CN103588851A - Novel hecogenin compound and extract and preparation method and application thereof - Google Patents

Novel hecogenin compound and extract and preparation method and application thereof Download PDF

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CN103588851A
CN103588851A CN201310574778.9A CN201310574778A CN103588851A CN 103588851 A CN103588851 A CN 103588851A CN 201310574778 A CN201310574778 A CN 201310574778A CN 103588851 A CN103588851 A CN 103588851A
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extract
ethanol
water
haike
xin
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CN103588851B (en
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张鹏
吴红华
田晓轩
付爱珍
吴学芹
朱彦
高秀梅
刘二伟
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Tianjin University of Traditional Chinese Medicine
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Abstract

The invention relates to a novel hecogenin compound with a structural formula (I): (novel hecogenin,3-O-beta-D-glucose-(1-2)-[beta-D-glucose-(1-3)]-beta-D-glucose-(1-4)-beta-D-galactoside); after the reflowing extraction of C.tuberosum harb ethanol, obtained crude extract is implemented with column chromatography by macroporous resin, and is gradually eluted by water and ethanol; the obtained 70% ethanol eluate is implemented with ODS column chromatography, and is eluted by water and 100% acetonitrile; the 70% ethanol eluate has the purpose of inducing platelet aggregation activity aspect.

Description

Xin Haike saponin compound and extract and preparation method thereof and application
Technical field
The present invention relates to a kind of Liliaceae bracketplant genus plants extract and preparation method thereof and application, especially Xin Haike saponin compound and extract and preparation method thereof and application.
Background technology
Platelet aggregation and thrombosis are the major reasons of ischemic cardiac brain injury, and platelet aggregation-against treatment contributes to reduce the generation of cardiovascular and cerebrovascular adverse events.The antiplatelet aggregative activity of steroidal saponin is that it is as treatment diseases of cardiovascular and cerebrovascular systems medicine, one of mechanism of action of the illnesss such as treatment atherosclerosis, myocardial infarction.
Steroidal saponin is the important activeconstituents of a class in plant, as the medicine for the treatment of diseases of cardiovascular and cerebrovascular systems, is widely used clinically, and its indication comprises the sequela of coronary heart disease, myocardial ischemia, cerebral arteriosclerosis and cerebral thrombosis etc.The medicine having gone on the market comprises: DIAOXINXUE KANG JIAONANG (Dioscorea panthaica Prain et Burkill steroidal saponin), XINNAOSHUTONG JIAONANG (Steroidal Saponin of Tribulus Terrestri L), perhexiline sheet (Rhizome of Peltate Yam rhizome saponin(e).DIAOXINXUE KANG JIAONANG was successfully got permission European Union's registration listing in 2012.First therapeutic pharmaceuticals ，Ye Shi EU member country with independent intellectual property right that Zhe Shi China successfully enters EU market obtains first plant amedica of the market access in addition.
It is Liliaceae (Liliacea) per nnial herb that bracketplant belongs to (Chlorophytum), and the whole world approximately has 215 kinds.The multiple pharmacologically actives such as bracketplant platymiscium has anti-diabetic, regulates immunity of organisms, reducing blood-fat, anti-oxidant, antifatigue, antalgic and inflammation relieving, its chemical composition comprises: the compositions such as steroidal saponin (main component), triterpene, flavones, sterol, lipid acid.
C.tuberosum is Liliaceae bracketplant platymiscium, and the popular name of C.borivilianum and C.tuberosum is safed musli.C.tuberosum is annual herb plant, gathers herb ， India and Africa mainly as nourishing drug use between florescence or fruiting period, has the effectiveness such as enhancing body immunizing power, cardiac stimulant, sexual desire promoting, treatment dysentery, lactagogue.Yet so far, also nobody carried out research to its chemical composition.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of Xin Haike saponin compound.
Another technical problem to be solved by this invention is to provide the extract that contains above-mentioned Xin Haike saponin compound.
Another technical problem to be solved by this invention is to provide the preparation method of above-mentioned Xin Haike saponin compound.
Another technical problem to be solved by this invention is to provide the preparation method of the extract that contains above-mentioned Xin Haike saponin compound.
Another technical problem to be solved by this invention is to provide the application of the extract that contains above-mentioned Xin Haike saponin compound.
