hz, H-2' and H-6 '), 7.39 (2H, t, j=7.5, 7.7hz, H-3' and H-5 '), 7.30 (1H, d, j=7.4 hz, H-4 '); 1 hydrogen signal to trans double bond: delta H 7.19 (1 h, d, j=16.5 hz, h- α), 6.34 (1 h, d, j=16.7 hz, h- β); 6 methyl hydrogen atom signals: delta H 1.77(3H,s,H-7”’)，1.74(3H,s,H-7”)，0.90(3H,d, j=6.6 hz, H-9 '"), 0.84 (6H, t, j=4.8, 6.6hz, H-9", and H-10' "), 0.40 (3H, d, j=6.6 hz, H-10"). 13 C-NMR(125MHz,CDCl 3 ) The spectrum shows two sets of carbon signals delta C 139.6 (C-1 "), 125.1 (C-2"), 39.4 (C-3 "), 43.5 (C-4"), 23.6 (C-5 "), 30.7 (C-6), 23.6 (C-7), 27.3 (C-8"), 21.9 (C-9), 17.7 (C-10 "), and delta C 139.6 23.7 (C-5 '), 30.7 (C-6'), 23.7 (C-5 '), 30.7 (C-6'), 23.6 (C-7 '), 29.2 (C-8'), 21.7 (C-9 '), 16.6 (C-10') are substantially identical to the reported signals for menthenyl groups, and the compound is determined to contain two p-menthenyl fragments. Delta of the remaining 14 carbons of the aromatic zone C 126.3 128.8 is the carbon signal on the AA 'BB' system, indicating that it is the carbon on the monosubstituted benzene ring, combined with HSQC to determine delta H 7.19 (1H, d, J=16.5 Hz) and 6.34 (1H, d, J=16.7 Hz) are attached to C- α and C- β, respectively, indicating δ C 129.9 134.0 is carbon on the trans double bond; delta H 6.30 (1H, s) and delta C 105.2 linkages indicating that the remaining 6 carbons are a pentasubstituted benzene ring, delta C 155.2 156.7 shows that the benzene ring has two hydroxyl groups attached, which is a fragment of trans-stilbene as determined in the incorporated literature.
The structure of the compound Reflexanbene J and the position of attachment of each group were determined by two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR). H-4 is related to C-3 and C-5 in the HMBC spectra, indicating that two hydroxyl groups are attached to C-3 and C-5, respectively; h-alpha is associated with C-1, C-2 and C-6, H-3 'and H-4' are associated with C-2, H-3 '"and H-4'" are related to C-6, two p-menthenyl groups are illustrated as symmetrically substituted on the C-2 and C-6 benzene rings. NOESY spectra show that H-3 '"and H-4'" have NOE relationship, H-3 '"and H-4'" are on the same side, and similarly H-3 '"and H-4'" have no NOE relationship, and are on opposite sides, and the compound is found to be in 3 'R4' R3 '"R4'" S configuration according to comparison of the theoretical value and the actual value of ECD. In summary, the structure of the compound is determined to be 3, 5-dihydroxy-2, 6-bis- [ (3 "R4" R3 '"R4'" S) -p-menthenyl ] -trans-stilbene, and no report that the structure is the same is found through searching by a scibinder database, so the compound is determined to be a novel compound, named as Reflexanbene J, and the molecular structural formula is:
TABLE 2-8 Reflexanbene J 1 H-NMR 13 C-NMR data (in CDCl) 3 )
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The compound Reflexanbene H (3, 5-dihydroxy-4- [ (3 "S4" R) p-menthenyl]Trans-stilbene) as a reddish brown oil, readily soluble in chloroform, and having an excimer ion peak M/z349.2160[ M+H ] given by high resolution mass spectrum HR-ESI-MS] + (calculated for C 24 H 29 O 2 349.2162) and the molecular formula of the compound is presumed to be C 24 H 28 O 2 The unsaturation was 11. The ultraviolet spectrum shows the maximum absorption wavelength (lambda) of the compound Reflexanbene H in methanol max ) 211nm and 314nm. IR spectrum shows hydroxyl groups (3417 cm) -1 ) And benzene rings (1439, 1449, 1567, 1577, 1620 cm) -1 ) Is not limited to the absorption of (a).
