CN104086559B - A kind of crystal formation of diterpene-kind compound - Google Patents
A kind of crystal formation of diterpene-kind compound Download PDFInfo
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- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
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Abstract
The invention provides the crystal formation of compound shown in formula II, this crystal formation is rhombic system, and space group is P212121, cell parameter isα＝90.00°，β＝90.00°，γ=90.00 °, Z=4, unit cell volume isThe present invention also provides preparation method and the purposes of this crystal formation. In motherwort, separate the formula II crystal formation compound obtaining platelet aggregation is had to certain inhibitory action, under same concentration (0.1mM), than medicament for resisting platelet aggregation " bisulfate clopidogrel " better effects if, for the exploitation of the natural medicament for resisting platelet aggregation of novelty provides new selection.
Description
Technical field
The present invention relates to a kind of novel crystal forms of diterpene-kind compound.
Background technology
Motherwort begins to be loaded in Shennong's Herbal, and original name motherwort, is listed in top grade. In Compendium of Material MedicaIn be referred to as " panacea of blood man ", cure mainly " threatened abortion difficult labour, retention of afterbirth, bruise, blood wind, blood pain,Metrorrhagia and metrostaxis, hematuria ", be gynaecology's multiparity key medicine.
In motherwort, mainly contain alkaloid, diterpene, flavones, iridoid glycoside, benzyl carbinol glycosides, volatilizationThe compositions such as oil, peptide class and fatty acid, what activity research was more at present is mainly alkaloid and Diterpenes:
(1) alkaloid: mainly comprise stachydrine, leonurine, motherwort pyridine and leonurinine etc. ExistingGeneration research shows that motherwort total alkaloid has a therapeutic action to cerebral ischemia re-pouring injured, wherein, and motherwortAlkali not only can be protected by antioxidation the cardiac muscle cell of ischemic injuries, also has neuroprotective activity.
(2) diterpene: in recent years its study of active components is increased, wherein LeoheteroninA andThe obvious acetylcholine esterase inhibition activity of LeopersinG, is having well aspect treatment Alzheimer diseaseProspect; Prehispanolone is a kind of blood platelet antagonism factor, can suppress platelet aggregation; LeojaponinCerebral cortex cell to injured with glucose deprivation has protective effect; The labdane type diterpenoids tool that Khan obtainsThere is antiinflammatory action etc.
Although along with the intensification of people to motherwort understanding and the progress of modern science and technology, motherwort drug effectThe research of position and active component is carried out rapidly, Diterpenes chemical constitution study more, activity research phaseTo less. It is relevant that antiplatelet aggregative activity and traditional effect of motherwort " are invigorated blood circulation ", but relevant anti-freezingThe research of blood component extremely lacks. As can be seen here, from motherwort, obtaining anticoagulant composition has wellProspect and researching value.
Summary of the invention
The object of the present invention is to provide a kind of novel crystal forms of furans lactonic ring analog derivative.
The invention provides the crystal formation of compound shown in formula II,
This crystal formation is rhombic system, and space group is P212121, cell parameter isα＝90.00°，β＝90.00°，γ＝90.00°，Z＝4，Unit cell volume is
Further, the specific rotatory power of described crystal formation Chinese style I compound is [α]20 D-58.8。
The present invention also provides the preparation method of above-mentioned crystalline form I, and it comprises following operating procedure:
(1) take motherwort medicinal material, pulverize, add 95%v/v alcohol extract, extract recovered under reduced pressure is moltenAfter agent, obtain ethanol extract;
(2) by ethanol extract aqueous dispersion, be extracted with ethyl acetate successively, combined ethyl acetate part,Decompression and solvent recovery, obtains ethyl acetate extract;
(3) adopt silica gel column chromatography to separate ethyl acetate extract, benzinum: acetone=(100:1)～(0:1) gradient elution, gets anti-phase polystyrene type resin in benzinum-acetone=5:1 wash-out partPost, 50-100%v/v methanol aqueous solution gradient elution, collects 70% methanol-eluted fractions part, then by middle pressureLiquid chromatogram, octadecyl silane chromatographic column, 50-95%v/v methanol aqueous solution gradient elution, collects70～75% methanol aqueous solution wash-out parts, reclaim solvent, and residue is again through SephadexLH-20 glucanGel column chromatography separates, and Shi You Mi ﹕ tri-Lv Jia Wan ﹕ methyl alcohol=5 ﹕ 5 ﹕ 1 wash-outs are collected eluent,Reclaim solvent, anti-phase liquid phantom preparing chromatogram on residue, 82%v/v methanol aqueous solution wash-out, collects respectivelyThe eluent of different appearance times, can obtain formula II compound;
(4) modus ponens II compound, adds acetone, be placed on subsequently in the drier that drier is housed,Be placed to crystal and separate out, obtain crystal formation.
Further, the mass volume ratio of formula II compound and acetone is: 10mg:0.1-1ml.
Further, the mass volume ratio of formula II compound and acetone is: 10mg:0.3-0.5ml.
Preferably, the mass volume ratio of formula II compound and acetone is: 10mg:0.4ml.
Further, described drier is selected from discolour silica gel. The present invention also provides above-mentioned crystalline form I in systemPurposes in standby platelet aggregation-against class medicine.
The present invention also provides a kind of pharmaceutical composition, and it is the preparation that contains above-mentioned crystal formation.
The present invention can adopt preparation technique means or the pharmaceutical methods of this area routine, by crystalline substance of the present inventionType is prepared into suitable medicine type, comprises: tablet, injection, tincture, suppository, capsule, creamAgent (ointment, cream), eye-drops preparations, pill, implant, syrup, mist agent (aerosol,Powder spray, spray), film, granule, oral solution (oral suspensions, Orally taken emulsion),Powder, aural preparations, nasal formulations, lotion (irrigation, enema), liniment (paint, plastics),Gel, patch etc.; Be preferably tablet, capsule, eye-drops preparations. Wherein, described tablet is selected from and containsSheet, sublingual tablet, mouth paster, chewable tablets, dispersing tablet, fuse, effervescent tablet, sustained release tablets, controlled releaseSheet, enteric coatel tablets etc.; Described injection is selected from injection, transfusion, freeze-dried powder, emulsion, implant, micro-Ball preparation, pellet preparations etc.; Described capsule is selected from hard shell capsules, soft capsule, spansule, controlled release glueCapsule and capsulae enterosolubilis etc.; Described eye-drops preparations is selected from eye drops, eyewash, intraocular injection solution, eye ointmentAgent, cream, gel for eye, eye mask agent, eye pill, intraocular insertion agent etc. for eye; Described pillBe selected from dripping pill, sugar-pill etc.; Described granule is selected from mix suspension grain, effervescence granular, enteric coated particles, slowly-releasingParticle, controlled release granule etc.; Described aural preparations is selected from auristilla, ear lotion, for ear, spray, ear are usedOintment, cream for ear, gel for ear, earplug, powder for ear, pill etc. for ear; Described noseWith preparation be selected from nasal drop, collunarium, nasal spray, for nose, ointment, gel for nose, nose are usedPowder, powder nose inhalant, stylus etc. for nose.