For solving the problems of the technologies described above, technical scheme of the present invention is:
A saponin compound, the new hecogenin of ﹛, 3-O-β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 3)]-β-D-Glucose-(1 → 4)-β-D-half lactose glycosides ﹜, has following structural formula (I):
Figure BDA0000413434670000021
Preferably, the physical chemistry of above-mentioned Xin Haike saponin compound and Spectroscopic Properties: white powder (methyl alcohol), nuclear magnetic data is:
1h-NMR (C 5d 5n, 400MHz) δ 0.64 (3H, s, Me-19), 1.06 (3H, s, Me-18), 1.06 (3H, d, J=4.4Hz, Me-27), 1.36 (3H, d, J=6.8Hz, Me-21), 2.21 (1H, dd, J 1=14.4, J 2=4.4Hz, H-11 eq), 2.35 (1H, t, J=13.6Hz, H-11 ax), 2.73 (1H, t, J=6.8Hz, H-17), 3.36 (1H, br d, J=10.8Hz, H-26 ax), 4.86 (1H, J=7.6Hz, Gal-1'), 5.15 (1H, J=8Hz, Glc-1 "), 5.30 (1H, J=8Hz, Glc-1''''), 5.59 (1H, J=7.6Hz, Glc-1'''); Carbon spectrum data are shown in embodiment part table 1.
The preparation method of above-mentioned Xin Haike saponin compound, concrete steps are as follows:
(1) after C.tuberosum herb alcohol reflux, extracting solution simmer down to crude extract medicinal extract;
(2) dilution of gained crude extract medicinal extract adding distil water disperses and filters to obtain settled solution, and through macroporous resin column chromatography, water-ethanol gradient elution, obtains 70% ethanol elution thing;
(3) by 70% ethanol elution thing through ODS column chromatography, water-100% acetonitrile wash-out, collect 50% acetonitrile washing separation of flow part, obtained component is through silica gel column chromatography, methylene chloride-methanol-water elution, obtains Xin Haike saponin compound.
Preferably, the preparation method of above-mentioned Xin Haike saponin compound, concrete steps are as follows:
(1) 8 times of amount 50%(v/v for C.tuberosum herb) alcohol reflux is 3 times, and each 2 hours, united extraction liquid, decompression recycling ethanol, to without alcohol taste, obtains crude extract medicinal extract;
(2) dilution of gained crude extract medicinal extract adding distil water is dispersed to 4L, and after filter paper filters, settled solution is through D101 macroporous resin column chromatography, and water-95% ethanol (v/v) gradient elution, obtains 30%, 50%, 70%, 95%4 ethanol elution thing;
(3) by 70% ethanol elution thing through ODS column chromatography, water-100% acetonitrile wash-out, collect 50% acetonitrile washing separation of flow part, obtained component is through 40-63 μ m silica gel column chromatography, methylene chloride-methanol-water 6.2:2.8:1(v/v/v) wash-out, obtain this Xin Haike saponin compound.
An extract that contains above-mentioned Xin Haike saponin compound, is obtained by following method:
(1) after C.tuberosum herb alcohol reflux, extracting solution simmer down to crude extract medicinal extract;
(2) dilution of gained crude extract medicinal extract adding distil water disperses and filters to obtain settled solution, and through macroporous resin column chromatography, water-ethanol gradient elution, obtains 70% ethanol elution thing.
The preparation method of said extracted thing, concrete steps are as follows:
(1) after C.tuberosum herb alcohol reflux, extracting solution simmer down to crude extract medicinal extract;
(2) dilution of gained crude extract medicinal extract adding distil water disperses and filters to obtain settled solution, and through macroporous resin column chromatography, water-ethanol gradient elution, obtains 70% ethanol elution thing, i.e. target extract.
Preferably, the preparation method of said extracted thing, concrete steps are as follows:
(1) 8 times of amount 50%(v/v for C.tuberosum herb) alcohol reflux is 3 times, and each 2 hours, united extraction liquid, decompression recycling ethanol, to without alcohol taste, obtains crude extract medicinal extract;
(2) dilution of gained crude extract medicinal extract adding distil water is dispersed to 4L, and after filter paper filters, settled solution is through D101 macroporous resin column chromatography, and water-95% ethanol (v/v) gradient elution, obtains 70% ethanol elution thing, i.e. target extract.
The invention discloses the application of extract aspect induced platelet aggregation activity that contains above-mentioned Xin Haike saponin compound.
Preferably, the invention discloses the application of extract in preparing blood-clotting agent that contains above-mentioned Xin Haike saponin compound.
The pharmaceutical composition with said extracted thing, comprises and treats and/or prevents the extract of significant quantity and the optional acceptable vehicle of pharmacy.