Compounds of the Reflexanbene H 1 H-NMR(500MHz，CDCl 3 ) The spectrum shows a signal of 1 benzene ring monosubstituted: delta H 7.48 (2H, d, j=7.4 hz, H-3' and H-5 '), 7.35 (2H, t, j=7.5, 7.8hz, H-2' and H-6 '), 7.26 (1H, m, H-4 '); 1 hydrogen signal to trans double bond: delta H 7.03 (2h, dd, j=16.3, 16.3hz, H- α and H- β); hydrogen signal delta of 2 benzene rings substituted by 1,3,4,5 position 4 H 6.62 (1H, m, H-6), 6.52 (1H, m, H-2); 3 methyl hydrogen atom signals: delta H 1.79 (3H, s, H-7 "), 0.88 (6H, dd, j=6.9, 7.0hz, H-9", and H-10 "). 13 C-NMR(125MHz，CDCl 3 ) The spectrum shows that there is a group of delta C 140.4 10 carbon signals of 124.4,43.6,35.7,30.7,23.7, 27.9,22.1,21.7, 16.4, found in the literature to be a set of p-menthenyl fragment signals, δ C 128.7 and 126.5 are carbon signals, delta, on the AA 'BB' system C 155.1 and 157.0 are carbon signals of the hydroxyl groups attached to the benzene ring, which are substantially consistent with the signals reported in the literature for 3, 5-dihydroxy-trans-stilbene. Display delta in HMBC spectra H 6.62 (1H, m, H-6) and delta C 116.7 157.0, 104.2 and 128.1, 6.52 (1H, m, H-2) and delta C 116.7 155.1, 106.2 and 128.1, which illustrate the ligation of the menthenyl fragment to C-4. NOESY spectra show that H-3 'and H-4' have NOE relationship, which indicates that H-3 'and H-4' are on the same side, and the compound is found to be in 3 'S4' R configuration according to comparison of theoretical value and actual value of ECD. In summary, the structure of the compound was determined to be 3, 5-dihydroxy-4- [ (3 "S4" R) p-menthenyl]Trans-stilbene, which is identified as a new compound by the scibinder database search, is named Reflexanbene H, with the molecular structural formula:
tables 2-6 Reflexanbene H 1 H-NMR 13 C-NMR data (in CDCl) 3 )
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The compound Reflexanbene K (3, 5-dimethoxy-trans-stilbene) is white rod-shaped crystal, and the excimer ion peak M/z241.1218[ M+H ] given by high resolution mass spectrum HR-ESI-MS] + (calculated for C 16 H 17 O 2 241.1223) and the molecular formula of the compound is presumed to be C 16 H 16 O 2 Unsaturation degree9. The ultraviolet spectrum shows the maximum absorption wavelength (lambda) of the compound Reflexanbene K in methanol max ) 211nm,299nm and 306nm. IR spectrum shows benzene rings (1423, 1455,1497,1592 cm) -1 ) Is not limited to the absorption of (a).
Compounds of the Reflexanbene K 1 H-NMR(500MHz，CDCl 3 ) The spectrum shows a signal of 1 benzene ring monosubstituted: delta H 7.51 (2H, d, j=7.85 hz, H-2' and H6 '), 7.36 (2H, t, j=7.7, 7.5hz, H-3' and H-5 '), 7.25 (1H, m, H-4 '); 1 hydrogen signal to trans double bond: delta H 7.10 (2h, dd, j=16.25, 16.25hz, H- α and H- β); hydrogen signal delta of 1,3, 5-position 3 substituted benzene ring H 6.67 (2H, d, j=2.0 hz, H-2 and H-6), 6.39 (1H, m, H-4); 2 methoxy hydrogen atom signals: delta H 3.82(6H,s,3-OCH 3 And 5-OCH 3 )。 13 C-NMR(125MHz，CDCl 3 ) The spectra and DEPT-135 spectra give 16 carbon signals: 4 quaternary carbons, 2 primary carbons and 10 tertiary carbons, wherein the signal of two primary carbons is delta C 55.4. The aromatic region has 14 carbons in total, and after removing 2 carbons on the trans double bond, 12 carbons remain, presumably two benzene rings, δ C 161.0 has two quaternary carbon signals, indicating that the benzene ring has two methoxy groups symmetrically substituted, delta C 128.7 and 126.6 are carbon signals on the AA 'BB' system. The compound was identified by reference as 3, 5-dimethoxy-trans-stilbene (3, 5-dimethoxy-tilbene), designated as Reflexanbene K, having the molecular structural formula:
Tables 2-10 Reflexanbene K Compounds 1 H-NMR 13 C-NMR data (in CDCl) 3 )
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2. Activity experiments
The compounds Reflexanbene D, reflexanbene G, reflexanbene J, reflexanbene H and Reflexanbene K prepared by the invention have anti-inflammatory effect through experiments, and related experimental data are as follows:
1. experimental materials
Mouse mononuclear macrophage RAW264.7 was purchased from marsupunorace life technologies limited; fetal bovine serum was purchased from Gibco company.
2. Cell culture
RAW264.7 cells were cultured in DMEM high-sugar medium containing 10% heat-inactivated fetal calf serum, 100U/mL penicillin, 100. Mu.g/mL streptomycin, and the dishes were placed at 37℃with 5% CO 2 Culturing in saturated humidity incubator, changing culture solution every 1-2 days, and digestion and passage with trypsin when cell fusion degree reaches above 80%.