In motherwort, separating the formula II crystal formation compound obtaining has certain inhibitory action to platelet aggregation,Under same concentration (0.1mM), than medicament for resisting platelet aggregation " bisulfate clopidogrel " better effects if,For the exploitation of the natural medicament for resisting platelet aggregation of novelty provides new selection.
Below by detailed description of the invention, the present invention is described in further detail, but do not limit thisBright, those skilled in the art can make according to the present invention various changes and distortion, only otherwise depart from thisBright spirit, all should belong to the scope of claims of the present invention.
Brief description of the drawings
The single crystal diffraction structure chart of Fig. 1 formula II compound crystal formation
Detailed description of the invention
Embodiment 1 (-)-(3R, 5S, 7R, 8R, 9R, 10S, 13R, 15R)-3-acetoxyl group-7-hydroxyl-15-ethyoxyl-9,13; Extraction, separation and purification and the knot of 15,16-bis-epoxy Ladanum alkane-6-ketone (formula II compound crystal formation)Structure qualification
(1) experiment material:
1. medicinal material
Motherwort is picked up from Wenjiang District, Chengdu, Sichuan, teaches through the Li Min of Chinese traditional medicine identification teaching and research room of Chengdu University of Traditional Chinese MedicineAward the herb that is accredited as labiate motherwort LeonurusjaponicusHoutt..
2. reagent and filler
Column chromatography silica gel, 200～300 orders (SILVER REAGENT), are purchased from Qingdao Haiyang silica-gel desiccant factory;
Tlc silica gel G, GF254And H (chemical pure), be purchased from Qingdao Haiyang silica-gel desiccant factory;
MCIgelCHP20P, 75～150 μ m, for anti-phase polystyrene type resin, are purchased from MitsubishiChemical company;
SephadexLH-20 sephadex, is purchased from Amersham company of Sweden;
GF254Silica gel Preparative TLC, is purchased from Yantai Jiang You silica gel development corporation, Ltd.;
Chromatogram methyl alcohol, 4L/ bottle, is purchased from Fisher company of the U.S.;
The AR such as benzinum, ethyl acetate, n-butanol, acetone, methyl alcohol, are purchased from Chengdu section dragonChemical reagent factory.
3. laboratory apparatus
Cometro6000 high performance liquid chromatograph (U.S. Cometro);
WatersSynaptG2HDMS high-resolution flight time mass spectrum (U.S. Waters);
Bruker-AV-400 NMR (Switzerland Bruker);
Vector22FT-IR infrared spectrometer (Switzerland Bruker);
ShimadzuUV-260 spectrophotometry instrument (Japanese Shimadzu);
Perkin-Elmer341 polariscopy instrument (U.S. PerkinElmer);
BP211D 100,000/electronic balance (Switzerland Sartorius);
R-210 rotary evaporator (Switzerland BUCHI);
DZG-6050 type vacuum drying chamber (the gloomy letter in Shanghai).
(2) extraction of medicinal material: take motherwort medicinal material 20Kg, pulverize, add 95% ethanol (3 × 160L)Extract 72 hours, extract 3 times, extract after decompression and solvent recovery, obtains ethanol extract 1.2 after mergingKg；
(3) separation and purification of composition:
1. take motherwort medicinal material 20kg, pulverize, add 95%v/v ethanol 160L and extract 3 times, extractAfter decompression and solvent recovery, obtain ethanol extract 1.2kg;
2. by ethanol extract aqueous dispersion, use successively ethyl acetate, extracting n-butyl alcohol, combined ethyl acetatePart, decompression and solvent recovery, obtains ethyl acetate extract 400g;
3. adopt silica gel column chromatography to separate ethyl acetate extract, benzinum: acetone=(100:1)～(0:1)Gradient elution, gets benzinum-acetone=5:1 wash-out part (H) 8.2g, upper anti-phase polystyrene type resin postMCIgelCHP20P, 50-100%v/v methanol aqueous solution gradient elution, collects 70% methanol-eluted fractions part(H-3) 2.1g, then by anti-phase RP-C18 medium pressure liquid chromatography, 50-95%v/v methanol aqueous solution gradientWash-out, thin layer is followed the trail of, and collects 70～75% eluents (H-3-4) that contain target compound, reclaims solvent,Residue separates through SephadexLH-20 dextran gel column chromatography again, Shi You Mi ﹕ tri-Lv Jia Wan ﹕ firstAlcohol=5 ﹕ 5 ﹕ 1 wash-outs, thin-layer chromatography is followed the trail of, and collects and contains target compound cut, reclaims solvent,Residue separates through anti-phase liquid phantom preparing chromatogram, 82%v/v methanol aqueous solution wash-out, and collection retention time istRThe target product of=48min, except desolventizing, gained solid content is for subsequent use.
4. get solid content 10mg, be placed in the glass sample bottle of 2ml, add 0.4ml acetone. Subsequently willSample bottle is put into the drier that discolour silica gel is housed, and places until separate out colourless needle, obtains formula IIizationCompound crystal formation.