The acceptable vehicle of above-mentioned pharmacy can be the vehicle of any routine in field of pharmaceutical preparations, the selection of particular excipient will be depended on administering mode or disease type and the state that is used for the treatment of particular patient, for the preparation method of the suitable drug composition of specific administration pattern completely in pharmaceutical field technician's ken.For example, can be used as thinner, carrier, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier and lubricant etc. that the acceptable vehicle of pharmacy comprises pharmaceutical field routine, if desired, can also in pharmaceutical composition, add flavouring agent, preservative and sweetener etc.
Above-mentioned composition can be made the various ways such as tablet, pulvis, granule, capsule, oral liquid, paste, creme, injectable emulsion, aseptic powder needle for injection.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.
The invention has the beneficial effects as follows:
Xin Haike saponin compound of the present invention extracts and obtains in the herb of C.tuberosum, and its 70% ethanol elution thing has induced platelet aggregation activity, can further be applied to the preparation of haemostatic medicament in body, has important clinical value.
Accompanying drawing explanation
Fig. 1 is the HMBC correlogram of Xin Haike saponin compound;
Embodiment
In order to make those skilled in the art better understand technical scheme of the present invention, below in conjunction with embodiment, technical scheme of the present invention is described in further detail.
Laboratory apparatus and reagent: Fourier transform nuclear magnetic resonance spectrometer (Switzerland Bruker company, AVIII type 400MHz and 600MHz); Developer: 10% sulfuric acid ethanol; A reagent (1% aubepine ethanolic soln); E reagent (1% paradimethy laminobenzaldehyde methanol solution); Aniline-pentanoic-phosphoric acid solution.
Embodiment 1
The preparation (extraction separation process) of activeconstituents monomer Xin Haike saponin(e:
The herb of C.tuberosum is provided by the Africa foreign student Abdulai Jawo Bah of International School of Tianjin University Of Traditional Chinese Medicine, is taken back about 1.5kg by Africa, with 8 times, measure 50% alcohol reflux 3 times, each 2 hours, merge 50% ethanol extract, decompression recycling ethanol is extremely without alcohol taste.Adding distil water is diluted to 4L, filtration is by macroporous resin column chromatography, water-ethanol gradient elution (water-95% ethanol, v/v), vacuum distillation recovered solvent, obtains following five components: 30% ethanol elution thing 60g, 50% ethanol elution thing 20g, 70% ethanol elution thing 2.7g, 95% ethanol elution thing 1.4g.
70% ethanol elution thing obtains 5 components through ODS column chromatography (water-100% acetonitrile gradient wash-out), wherein component Fr23-25 is again through silica gel column chromatography (eluting solvent: methylene dichloride: methyl alcohol: water=6.2:2.8:1) obtain the about 7mg of above-mentioned Xin Haike saponin compound (chemical compounds I), physical behavior is white powder (methyl alcohol).
Above-mentioned Xin Haike saponin(e new compound, 10% sulfuric acid ethanol displaing yellow spot, A reagent colour development is yellow spotting, and E reagent does not develop the color, and infers that this compound may be spirostane alcohol saponins.ESI-MS provides molecular ion peak m/z:1079.51[M+H] +, 1123.48[M+HCOO] -, infer that thus molecular weight is 1078.In negative ion second order ms (take m/z1077 as parent ion), main fragmention is m/z:1077.67[M-H] -, 915.53[M-H-162] -, 753.30[M-H-162-162] -, 591.30[M-H-162-162-162] -; In positive ion second order ms (take m/z1079 as parent ion), main fragment ion peak is m/z:431.16[M+H-162-162-162-162] +, 593.26[M+H-162-162-162] +, 755.23[M+H-162-162] +.According to ESI-MS 2mass-spectrometric data, infers and in this compound, contains four hexoses.