3. Influence of drugs on macrophage Activity (MTT)
Culturing logarithmic growth phase cells in 96-well plate with 100 μl (containing 2000 cells) per well, placing at 37deg.C and 5% CO 2 The culture was carried out in an incubator for 12 hours, the medium was carefully removed, and then dilutions containing different concentrations of the test compound were added, 5 dose groups were set, each group being provided with 3 duplicate wells, 200 μl per well. Adding the control group with the same volume of solvent as the administration group, standing at 37deg.C and 5% CO 2 The culture was carried out in an incubator for 24 hours, taking care to remove the medium, and adding 20. Mu.L (1 mg/mL) of MTT solution and 100. Mu.L of serum-free medium per well. 37 ℃ and 5% CO 2 Incubate for 4h in incubator, discard supernatant, add 150 μl of DMSO per well to dissolve formazan particles, gently shake dissolve, measure absorbance (OD) with enzyme-linked immunosorbent assay at 570nm wavelength, use solvent control treated cells as control, and calculate cell viability.
4. Effect of drugs on LPS-induced NO and IL-6 Release from RAW264.7 cells
4.1. Collection of samples
Culturing logarithmic phase cells in 96-well plate with 100 μl (containing 1×10) 5 Individual cells), placed at 37℃in 5% CO 2 The culture was carried out in an incubator for 12 hours, the medium was carefully removed, and then 200. Mu.L of the drug diluted with 1. Mu.g/mL LPS in the medium was added, 3 dose groups were set, each group was set with 3 duplicate wells, and 200. Mu.L of each well. 200. Mu.L of DMEM medium without fetal bovine serum is added to the blank group, 200. Mu.L of medium with 1. Mu.g/mL LPS is added to the control group, and the mixture is placed at 37 ℃ and 5% CO 2 Culturing in incubator for 24 hr, transferring the cell culture supernatant to centrifuge tube, and storing in refrigerator at-80deg.C.
Preparation of Griess reagent
(1) The 85% phosphoric acid solution was prepared into 100mL of a 5% phosphoric acid solution with distilled water.
(2) 0.05g of naphthalene ethylenediamine hydrochloride is precisely weighed and dissolved in 50mL of 5% phosphoric acid solution, namely 0.1% naphthalene ethylenediamine hydrochloride solution, 0.50g of sulfanilamide is precisely weighed and dissolved in 50mL of 5% phosphoric acid solution, namely 1% sulfanilamide solution, and the mixture is stored in a dark place at 4 ℃ and mixed according to a ratio of 1:1 before use.
(3) 0.13799g of NaNO is precisely weighed 2 Dissolving in 10mL double distilled water to obtain 200mM NaNO 2 The solution was stored at 4℃in the dark.
Determination of NO Release amount
(1) Standard substance: standards were diluted to 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 and 0.15625 μm solutions in DMEM medium.
(2) Sample: the resulting cell culture broth was collected.
(3) After adding 100. Mu.L of standard solution or sample to be measured to each well, 100. Mu.L of Griess reagent was added.
(4) After standing at room temperature for 15 minutes, absorbance at 540nm was measured with a microplate reader.
Determination of IL-6 Release amount
The concentration of IL-6 in the samples was measured using the double antibody Sandwich method (Sandwich). The sample and the standard substances with different concentrations are respectively added into the corresponding enzyme-labeled plate holes according to 100 mu L/hole, the membrane sealing plate is sealed, the plate is incubated for 90 minutes at room temperature, the plate is washed for 6 times, and the plate is placed on absorbent paper for beating to be dry each time. 100 μl of detection antibody was added to each well, the plates were sealed, incubated at room temperature for 30 min, and washed 6 times. 100 mu L of streptavidin marked by horseradish peroxidase is added, membrane sealing plates are closed, incubation is carried out for 30 minutes at room temperature, and the plates are washed for 6 times. 100 mu L of signal enhancer is added, membrane sealing plates are sealed, the plates are incubated for 15 minutes at room temperature, and the plates are washed 6 times. Then, 100. Mu.L of horseradish peroxidase-labeled streptavidin was added again, membrane-sealed plates were sealed and incubated for 15 minutes at room temperature. 100 mu L of a color developing agent TMB solution is added, a membrane sealing plate is sealed, and the mixture is incubated for 15 minutes at room temperature in a dark place. 100. Mu.L of a stop solution was added, and immediately after mixing, the OD at the maximum absorption wavelength of 450nm and the reference wavelength of 570nm was measured, and the measured OD after calibration was a measured value of 450nm minus a measured value of 570 nm.