(4) Structural Identification of composition: clear crystal; Without blackening, iodine displaing yellow, spray 10% sulfuric acid ethanolSolution is aobvious orange-yellow at 105 DEG C; By HRESIMSm/z461.2520[M+Na]+Can determine moleculeFormula is C24H38O7. Compound14 unimodal signals of methyl of H-NMR demonstration (δ: 0.90,0.95,1.35,2.04), a methyl signals δ who is connected with methineH1.12(d,J＝6.6Hz,H3-17), one withThe methyl δ that methylene is connectedH1.15(t,J＝7.2Hz,H3-22), three low connects oxygen methine signal δH5.10(dd,J＝6.0,3.6Hz,H-15),4.46(dd,J＝3.0,1.8Hz,H-3),3.78(d, J=10.8Hz, H-7); Compound13C-NMR and DEPT spectrum show 24 carbon signals, DEPT spectrum shows that these carbon signals are respectively 6 methyl, 7 methylene (comprising 1 even oxygen carbon), 5 timesMethyl and 6 quaternary carbon signals, wherein have 1 carbonyl (δC212.5) and two oxygen carbon (δ of companyC92.7,91.4). In conjunction with above information and bibliography (PlantaMed.2008,74:1288 – 1290.), deterministicThe planar structure of compound is 3-acetoxyl group-7-hydroxyl-15-ethyoxyl-9,13; 15,16-bis-epoxy Ladanum alkane-6-ketone. Adopt subsequently 2DNMR experiment to confirm. The relative configuration of compound is passed through NOESYSpectrum is inferred, H-5 and H-7 and H3-18 is relevant, H-7 again with H3-17 is relevant, shows H-5, H-7,H3-18,H3-17 homonymies (α direction) at ring, in like manner, H3-20 and H-8, H2-11,H3-19 is relevant,Show that they are positioned at opposite side (β direction). Further adopt the experiment of X-ray single crystal diffraction to determine absolute configuration,As shown in Figure 1. Therefore, the structure of this compound is defined as (-)-(3R, 5S, 7R, 8R, 9R, 10S, 13R, 15R)-3-acetoxyl group-7-hydroxyl-15-ethyoxyl-9,13; 15,16-bis-epoxy Ladanum alkane-6-ketone, concrete wave spectrumData are in table 1.
(5) proton nmr spectra (1H-NMR): Bruker-AV-600spectrometer measures, dataIn table 1.
(6) carbon-13 nmr spectra (13C-NMR): Bruker-AV-600spectrometer measures, dataIn table 2.
(7) single crystal diffraction data are in table 3.
Table 1.1H-NMR (600MHz) nuclear magnetic data (solvent: Me2CO-d6；δ：ppm；J：Hz)
Table 2.13C-NMR (150MHz) nuclear magnetic data (is measured solvent: Me2CO-d6；δ：ppm)
Table 3 single crystal diffraction data
Reference examples 2 (-)-(5S, 7R, 8R, 9R, 10S, 13R, 15R)-7-hydroxyl-15-ethyoxyl-9,13; 15,16-dicycloExtraction, separation and purification and the Structural Identification of oxygen Ladanum alkane-6-ketone (formula III compound)
(1) experiment material: with embodiment 1
(2) extraction of medicinal material: take motherwort medicinal material 20Kg, pulverize, add 95% ethanol (3 × 160L)Extract 72 hours, extract 3 times, extract after decompression and solvent recovery, obtains ethanol extract 1.2 after mergingKg；
(3) separation and purification of composition:
1. take motherwort medicinal material 20kg, pulverize, add 95%v/v ethanol 160L and extract 3 times, extractAfter decompression and solvent recovery, obtain ethanol extract 1.2kg;
2. by ethanol extract aqueous dispersion, use successively ethyl acetate, extracting n-butyl alcohol, combined ethyl acetatePart, decompression and solvent recovery, obtains ethyl acetate extract 400g;
3. adopt silica gel column chromatography to separate ethyl acetate extract, benzinum: acetone=(100:1)～(0:1)Gradient elution, gets benzinum-acetone=5:1 wash-out part (H) 8.2g, upper anti-phase polystyrene type resin postMCIgelCHP20P, 50-100%v/v methanol aqueous solution gradient elution, collects 70% methanol-eluted fractions part(H-3) 2.1g, then by anti-phase RP-C18 medium pressure liquid chromatography, 50-95%v/v methanol aqueous solution gradientWash-out, thin layer is followed the trail of, and collects 70～75% eluents (H-3-4) that contain target compound, reclaims solvent,Residue separates through SephadexLH-20 dextran gel column chromatography again, Shi You Mi ﹕ tri-Lv Jia Wan ﹕ methyl alcohol=5 ﹕ 5 ﹕ 1 wash-outs, thin-layer chromatography is followed the trail of, and collects and contains target compound cut, reclaims solvent, residueThing separates through anti-phase liquid phantom preparing chromatogram, 82%v/v methanol aqueous solution wash-out, and collection retention time is tR＝28The target product of min, purifying obtains 16.5mg.
(4) Structural Identification of composition: colorless oil; [α]20 D-92.2 (c0.15, MeOH); Molecular formula:C22H36O5. Its NMR data are more similar to formula II compound. The NMR number of comparative compound III and IIAccording to, the acetyl group in II and even oxygen methine are by the MU (δ in IIIH1.30 and 1.13, δC43.2ppm) substitute. In addition, for II, the C-1 in III, C-5 (the Δ δ that deshielded respectivelyC+6.5,+ 5.0ppm), C-2, C-4 conductively-closed (Δ δC-3.8 ,-3.4ppm), disclose 3 that compound III is II and take offAcetoxyl group analog, and test and obtained further confirmation by 2DNMR. The configuration of compd B isNOESY spectrum by relatively B and A and optical value are determined. Infer that structure is(-)-(5S, 7R, 8R, 9R, 10S, 13R, 15R)-7-hydroxyl-15-ethyoxyl-9,13; 15,16-bis-epoxy Ladanum alkane-6-ketone.
(5) proton nmr spectra (1H-NMR): Bruker-AV-600spectrometer measures, dataIn table 1.
(6) carbon-13 nmr spectra (13C-NMR): Bruker-AV-600spectrometer measures, dataIn table 2.
