CN114956965A - Stilbene compound separated from lindera reflexa hemsl and preparation method and application thereof - Google Patents
Stilbene compound separated from lindera reflexa hemsl and preparation method and application thereof Download PDFInfo
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- CN114956965A CN114956965A CN202210379901.0A CN202210379901A CN114956965A CN 114956965 A CN114956965 A CN 114956965A CN 202210379901 A CN202210379901 A CN 202210379901A CN 114956965 A CN114956965 A CN 114956965A
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- 241000012106 Lindera reflexa Species 0.000 title claims abstract description 98
- -1 Stilbene compound Chemical class 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- PJANXHGTPQOBST-VAWYXSNFSA-N Stilbene Natural products C=1C=CC=CC=1/C=C/C1=CC=CC=C1 PJANXHGTPQOBST-VAWYXSNFSA-N 0.000 title claims abstract description 19
- 235000021286 stilbenes Nutrition 0.000 title claims abstract description 9
- 150000001875 compounds Chemical class 0.000 claims abstract description 101
- 239000000284 extract Substances 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 16
- 238000000605 extraction Methods 0.000 claims abstract description 10
- 229940124599 anti-inflammatory drug Drugs 0.000 claims abstract description 8
- 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 claims abstract description 7
- 229930003944 flavone Natural products 0.000 claims abstract description 7
- 150000002212 flavone derivatives Chemical class 0.000 claims abstract description 7
- 235000011949 flavones Nutrition 0.000 claims abstract description 7
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 154
- 239000003480 eluent Substances 0.000 claims description 63
- 239000011347 resin Substances 0.000 claims description 61
- 229920005989 resin Polymers 0.000 claims description 61
- 238000001514 detection method Methods 0.000 claims description 49
- 238000010828 elution Methods 0.000 claims description 48
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 40
- 238000004809 thin layer chromatography Methods 0.000 claims description 35
- 238000001179 sorption measurement Methods 0.000 claims description 34
- 238000004262 preparative liquid chromatography Methods 0.000 claims description 32
- 238000005070 sampling Methods 0.000 claims description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- 238000005406 washing Methods 0.000 claims description 27
- 239000003208 petroleum Substances 0.000 claims description 26
- NPOMSUOUAZCMBL-UHFFFAOYSA-N dichloromethane;ethoxyethane Chemical compound ClCCl.CCOCC NPOMSUOUAZCMBL-UHFFFAOYSA-N 0.000 claims description 25
- 239000003795 chemical substances by application Substances 0.000 claims description 24
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 24
- 239000012046 mixed solvent Substances 0.000 claims description 24
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 22
- 229930003935 flavonoid Natural products 0.000 claims description 22
- 150000002215 flavonoids Chemical class 0.000 claims description 22
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- 239000000741 silica gel Substances 0.000 claims description 22
- 229910002027 silica gel Inorganic materials 0.000 claims description 22
- PJANXHGTPQOBST-UHFFFAOYSA-N stilbene Chemical class C=1C=CC=CC=1C=CC1=CC=CC=C1 PJANXHGTPQOBST-UHFFFAOYSA-N 0.000 claims description 18
- 238000012856 packing Methods 0.000 claims description 17
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 16
- 238000010438 heat treatment Methods 0.000 claims description 15
- 229910052739 hydrogen Inorganic materials 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 11
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- 239000000203 mixture Substances 0.000 claims 1
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 8
- 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 7
- 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 7
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 17
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- 239000001257 hydrogen Substances 0.000 description 11
- 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
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- 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
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- 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
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- NOOLISFMXDJSKH-UTLUCORTSA-N (+)-Neomenthol Chemical compound CC(C)[C@@H]1CC[C@@H](C)C[C@@H]1O NOOLISFMXDJSKH-UTLUCORTSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- NOOLISFMXDJSKH-UHFFFAOYSA-N DL-menthol Natural products CC(C)C1CCC(C)CC1O NOOLISFMXDJSKH-UHFFFAOYSA-N 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
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000012258 culturing Methods 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
- 239000003921 oil Substances 0.000 description 3
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 3
- 229940124530 sulfonamide Drugs 0.000 description 3
- ZCUQOPGIJRGJDA-UHFFFAOYSA-N 1-naphthalen-1-ylethane-1,2-diamine Chemical compound C1=CC=C2C(C(N)CN)=CC=CC2=C1 ZCUQOPGIJRGJDA-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 241000546273 Lindera <angiosperm> Species 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
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- 239000001963 growth medium Substances 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 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
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- 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
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- 241000702141 Corynephage beta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
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- 208000007882 Gastritis Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000218195 Lauraceae Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 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
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- 229930013930 alkaloid Natural products 0.000 description 1
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- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 208000023652 chronic gastritis Diseases 0.000 description 1
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- 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
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- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
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- 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
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- 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
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- 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
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- 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
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- 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)
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Abstract
A stilbene compound isolated from lindera reflexa Hemsl comprises compounds Reflexanbene D (3, 5-dihydroxy-4- [ (1 '' S3 '' S4 '' S) 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 '' 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 comprises the following steps: (1) preparing lindera reflexa total flavone extract; (2) separating by silica gel chromatography; (3) and (4) directionally separating the target compound. The method has simple extraction and separation process, easy operation and obvious anti-inflammatory effect, can be used for preparing anti-inflammatory drugs, develops the new medicinal application of lindera reflexa hemsl, 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 hemsl and a preparation method and application thereof.
Background
Lindera reflexa Hemsl, which is a common medicine used by folks for treating chronic gastritis and gastric ulcer, is mainly distributed in Dabie mountain areas in Henan province and is a dry root of Lindera reflexa Hemsl belonging to Lindera genus Lindera of Lauraceae. The chemical components of lindera reflexa hemsl mainly comprise volatile oil, alkaloids, flavonoids, stilbenes and the like. The pinosylvin is one of stilbene compounds with high content in lindera reflexa hemsl, and researches show that the pinosylvin has good anti-inflammatory and antioxidant activity. With the continuous and intensive research on the chemical components of lindera reflexa Hemsl, how to separate more new compounds from lindera reflexa Hemsl, expanding the application range of lindera reflexa Hemsl and making clear the material basis for lindera reflexa to exert the medicinal effect are important problems concerned by those skilled in the art, but the invention firstly finds Reflexanbene D (stilbene compound 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) has obvious anti-inflammatory effect on menthenyl ] -trans-stilbene) and Reflexanbene K (3, 5-dimethoxy-trans-stilbene), and has no public report so far.
Disclosure of Invention
In view of the above situation, in order to overcome the defects of the prior art, the present invention aims to provide a stilbene compound separated from lindera reflexa, a preparation method and an application thereof, which can effectively solve the problems of preparation of a new compound from lindera reflexa, preparation of a new anti-inflammatory drug and new medicinal applications of lindera reflexa.
The technical scheme of the invention is that the stilbene compound separated from lindera reflexa Hemsl comprises compounds Reflexanbene D (3, 5-dihydroxy-4- [ (1 ' S3 ' S4 ' S) p-menthol group ] -trans-stilbene), Reflexanbene G (3, 5-dimethoxy-2- [ (3 ' R4 ' R) p-menthenyl group ] -trans-stilbene), Reflexanbene J (3, 5-dihydroxy-2, 6-bis- [ (3 ' R4 ' R3 ' R4 ' S) -p-menthenyl group ] -trans-stilbene), Reflexanbene H (3, 5-dihydroxy-4- [ (3 ' S4 ' R) p-menthenyl group ] -trans-stilbene) and Reflexanbene K (3, 5-dimethoxy-trans-stilbene), the molecular structural formulas are respectively:
the preparation method comprises the following steps:
(1) preparing a lindera reflexa total flavone extract: extracting lindera reflexa with ethanol, performing wet column packing with macroporous adsorption resin, performing sample adsorption on the lindera reflexa ethanol extract, washing with pure water to remove impurities, collecting ethanol eluent, and recovering ethanol under reduced pressure to obtain purified lindera reflexa total flavonoids;
(2) silica gel chromatographic separation: weighing purified lindera reflexa Hemsl total flavonoids, ultrasonically dissolving with methanol, performing silica gel column chromatography, eluting with petroleum ether-dichloromethane-methanol system, combining with thin layer chromatography, and mixing the same fractions to obtain 7 fractions Fr.1-Fr.7;
(3) directional separation of the target compound: silica gel column chromatography of components Fr.1 and Fr.3 in a petroleum ether-dichloromethane system followed by separation and purification of the semi-preparative liquid phase using MCI medium pressure preparative columns and solvent recovery under reduced pressure yielded compounds Reflexanbene D (3, 5-dihydroxy-4- [ (1 "S3" S4 "S) on menthol ] -trans-stilbene), Reflexanbene G (3, 5-dimethoxy-2- [ (3" R4 "R) on menthenyl ] -trans-stilbene), Reflexanbene J (3, 5-dihydroxy-2, 6-bis- [ (3" R4 "R3 '" R4' "S) -on menthenyl ] -trans-stilbene), Reflexanbene H (3, 5-dihydroxy-4- [ (3" S4 "R) on menthenyl ] -trans-stilbene) and Reflexanbene K (3), 5-dimethoxy-trans-stilbene).