5. Experimental results
5.1. Influence of drugs on macrophage viability
After 24h co-culture of drug with RAW264.7 cells, the effect of Reflexanbene K (< 100 μm) on cell viability was small, and when the concentration of each compound was below 25 μm, the cell viability was 80% or more and cytotoxicity was small (fig. 38).
5.2. Effect of drugs on LPS-induced NO and IL-6 Release from RAW264.7 cells
The experimental results show that after different concentrations of Reflexanbene J and Reflexanbene H act on the inflammatory cell model for 24H, the release amount of NO decreases significantly and shows a certain dose dependency, and as the concentration increases, the release amount of NO decreases (fig. 39). After the isolated 5 compounds acted on the cell inflammation model for 24 hours, the release amount of IL-6 was reduced, and the release amount was extremely significantly different from that of the model group when the concentration reached 20 mu M (p < 0.01), wherein the effect of reducing IL-6 after the concentration reached 20 mu M was close to that of positive control dexamethasone (Dex), and a good anti-inflammatory effect was shown (figure 40).
In conclusion, the compounds Reflexanbene J and Reflexanbene H which are separated from lindera reflexa and have anti-inflammatory effects are found through experiments, the compounds Reflexanbene J and Reflexanbene H can effectively inhibit the release level of LPS-induced RAW264.7 cell NO, and the 5 compounds which are separated, particularly the compound Reflexanbene D, can effectively inhibit the release level of inflammatory factor IL-6. Therefore, the invention has application value for preparing anti-inflammatory drugs, exploits the new application of lindera reflexa hemsl medicinal materials, provides technical support for preparing anti-inflammatory drugs, and has very good application prospect. The raw materials are rich, the preparation method is easy to operate and popularize and apply, and is a great innovation in preparing the anti-inflammatory medicament, and the economic and social benefits are obvious.
Claims (6)
1. The stilbene compound separated from lindera Reflexanbene is characterized by comprising compounds Reflexanbene D and G, J, H, and the molecular structural formulas are respectively as follows:
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2. the method for preparing the stilbene compound separated from lindera reflexa as claimed in claim 1, which is characterized by comprising the following steps:
(1) Preparing lindera reflexa extract solution: pulverizing lindera reflexa hemsl medicinal materials into powder, adding ethanol with the volume concentration of 70% for ultrasonic extraction for 3-5 times, wherein the amount of ethanol added with 70% for each extraction is 10-14 times of the weight of the lindera reflexa hemsl, and extracting for 0.8-1.2 h each time; combining the extracting solutions, recovering ethanol under reduced pressure until the extracting solutions have no alcohol smell, obtaining concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into lindera reflexa Hemsl extract solution with the mass concentration of 0.04-0.06 mg/mL for later use;
(2) Resin treatment and column filling: soaking macroporous adsorption resin in 95% ethanol for 10-14 hr, washing with 95% ethanol until the ethanol eluate is mixed with water to form no white turbidity, washing with distilled water until no ethanol smell exists, and wet packing the macroporous adsorption resin in the ratio of resin column diameter to column height of 1:8;
(3) Preparation of purified lindera reflexa total flavonoids: taking the lindera reflexa extract solution prepared in the step (1), loading the sample with the loading amount which is 3-5 times of the weight of the macroporous adsorption resin, wherein the loading flow rate is 0.5-1.5 mL/min, standing for 1.9-2.1 h after loading is finished, flushing impurities with pure water with the weight which is 3-5 times of that of the macroporous adsorption resin, eluting with 70% ethanol with the volume concentration which is 3-5 times that of the macroporous adsorption resin, collecting eluent, recovering the ethanol under reduced pressure, and obtaining purified lindera reflexa total flavonoids;
(4) Preparing monomer components: separating 500g of lindera reflexa total flavonoids prepared in the step (3) by silica gel column chromatography, and sequentially carrying out gradient elution by using petroleum ether-dichloromethane mixed solvents with volume ratios of 100:0, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1 and 1:1, wherein the volume of eluent used by each gradient is 12.5L, 16.8L, 20L, 28L, 100L, 70.8L, 67.5L and 65.5L in sequence, and the flow rate is 18-22 mL/min; then, carrying out gradient elution by using methylene dichloride-methanol mixed solvents with volume ratios of 100:0, 100:1, 50:1, 10:1, 5:1, 3:1, 1:1 and 0:100, wherein the volume of eluent used by each gradient is 46L, 48.5L, 97L, 70.5L, 71L, 69L, 38L and 49L in sequence, the flow rate is 90-110 mL/min, each 500mL is one fraction, 1740 fractions are collected in total, each fraction is separated by silica gel thin layer chromatography detection, a GF254 thin layer plate is used, petroleum ether-methylene dichloride with volume ratio of 1:3, methylene dichloride-methanol with volume ratio of 50:1 and methylene dichloride-methanol with volume ratio of 3:1 are respectively used as developing agents, anisaldehyde-concentrated sulfuric acid is heated at 105 ℃ for 3-5min, and according to the detection result of thin layer chromatography, 190-278, fraction-324, fraction 767-810, fraction 811-887, fraction 926-983, fraction 6-6.1427 and Fr 7-7 are respectively combined;