Reference examples 3 (+)-(5S, 7R, 8R, 9R, 10S, 13S, 15R)-7-hydroxyl-15-ethyoxyl-9,13; 15,16-bis-epoxyExtraction, separation and purification and the Structural Identification of Ladanum alkane-6-ketone (formula IV compound)
(1) experiment material: with embodiment 1
(2) extraction of medicinal material: take motherwort medicinal material 20Kg, pulverize, add 95% ethanol (3 × 160L)Extract 72 hours, extract 3 times, extract after decompression and solvent recovery, obtains ethanol extract 1.2 after mergingKg；
(3) separation and purification of composition:
1. take motherwort medicinal material 20kg, pulverize, add 95%v/v ethanol 160L and extract 3 times, extractAfter decompression and solvent recovery, obtain ethanol extract 1.2kg;
2. by ethanol extract aqueous dispersion, use successively ethyl acetate, extracting n-butyl alcohol, combined ethyl acetatePart, decompression and solvent recovery, obtains ethyl acetate extract 400g;
3. adopt silica gel column chromatography to separate ethyl acetate extract, benzinum: acetone=(100:1)～(0:1)Gradient elution, gets benzinum-acetone=5:1 wash-out part (H) 8.2g, upper anti-phase polystyrene type resin postMCIgelCHP20P, 50-100%v/v methanol aqueous solution gradient elution, collects 70% methanol-eluted fractions part(H-3) 2.1g, then by anti-phase RP-C18 medium pressure liquid chromatography, 50-95%v/v methanol aqueous solution gradientWash-out, thin layer is followed the trail of, and collects 70～75% eluents (H-3-4) that contain target compound, reclaims solvent,Residue separates through SephadexLH-20 dextran gel column chromatography again, Shi You Mi ﹕ tri-Lv Jia Wan ﹕ firstAlcohol=5 ﹕ 5 ﹕ 1 wash-outs, thin-layer chromatography is followed the trail of, and collects and contains target compound cut, reclaims solvent,Residue separates through anti-phase liquid phantom preparing chromatogram, 82%v/v methanol aqueous solution wash-out, and collection retention time istRThe target product of=32min, purifying obtains 9mg.
(4) Structural Identification of composition: colorless oil, its Spectral Characteristic and compound III are closely similar,But there is contrary specific rotation { [α]20 D+ 114.9 (c0.20, MeOH) }. comprehensively compare the HR of III and IV-ESI-MS and 2D-NMR data, show that both have identical planar structure. Further compare two in detailPerson is at identical deuterated solvent1H and13C-NMR data, find that both fine differences are: with respect to III, H-11b in compounds Ⅳ, H-16a, H-16b, C-15 and C-16 signal move Δ δ to High-Field respectively-0.13 ,-0.08 ,-0.06 ,-0.4 and-0.5ppm. On the contrary, H-11a, H-12b, H-14a, H-14b,C-11 and C-12 signal are respectively to low mobile Δ δ+0.08 ,+0.07 ,+0.03 ,+0.06 ,+0.6 and+0.3Ppm. More than show that compounds Ⅳ is the 13S-epimer of compound III, and adopt NOESY to carry outConfirmation. In NOESY spectrum, H2-14 and H3-17 is relevant, H2-12 and H2-16 relevant confirmation C-13 are SConfiguration. Therefore, compounds Ⅳ is defined as (+)-(5S, 7R, 8R, 9R, 10S, 13S, 15R)-7-hydroxyl-15-ethyoxyl-9,13; 15,16-bis-epoxy Ladanum alkane-6-ketone.
(5) proton nmr spectra (1H-NMR): Bruker-AV-600spectrometer measures, dataIn table 1.
(6) carbon-13 nmr spectra (13C-NMR): Bruker-AV-600spectrometer measures, dataIn table 2.
Reference examples 4 (-)-(5S, 7R, 8R, 9R, 10S, 13S, 15S)-7-hydroxyl-15-methoxyl group-9,13; 15,16-dicycloExtraction, separation and purification and the Structural Identification of oxygen Ladanum alkane-6-ketone (formula V compound)
(1) experiment material: with embodiment 1
(2) extraction of medicinal material: take motherwort medicinal material 20Kg, pulverize, add 95% ethanol (3 × 160L)Extract 72 hours, extract 3 times, extract after decompression and solvent recovery, obtains ethanol extract 1.2 after mergingKg；
(3) separation and purification of composition:
1. take motherwort medicinal material 20kg, pulverize, add 95%v/v ethanol 160L and extract 3 times, extractAfter decompression and solvent recovery, obtain ethanol extract 1.2kg;
2. by ethanol extract aqueous dispersion, use successively ethyl acetate, extracting n-butyl alcohol, combined ethyl acetatePart, decompression and solvent recovery, obtains ethyl acetate extract 400g;
3. adopt silica gel column chromatography to separate ethyl acetate extract, benzinum: acetone=(100:1)～(0:1)Gradient elution, gets benzinum-acetone=5:1 wash-out part (H) 8.2g, upper anti-phase polystyrene type resin postMCIgelCHP20P, 50-100%v/v methanol aqueous solution gradient elution, collects 70% methanol-eluted fractions part(H-3) 2.1g, then by anti-phase RP-C18 medium pressure liquid chromatography, 50-95%v/v methanol aqueous solution gradientWash-out, thin layer is followed the trail of, and collects 65～70% eluents (H-3-3) that contain target compound, reclaims solvent,Residue is again through Preparative TLC chromatographic isolation, and Shi You Mi ﹕ acetone=5 ﹕ 1 launches, scraping target product lookBands of a spectrum, with acetone 30ml wash-out, collect eluent, reclaim solvent, and residue is prepared look through anti-phase liquid phaseSpectrum separates, 78%v/v methanol aqueous solution wash-out, and collection retention time is tRThe target product of=37min,Purifying obtains 1.5mg.
(4) Structural Identification of composition: colorless oil, molecular formula: C21H34O5. Its chromatogram characteristicShowing that it and II, III, IV structure are similar, is another pair of volution Ladanum alkane diterpene. Comparative compoundThe NMR data of V and IV, show that ethyoxyl in IV is by the methoxy substitution (δ in VH3.26ppm,δC54.7ppm). Meanwhile, H2-14 and H-15 between coupling constant diminished by 6.0 in IV and 4.2HzFor 5.6 in V and 1.2Hz, and in compound V, C-15 and C-16 have moved Δ δ to low field respectivelyC+1.9 and+2.9ppm. this show that compound V is 15 'beta '-methoxy derivatives of compounds Ⅳ. Adopt subsequently 2D-NMR experiment is confirmed. Adopt II, III, the definite method of IV configuration, determined compound VAbsolute configuration. Therefore, the structure of compound V is defined as (-)-(5S, 7R, 8R, 9R, 10S, 13S, 15S)-7-hydroxyl-15-methoxyl group-9,13; 15,16-bis-epoxy Ladanum alkane-6-ketone.