1h-NMR (C 5d 5n, 400MHz) spectrum provide four methyl signals, two unimodal chemical shifts are δ 0.64 (3H, s, Me-19) and 1.06 (3H, s, Me-18), two bimodal chemical shifts are 1.06 (3H, d, J=4.4Hz, Me-27) and 1.36 (3H, d, J=6.8Hz, Me-21).δ 2.73 (1H, t, J=6.8Hz) is the hydrogen signal on 17 tertiary carbons of spirostane alcohol; δ 3.36 (1H, br d, J=10.8Hz, H-26 ax) be the upright hydrogen signal on spirostane alcohol F encircles 26.Due to 12 carbonyl substituted, make 11 carbon become latent chiral carbon, wherein δ 2.21 (1H, dd, J 1=14.4, J 2=4.4Hz) be 11 calm hydrogen signals, 2.35 (1H, t, J=13.6Hz) are 11 upright hydrogen signals.Above hydrogen spectrum data are basically identical with new hecogenin hydrogen spectrum data, infer that its aglycon parent nucleus is new hecogenin.Hydrogen spectrum gives four sugared terminal hydrogen signal δ 4.86 (1H, J=7.6Hz), 5.15 (1H, J=8Hz), 5.30 (1H, J=8Hz) and 5.59 (1H, J=7.6Hz).
13c-NMR (C 5d 5n, 100MHz) spectrum provides 51 carbon signals.δ 109.7 is characteristic signals of 22 spiral shell carbon atoms of spirostane alcohol compound; Low place δ 212.7 shows in molecular structure have carbonyl substituted; δ 79.7 and 65.1 is respectively 16 of spirostane alcohol and 26 carbon signals; Carbon-13 nmr spectra high field region shows that four methyl carbon signals are respectively δ 16.2,11.6,13.7,16.0, and above data, in conjunction with ESI-MS 2the fragment ion peak of middle m/z431.19, further infers that aglycon parent nucleus is Xin Haike saponin(e.In carbon spectrum, show that δ 102.3,104.5,104.8,105.0 is four sugared end group carbon signals, in conjunction with four sugared terminal hydrogen signal coupling constants, be all about 8Hz, infer that four sugar may be β-D configuration.
As shown in Figure 1, according to HMBC spectrum, further inferring the sugared order of connection, there is distant relation in terminal hydrogen signal δ 4.86 and the aglycon C-3 position carbon signal δ 76.9 of semi-lactosi, proves that semi-lactosi is directly connected on 3 of aglycons.There is distant relation in the terminal hydrogen signal δ 5.15 of glucose and the C-4' position δ 80.2 of semi-lactosi, proves that this glucose is connected on the 4' position of semi-lactosi.There is distant relation in glucose terminal hydrogen signal δ 5.30 and glucose 3'' position, inner side carbon signal δ 88.5, therefore infer that it is connected in glucose C-3'' position, inner side, there is distant relation with glucose 2'' position, inner side carbon signal δ 81.4 in the terminal hydrogen signal δ 5.59 of another outside glucose, the order of connection of inferring accordingly sugar chain is β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 3)]-β-D-Glucose-(1 → 4)-β-D-gala pyranose, (25R)-3 β of these sugar chain part NMR data and bibliographical information-[(O-β-D-glucopyranosyl-(1 → 2)-O-[β-D-glucopyranosyl-(1 → 3)]-O-β-D-glucopyranosyl-(1 → 4)-β-D-galactopyranosyl)-oxy]-5 α-spirostan-12-one corresponding data is basically identical, thus, sugar chain part-structure is substantially definite.
In order to confirm the structure of aglycon part in this compound and sugared kind, it has been carried out to acid hydrolysis, described acid hydrolytic reaction is: take the new hecogenin 1mg of compound and be dissolved in 10ml trifluoroacetic acid aqueous solution (2M), 95 ℃ of reflux 3 hours, cooling after, with methylene dichloride 10ml extraction three times, after merging, reclaim methylene dichloride, obtain this compound aglycon part, be total to thin layer with corresponding aglycon, determine the structure of aglycon.After water layer reclaims, with 18cm circular filter paper, do paper chromatography experiment, with standard sugar contrast, determine sugared kind.Developping agent: propyl carbinol: acetic acid: water=4:1:5, developer: aniline-pentanoic-phosphoric acid.
Nuclear magnetic data is as follows:
1h-NMR (C 5d 5n, 400MHz) δ 0.64 (3H, s, Me-19), 1.06 (3H, s, Me-18), 1.06 (3H, d, J=4.4Hz, Me-27), 1.36 (3H, d, J=6.8Hz, Me-21), 2.21 (1H, dd, J 1=14.4, J 2=4.4Hz, H-11 eq), 2.35 (1H, t, J=13.6Hz, H-11 ax), 2.73 (1H, t, J=6.8Hz, H-17), 3.36 (1H, br d, J=10.8Hz, H-26 ax), 4.86 (1H, J=7.6Hz, Gal-1'), 5.15 (1H, J=8Hz, Glc-1 "), 5.30 (1H, J=8Hz, Glc-1''''), 5.59 (1H, J=7.6Hz, Glc-1'''); Compound carbon spectrum data are in Table 1.