Eluting the component Fr.1 by semi-preparative liquid chromatography with methanol-water with a volume ratio of 98:5 as eluent at a flow rate of 2-5 mL/min, detecting the wavelength of 297nm, and collecting the peak with an eluting time of 22.30min to obtain a compound, namely Reflexanbene G;
subjecting a component Fr.3 to medium-pressure MCI column chromatography, sequentially carrying out gradient elution by using methanol-water mixed solvents with volume ratios of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0, using 4L eluent for each gradient, wherein the flow rate is 12-17 mL/min, each 250mL is a fraction, collecting 96 fractions, carrying out detection analysis on each fraction by silica gel thin-layer chromatography, using a GF254 thin-layer plate, using petroleum ether-dichloromethane with volume ratio of 1:3 as a developing agent, using an anisaldehyde-concentrated sulfuric acid solution as a color reagent, heating at 105 ℃ for 3-5min, respectively combining fractions 69-80, 85-90 and 91-96 according to the detection result of the thin-layer chromatography to obtain 3 subcomponents Fr.3-1, fr.3-2 and Fr.3-3, respectively carrying out semi-preparation liquid chromatography on the subcomponent.3-2 by using methanol-water with volume ratio of 98:2 as eluent, and using the flow rate of 2-5 mL/min, detecting wavelength of the eluent, and respectively obtaining a reflbenne compound with detection wavelength of 10.00 nm and a reflbenne J compound; performing semi-preparative liquid chromatography on the subcomponent Fr.3-3, eluting with methanol-water with a volume ratio of 95:5 as eluent at a flow rate of 2-5 mL/min, detecting wavelength 297nm, and collecting peak with elution time of 17.90min to obtain a compound Reflexanbene D; performing semi-preparative liquid chromatography on the subcomponent Fr.3-3, eluting with methanol-water with a volume ratio of 85:15 as eluent at a flow rate of 2-5 mL/min, detecting wavelength 297nm, and collecting peak with eluting time of 15.37min to obtain a compound Reflexanbene K;
The molecular structural formula of the compound Reflexanbene K is as follows:
。
3. the method for preparing the stilbene compound separated from lindera reflexa according to claim 2, which is characterized by comprising the following steps:
(1) Preparing lindera reflexa extract solution: pulverizing lindera reflexa Hemsl into powder, adding 70% ethanol by volume concentration, extracting with ultrasound for 3 times for 1 hr, wherein the amount of 70% ethanol added is 12 times of lindera Hemsl by weight; combining the extracting solutions, recovering ethanol under reduced pressure until the extracting solutions have no alcohol smell, obtaining concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into lindera reflexa Hemsl extract solution with the mass concentration of 0.05mg/mL for later use;
(2) Resin treatment and column filling: soaking macroporous adsorption resin in 95% ethanol for 12h, washing with 95% ethanol until ethanol eluent is mixed with water to form no white turbidity, washing with distilled water until no alcohol smell exists, and packing macroporous adsorption resin in a wet method according to the ratio of resin column diameter to column height of 1:8;
(3) Preparation of purified lindera reflexa total flavonoids: taking lindera reflexa extract solution prepared in the step (1), loading the solution with the loading amount being 4 times of the weight of the macroporous adsorption resin, wherein the loading flow rate is 1mL/min, standing for 2 hours after loading is finished, flushing impurities with pure water with the weight being 4 times of the weight of the macroporous adsorption resin, eluting with 70% ethanol with the volume concentration being 4 times of the weight of the macroporous adsorption resin, collecting eluent, and recovering the ethanol under reduced pressure to obtain purified lindera reflexa total flavonoids;