Application of the stilbene compounds Reflexanbene D, Reflexanbene G, Reflexanbene J, Reflexanbene H and Reflexanbene K prepared by the method in preparation of anti-inflammatory drugs.
The extraction and separation process is simple and easy to operate, and stilbene compounds Reflexanbene D (3, 5-dihydroxy-4- [ (1 ' S3 ' S4 ' S) p-menthol group ] -trans-stilbene), Reflexanbene G (3, 5-dimethoxy-2- [ (3 ' R4 ' R) p-menthenyl group ] -trans-stilbene), Reflexanbene J (3, 5-dihydroxy-2, 6-bis- [ (3 ' R4 ' R3 ' R4 ' S) -p-menthenyl group ] -trans-stilbene), Reflexanbene H (3, 5-dihydroxy-4- [ (3 ' S4 ' R) p-menthenyl group ] -trans-stilbene) and Reflexanbene K (3, 5-dimethoxy-trans-stilbene), has obvious anti-inflammatory effect, can be used for preparing anti-inflammatory drugs, develops the new medicinal application of lindera reflexa hemsl, has practical clinical significance and has obvious economic and social benefits.
Drawings
Fig. 1 is a molecular structural diagram of Reflexanbene D, Reflexanbene G, Reflexanbene J, Reflexanbene H and Reflexanbene K, which are Reflexanbene compounds extracted by the present invention.
FIG. 2 shows Reflexanbene D of the present invention 1 H-NMR chart.
FIG. 3 shows Reflexanbene D of the present invention 13 C-NMR chart.
FIG. 4 is a DEPT135 diagram of Reflexanbene D of the present invention.
FIG. 5 is a diagram of HSQC of Reflexanbene D of the present invention.
FIG. 6 is a HMBC diagram of Reflexanbene D of the present invention.
FIG. 7 shows Reflexanbene D of the present invention 1 H- 1 H COSY picture.
FIG. 8 is a NOESY diagram of Reflexanbene D of the present invention.
FIG. 9 is a HR-ESI-MS diagram of Reflexanbene D of the present invention.
Fig. 10 is an ECD diagram of Reflexanbene D of the present invention.
FIG. 11 shows Reflexanbene G of the present invention 1 H-NMR chart.
FIG. 12 shows Reflexanbene G of the present invention 13 C-NMR chart.
FIG. 13 is a diagram of HSQC of Reflexanbene G of the present invention.
FIG. 14 shows a HMBC diagram of Reflexanbene G according to the present invention.
FIG. 15 shows Reflexanbene G of the present invention 1 H- 1 H COSY picture.
FIG. 16 is a NOESY chart of Reflexanbene G of the present invention.
FIG. 17 is a HR-ESI-MS diagram of Reflexanbene G according to the present invention.
Fig. 18 is an ECD diagram of Reflexanbene G of the present invention.
FIG. 19 shows Reflexanbene J of the present invention 1 H-NMR chart.
FIG. 20 shows Reflexanbene J of the present invention 13 C-NMR chart.
FIG. 21 is a DEPT135 diagram of Reflexanbene J of the present invention.
FIG. 22 is a diagram of HSQC of Reflexanbene J of the present invention.
FIG. 23 is a HMBC diagram of Reflexanbene J of the present invention.
FIG. 24 shows Reflexanbene J of the present invention 1 H- 1 H COSY picture.
FIG. 25 is a NOESY chart of Reflexanbene J of the present invention.
FIG. 26 is a HR-ESI-MS diagram of Reflexanbene J according to the present invention.
Fig. 27 is an ECD diagram of Reflexanbene J of the present invention.
FIG. 28 shows Reflexanbene H of the present invention 1 H-NMR chart.
FIG. 29 shows Reflexanbene H of the present invention 13 C-NMR chart.
FIG. 30 is a diagram of HSQC of Reflexanbene H of the present invention.
FIG. 31 shows a HMBC diagram of Reflexanbene H according to the present invention.
FIG. 32 is a NOESY diagram of Reflexanbene H of the present invention.
FIG. 33 is a HR-ESI-MS graph of Reflexanbene H according to the present invention.
Fig. 34 is an ECD diagram of Reflexanbene H of the present invention.
FIG. 35 shows Reflexanbene K of the present invention 1 H-NMR chart.
FIG. 36 shows Reflexanbene K of the present invention 13 C-NMR chart.
FIG. 37 is a HR-ESI-MS diagram of Reflexanbene K according to the present invention.
FIG. 38 is a graph of the effect of drug on macrophage viability.
FIG. 39 is a graph of the effect of drugs on the amount of NO released from macrophages.
FIG. 40 is a graph showing the effect of drugs on the release of IL-6 from macrophages.
Detailed Description
The following detailed description of embodiments of the invention refers to the accompanying drawings and examples.
Example 1
The invention relates to a preparation method of stilbene compounds Reflexanbene D, Reflexanbene G, Reflexanbene J, Reflexanbene H and Reflexanbene K separated from lindera reflexa Hemsl, wherein the molecular structural formulas of the compounds Reflexanbene D, Reflexanbene G, Reflexanbene J, Reflexanbene H and Reflexanbene K are respectively as follows:
the preparation method comprises the following steps:
(1) preparing a lindera reflexa hemsl extract solution: crushing lindera reflexa Hemsl medicinal materials into powder, adding ethanol with the volume concentration of 70% for ultrasonic extraction for 3-5 times, wherein the extraction time is 0.8-1.2 h, and the amount of the ethanol with the volume concentration of 70% added for each time is 10-14 times of the weight of the lindera reflexa Hemsl; combining the extracting solutions, recovering ethanol under reduced pressure until the extracting solution has no alcohol smell to obtain a concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into a 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 per 1 mL) for later use;
(2) resin treatment and column packing: soaking macroporous adsorbent resin in 95% ethanol for 10-14h, washing with 95% ethanol until ethanol eluate is not white turbid, washing with distilled water until no ethanol smell is produced, and wet packing the macroporous adsorbent resin into column according to the ratio of the diameter of the resin column to the height of the column of 1: 8;
(3) preparing purified lindera reflexa total flavonoids: sampling the lindera reflexa hemsl extract solution prepared in the step (1), wherein the sampling amount is 3-5 times of the weight of macroporous adsorption resin, the sampling flow rate is 0.5-1.5 mL/min, standing for 1.9-2.1 h after sampling is finished, washing impurities with pure water 3-5 times of the weight of macroporous adsorption resin, eluting with 70% ethanol with volume concentration 3-5 times of the macroporous adsorption resin, collecting eluent, and recovering ethanol under reduced pressure to obtain purified lindera reflexa total flavonoids;
(4) preparing a monomer component: subjecting 500g of lindera reflexa total flavonoids prepared in the step (3) to silica gel column chromatography, and performing gradient elution by using a petroleum ether-dichloromethane mixed solvent with the volume ratio of 100:0, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1 and 1:1 in sequence, wherein the volume of eluent used in 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 performing gradient elution by using dichloromethane-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 used as one fraction, totally 1740 fractions are collected, each fraction is detected and separated by silica gel thin layer chromatography, petroleum ether-dichloromethane with volume ratio of 1:3, dichloromethane-methanol with volume ratio of 50:1 and dichloromethane-methanol with volume ratio of 3:1 are used as developing agents, anisaldehyde-concentrated sulfuric acid is used as a color developing agent, heating is performed at 105 ℃ for 3-5min, and according to the detection result of the thin layer chromatography, the fractions are respectively 190-278, 278 and 49, The fractions 279-324, 325-454, 767-810, 811-887, 926-983 and 1316-1428 to obtain 7 components Fr.1-Fr.7;
subjecting the component Fr.1 to semi-preparative liquid chromatography, eluting with methanol-water as an eluent with a volume ratio of 98:5, with a flow rate of 2-5 mL/min and a detection wavelength of 297nm, and collecting a peak with an elution time of 22.30min to obtain a compound Reflexanbene G (3, 5-dimethoxy-2- [ (3 'R4' R) p-menthenyl ] -trans-stilbene);
subjecting component Fr.3 to medium pressure MCI column chromatography, gradient eluting with methanol-water mixed solvent at volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0 sequentially, collecting 96 fractions with each gradient eluting with 4L of eluent at flow rate of 12-17 mL/min and one fraction per 250mL, detecting and analyzing each fraction by silica gel thin layer chromatography, subjecting each fraction to GF254 thin layer chromatography, developing with petroleum ether-dichloromethane at volume ratio of 1:3 as developing agent, subjecting anisaldehyde-concentrated sulfuric acid solution as developer, heating at 105 deg.C for 3-5min, combining fractions 69-80, 85-90 and 91-96 respectively according to detection result of thin layer chromatography to obtain 3 subcomponents Fr.3-1, Fr.3-2 and Fr.3-3, subjecting subcomponent Fr.3-2 to semi-preparative liquid chromatography, eluting with methanol-water at volume ratio of 98:2, the flow rate is 2-5 mL/min, the detection wavelength is 297nm, and peaks with the elution time of 10.44min and 20.00min are respectively collected to obtain a compound Reflexanbene H (3, 5-dihydroxy-4- [ (3 'S4' R) to menthenyl ] -trans-stilbene) and a compound Reflexanbene J (3, 5-dihydroxy-2, 6-bis- [ (3 'R4' R3 '-R4' -S) -to menthenyl ] -trans-stilbene); performing semi-preparative liquid chromatography on the subcomponent Fr.3-3, eluting with methanol-water as an eluent at a volume ratio of 95:5 at a flow rate of 2-5 mL/min and a detection wavelength of 297nm, and collecting a peak with an elution time of 17.90min to obtain a compound Reflexanbene D (3, 5-dihydroxy-4- [ (1 ' S3 ' S4 ' S) p-menthol-group ] -trans-stilbene); and (3) performing semi-preparative liquid chromatography on the subcomponent Fr.3-3, eluting with methanol-water serving as an eluent in a volume ratio of 85:15 at a flow rate of 2-5 mL/min and a detection wavelength of 297nm, and collecting a peak with an elution time of 15.37min to obtain a compound Reflexanbene K (3, 5-dimethoxy-trans-stilbene).