Experimental result confirms that this compound aglycon parent nucleus is new hecogenin, and in sugar chain, sugar consists of β-D glucose and β-D semi-lactosi.
Except F ring, this Xin Haike saponin compound is compared with hecogenin 3-O-β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 3)]-β-D-Glucose-(1 → 4)-β-D-synthesis in document, and carbon spectrum data are basically identical.According to 1h-NMR, 13c-NMR, HSQC, HMBC data, belong to its carbon, hydrogen signal, as shown in table 1 below.Comprehensive its physico-chemical property, NMR and ESI-MS data determine that its structure is for α-spiral shell steroid-12-ketone 3-O-β-D-Glucose-(1 → 2), beta-hydroxy-5, (25S)-3-[β-D-Glucose-(1 → 3)]-β-D-Glucose-(1 → 4)-β-D-synthesis.
NMR attribution data (the in C of table 1 compound 1 5d 5n)
Figure BDA0000413434670000081
Figure BDA0000413434670000091
According to above-mentioned physico-chemical property and Spectral Characteristic, judge that its structure is as follows:
Figure BDA0000413434670000092
This compound has no bibliographical information, for separation first obtains.
Embodiment 2
Xin Haike saponin compound of the present invention and the impact of C.tuberosum extract on platelet aggregation
Utilize extracorporeal platelet aggregation active testing system to test component and the impact of part of compounds on platelet aggregation that C.tuberosum50% ethanol extraction obtains through macroporous resin column separation, result shows that 70% ethanol elution thing has the activity that induced platelet is assembled, and 95% ethanol elution thing has and suppresses active the platelet aggregation of ADP induction.
1 laboratory apparatus and reagent
Laboratory apparatus:
Flexstation3 microplate reader (Molecular Device); Supercentrifuge (U.S. Thermo Electron Corporation company, Thermo-RC6 +); 96 hole enzyme plates, and ten thousand/balance (METTLER TOLEDO instrument Shanghai company limited, AL204), thrombocyte mirror; Oscillating agitator (Jintan City Medical Instruments factory, XH-C); Liquid-transfering gun (1000 μ l, 200 μ l, 20 μ l, 10 μ l, 2.5 μ l, U.S. Eppendorf company); Surgical scissors, small size mosquito forceps, aseptic cotton, 10ml disposable syringe.
Experiment reagent:
Chloral Hydrate (analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group); Prostaglandin(PG) (prostaglandin, PGE1, U.S. sigma company); Trisodium Citrate, glucose, acetylsalicylic acid, NaCl, NaHCO 3, CaCl 2, KH 2pO 4, Tris-base, MgCl 2being U.S. sigma company produces.
Reagent compound method:
1 * ACD preparation: 38mM citric acid, 75mM Trisodium Citrate, 124mM glucose;
1 * BufferA(PH7.4) preparation: 130mM NaCl, 10mM Trisodium Citrate, 9mM NaHCO 3, 6mM glucose, 0.9mM MgCl 2, 0.81mM KH 2pO 4, 10mM Tris-base;
CaCl 2solution: 180mM;
Aggregation system (200 μ l): wherein volume of platelets 100 μ L OD values are about 1; Anticoagulant 50 μ l; ADP50 μ L(20 μ M, it act as induced platelet and assembles); Hatch for 37 ℃.
2 sample preparations
Sample preparation: by C.tuberosum DMSO or water dissolution for each component, component preparation 10mg/ml, monomeric compound is mixed with 20mM, then is diluted to and tests required concentration with Buffer A.
The preparation of positive control sample: aspirin solution (10mM, Buffer A dissolves).
3 experimental implementation processes
3.1SD rats in vitro abdomen arterial blood extracting
SD rat is weighed, and with 10% chloral hydrate anesthesia (0.5mL/100g rat), near anus place, carries skin, directly opens 1-shaped open, cuts off integumentary musculature, exposes abdominal cavity, by the frictional force of cotton, peels off intestinal tube.With small size mosquito forceps, clamp the right artery of ilium, use through the 10ml of ACD rinse syringe (ACD account for whole blood 15%), take centripetal end 1～3mm place, abdominal aortic bifurcation place is optimum puncturing point, punctures and gets blood.The desirable blood 10mL of 200g rat.