(4) Preparing monomer components: separating 500g of lindera reflexia total flavonoids prepared in the step (3) by silica gel column chromatography, and sequentially carrying out gradient elution by using petroleum ether-dichloromethane mixed solvents with volume ratios of 100:0, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1 and 1:1, wherein the volume of eluent used by each gradient is 12.5L, 16.8L, 20L, 28L, 100L, 70.8L, 67.5L and 65.5L in sequence, and the flow rate is 20mL/min; then carrying out gradient elution by using methylene dichloride-methanol mixed solvents with volume ratios of 100:0, 100:1, 50:1, 10:1, 5:1, 3:1, 1:1 and 0:100, wherein the volume of eluent used by each gradient is 46L, 48.5L, 97L, 70.5L, 71L, 69L, 38L and 49L in sequence, the flow rate is 100mL/min, each 500mL is one fraction, 1740 fractions are collected in total, each fraction is separated by silica gel thin layer chromatography detection, a GF254 thin layer plate is used, petroleum ether-methylene dichloride with volume ratio of 1:3, methylene dichloride-methanol with volume ratio of 50:1 and methylene dichloride-methanol with volume ratio of 3:1 are respectively used as developing agents, anisaldehyde-concentrated sulfuric acid is heated at 105 ℃ for 3-5min, and according to the detection result of thin layer chromatography, the fractions 190-278, the fractions of fractions 279-324, the fractions 767-325, the fractions 811-887, the fractions 926-983 and the fractions of 131887.7-1428 are respectively combined to obtain Fr 7.7 fractions;
Eluting the component Fr.1 by semi-preparative liquid chromatography with methanol-water with volume ratio of 98:5 as eluent at flow rate of 3mL/min, detecting wavelength of 297nm, and collecting peak with eluting time of 22.30min to obtain compound Reflexanbene G;
subjecting a component Fr.3 to medium-pressure MCI column chromatography, sequentially performing gradient elution by using methanol-water mixed solvents with volume ratios of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0, performing semi-preparative liquid chromatography on each of the gradient fractions by using 4L eluent with a flow rate of 15mL/min and each of 250mL as a fraction, collecting 96 fractions, performing silica gel thin-layer chromatography detection analysis on each of the fraction, performing detection by using a GF254 thin-layer plate, using petroleum ether-dichloromethane with a volume ratio of 1:3 as a developing agent, using an anisaldehyde-concentrated sulfuric acid solution as a color-developing agent, heating at 105 ℃ for 3-5min, respectively combining fractions 69-80, 85-90 and 91-96 according to the detection result of the thin-layer chromatography to obtain 3 subfractions Fr.3-1, fr.3-2 and Fr.3-3, performing elution by using methanol-water with a volume ratio of 98:2 as eluent with a flow rate of 3mL/min, and respectively collecting the compounds with detection wavelength of 297nm and 20.00 peak and reflne, thereby obtaining the reflne; eluting the subcomponent Fr.3-3 with methanol-water with volume ratio of 95:5 as eluent at flow rate of 3mL/min, detecting wavelength of 297nm, and collecting peak with elution time of 17.90min to obtain compound Reflexanbene D; and (3) performing semi-preparative liquid chromatography on the subfractions Fr.3-3, eluting with methanol-water with a volume ratio of 85:15 as eluent at a flow rate of 3mL/min, detecting the wavelength of 297nm, and collecting the peak with an elution time of 15.37min to obtain a compound, namely Reflexanbene K.
4. The method for preparing the stilbene compound separated from lindera reflexa according to claim 2, which is characterized by comprising the following steps:
(1) Preparing lindera reflexa extract solution: pulverizing lindera reflexa Hemsl into powder, adding 70% ethanol by volume concentration, ultrasonic extracting for 3 times, extracting for 0.8 hr each time, wherein the amount of 70% ethanol added each time is 10 times of the weight of lindera reflexa Hemsl; combining the extracting solutions, recovering ethanol under reduced pressure until the extracting solutions have no alcohol smell, obtaining concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into lindera reflexa Hemsl extract solution with the mass concentration of 0.04mg/mL for later use;
(2) Resin treatment and column filling: soaking macroporous adsorption resin in 95% ethanol for 10h, washing with 95% ethanol until the ethanol eluent is mixed with water to form no white turbidity, washing with distilled water until no alcohol smell exists, and packing the macroporous adsorption resin in a wet method according to the ratio of the diameter of the resin column to the height of the column of 1:8;
(3) Preparation of purified lindera reflexa total flavonoids: taking lindera reflexa extract solution prepared in the step (1), loading the solution with the loading amount which is 3 times of the weight of the macroporous adsorption resin, wherein the loading flow rate is 0.5mL/min, standing for 1.9h after loading is finished, flushing impurities with pure water with the weight 3 times of the weight of the macroporous adsorption resin, eluting with 70% ethanol with the volume concentration which is 3 times of the weight of the macroporous adsorption resin, collecting eluent, and recovering ethanol under reduced pressure to obtain purified lindera reflexa total flavonoids;