(5) proton nmr spectra (1H-NMR): Bruker-AV-600spectrometer measures, dataIn table 1.
(6) carbon-13 nmr spectra (13C-NMR): Bruker-AV-600spectrometer measures, dataIn table 2.
Reference examples 5 (-)-(3R, 5S, 7R, 8R, 9R, 10S, 13S, 15S)-3-acetoxyl group-7-hydroxyl-15-methoxyl group-9,13; Extraction, separation and purification and the structure mirror of 15,16-bis-epoxy Ladanum alkane-6-ketone (formula VI compound)Fixed
(1) experiment material: with embodiment 1
(2) extraction of medicinal material: take motherwort medicinal material 20Kg, pulverize, add 95% ethanol (3 × 160L)Extract 72 hours, extract 3 times, extract after decompression and solvent recovery, obtains ethanol extract 1.2 after mergingKg；
(3) separation and purification of composition:
1. take motherwort medicinal material 20kg, pulverize, add 95%v/v ethanol 160L and extract 3 times, extractAfter decompression and solvent recovery, obtain ethanol extract 1.2kg;
2. by ethanol extract aqueous dispersion, use successively ethyl acetate, extracting n-butyl alcohol, combined ethyl acetatePart, decompression and solvent recovery, obtains ethyl acetate extract 400g;
3. adopt silica gel column chromatography to separate ethyl acetate extract, benzinum: acetone=(100:1)～(0:1)Gradient elution, gets benzinum-acetone=5:1 wash-out part (H) 8.2g, upper anti-phase polystyrene type resin postMCIgelCHP20P, 50-100%v/v methanol aqueous solution gradient elution, collects 70% methanol-eluted fractions part(H-3) 2.1g, then by anti-phase RP-C18 medium pressure liquid chromatography, 50-95%v/v methanol aqueous solution gradientWash-out, thin layer is followed the trail of, and collects 70～75% eluents (H-3-4) that contain target compound, reclaims solvent,Residue separates through SephadexLH-20 dextran gel column chromatography again, Shi You Mi ﹕ tri-Lv Jia Wan ﹕ firstAlcohol=5 ﹕ 5 ﹕ 1 wash-outs, thin-layer chromatography is followed the trail of, and collects and contains target compound cut, reclaims solvent,Residue separates through anti-phase liquid phantom preparing chromatogram, 82%v/v methanol aqueous solution wash-out, and collection retention time istRThe target product of=54min, purifying obtains 1.6mg.
(4) Structural Identification of composition: colorless oil, HREI-MSm/z447.2366[M+Na]+Prompting molecular composition is C23H36O7(calcdforC23H36O7Na, 447.2359). Comparative compound VI andThe NMR data of V, show that the 3-acetoxyl group that this compound VI is V replaces analog. At 1D-NMRIn spectrum, an oxygen methine (δ of company in compound VIH4.47ppm,δC78.4ppm) He one acetylOxygen base (δH2.06ppm,δC170.2,21.2ppm) replace the methylene in compound V. Meanwhile,In compound VI C-1 and C-5 respectively to High-Field move Δ δ-6.3 and-5.1ppm, and C-2 and C-4 respectively toLow mobile Δ δC+ 3.9 and+3.4ppm, prompting C-3 be acetoxyl group replace. The judgement of compound structure typeMethod and II, III, IV, V are identical. Therefore, the structure of compound VI be defined as (-)-(3R, 5S, 7R, 8R,9R, 10S, 13S, 15S)-3-acetoxyl group-7-hydroxyl-15-methoxyl group-9,13; 15,16-bis-epoxy Ladanum alkane-6-Ketone.
(5) proton nmr spectra (1H-NMR): Bruker-AV-600spectrometer measures, dataIn table 1.
(6) carbon-13 nmr spectra (13C-NMR): Bruker-AV-600spectrometer measures, dataIn table 2.
Reference examples 6 (+)-(3R, 5S, 7R, 8R, 9R, 10S, 13S, 15R)-3-acetoxyl group-7-hydroxyl-15-ethyoxyl-9,13; Extraction, separation and purification and the structure mirror of 15,16-bis-epoxy Ladanum alkane-6-ketone (formula VII compound)Fixed
(1) experiment material: with embodiment 1
(2) extraction of medicinal material: take motherwort medicinal material 20Kg, pulverize, add 95% ethanol (3 × 160L)Extract 72 hours, extract 3 times, extract after decompression and solvent recovery, obtains ethanol extract 1.2 after mergingKg；
(3) separation and purification of composition:
1. take motherwort medicinal material 20kg, pulverize, add 95%v/v ethanol 160L and extract 3 times, extractAfter decompression and solvent recovery, obtain ethanol extract 1.2kg;
2. by ethanol extract aqueous dispersion, use successively ethyl acetate, extracting n-butyl alcohol, combined ethyl acetatePart, decompression and solvent recovery, obtains ethyl acetate extract 400g;
3. adopt silica gel column chromatography to separate ethyl acetate extract, benzinum: acetone=(100:1)～(0:1)Gradient elution, gets benzinum-acetone=5:1 wash-out part (H) 8.2g, upper anti-phase polystyrene type resin postMCIgelCHP20P, 50-100%v/v methanol aqueous solution gradient elution, collects 70% methanol-eluted fractions part(H-3) 2.1g, then by anti-phase RP-C18 medium pressure liquid chromatography, 50-95%v/v methanol aqueous solution gradientWash-out, thin layer is followed the trail of, and collects 70～75% eluents (H-3-4) that contain target compound, reclaims solvent,Residue separates through SephadexLH-20 dextran gel column chromatography again, Shi You Mi ﹕ tri-Lv Jia Wan ﹕ firstAlcohol=5 ﹕ 5 ﹕ 1 wash-outs, thin-layer chromatography is followed the trail of, and collects and contains target compound cut, reclaims solvent,Residue separates through anti-phase liquid phantom preparing chromatogram, 82%v/v methanol aqueous solution wash-out, and collection retention time istRThe target product of=51min, purifying obtains 15mg.