Example 2
The invention relates to a preparation method of stilbene compounds Reflexanbene D, Reflexanbene G, Reflexanbene J, Reflexanbene H and Reflexanbene K separated from lindera reflexa Hemsl, which comprises the following steps:
(1) preparing a lindera reflexa hemsl extract solution: crushing lindera reflexa hemsl medicinal materials into powder, adding ethanol with the volume concentration of 70%, and performing ultrasonic extraction for 3 times, wherein the extraction time is 1 hour each time, and the amount of the ethanol added with 70% each time is 12 times of the weight of lindera reflexa hemsl; mixing the extracting solutions, recovering ethanol under reduced pressure until the extracting solution has no alcohol smell to obtain a concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into a lindera reflexa hemsl extract solution with the mass concentration of 0.05mg/mL for later use;
(2) resin treatment and column packing: soaking macroporous adsorbent resin in 95% ethanol for 12 hr, washing with 95% ethanol until the ethanol eluate is not white and turbid, washing with distilled water until no ethanol smell is produced, and wet packing the macroporous adsorbent resin into column according to the ratio of the diameter of the resin column to the height of the column of 1: 8;
(3) preparing purified lindera reflexa total flavonoids: sampling the lindera reflexa hemsl extract solution prepared in the step (1), wherein the sampling amount is 4 times of the weight of macroporous adsorption resin, the sampling flow rate is 1mL/min, standing for 2h after sampling is finished, washing impurities by pure water 4 times of the weight of macroporous adsorption resin, eluting by 70% ethanol with volume concentration 4 times of the weight of macroporous adsorption resin, collecting eluent, and recovering ethanol under reduced pressure to obtain purified lindera reflexa total flavone;
(4) preparing a monomer component: performing chromatographic separation on 500g of lindera reflexa total flavonoids prepared in the step (3) by using a silica gel column, and performing 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 in sequence, wherein the volume of eluent used for each gradient is 12.5L, 16.8L, 20L, 28L, 100L, 70.8L, 67.5L and 65.5L in sequence, and the flow rate is 20 mL/min; then performing gradient elution by using dichloromethane-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 used as one fraction, 1740 fractions are totally collected, each fraction is detected and separated by silica gel thin layer chromatography, petroleum ether-dichloromethane with volume ratio of 1:3, dichloromethane-methanol with volume ratio of 50:1 and dichloromethane-methanol with volume ratio of 3:1 are respectively used as developing agents, anisaldehyde-concentrated sulfuric acid is used as a color developing agent, heating is performed at 105 ℃ for 3-5min, and according to the detection result of the thin layer chromatography, the fractions are respectively 190-, The fractions 325 & 454, 767 & 810, 811 & 887, 926 & 983, 1316 & 1428, to obtain 7 fractions of the components Fr.1-Fr.7;
subjecting the component Fr.1 to semi-preparative liquid chromatography, eluting with methanol-water with a volume ratio of 98:5 as an eluent at a flow rate of 3mL/min and a detection wavelength of 297nm, and collecting a peak with an elution time of 22.30min to obtain a compound Reflexanbene G;
subjecting component Fr.3 to medium pressure MCI column chromatography, gradient eluting with methanol-water mixed solvent at volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0 sequentially, collecting 96 fractions with 4L eluent at flow rate of 15mL/min and one fraction per 250mL, detecting and analyzing each fraction by silica gel thin layer chromatography, subjecting GF254 thin layer plate with petroleum ether-dichloromethane at volume ratio of 1:3 as developing agent, subjecting anisaldehyde-concentrated sulfuric acid solution as developer, heating at 105 deg.C for 3-5min, combining fractions 69-80, 85-90 and 91-96 respectively according to detection result of thin layer chromatography to obtain 3 subcomponents Fr.3-1, Fr.3-2 and Fr.3-3, subjecting subcomponent Fr.3-2 to semi-preparative liquid chromatography, eluting with methanol-water at volume ratio of 98:2 as eluent, the flow rate is 3mL/min, the detection wavelength is 297nm, and peaks with the elution time of 10.44min and 20.00min are respectively collected to obtain a compound Reflexanbene H and a compound Reflexanbene J; performing semi-preparative liquid chromatography on the subfraction Fr.3-3, eluting with methanol-water with a volume ratio of 95:5 as eluent at a flow rate of 3mL/min and a detection wavelength of 297nm, and collecting the peak with an elution time of 17.90min to obtain a compound Reflexanbene D; performing semi-preparative liquid chromatography on the subfraction Fr.3-3, eluting with methanol-water with volume ratio of 85:15 as eluent at flow rate of 3mL/min and detection wavelength of 297nm, and collecting the peak with elution time of 15.37min to obtain a compound Reflexanbene K.
Example 3
The invention relates to a preparation method of stilbene compounds Reflexanbene D, Reflexanbene G, Reflexanbene J, Reflexanbene H and Reflexanbene K separated from lindera reflexa Hemsl, which comprises the following steps:
(1) preparing a lindera reflexa hemsl extract solution: crushing lindera reflexa Hemsl medicinal materials into powder, adding ethanol with volume concentration of 70%, and performing ultrasonic extraction for 3 times, wherein the extraction time is 0.8h, and the amount of the ethanol added with 70% is 10 times of the weight of lindera reflexa Hemsl; mixing the extracting solutions, recovering ethanol under reduced pressure until the extracting solution has no alcohol smell to obtain a concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into a lindera reflexa hemsl extract solution with the mass concentration of 0.04mg/mL for later use;
(2) resin treatment and column packing: soaking macroporous adsorption resin in 95% ethanol with volume concentration for 10h, washing with 95% ethanol with volume concentration until ethanol eluate is not white and turbid when being mixed with water, washing with distilled water until no alcohol smell exists, and packing the macroporous adsorption resin into a column by a wet method according to the ratio of the diameter of the resin column to the height of the resin column of 1: 8;
(3) preparing purified lindera reflexa total flavonoids: sampling the lindera reflexa hemsl extract solution prepared in the step (1), wherein the sampling amount is 3 times of the weight of the macroporous adsorption resin, the sampling flow rate is 0.5mL/min, standing for 1.9h after sampling is finished, washing impurities with pure water 3 times of the weight of the macroporous adsorption resin, eluting with 70% ethanol with volume concentration 3 times of the amount of the macroporous adsorption resin, collecting eluent, and recovering ethanol under reduced pressure to obtain purified lindera reflexa total flavonoids;
(4) preparing a monomer component: subjecting 500g of lindera reflexa total flavonoids prepared in the step (3) to silica gel column chromatography, and performing gradient elution by using a petroleum ether-dichloromethane mixed solvent with the volume ratio of 100:0, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1 and 1:1 in sequence, wherein the volume of eluent used in 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 mL/min; then performing gradient elution by using dichloromethane-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 used as one fraction, 1740 fractions are totally collected, each fraction is detected and separated by silica gel thin layer chromatography, petroleum ether-dichloromethane with volume ratio of 1:3, dichloromethane-methanol with volume ratio of 50:1 and dichloromethane-methanol with volume ratio of 3:1 are respectively used as developing agents, anisaldehyde-concentrated sulfuric acid is used as a color developing agent, heating is performed at 105 ℃ for 3-5min, and according to the detection result of the thin layer chromatography, the fractions are respectively 190-, The fractions 325 & 454, 767 & 810, 811 & 887, 926 & 983, 1316 & 1428, to obtain 7 fractions of the components Fr.1-Fr.7;
subjecting the component Fr.1 to semi-preparative liquid chromatography, eluting with methanol-water with a volume ratio of 98:5 as an eluent at a flow rate of 2mL/min and a detection wavelength of 297nm, and collecting a peak with an elution time of 22.30min to obtain a compound Reflexanbene G;
subjecting component Fr.3 to medium pressure MCI column chromatography, gradient eluting with methanol-water mixed solvent at volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0 sequentially, collecting 96 fractions with 4L eluent at flow rate of 12mL/min and one fraction per 250mL, detecting and analyzing each fraction by silica gel thin layer chromatography, subjecting GF254 thin layer plate with petroleum ether-dichloromethane at volume ratio of 1:3 as developing agent, subjecting anisaldehyde-concentrated sulfuric acid solution as developer, heating at 105 deg.C for 3-5min, combining fractions 69-80, 85-90 and 91-96 respectively according to detection result of thin layer chromatography to obtain 3 subcomponents Fr.3-1, Fr.3-2 and Fr.3-3, subjecting subcomponent Fr.3-2 to semi-preparative liquid chromatography, eluting with methanol-water at volume ratio of 98:2 as eluent, the flow rate is 2mL/min, the detection wavelength is 297nm, and peaks with the elution time of 10.44min and 20.00min are respectively collected to obtain a compound Reflexanbene H and a compound Reflexanbene J; performing semi-preparative liquid chromatography on the subfraction Fr.3-3, eluting with methanol-water with a volume ratio of 95:5 as eluent at a flow rate of 2mL/min and a detection wavelength of 297nm, and collecting the peak with an elution time of 17.90min to obtain a compound Reflexanbene D; performing semi-preparative liquid chromatography on the subfraction Fr.3-3, eluting with methanol-water with a volume ratio of 85:15 as eluent at a flow rate of 2mL/min and a detection wavelength of 297nm, and collecting the peak with an elution time of 15.37min to obtain a compound Reflexanbene K.