(4) Preparing monomer components: separating 500g of lindera reflexia total flavonoids prepared in the step (3) by silica gel column chromatography, and sequentially carrying out gradient elution by using petroleum ether-dichloromethane mixed solvents with volume ratios of 100:0, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1 and 1:1, wherein the volume of eluent used by each gradient is 12.5L, 16.8L, 20L, 28L, 100L, 70.8L, 67.5L and 65.5L in sequence, and the flow rate is 18mL/min; then carrying out gradient elution by using methylene dichloride-methanol mixed solvents with volume ratios of 100:0, 100:1, 50:1, 10:1, 5:1, 3:1, 1:1 and 0:100, wherein the volume of eluent used by each gradient is 46L, 48.5L, 97L, 70.5L, 71L, 69L, 38L and 49L in sequence, the flow rate is 90mL/min, each 500mL is one fraction, 1740 fractions are collected in total, each fraction is separated by silica gel thin layer chromatography detection, GF254 thin layer plates are used as developing agents, petroleum ether-methylene dichloride with volume ratio of 1:3, methylene dichloride-methanol with volume ratio of 50:1 and methylene dichloride-methanol with volume ratio of 3:1 are used as developing agents, anisaldehyde-concentrated sulfuric acid is heated at 105 ℃ for 3-5min, and according to the detection result of thin layer chromatography, fractions 190-278, fractions 279-324, fractions 767-454, fractions 811-887, fractions-983 and fractions 1317.7-1427 are combined respectively, so as to obtain Fr-7.7 fractions;
Eluting the component Fr.1 by semi-preparative liquid chromatography with methanol-water with volume ratio of 98:5 as eluent at flow rate of 2mL/min, detecting wavelength of 297nm, and collecting peak with eluting time of 22.30min to obtain compound Reflexanbene G;
subjecting a component Fr.3 to medium-pressure MCI column chromatography, sequentially performing gradient elution by using methanol-water mixed solvents with volume ratios of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0, performing semi-preparative liquid chromatography on each of the gradient fractions by using 4L eluent with flow rate of 12mL/min and each of the gradient fractions of 250mL, collecting 96 fractions, performing silica gel thin-layer chromatography detection analysis on each of the fraction fractions, performing detection by using a GF254 thin-layer plate, using petroleum ether-dichloromethane with volume ratio of 1:3 as a developing agent, using an anisaldehyde-concentrated sulfuric acid solution as a color-developing agent, heating at 105 ℃ for 3-5min, respectively combining fractions 69-80, 85-90 and fractions 91-96 according to the detection result of the thin-layer chromatography to obtain 3 subfractions Fr.3-1, fr.3-2 and Fr.3-3, performing elution by using methanol-water with volume ratio of 98:2 as eluent with flow rate of 2mL/min, and respectively collecting the compounds with detection wavelength of 297nm and 20.00 peak and reflne, thereby obtaining the reflne; eluting the subcomponent Fr.3-3 with methanol-water with volume ratio of 95:5 as eluent at flow rate of 2mL/min, detecting wavelength of 297nm, and collecting peak with elution time of 17.90min to obtain compound Reflexanbene D; and (3) performing semi-preparative liquid chromatography on the subfractions Fr.3-3, eluting with methanol-water with a volume ratio of 85:15 as eluent at a flow rate of 2mL/min, detecting the wavelength of 297nm, and collecting the peak with an elution time of 15.37min to obtain a compound, namely Reflexanbene K.
5. The method for preparing the stilbene compound separated from lindera reflexa according to claim 2, which is characterized by comprising the following steps:
(1) Preparing lindera reflexa extract solution: pulverizing lindera reflexa Hemsl into powder, adding 70% ethanol by volume concentration, ultrasonic extracting for 5 times for 1.2 hr each time, wherein the amount of 70% ethanol added each time is 14 times of the weight of lindera reflexa Hemsl; combining the extracting solutions, recovering ethanol under reduced pressure until the extracting solutions have no alcohol smell, obtaining concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into lindera reflexa Hemsl extract solution with the mass concentration of 0.06mg/mL for later use;
(2) Resin treatment and column filling: soaking macroporous adsorption resin in 95% ethanol for 14h, washing with 95% ethanol until ethanol eluent is mixed with water to form no white turbidity, washing with distilled water until no alcohol smell exists, and packing macroporous adsorption resin in a wet method according to the ratio of resin column diameter to column height of 1:8;
(3) Preparation of purified lindera reflexa total flavonoids: taking the lindera reflexa extract solution prepared in the step (1), loading the sample with the loading amount which is 5 times of the weight of the macroporous adsorption resin, standing for 2.1h after loading, flushing impurities with pure water with the weight which is 5 times of that of the macroporous adsorption resin, eluting with 70% ethanol with the volume concentration which is 5 times of that of the macroporous adsorption resin, collecting eluent, and recovering ethanol under reduced pressure to obtain purified lindera reflexa total flavonoids;