(4) Structural Identification of composition: colorless oil; Molecular formula: C24H38O7,M＝438,HRESIMSm/z461.2508[M+Na]+；1HNMR(600MHz,CD3COCD3)δ:0.89(3H,s,H3-20),0.94(3H,s,H3-18),1.34(3H,s,H3-19),1.17(3H,d,J＝6.6Hz,H3-17),5.10(1H,dd,J＝5.4,3.6Hz,H-15),4.46(1H,brs,H-3),3.28(1H,s,H-5),3.89(1H,d,J＝7.8Hz,H-16a),3.67(1H,d,J＝7.8Hz,H-16b),3.71,3.82(2H,2m,MeCH2O),1.15(3H,t,J＝7.2Hz,MeCH2O)；13CNMR(150MHz,CD3COCD3)δ:26.2(C-1),22.9(C-2),78.5(C-3),36.6(C-4),52.9(C-5),212.7(C-6),78.4(C-7),48.2(C-8),92.7(C-9),48.5(C-10),30.4(C-11),40.6(C-12),91.5(C-13),47.8(C-14),104.0(C-15),75.6(C-16),20.1(C-17),27.1(C-18),22.5(C-19),13.6(C-20),170.2(MeCO),21.2(MeCO),63.4(MeCH2O),15.7(MeCH2O). According to above data, reallyDetermine compound VII for (+)-(3R, 5S, 7R, 8R, 9R, 10S, 13S, 15R)-3-acetoxyl group-7-hydroxyl-15-ethyoxyl-9,13; 15,16-bis-epoxy Ladanum alkane-6-ketone.
Reference examples 7 (-)-(3R, 5S, 7R, 8R, 9R, 10S, 13S, 15S)-3-acetoxyl group-7-hydroxyl-15-ethyoxyl-9,13; Extraction, separation and purification and the structure mirror of 15,16-bis-epoxy Ladanum alkane-6-ketone (formula VIII compound)Fixed
(1) experiment material: with embodiment 1
(2) extraction of medicinal material: take motherwort medicinal material 20Kg, pulverize, add 95% ethanol (3 × 160L)Extract 72 hours, extract 3 times, extract after decompression and solvent recovery, obtains ethanol extract 1.2 after mergingKg；
(3) separation and purification of composition:
1. take motherwort medicinal material 20kg, pulverize, add 95%v/v ethanol 160L and extract 3 times, extractAfter decompression and solvent recovery, obtain ethanol extract 1.2kg;
2. by ethanol extract aqueous dispersion, use successively ethyl acetate, extracting n-butyl alcohol, combined ethyl acetatePart, decompression and solvent recovery, obtains ethyl acetate extract 400g;
3. adopt silica gel column chromatography to separate ethyl acetate extract, benzinum: acetone=(100:1)～(0:1)Gradient elution, gets benzinum-acetone=5:1 wash-out part (H) 8.2g, upper anti-phase polystyrene type resin postMCIgelCHP20P, 50-100%v/v methanol aqueous solution gradient elution, collects 70% methanol-eluted fractions part(H-3) 2.1g, then by anti-phase RP-C18 medium pressure liquid chromatography, 50-95%v/v methanol aqueous solution gradientWash-out, thin layer is followed the trail of, and collects 70～75% eluents (H-3-4) that contain target compound, reclaims solvent,Residue separates through SephadexLH-20 dextran gel column chromatography again, Shi You Mi ﹕ tri-Lv Jia Wan ﹕ firstAlcohol=5 ﹕ 5 ﹕ 1 wash-outs, thin-layer chromatography is followed the trail of, and collects and contains target compound cut, reclaims solvent,Residue separates through anti-phase liquid phantom preparing chromatogram, 82%v/v methanol aqueous solution wash-out, and collection retention time istRThe target product of=58min, purifying obtains 22mg.
(4) Structural Identification of composition: colorless oil; Molecular formula: C24H38O7,M＝438,HRESIMSm/z461.2508[M+Na]+；1HNMR(600MHz,CD3COCD3)δ:0.90(3H,s,H3-17),0.94(3H,s,H3-18),1.34(3H,s,H3-19),1.19(3H,d,J＝6.4Hz,H3-20),5.19(1H,brd,J＝5.2Hz,H-15),4.47(1H,brs,H-3),3.27(1H,s,H-5),4.08(1H,d,J＝8.8Hz,H-16a),3.90(1H,d,J＝8.8Hz,H-16b),3.40,3.46(2H,2m,MeCH2O),1.13(3H,t,J＝6.8Hz,MeCH2O)；13CNMR(CD3COCD3,100MHz)δ:26.2(C-1),22.9(C-2),78.9(C-3),36.5(C-4),52.5(C-5),212.6(C-6),78.4(C-7),47.4(C-8),93.1(C-9),48.7(C-10),29.6(C-11),39.3(C-12),92.0(C-13),48.7(C-14),104.7(C-15),78.2(C-16),20.3(C-17),27.1(C-18),22.5(C-19),13.8(C-20),170.2(MeCO),63.1(MeCH2O)15.7(MeCH2O), 21.2 (MeCO). According to above data, deterministicCompound VIII is (-)-(3R, 5S, 7R, 8R, 9R, 10S, 13S, 15S)-3-acetoxyl group-7-hydroxyl-15-ethyoxyl-9,13; 15,16-bis-epoxy Ladanum alkane-6-ketone.
Reference examples 8 (+)-(5S, 7R, 8R, 9R, 10S, 13S, 15R)-7-hydroxyl-15-methoxyl group-9,13; 15,16-dicycloOxygen Ladanum alkane-6, extraction, separation and purification and the Structural Identification of 16-diketone (formula IX compound)
(1) experiment material: with embodiment 1
(2) extraction of medicinal material: take motherwort medicinal material 20Kg, pulverize, add 95% ethanol (3 × 160L)Extract 72 hours, extract 3 times, extract after decompression and solvent recovery, obtains ethanol extract 1.2 after merging Kg；
(3) separation and purification of composition:
1. take motherwort medicinal material 20kg, pulverize, add 95%v/v ethanol 160L and extract 3 times, extractAfter decompression and solvent recovery, obtain ethanol extract 1.2kg;
2. by ethanol extract aqueous dispersion, use successively ethyl acetate, extracting n-butyl alcohol, combined ethyl acetatePart, decompression and solvent recovery, obtains ethyl acetate extract 400g;
3. adopt silica gel column chromatography to separate ethyl acetate extract, benzinum: acetone=(100:1)～(0:1)Gradient elution, gets benzinum-acetone=5:1 wash-out part (H) 8.2g, upper anti-phase polystyrene type resin postMCIgelCHP20P, 50-100%v/v methanol aqueous solution gradient elution, collects 70% methanol-eluted fractions part(H-3) 2.1g, then by anti-phase RP-C18 medium pressure liquid chromatography, 50-95%v/v methanol aqueous solution gradientWash-out, thin layer is followed the trail of, and collects 65～70% eluents (H-3-3) that contain target compound, reclaims solvent,Residue is again through Preparative TLC chromatographic isolation, and Shi You Mi ﹕ acetone=5 ﹕ 1 launches, scraping target product lookBands of a spectrum, with acetone 30ml wash-out, collect eluent, reclaim solvent, and residue is prepared look through anti-phase liquid phaseSpectrum separates, 78%v/v methanol aqueous solution wash-out, and collection retention time is tRThe target product of=40min,Purifying obtains 2.4mg.