Example 4
The invention relates to a preparation method of stilbene compounds Reflexanbene D, Reflexanbene G, Reflexanbene J, Reflexanbene H and Reflexanbene K separated from lindera reflexa Hemsl, which comprises the following steps:
(1) preparing a lindera reflexa hemsl extract solution: crushing lindera reflexa Hemsl medicinal materials into powder, adding ethanol with the volume concentration of 70%, and performing ultrasonic extraction for 5 times, wherein the extraction time is 1.2h, and the amount of the ethanol added with 70% is 14 times of the weight of lindera reflexa Hemsl; mixing the extracting solutions, recovering ethanol under reduced pressure until the extracting solution has no alcohol smell to obtain a concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into a lindera reflexa hemsl extract solution with the mass concentration of 0.06mg/mL for later use;
(2) resin treatment and column packing: soaking macroporous adsorption resin in 95% ethanol with volume concentration for 14h, washing with 95% ethanol with volume concentration until ethanol eluate is not white and turbid when being mixed with water, washing with distilled water until no alcohol smell exists, and packing the macroporous adsorption resin into a column by a wet method according to the ratio of the diameter of the resin column to the height of the resin column of 1: 8;
(3) preparing purified lindera reflexa total flavonoids: sampling the lindera reflexa hemsl extract solution prepared in the step (1), wherein the sampling amount is 5 times of the weight of macroporous adsorption resin, the sampling flow rate is 1.5mL/min, standing for 2.1h after sampling is finished, washing impurities by pure water 5 times of the weight of macroporous adsorption resin, eluting by 70% ethanol with volume concentration 5 times of that of macroporous adsorption resin, collecting eluent, and recovering ethanol under reduced pressure to obtain purified lindera reflexa total flavone;
(4) preparing a monomer component: subjecting 500g of lindera reflexa total flavonoids prepared in the step (3) to silica gel column chromatography, and performing gradient elution by using a petroleum ether-dichloromethane mixed solvent with the volume ratio of 100:0, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1 and 1:1 in sequence, wherein the volume of eluent used in each gradient is 12.5L, 16.8L, 20L, 28L, 100L, 70.8L, 67.5L and 65.5L in sequence, and the flow rate is 22 mL/min; then performing gradient elution by using dichloromethane-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 used as one fraction, 1740 fractions are totally collected, each fraction is detected and separated by silica gel thin layer chromatography, petroleum ether-dichloromethane with volume ratio of 1:3, dichloromethane-methanol with volume ratio of 50:1 and dichloromethane-methanol with volume ratio of 3:1 are respectively used as developing agents, anisaldehyde-concentrated sulfuric acid is used as a color developing agent, heating is performed at 105 ℃ for 3-5min, and according to the detection result of the thin layer chromatography, the fractions are respectively 190-, The fractions 325 & 454, 767 & 810, 811 & 887, 926 & 983, 1316 & 1428, to obtain 7 fractions of the components Fr.1-Fr.7;
subjecting the component Fr.1 to semi-preparative liquid chromatography, eluting with methanol-water with a volume ratio of 98:5 as an eluent at a flow rate of 5mL/min and a detection wavelength of 297nm, and collecting a peak with an elution time of 22.30min to obtain a compound Reflexanbene G;
subjecting component Fr.3 to medium pressure MCI column chromatography, gradient eluting with methanol-water mixed solvent at volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0 sequentially, collecting 96 fractions with 4L eluent at flow rate of 17mL/min and one fraction per 250mL, detecting and analyzing each fraction by silica gel thin layer chromatography, subjecting GF254 thin layer plate with petroleum ether-dichloromethane at volume ratio of 1:3 as developing agent, subjecting anisaldehyde-concentrated sulfuric acid solution as developer, heating at 105 deg.C for 3-5min, combining fractions 69-80, 85-90 and 91-96 respectively according to detection result of thin layer chromatography to obtain 3 subcomponents Fr.3-1, Fr.3-2 and Fr.3-3, subjecting subcomponent Fr.3-2 to semi-preparative liquid chromatography, eluting with methanol-water at volume ratio of 98:2 as eluent, the flow rate is 5mL/min, the detection wavelength is 297nm, and peaks with the elution time of 10.44min and 20.00min are respectively collected to obtain a compound Reflexanbene H and a compound Reflexanbene J; performing semi-preparative liquid chromatography on the subfraction Fr.3-3, eluting with methanol-water with volume ratio of 95:5 as eluent at flow rate of 5mL/min and detection wavelength of 297nm, and collecting the 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 5mL/min and a detection wavelength of 297nm, and collecting a peak with an elution time of 15.37min to obtain a compound Reflexanbene K.
The stilbene 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) which were prepared by the process described in examples 1 to 4 of the present invention were structurally characterized, the compounds Reflexanbene D are all the same in structure, Reflexanbene G are all the same in structure, Reflexanbene J are all the same in structure, Reflexanbene H is also all the same in structure, Reflexanbene K is also all the same in structure, has an anti-inflammatory effect, is effectively used for preparing anti-inflammatory drugs, and obtains the same or similar results after repeated experiments, and the related information is as follows:
structural identification of compounds
By nuclear magnetic resonance spectroscopy ( 1 H-NMR、 13 C-NMR) and high resolution mass spectrometry (HR-ESI-MS) spectral techniques, wherein:
the compound Reflexanbene D3, 5-dihydroxy-4- [ (1 "S3" S4 "S) p-menthol]Trans-stilbene) is a tan oil, and the peak M/z of an excimer ion given by a high-resolution mass spectrum HR-ESI-MS is 367.2267[ M + H ]] + (calculated for C 24 H 31 O 3 367.2268), the molecular formula of the compound is presumed to be C 24 H 30 O 3 The unsaturation degree was 10. The ultraviolet spectrum shows the maximum absorption wavelength (. lamda.) of the compound Reflexanbene D in methanol max ) 212nm, 237nm and 314 nm. IR spectrum showing hydroxyl group (3431 cm) -1 ) And benzene rings (1422,1505,1579,1616 cm) -1 ) Absorption of (2).
Process for preparing compound Reflexanbene D 1 H-NMR(500MHz，CDCl 3 ) The spectrum shows a signal in which 1 benzene ring is monosubstituted: delta H 7.47(2H, d, J ═ 7.3Hz, 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); 1 hydrogen signal delta of 1, 3, 4, 5-position 4-substituted benzene ring H 6.62(1H, d, J ═ 1.3Hz, H-6), 6.45(1H, d, J ═ 1.4Hz, H-2); group 3 methyl hydrogen atom signals: 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 (125MHz, CDCl3) and DEPT-135 spectra gave 24 carbon signals, 6 quaternary carbons, 3 secondary carbons, 3 primary carbons and 12 tertiary carbons, 14 of which were olefinic carbon signals: delta. for the preparation of a coating 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 "). Based on the above hydrocarbon nuclear magnetic data and references, compound Reflexanbene D was identified as a parent core of 3, 5-dihydroxy-trans-stilbene with a 10 carbon substituent attached, where 3 carbons were methyl carbons.