(4) Preparing monomer components: separating 500g of lindera reflexia total flavonoids prepared in the step (3) by silica gel column chromatography, and sequentially carrying out gradient elution by using petroleum ether-dichloromethane mixed solvents with volume ratios of 100:0, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1 and 1:1, wherein the volume of eluent used by each gradient is 12.5L, 16.8L, 20L, 28L, 100L, 70.8L, 67.5L and 65.5L in sequence, and the flow rate is 22mL/min; then carrying out gradient elution by using methylene dichloride-methanol mixed solvents with volume ratios of 100:0, 100:1, 50:1, 10:1, 5:1, 3:1, 1:1 and 0:100, wherein the volume of eluent used by each gradient is 46L, 48.5L, 97L, 70.5L, 71L, 69L, 38L and 49L in sequence, the flow rate is 110mL/min, each 500mL is one fraction, 1740 fractions are collected in total, each fraction is separated by silica gel thin layer chromatography detection, GF254 thin layer plates are used as developing agents, petroleum ether-methylene dichloride with volume ratio of 1:3, methylene dichloride-methanol with volume ratio of 50:1 and methylene dichloride-methanol with volume ratio of 3:1 are used as developing agents, anisaldehyde-concentrated sulfuric acid is heated at 105 ℃ for 3-5min, and according to the detection result of thin layer chromatography, fractions 190-278, fractions 279-324, fractions 767-454, fractions 811-887, fractions-983 and fractions 1317.7-1427 are combined respectively, so as to obtain Fr-7.7 fractions;
Eluting the component Fr.1 by semi-preparative liquid chromatography with methanol-water with volume ratio of 98:5 as eluent at flow rate of 5mL/min, detecting wavelength of 297nm, and collecting peak with eluting time of 22.30min to obtain compound Reflexanbene G;
subjecting a component Fr.3 to medium-pressure MCI column chromatography, sequentially performing gradient elution by using methanol-water mixed solvents with volume ratios of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0, performing semi-preparative liquid chromatography on each of the gradient fractions by using 4L eluent with flow rate of 17mL/min and each of the gradient fractions of 250mL, collecting 96 fractions, performing silica gel thin-layer chromatography detection analysis on each of the fraction fractions, performing detection by using a GF254 thin-layer plate, using petroleum ether-dichloromethane with volume ratio of 1:3 as a developing agent, using an anisaldehyde-concentrated sulfuric acid solution as a color-developing agent, heating at 105 ℃ for 3-5min, respectively combining fractions 69-80, 85-90 and fractions 91-96 according to the detection result of the thin-layer chromatography to obtain 3 subfractions Fr.3-1, fr.3-2 and Fr.3-3, performing elution by using methanol-water with volume ratio of 98:2 as eluent with flow rate of 5mL/min, and respectively collecting the compounds with detection wavelength of 297nm and 20.00 peak and reflne, thereby obtaining the reflne; eluting the subcomponent Fr.3-3 with methanol-water with volume ratio of 95:5 as eluent at flow rate of 5mL/min, detecting wavelength of 297nm, and collecting peak with elution time of 17.90min to obtain compound Reflexanbene D; and (3) performing semi-preparative liquid chromatography on the subfractions Fr.3-3, eluting with methanol-water with a volume ratio of 85:15 as eluent, and collecting peaks with an elution time of 15.37min at a detection wavelength of 297nm at a flow rate of 5mL/min to obtain a compound, namely Reflexanbene K.
6. The use of stilbene compounds isolated from lindera reflexa as claimed in claim 1 for the preparation of anti-inflammatory drugs.
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| CN102349945A (en) * | 2011-10-19 | 2012-02-15 | 河南中医学院 | Method for extracting purified total flavonoids from lindera reflexa hemsl by means of macroporous absorption resin |
| CN104628531A (en) * | 2015-02-13 | 2015-05-20 | 河南中医学院 | Compound SJ-11 extracted from lindera reflexa hemsl as well as preparation method and application of compound SJ-11 |
| CN112094176A (en) * | 2020-07-22 | 2020-12-18 | 河南中医药大学 | Stilbene compound extracted from lindera reflexa hemsl and preparation method and application thereof |
| CN113842390A (en) * | 2021-10-26 | 2021-12-28 | 河南中医药大学 | Lindera reflexa hemsl active ingredient composition with effect of preventing and treating gastric ulcer as well as preparation method and application thereof |
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| CN102349945A (en) * | 2011-10-19 | 2012-02-15 | 河南中医学院 | Method for extracting purified total flavonoids from lindera reflexa hemsl by means of macroporous absorption resin |
| CN104628531A (en) * | 2015-02-13 | 2015-05-20 | 河南中医学院 | Compound SJ-11 extracted from lindera reflexa hemsl as well as preparation method and application of compound SJ-11 |
| CN112094176A (en) * | 2020-07-22 | 2020-12-18 | 河南中医药大学 | Stilbene compound extracted from lindera reflexa hemsl and preparation method and application thereof |
| CN113842390A (en) * | 2021-10-26 | 2021-12-28 | 河南中医药大学 | Lindera reflexa hemsl active ingredient composition with effect of preventing and treating gastric ulcer as well as preparation method and application thereof |
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