(4) Structural Identification of composition: colorless oil, HRESIMSm/z403.2094[M+Na]+CarryShow that molecular formula is C21H32O6. The NMR data of comparative compound IX and IV, find that their difference is mainBe: in compound IX, contain interior ester carbonyl group, and the methoxyl group (δ of 15 in compound IXH3.54ppm,δC57.6ppm) substitute the ethyoxyl in IV. This inference further confirms by 2DNMR. At HMBCIn spectrum, H2-12 and C-9, C-11, C-14 is relevant with C-16, H2-14 and C-12, C-13, C-15 and C-16Relevant, H-15 and C-14, C-16 is relevant with C-21, illustrates that ester carbonyl group is positioned at C-16 position. Pass through NOESYSpectrum, the features such as coupling constant and specific rotation can be judged C-5 in compound F 17-hydroxy-corticosterone, C-7, C-8, C-9, C-10,The absolute configuration of C-13 is identical with C. In addition J in compound IX,14,15Be 6.0 and 4.8Hz, and haveThe compound ii of 15 α-configuration, III, IV, VII close (J=6.0,4.2Hz), and be greater than the change of 15 beta configurationsCompound V, VI, VIII (J=5.4,1.2Hz). Therefore, the configuration of C-15 and compound ii in compound IX,III, IV, VII are identical. The structure of compound IX is defined as (+)-(5S, 7R, 8R, 9R, 10S, 13S, 15R)-7-hydroxyl-15-methoxyl group-9,13; 15,16-bis-epoxy Ladanum alkane-6,16-diketone.
(5) proton nmr spectra (1H-NMR): Bruker-AV-600spectrometer measures, dataIn table 1.
(6) carbon-13 nmr spectra (13C-NMR): Bruker-AV-600spectrometer measures, dataIn table 2.
Illustrate beneficial effect of the present invention by test example below.
Test example 1 has two volution labdane type diterpenoids (chemical combination shown in formula II crystal formation and III of 13R configurationThing) platelet aggregation-against test
(1) experiment material:
1. medicine
Test-compound formula II crystal formation and formula III compound are configured to the stock solution of 10mM with DMSO,-10 DEG C of preservations, become 0.1mM with normal saline dilution before use. Positive drug (clopidogrel bisulfate tablet),Collocation method is the same.
2. animal
SD rat, clean level, male and female are regardless of, and body weight 200～240g, is tested by Chengdu University of Traditional Chinese MedicineZooscopy center provides. The quality of production quality certification number: SCXK (river) 2008-11.
3. reagent
Adenosine diphosphate (ADP) disodium (adenosine-5 '-diphosphate, ADP), sigma company of the U.S. produces,Domestic packing, 1g/ bottle.
Dimethyl sulfoxide (DMSO) (DMSO), analyzes purely, and 500ml/ bottle, is purchased from Chengdu Ke Long chemical reagent factory.
4. laboratory apparatus
Multichannel platelet aggregation instrument (APACT/2 type, German blue ripple)
Electronic balance (ESJ120-4 type, Longteng Electronic Weighing Instrument Co., Ltd., Shenyang);
Water-bath, low temperature refrigerator etc.
(2) experimental technique:
Select adult, healthy SD rat, male and female are regardless of, and after conforming, femoral artery blood sampling, with 3.8%The ratio anti-freezing of sodium citrate and blood 1:9, then, with the centrifugal 10min of 800r/min, prepares rich bloodPlatelet blood plasma (platelet-richplasma, PRP), remainder is with the centrifugal 10min system of 3000r/minStandby platelet poor plasma (platelet-poorplasma, PPP). With PPP zeroing, get 200 μ lPRP and addEnter 37 DEG C of incubation 5min in opacity tube, add the tested drug solution of 20 μ l, make final concentration be 0.1MM, blank group adds 1%DMSO solution. Mix rear continuation incubation 5min, then add ADP20μ l (final concentration is 0.05mg/ml) induces gathering. With platelet aggregation instrument detection blood platelet MA,Calculate L-Arginine (AIR) by following formula:
AIR=[(blank group MA-administration group MA)/blank group is maximum poly-Collection rate] × 100%
3. experimental result and evaluation:
The results are shown in Table 4.
The impact on extracorporeal platelet aggregation of table 4 formula II crystal formation and formula III compound ()
Note: with the comparison of physiological saline group,*P<0.05。
Find by research: in the time that final concentration is 0.1mM, formula II crystal formation and formula III compound are to externalThe platelet aggregation of ADP induction has obvious inhibitory action (P < 0.05), and inhibiting rate is respectively 19.59% He15.27%, with positive controls inhibiting rate 16.91% quite, even higher.
Test example 2 has two volution labdane type diterpenoids (chemical combination shown in formula V, VI, IX of 13S configurationThing) platelet aggregation-against test
(1) experiment material:
1. medicine
Test-compound (-)-(5S, 7R, 8R, 9R, 10S, 13S, 15S)-7-hydroxyl-15-methoxyl group-9,13; 15,16-is twoEpoxy Ladanum alkane-6-ketone (V), (-)-(3R, 5S, 7R, 8R, 9R, 10S, 13S, 15S)-3-acetoxyl group-7-hydroxylBase-15-methoxyl group-9,13; 15,16-bis-epoxy Ladanum alkane-6-ketone (VI),(+)-(5S, 7R, 8R, 9R, 10S, 13S, 15R)-7-hydroxyl-15-methoxyl group-9,13; 15,16-bis-epoxy Ladanum alkane-6,16-diketone (IX), is configured to the stock solution of 10mM with DMSO ,-10 DEG C of preservations, use before useNormal saline dilution becomes 0.1mM. Positive drug (clopidogrel bisulfate tablet), collocation method is the same.