The structure of the compound Reflexanbene D and the position of the linkage of each group were determined by two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR). In the HMBC spectrum, H-9 'and H-10' are related to each other and are simultaneously related to C-8 ', and are determined to be isopropyl structural fragments, H-9', H-10 ', and H-8' are all related to C-4 ', which shows that the isopropyl structural fragments are connected to C-4', and 3 carbons and delta of the isopropyl fragments are removed from 10 carbons of the substituent group C 29.3 (C-7') and also 6 carbon atoms, wherein H-5 "is associated with C-4", C-6 "and C-1", H-3 "is associated with C-2", C-4 "and C-1", 1 H- 1 h COSY showed that H-3 "had a signal correlation with H-2" and H-4 "and H-5" had a signal correlation with H-4 "and H-6", indicating that the remaining six carbons were a six-membered ring; h-7 "is related to C-1", C-6 "and C-2", indicating delta C 29.3(C-7 ') of the methyl carbon to C-1' with a DEPT-135 spectrum showing delta C 74.7 (C-1') is a quaternary carbon having a chemical shift toward lower fields, which is judged to be linked to a hydroxyl group, and the 10 carbon substituent is a structural fragment of p-menthol, as determined by reference; analysis was performed in combination with signals in HMBC spectra in which H-3 'is associated with C-4 and H-2 and H-6 are associated with C- α, to determine the attachment of C-3' to menthol to C-4 on the phenyl ring. The NOESY spectrum shows that H-3 is in NOE relation with H-4 and H-7, which indicates that H-3, H-4 and H-7 are in the same side, and the compound is found to be in the S configuration of 1 ' S3 ' S4 ' according to the comparison of the theoretical value and the actual value of ECD. In conclusion, the structure of the compound is determined to be 3, 5-dihydroxy-4- [ (1 ' S3 ' S4 ' S) to menthol group]Trans-stilbene, which is not reported to be structurally identical by the search of the Scfiner database, and is therefore determined to be a new compound, named Reflexanbene D, with the molecular formula:
method for preparing compound Reflexanbene D in Table 1 1 H-NMR and 13 C-NMR data (in CDCl) 3 )
Reflexanbene G (3, 5-dimethoxy-2- [ (3 "R4" R) p-menthenyl]Trans-stilbene) is light yellow oily substance, is easy to dissolve in chloroform, and has an excimer ion peak M/z 377.2476[ M + H ] given by high resolution mass spectrum HR-ESI-MS] + (calculated for C 26 H 33 O 2 377.2475), the molecular formula of which is presumed to be C 26 H 32 O 2 . Ultraviolet spectrum UV shows the maximum absorption wavelength (lambda) of the compound Reflexanbene G in methanol solution max ) 214nm and 299 nm. IR spectrum showing hydroxyl group (3420 cm) -1 ) And benzene rings (1459,1576 cm) -1 ) Absorption of (2).
Process for preparation of compound Reflexanbene G 1 H-NMR(CDCl 3 500MHz) shows a pair of trans double bond hydrogen signals: delta H 7.84(1H, d, J ═ 15.6Hz, H- α), 6.84(1H, d, J ═ 16.2Hz, H- β); 1 hydrogen signal on the monosubstituted phenyl ring: delta H 7.43(2H, d, J ═ 7.5Hz, 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 ) (ii) a 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 spectrum 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 the carbon signals on the monosubstituted phenyl rings; delta C 55.8(3-OCH 3 ) And 55.3 (5-OCH) 3 ) Is a methoxy groupCarbon signal of (c); delta C 23.6(C-7 "), 21.7 (C-9"), 16.2(C-10 ") are the 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") is 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 spectrum shows, delta H 3.85(3H, s) is remotely related to C-5, δ H 3.77(3H, s) is remotely related to C-3, indicating that two methoxy groups are attached at the C-5 and C-3 positions, respectively; the existence of remote correlation signals between H-6 and C-5 and C-alpha, the absence of remote correlation signals between H-4 and C-alpha, the existence of remote correlation signals between H-2 'and H-4' and C-2, which indicate that the menthenyl fragment is connected with C-2, the NOESY spectrum shows that the relation between H-3 'and H-4' does not exist, which indicate that the two are on opposite sides, the ECD data are compared with the calculated value to find that the compound has the 3 'R4' R configuration, and the data are combined to identify that the compound has the 3, 5-dimethoxy-2- [ (3 'R4' R) and the menthenyl fragment]-trans-stilbene. The compound is found to be unreported by searching a Scifinder database, so that the compound is determined to be a novel compound, namely Reflexanbene G, and the molecular structural formula is as follows:
TABLE 2 of the Compound Reflexanbene G 1 H-NMR and 13 C-NMR data (in CDCl) 3 )
The compound Reflexanbene J (3, 5-dihydroxy-2, 6-bis- [ (3 "R4" R3 '"R4'" S) -p-menthenyl]-transFormula-stilbene) is reddish brown oil, and the excimer peak M/z485.3403[ M + H ] is given by high-resolution mass spectrum HR-ESI-MS] + (calculated for C 34 H 45 O 2 485.3414), the molecular formula of the compound is presumed to be C 34 H 44 O 2 The unsaturation degree was 13. The ultraviolet spectrum shows the maximum absorption wavelength (. lamda.) of the compound Reflexanbene J in methanol max ) 203nm and 296 nm. IR spectrum showing hydroxyl group (3434 cm) -1 ) And benzene rings (1446,1599 cm) -1 ) Absorption of (2).
Of the compound Reflexanbene J 1 H-NMR(500MHz，CDCl 3 ) The spectrum shows a signal in which 1 benzene ring is monosubstituted: delta H 7.45(2H, d, J ═ 7.4Hz, H-2 ', and H-6 '), 7.39(2H, t, J ═ 7.5,7.7Hz, H-3 ', and H-5 '), 7.30(1H, d, J ═ 7.4Hz, H-4 '); 1 hydrogen signal to trans double bond: delta H 7.19(1H, d, J ═ 16.5Hz, H- α), 6.34(1H, d, J ═ 16.7Hz, 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 ═ 6.6Hz, H-9 "'), 0.84(6H, t, J ═ 4.8,6.6Hz, H-9", and H-10 "'), 0.40(3H, d, J ═ 6.6Hz, 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(C-1 '), 124.8 (C-2'), 40.8(C-3 '), 44.4 (C-4'), 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 essentially identical to literature-reported signals for menthenyl, and the compound is determined to contain two p-menthenyl fragments. Of the remaining 14 carbons of the aromatic region, delta C 126.3, 128.8 are the carbon signals on the AA 'BB' system, indicating that they are the carbons on the monosubstituted phenyl ring, determining delta in combination with HSQC H 7.19(1H, d, J ═ 16.5Hz) and 6.34(1H, d, J ═ 16.7Hz) were attached to C- α and C- β, respectively, indicating δ C 129.9, 134.0 are carbons on the trans double bond; delta H 6.30(1H, s) and δ C 105.2 linkage, indicating that the remaining 6 carbons is a penta-substituted benzene ring, δ C 155.2，156.7 shows that the benzene ring is connected with two hydroxyl groups, and the combination literature determines that the benzene ring is a trans-stilbene fragment.
The structure of the compound Reflexanbene J and the position of the linkage 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 an HMBC spectrum, and two hydroxyl groups are respectively connected to C-3 and C-5; h-alpha is related to C-1, C-2 and C-6, H-3 'and H-4' are related to C-2, H-3 'and H-4' are related to C-6, and two p-menthenyl groups are symmetrically substituted on C-2 and C-6 of a benzene ring. The NOESY spectrum shows that the H-3 'and the H-4' have NOE relation, the H-3 'and the H-4' are positioned at the same side, and the H-3 'and the H-4' have no NOE relation, which indicates that the compounds are positioned at the opposite side, and the compounds are found to be in 3 'R4' R3 'R4' S configuration according to the comparison of the theoretical value and the actual value of the ECD. In conclusion, 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 a report with the same structure is not found through the search of a Scfiner database, so that the compound is determined to be a new compound, namely Reflexanbene J, and the molecular structural formula is as follows:
tables 2-8 of the Compound Reflexanbene J 1 H-NMR and 13 C-NMR data (in CDCl) 3 )
Reflexanbene H (3, 5-dihydroxy-4- [ (3 "S4" R) p-menthenyl]Trans-stilbene) is a reddish brown oil, is easily soluble in chloroform, and has 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) The compound is presumed to have the formula C 24 H 28 O 2 The unsaturation degree was 11. The ultraviolet spectrum shows the maximum absorption wavelength (. lamda.) of the compound Reflexanbene H in methanol max ) 211nm and 314 nm. IR spectrum showing hydroxyl groups (3417 cm) -1 ) And a benzene ring (1439, 1449, 1567, 1577, 1620cm -1 ) Absorption of (2).
Process for preparing compound Reflexanbene H 1 H-NMR(500MHz，CDCl 3 ) The spectrum shows a signal in which 1 benzene ring is monosubstituted: delta H 7.48(2H, d, J ═ 7.4Hz, 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 signals delta of 21, 3, 4, 5-position 4-substituted benzene rings 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 ) Spectrum shows, there is a set of delta C 140.4, 124.4,43.6,35.7,30.7,23.7, 27.9,22.1,21.7, 16.4, which in combination with the literature is found to be a set of signals for menthenyl fragments, delta C 128.7 and 126.5 are the carbon signals on the AA 'BB' system, delta C 155.1 and 157.0 are carbon signals of the attached hydroxyl group on the benzene ring, which is substantially consistent with the signals reported in the literature for 3, 5-dihydroxy-trans-stilbene. Display of δ in HMBC spectra H 6.62(1H, m, H-6) and δ C 116.7, 157.0, 104.2 and 128.1 correlations, 6.52(1H, m, H-2) with δ C 116.7, 155.1, 106.2 and 128.1, which indicate the attachment of the menthenyl fragment to C-4. The NOESY spectrum shows that H-3 'and H-4' have NOE relation, which indicates that H-3 'and H-4' are in the same side, and the compound is found to be in 3 'S4' R configuration according to the comparison of the theoretical value and the actual value of ECD. In conclusion, the structure of the compound is determined to be 3, 5-dihydroxy-4- [ (3 'S4' R) p-menthenyl]Trans-stilbene, which is not reported to be identical in structure by the search of the Scfiner database, and is therefore determined to be a new compound, named Reflexanbene H, with the molecular structural formula:
method for producing the compound Reflexanbene H of tables 2-6 1 H-NMR and 13 C-NMR data (in CDCl) 3 )
Reflexanbene K (3, 5-dimethoxy-trans-stilbene) is white rod-shaped crystal, and the excimer ion peak M/z241.1218[ M + H ] is given by high resolution mass spectrum HR-ESI-MS] + (calculated for C 16 H 17 O 2 241.1223), the molecular formula of the compound is presumed to be C 16 H 16 O 2 The unsaturation degree was 9. The ultraviolet spectrum shows the maximum absorption wavelength (. lamda.) of the compound Reflexanbene K in methanol max ) 211nm, 299nm and 306 nm. Infrared spectrum IR shows benzene ring (1423,1455,1497,1592 cm) -1 ) Absorption of (2).
Process for preparing compound Reflexanbene K 1 H-NMR(500MHz，CDCl 3 ) The spectra show a signal with 1 single substitution of the benzene rings: delta H 7.51(2H, d, J ═ 7.85Hz, 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); 1 hydrogen signal delta of 1, 3, 5-position 3-substituted benzene ring H 6.67(2H, d, J ═ 2.0Hz, 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 the DEPT-135 spectra give a 16 carbon signal: 4 quaternary carbons, 2 primary carbons and 10 tertiary carbons, where the signal for two primary carbons is δ C 55.4. The aromatic region has a total of 14 carbons, and 12 carbons remain after removal of 2 carbons from the trans double bond, presumably two benzene rings, δ C 161.0 has two quaternary carbon creditsNumber, indicates that there are two methoxy groups symmetrically substituted on the phenyl ring, δ C 128.7 and 126.6 are carbon signals on the AA 'BB' system. The compound is determined to be 3, 5-dimethoxy-trans-stilbene (3,5-dimethoxystilbene) by combining references, is named as Reflexanbene K, and has a molecular structural formula as follows:
TABLE 2-10 of the Compound Reflexanbene K 1 H-NMR and 13 C-NMR data (in CDCl) 3 )
Second, Activity test
The compounds Reflexanbene D, Reflexanbene G, Reflexanbene J, Reflexanbene H and Reflexanbene K prepared by the invention have anti-inflammatory action through experimental findings, and the related experimental data are as follows:
1. experimental Material
Mouse mononuclear macrophage RAW264.7 was purchased from Wuhan Punuoise Life technologies, Inc.; fetal bovine serum was purchased from Gibco.
2. Cell culture
RAW264.7 cells were cultured in DMEM high-sugar medium containing 10% heat-inactivated fetal bovine serum, 100U/mL penicillin, and 100. mu.g/mL streptomycin, and the culture dishes were placed at 37 ℃ and 5% CO 2 Culturing in saturated humidity incubator, changing culture medium every 1-2 days, and digesting with trypsin for passage when the cell fusion degree reaches above 80%.
3. Effect of drugs on macrophage viability (MTT)
Culturing logarithmic growth phase cells in 96-well plate at a temperature of 37 deg.C and 5% per well with a volume of 100 μ L (2000 cells)CO 2 Incubate for 12h, carefully remove the medium, then add dilutions containing different concentrations of test compound, set up 5 dose groups, each set up 3 replicate wells, 200 μ Ι _ per well. Adding solvent with the same volume as the administered group into the control group, and standing at 37 deg.C and 5% CO 2 Incubate for 24h, carefully remove the medium, and add 20. mu.L (1mg/mL) MTT solution and 100. mu.L serum-free medium per well. 37 ℃ and 5% CO 2 After incubation in an incubator for 4h, the supernatant was discarded, formazan particles were solubilized by adding 150. mu.L of DMSO to each well, solubilized with gentle shaking, and their absorbance values (OD) were measured at a wavelength of 570nm using an enzyme-linked immunosorbent assay (ELISA) detector, and the solvent-control-treated cells were used as controls, and the cell survival rate was calculated.
4. Effect of drugs on LPS-induced NO and IL-6 Release amounts in RAW264.7 cells
4.1. Collection of samples
Cells in logarithmic growth phase were cultured in 96-well plates at 100. mu.L/well (containing 1X 10 cells) 5 Individual cells) were incubated at 37 ℃ with 5% CO 2 Incubate for 12h, carefully remove the medium, then add 200 μ L of drug diluted in 1 μ g/mL LPS-containing medium, set 3 dose groups, each set 3 replicate wells, 200 μ L per well. Adding 200 μ L DMEM medium without fetal calf serum into blank group, adding 200 μ L culture medium containing 1 μ g/mL LPS into control group, placing at 37 deg.C and 5% CO 2 Culturing in an incubator for 24h, transferring the cell culture supernatant to a centrifuge tube, and storing in a refrigerator at-80 ℃ for later use.
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 naphthyl ethylenediamine hydrochloride is precisely weighed and dissolved in 50mL of 5% phosphoric acid solution to obtain 0.1% naphthyl ethylenediamine hydrochloride solution, 0.50g of sulfanilamide is precisely weighed and dissolved in 50mL of 5% phosphoric acid solution to obtain 1% sulfanilamide solution, the sulfanilamide solution is stored at 4 ℃ in a dark place and is mixed according to the ratio of 1:1 before use.
(3) 0.13799g of NaNO were precisely weighed 2 Dissolving in 10mL of double distilled water to obtain 200mM NaNO 2 The solution was stored at 4 ℃ in the dark.
4.3. measurement of NO Release amount
(1) And (3) standard substance: the standards were diluted to 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 and 0.15625 μ M solutions with DMEM medium.
(2) Sample preparation: the resulting cell culture fluid was collected.
(3) After adding 100. mu.L of the standard solution or the sample to be tested 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 using a microplate reader.
Determination of IL-6 Release amounts
The concentration of IL-6 in the sample was determined by the double antibody Sandwich method (Sandwich). Respectively adding the sample and the standard substance with different concentrations into corresponding enzyme label plate holes according to 100 mu L/hole, sealing the plate and the membrane sealing plate, incubating for 90 minutes at room temperature, washing the plate for 6 times, and drying by patting on absorbent paper each time. Add 100. mu.L of detection antibody to each well, seal the plate with membrane, incubate for 30min at room temperature, wash the plate 6 times. Adding 100 mu L of streptavidin marked by horseradish peroxidase, sealing the plate with a membrane seal plate, incubating for 30 minutes at room temperature, and washing the plate for 6 times. Add signal enhancer 100. mu.L, seal plate membrane plate, incubate 15 minutes at room temperature, wash plate 6 times. And adding 100 mu L of streptavidin marked by horseradish peroxidase again, sealing the plate by a sealing membrane and incubating for 15 minutes at room temperature. Adding 100 mu L of color reagent TMB solution, sealing the plate with a plate film, keeping out of the light, and incubating for 15 minutes at room temperature. Adding 100 μ L of stop solution, mixing, immediately measuring OD values at 450nm maximum absorption wavelength and 570nm reference wavelength, and calibrating to obtain OD value of 450nm minus 570 nm.
5. Results of the experiment
5.1. Effect of drugs on macrophage Activity
After the drug and RAW264.7 cells were co-cultured for 24h, the effect of Reflexanbene K (<100 μ M) on cell viability was small, and when the concentration of each compound was less than 25 μ M, the cell viability was over 80% and cytotoxicity was small (fig. 38).
5.2. Effect of drugs on LPS-induced NO and IL-6 Release amounts in 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 is remarkably reduced, and certain dose dependence is shown, and the release amount of NO is reduced along with the increase of the concentration (FIG. 39). After 5 isolated compounds act on a cell inflammation model for 24 hours, the release amount of IL-6 is reduced, and the release amount is very different from that of a model group when the concentration reaches 20 mu M (p is less than 0.01), wherein after the concentration reaches 20 mu M, the effect of reducing IL-6 is close to that of a positive control drug dexamethasone (Dex), and the good anti-inflammatory effect is shown (figure 40).
In conclusion, experiments show that the compounds Reflexanbene J and Reflexanbene H can effectively inhibit the release level of NO induced by RAW264.7 cells by LPS, and the separated 5 compounds, particularly the compound Reflexanbene D can effectively inhibit the release level of inflammatory factor IL-6. Therefore, the invention has application value in preparing anti-inflammatory drugs, develops 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 the preparation method is a great innovation in preparing the anti-inflammatory medicament and has obvious economic and social benefits.