2. animal
SD rat, clean level, male and female are regardless of, and body weight 200～240g, is tested by Chengdu University of Traditional Chinese MedicineZooscopy center provides. The quality of production quality certification number: SCXK (river) 2008-11.
3. reagent
Adenosine diphosphate (ADP) disodium (adenosine-5 '-diphosphate, ADP), sigma company of the U.S. produces,Domestic packing, 1g/ bottle.
Dimethyl sulfoxide (DMSO) (DMSO), analyzes purely, and 500ml/ bottle, is purchased from Chengdu Ke Long chemical reagent factory.
4. laboratory apparatus
Multichannel platelet aggregation instrument (APACT/2 type, German blue ripple)
Electronic balance (ESJ120-4 type, Longteng Electronic Weighing Instrument Co., Ltd., Shenyang);
Water-bath, low temperature refrigerator etc.
(2) experimental technique:
Select adult, healthy SD rat, male and female are regardless of, and after conforming, femoral artery blood sampling, with 3.8%The ratio anti-freezing of sodium citrate and blood 1:9, then, with the centrifugal 10min of 800r/min, prepares rich bloodPlatelet blood plasma (platelet-richplasma, PRP), remainder is with the centrifugal 10min system of 3000r/minStandby platelet poor plasma (platelet-poorplasma, PPP). With PPP zeroing, get 200 μ lPRP and addEnter 37 DEG C of incubation 5min in opacity tube, add the tested drug solution of 20 μ l, make final concentration be 0.1MM, blank group adds 1%DMSO solution. Mix rear continuation incubation 5min, then add ADP20μ l (final concentration is 0.05mg/ml) induces gathering. With platelet aggregation instrument detection blood platelet MA,Calculate L-Arginine (AIR) by following formula:
AIR=[(blank group MA-administration group MA)/blank group is maximum poly-Collection rate] × 100%
3. experimental result and evaluation:
The results are shown in Table 5.
The impact on extracorporeal platelet aggregation of table 5 compound V, VI and IX ()
Find by research: in the time that final concentration is 0.1mM, test-compound compound(-)-(5S, 7R, 8R, 9R, 10S, 13S, 15S)-7-hydroxyl-15-methoxyl group-9,13; 15,16-bis-epoxy Ladanum alkane-6-ketone (V), (-)-(3R, 5S, 7R, 8R, 9R, 10S, 13S, 15S)-3-acetoxyl group-7-hydroxyl-15-methoxyl group-9,13; 15,16-bis-epoxy Ladanum alkane-6-ketone (VI), (+)-(5S, 7R, 8R, 9R, 10S, 13S, 15R)-7-hydroxylBase-15-methoxyl group-9,13; 15,16-bis-epoxy Ladanum alkane-6,16-diketone (IX) is induced external ADPPlatelet aggregation has certain inhibitory action, and wherein the effect trend of VI is obvious.
From above test example, in motherwort, separating the crystal formation obtaining has certain pressing down to platelet aggregationMake use, wherein with formula II crystal formation compound effect the best, under same concentration (0.1mM), than anti-bloodPlatelet is assembled medicine " bisulfate clopidogrel " better effects if, is the natural platelet aggregation inhibitor of noveltyThe exploitation of thing provides new selection.
Claims (10)
1. the crystal formation of compound shown in formula II, is characterized in that:
This crystal formation is rhombic system, and space group is P212121, cell parameter isα＝90.00°，β＝90.00°，γ=90.00 °, Z=4, structure cellVolume is
2. crystal formation according to claim 1, is characterized in that: described crystal formation Chinese style II compoundSpecific rotatory power is [α]20 D-58.8。
3. the preparation method of crystal formation described in claim 1 or 2, is characterized in that: it comprises following operationStep:
(1) take motherwort medicinal material, pulverize, add 95%v/v alcohol extract, extract recovered under reduced pressure is moltenAfter agent, obtain ethanol extract;
(2) by ethanol extract aqueous dispersion, be extracted with ethyl acetate successively, combined ethyl acetate part,Decompression and solvent recovery, obtains ethyl acetate extract;
(3) adopt silica gel column chromatography to separate ethyl acetate extract, benzinum: acetone=100:1～0:1Gradient elution, gets anti-phase polystyrene type resin post in benzinum-acetone=5:1 wash-out part, 50-100%v/vMethanol aqueous solution gradient elution, collects 70% methanol-eluted fractions part, then by medium pressure liquid chromatography, 18Alkyl linked silica gel chromatographic column, 50-95%v/v methanol aqueous solution gradient elution, collects 70～75% methanol-watersEluant solution part, reclaims solvent, and residue divides through SephadexLH-20 dextran gel column chromatography againFrom, Shi You Mi ﹕ tri-Lv Jia Wan ﹕ methyl alcohol=5 ﹕ 5 ﹕ 1 wash-outs, collect eluent, reclaim solvent, surplusAnti-phase liquid phantom preparing chromatogram on excess, 82%v/v methanol aqueous solution wash-out, collects respectively different appearance timesEluent, can obtain formula II compound;
(4) modus ponens II compound, adds acetone, be placed on subsequently in the drier that drier is housed,Be placed to crystal and separate out, obtain crystal formation.
4. preparation method according to claim 3, is characterized in that: formula II compound and acetoneMass volume ratio is: 10mg:0.1-1ml.
5. preparation method according to claim 4, is characterized in that: formula II compound and acetoneMass volume ratio is: 10mg:0.3-0.5ml.
6. preparation method according to claim 5, is characterized in that: formula II compound and acetoneMass volume ratio is: 10mg:0.4ml.
7. preparation method according to claim 3, is characterized in that: described drier is selected from variable colorSilica gel.
Described in claim 1 or 2 crystal formation in the purposes of preparing in platelet aggregation-against class medicine.
9. a pharmaceutical composition, is characterized in that: it is the claim 1 or 2 that contains effective doseThe preparation of described crystal formation.
10. pharmaceutical composition according to claim 9, is characterized in that: described preparation be selected from throughGastrointestinal absorption preparation or ejection preparation.
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