Claims (6)
1. A stilbene compound separated from lindera reflexa Hemsl is characterized by comprising compounds Reflexanbene D, G, J, H and K, and the molecular structural formulas are respectively as follows:
2. the method for preparing stilbene compounds separated from lindera reflexa hemsl according to claim 1, comprising the steps of:
(1) preparing a lindera reflexa hemsl extract solution: crushing lindera reflexa Hemsl medicinal materials into powder, adding ethanol with the volume concentration of 70% for ultrasonic extraction for 3-5 times, wherein the extraction time is 0.8-1.2 h, and the amount of the ethanol with the volume concentration of 70% added for each time is 10-14 times of the weight of the lindera reflexa Hemsl; combining the extracting solutions, recovering ethanol under reduced pressure until the extracting solution has no alcohol smell to obtain a concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into a lindera reflexa hemsl extract solution with the mass concentration of 0.04-0.06 mg/mL for later use;
(2) resin treatment and column packing: soaking macroporous adsorbent resin in 95% ethanol for 10-14h, washing with 95% ethanol until ethanol eluate is not white turbid, washing with distilled water until no ethanol smell is produced, and wet packing the macroporous adsorbent resin into column according to the ratio of the diameter of the resin column to the height of the column of 1: 8;
(3) preparing purified lindera reflexa total flavonoids: sampling the lindera reflexa hemsl extract solution prepared in the step (1), wherein the sampling amount is 3-5 times of the weight of macroporous adsorption resin, the sampling flow rate is 0.5-1.5 mL/min, standing for 1.9-2.1 h after sampling is finished, washing impurities with pure water 3-5 times of the weight of macroporous adsorption resin, eluting with 70% ethanol with volume concentration 3-5 times of the macroporous adsorption resin, collecting eluent, and recovering ethanol under reduced pressure to obtain purified lindera reflexa total flavonoids;
(4) preparing a monomer component: performing chromatographic separation on 500g of lindera reflexa total flavonoids prepared in the step (3) by using a silica gel column, and performing 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 in sequence, wherein the volume of eluent used for 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 performing gradient elution by using a dichloromethane-methanol mixed solvent 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, every 500mL is used as a fraction, totally 1740 fractions are collected, each fraction is detected and separated by silica gel thin layer chromatography, a GF254 thin layer plate is used, petroleum ether-dichloromethane with volume ratio of 1:3, dichloromethane-methanol with volume ratio of 50:1 and dichloromethane-methanol with volume ratio of 3:1 are used as developing agents, anisaldehyde-concentrated sulfuric acid is used as a color developing agent, the mixture is heated at 105 ℃ for 3-5min, and according to the detection result of the thin layer chromatography, the fractions are respectively 190-, The fractions 279-324, 325-454, 767-810, 811-887, 926-983 and 1316-1428 to obtain 7 components Fr.1-Fr.7;
subjecting the component Fr.1 to semi-preparative liquid chromatography, eluting with methanol-water with a volume ratio of 98:5 as an eluent at a flow rate of 2-5 mL/min and a detection wavelength of 297nm, and collecting a peak with an elution time of 22.30min to obtain a compound Reflexanbene G;
subjecting component Fr.3 to medium pressure MCI column chromatography, gradient eluting with methanol-water mixed solvent at volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0 sequentially, collecting 96 fractions with each gradient eluting with 4L of eluent at flow rate of 12-17 mL/min and one fraction per 250mL, detecting and analyzing each fraction by silica gel thin layer chromatography, subjecting each fraction to GF254 thin layer chromatography, developing with petroleum ether-dichloromethane at volume ratio of 1:3 as developing agent, subjecting anisaldehyde-concentrated sulfuric acid solution as developer, heating at 105 deg.C for 3-5min, combining fractions 69-80, 85-90 and 91-96 respectively according to detection result of thin layer chromatography to obtain 3 subcomponents Fr.3-1, Fr.3-2 and Fr.3-3, subjecting subcomponent Fr.3-2 to semi-preparative liquid chromatography, eluting with methanol-water at volume ratio of 98:2, the flow rate is 2-5 mL/min, the detection wavelength is 297nm, and peaks with the elution time of 10.44min and 20.00min are collected respectively to obtain a compound Reflexanbene H and a compound Reflexanbene J; performing semi-preparative liquid chromatography on the subfraction Fr.3-3, eluting with methanol-water with a volume ratio of 95:5 as an eluent at a flow rate of 2-5 mL/min and a detection wavelength of 297nm, and collecting a peak with an 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 an eluent at a flow rate of 2-5 mL/min and a detection wavelength of 297nm, and collecting a peak with an elution time of 15.37min to obtain a compound Reflexanbene K.
3. The method for preparing stilbene compounds separated from lindera reflexa Hemsl according to claim 2, comprising the steps of:
(1) preparing a lindera reflexa hemsl extract solution: crushing lindera reflexa hemsl medicinal materials into powder, adding ethanol with the volume concentration of 70%, and performing ultrasonic extraction for 3 times, wherein the extraction time is 1 hour each time, and the amount of the ethanol added with 70% each time is 12 times of the weight of lindera reflexa hemsl; mixing the extracting solutions, recovering ethanol under reduced pressure until the extracting solution has no alcohol smell to obtain a concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into a lindera reflexa hemsl extract solution with the mass concentration of 0.05mg/mL for later use;
(2) resin treatment and column packing: soaking macroporous adsorbent resin in 95% ethanol for 12 hr, washing with 95% ethanol until the ethanol eluate is not white and turbid, washing with distilled water until no ethanol smell is produced, and wet packing the macroporous adsorbent resin into column according to the ratio of the diameter of the resin column to the height of the column of 1: 8;
(3) preparing purified lindera reflexa total flavonoids: sampling the lindera reflexa hemsl extract solution prepared in the step (1), wherein the sampling amount is 4 times of the weight of macroporous adsorption resin, the sampling flow rate is 1mL/min, standing for 2h after sampling is finished, washing impurities by pure water 4 times of the weight of macroporous adsorption resin, eluting by 70% ethanol with volume concentration 4 times of the weight of macroporous adsorption resin, collecting eluent, and recovering ethanol under reduced pressure to obtain purified lindera reflexa total flavone;
(4) preparing a monomer component: performing chromatographic separation on 500g of lindera reflexa total flavonoids prepared in the step (3) by using a silica gel column, and performing 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 in sequence, wherein the volume of eluent used for each gradient is 12.5L, 16.8L, 20L, 28L, 100L, 70.8L, 67.5L and 65.5L in sequence, and the flow rate is 20 mL/min; then performing gradient elution by using dichloromethane-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 used as one fraction, 1740 fractions are totally collected, each fraction is detected and separated by silica gel thin layer chromatography, petroleum ether-dichloromethane with volume ratio of 1:3, dichloromethane-methanol with volume ratio of 50:1 and dichloromethane-methanol with volume ratio of 3:1 are respectively used as developing agents, anisaldehyde-concentrated sulfuric acid is used as a color developing agent, heating is performed at 105 ℃ for 3-5min, and according to the detection result of the thin layer chromatography, the fractions are respectively 190-, The fractions 325 & 454, 767 & 810, 811 & 887, 926 & 983, 1316 & 1428, to obtain 7 fractions of the components Fr.1-Fr.7;
subjecting the component Fr.1 to semi-preparative liquid chromatography, eluting with methanol-water at a volume ratio of 98:5 as eluent at a flow rate of 3mL/min and a detection wavelength of 297nm, and collecting the peak with elution time of 22.30min to obtain a compound Reflexanbene G;
subjecting component Fr.3 to medium pressure MCI column chromatography, gradient eluting with methanol-water mixed solvent at volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0 sequentially, collecting 96 fractions with 4L eluent at flow rate of 15mL/min and one fraction per 250mL, detecting and analyzing each fraction by silica gel thin layer chromatography, subjecting GF254 thin layer plate with petroleum ether-dichloromethane at volume ratio of 1:3 as developing agent, subjecting anisaldehyde-concentrated sulfuric acid solution as developer, heating at 105 deg.C for 3-5min, combining fractions 69-80, 85-90 and 91-96 respectively according to detection result of thin layer chromatography to obtain 3 subcomponents Fr.3-1, Fr.3-2 and Fr.3-3, subjecting subcomponent Fr.3-2 to semi-preparative liquid chromatography, eluting with methanol-water at volume ratio of 98:2 as eluent, the flow rate is 3mL/min, the detection wavelength is 297nm, and peaks with the elution time of 10.44min and 20.00min are respectively collected to obtain a compound Reflexanbene H and a compound Reflexanbene J; performing semi-preparative liquid chromatography on the subfraction Fr.3-3, eluting with methanol-water with a volume ratio of 95:5 as eluent at a flow rate of 3mL/min and a detection wavelength of 297nm, and collecting the peak with an elution time of 17.90min to obtain a compound Reflexanbene D; performing semi-preparative liquid chromatography on the subfraction Fr.3-3, eluting with methanol-water with volume ratio of 85:15 as eluent at flow rate of 3mL/min and detection wavelength of 297nm, and collecting the peak with elution time of 15.37min to obtain a compound Reflexanbene K.
4. The method for preparing stilbene compounds separated from lindera reflexa Hemsl according to claim 2, comprising the steps of:
(1) preparing a lindera reflexa hemsl extract solution: crushing lindera reflexa Hemsl medicinal materials into powder, adding ethanol with volume concentration of 70%, and performing ultrasonic extraction for 3 times, wherein the extraction time is 0.8h, and the amount of the ethanol added with 70% is 10 times of the weight of lindera reflexa Hemsl; mixing the extracting solutions, recovering ethanol under reduced pressure until the extracting solution has no alcohol smell to obtain a concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into a lindera reflexa hemsl extract solution with the mass concentration of 0.04mg/mL for later use;
(2) resin treatment and column packing: soaking macroporous adsorption resin in 95% ethanol with volume concentration for 10h, washing with 95% ethanol with volume concentration until ethanol eluate is not white and turbid when being mixed with water, washing with distilled water until no alcohol smell exists, and packing the macroporous adsorption resin into a column by a wet method according to the ratio of the diameter of the resin column to the height of the resin column of 1: 8;
(3) preparing purified lindera reflexa total flavonoids: sampling the lindera reflexa hemsl extract solution prepared in the step (1), wherein the sampling amount is 3 times of the weight of macroporous adsorption resin, the sampling flow rate is 0.5mL/min, standing for 1.9h after sampling is finished, washing impurities by pure water with the weight 3 times of that of the macroporous adsorption resin, eluting by 70% ethanol with the volume concentration 3 times of that of the macroporous adsorption resin, collecting eluent, and recovering ethanol under reduced pressure to obtain purified lindera reflexa total flavone;
(4) preparing a monomer component: performing chromatographic separation on 500g of lindera reflexa total flavonoids prepared in the step (3) by using a silica gel column, and performing 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 in sequence, wherein the volume of eluent used for 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 mL/min; then performing gradient elution by using dichloromethane-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 used as one fraction, 1740 fractions are totally collected, each fraction is detected and separated by silica gel thin layer chromatography, petroleum ether-dichloromethane with volume ratio of 1:3, dichloromethane-methanol with volume ratio of 50:1 and dichloromethane-methanol with volume ratio of 3:1 are respectively used as developing agents, anisaldehyde-concentrated sulfuric acid is used as a color developing agent, heating is performed at 105 ℃ for 3-5min, and according to the detection result of the thin layer chromatography, the fractions are respectively 190-, The fractions 325 & 454, 767 & 810, 811 & 887, 926 & 983, 1316 & 1428, to obtain 7 fractions of the components Fr.1-Fr.7;
subjecting the component Fr.1 to semi-preparative liquid chromatography, eluting with methanol-water with a volume ratio of 98:5 as an eluent at a flow rate of 2mL/min and a detection wavelength of 297nm, and collecting a peak with an elution time of 22.30min to obtain a compound Reflexanbene G;
subjecting component Fr.3 to medium pressure MCI column chromatography, gradient eluting with methanol-water mixed solvent at volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0 sequentially, collecting 96 fractions with 4L eluent at flow rate of 12mL/min and one fraction per 250mL, detecting and analyzing each fraction by silica gel thin layer chromatography, subjecting GF254 thin layer plate with petroleum ether-dichloromethane at volume ratio of 1:3 as developing agent, subjecting anisaldehyde-concentrated sulfuric acid solution as developer, heating at 105 deg.C for 3-5min, combining fractions 69-80, 85-90 and 91-96 respectively according to detection result of thin layer chromatography to obtain 3 subcomponents Fr.3-1, Fr.3-2 and Fr.3-3, subjecting subcomponent Fr.3-2 to semi-preparative liquid chromatography, eluting with methanol-water at volume ratio of 98:2 as eluent, the flow rate is 2mL/min, the detection wavelength is 297nm, and peaks with the elution time of 10.44min and 20.00min are respectively collected to obtain a compound Reflexanbene H and a compound Reflexanbene J; performing semi-preparative liquid chromatography on the subfraction Fr.3-3, eluting with methanol-water with a volume ratio of 95:5 as eluent at a flow rate of 2mL/min and a detection wavelength of 297nm, and collecting the peak with an elution time of 17.90min to obtain a compound Reflexanbene D; performing semi-preparative liquid chromatography on the subfraction Fr.3-3, eluting with methanol-water with a volume ratio of 85:15 as eluent at a flow rate of 2mL/min and a detection wavelength of 297nm, and collecting the peak with an elution time of 15.37min to obtain a compound Reflexanbene K.
5. The method according to claim 2, comprising the steps of:
(1) preparing a lindera reflexa hemsl extract solution: crushing lindera reflexa Hemsl medicinal materials into powder, adding ethanol with the volume concentration of 70% and carrying out ultrasonic extraction for 5 times, wherein the extraction time is 1.2h, and the amount of the added ethanol with the volume concentration of 70% is 14 times of the weight of lindera reflexa Hemsl; mixing the extracting solutions, recovering ethanol under reduced pressure until the extracting solution has no alcohol smell to obtain a concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into a lindera reflexa hemsl extract solution with the mass concentration of 0.06mg/mL for later use;
(2) resin treatment and column packing: soaking macroporous adsorption resin in 95% ethanol with volume concentration for 14h, washing with 95% ethanol with volume concentration until ethanol eluate is not white and turbid when being mixed with water, washing with distilled water until no alcohol smell exists, and packing the macroporous adsorption resin into a column by a wet method according to the ratio of the diameter of the resin column to the height of the resin column of 1: 8;
(3) preparing purified lindera reflexa total flavonoids: sampling the lindera reflexa hemsl extract solution prepared in the step (1), wherein the sampling amount is 5 times of the weight of macroporous adsorption resin, the sampling flow rate is 1.5mL/min, standing for 2.1h after sampling is finished, washing impurities by pure water 5 times of the weight of macroporous adsorption resin, eluting by 70% ethanol with volume concentration 5 times of that of macroporous adsorption resin, collecting eluent, and recovering ethanol under reduced pressure to obtain purified lindera reflexa total flavone;
(4) preparing a monomer component: performing chromatographic separation on 500g of lindera reflexa total flavonoids prepared in the step (3) by using a silica gel column, and performing 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 in sequence, wherein the volume of eluent used for each gradient is 12.5L, 16.8L, 20L, 28L, 100L, 70.8L, 67.5L and 65.5L in sequence, and the flow rate is 22 mL/min; then performing gradient elution by using dichloromethane-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 used as one fraction, 1740 fractions are totally collected, each fraction is detected and separated by silica gel thin layer chromatography, petroleum ether-dichloromethane with volume ratio of 1:3, dichloromethane-methanol with volume ratio of 50:1 and dichloromethane-methanol with volume ratio of 3:1 are respectively used as developing agents, anisaldehyde-concentrated sulfuric acid is used as a color developing agent, heating is performed at 105 ℃ for 3-5min, and according to the detection result of the thin layer chromatography, the fractions are respectively 190-, The fractions 325 & 454, 767 & 810, 811 & 887, 926 & 983, 1316 & 1428, to obtain 7 fractions of the components Fr.1-Fr.7;
subjecting the component Fr.1 to semi-preparative liquid chromatography, eluting with methanol-water with a volume ratio of 98:5 as an eluent at a flow rate of 5mL/min and a detection wavelength of 297nm, and collecting a peak with an elution time of 22.30min to obtain a compound Reflexanbene G;
subjecting component Fr.3 to medium pressure MCI column chromatography, gradient eluting with methanol-water mixed solvent at volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0 sequentially, collecting 96 fractions with 4L eluent at flow rate of 17mL/min and one fraction per 250mL, detecting and analyzing each fraction by silica gel thin layer chromatography, subjecting GF254 thin layer plate with petroleum ether-dichloromethane at volume ratio of 1:3 as developing agent, subjecting anisaldehyde-concentrated sulfuric acid solution as developer, heating at 105 deg.C for 3-5min, combining fractions 69-80, 85-90 and 91-96 respectively according to detection result of thin layer chromatography to obtain 3 subcomponents Fr.3-1, Fr.3-2 and Fr.3-3, subjecting subcomponent Fr.3-2 to semi-preparative liquid chromatography, eluting with methanol-water at volume ratio of 98:2 as eluent, the flow rate is 5mL/min, the detection wavelength is 297nm, and peaks with the elution time of 10.44min and 20.00min are respectively collected to obtain a compound Reflexanbene H and a compound Reflexanbene J; performing semi-preparative liquid chromatography on the subcomponent Fr.3-3, eluting with methanol-water with a volume ratio of 95:5 as an eluent at a flow rate of 5mL/min and a detection wavelength of 297nm, and collecting a peak with an elution time of 17.90min to obtain a compound Reflexanbene D; performing semi-preparative liquid chromatography on the subfraction Fr.3-3, eluting with methanol-water with volume ratio of 85:15 as eluent at flow rate of 5mL/min and detection wavelength of 297nm, and collecting the peak with elution time of 15.37min to obtain a compound Reflexanbene K.
6. Use of stilbene compounds isolated from lindera reflexa hemsl prepared by the method of any one of claims 2 to 5 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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