3.2 thrombocytes extract
Whole blood is through centrifugal (200 * g, 10min, room temperature), the supernatant liquor that separation obtains is platelet rich plasma (Platelet-rich plasma, PRP), in every milliliter of platelet rich plasma, add 2 μ LPGE1(500 * PGE1) to make the final concentration of prostaglandin(PG) be 1 μ M; Centrifugal (800 * g, 2650rpm, 10min, room temperature).Precipitation is used Buffer A washing the resuspended thrombocyte suspension that obtains, and under thrombocyte mirror, counts, and is adjusted into approximately 5 * 10 8individual/mL.
3.3 preliminary experiment
As there is aggreation in thrombocyte, the turbidity of corresponding thrombocyte system is that the OD value of sample can decline, therefore can characterize with the variation of OD value the situation (being turbidimetry) of platelet aggregation.Preliminary experiment is divided into three groups: positive group, negative group, control group (monitoring group).
Positive group:
Add 100 μ L thrombocyte suspension to 96 hole enzyme plates, every hole adds CaCl 2solution 1～2 μ L, (final concentration is 0.9～1.8mM).Use Flexstation3 microplate reader (Molecular Device) to carry out platelet aggregation test.Open Flexstation3 microplate reader, detect the OD value of 405nm place sample, if necessary, further adjust sample concentration and make OD in 1 left and right.Add respectively after 50 μ L positive drug (acetylsalicylic acid), in 37 ℃, hatch 10min and continue vibrations.Add 50 μ L ADP (final concentration 20 μ mol/L), microplate reader is set in 37 ℃ of every 45s readings once, the intermittent phase continues vibrations, stops experiment while no longer changing to OD value, finally obtains the time dependent kinetic curve of OD value.
Positive group background: 150 μ LbufferA+50 μ L positive drug;
Negative group:
Add 100 μ L thrombocyte suspension to 96 hole enzyme plates, every hole adds CaCl 2solution 1～2 μ L, (final concentration is 0.9～1.8mM).Use Flexstation3 microplate reader (Molecular Device) to carry out platelet aggregation test.Open Flexstation3 microplate reader, detect the OD value of 405nm place sample, if necessary, further adjust sample concentration and make OD in 1 left and right.Add respectively after 50 μ L BufferA, in 37 ℃, hatch 10min and continue vibrations.Add 50 μ L ADP (final concentration 20 μ mol/L), microplate reader is set in 37 ℃ of every 45s readings once, the intermittent phase continues vibrations, stops experiment while no longer changing to OD value, finally obtains the time dependent kinetic curve of OD value.
Background: 200 μ LbufferA
Control group:
Add 100 μ L thrombocyte suspension liquid to 96 hole enzyme plates, every hole adds CaCl 2solution 1～2 μ L, (final concentration is 0.9～1.8mM, Ca 2+concentration affects experimental result).Use Flexstation3 microplate reader (Molecular Device) to carry out platelet aggregation-against test.Open Flexstation3 microplate reader, detect the OD value of 405nm place sample, if necessary, further adjust sample concentration and make OD in 1 left and right.Add respectively after 50 μ L BufferA, in 37 ℃, hatch 10min and continue vibrations.Add 50 μ L BufferA again, microplate reader is set in 37 ℃ of every 45s readings once, the intermittent phase continues vibrations, stops experiment while no longer changing to OD value, finally obtains the time dependent kinetic curve of OD value.
Background: 200 μ L buffer A
3.4 formally experiments
Add 100 μ L thrombocyte suspension liquid to 96 hole enzyme plates, every hole adds CaCl 21～2 μ L, (final concentration is 0.9～1.8mM, Ca 2+concentration affects experimental result).Use Flexstation3 microplate reader (Molecular Device) to carry out platelet aggregation test.Open Flexstation3 microplate reader, detect the OD value of 405nm place sample, if necessary, further adjust sample concentration and make OD in 1 left and right.Add respectively after 50 μ L samples, in 37 ℃, hatch 10min and continue vibrations.Add 50 μ L ADP (final concentration 20 μ mol/L), microplate reader is set in 37 ℃ of every 45s readings once, the intermittent phase continues vibrations, stops experiment while no longer changing to OD value, finally obtains the time dependent kinetic curve of OD value.
Background: 150 μ Lbuffer A+50 μ L testing samples.
Platelet aggregation-against experiment is divided into five groups, is respectively positive group (for drug provision contrast to be measured), negative group (for detection of the calculating of thrombocyte the stress function and medicine inhibiting rate), Control group (monitoring spontaneous platelet aggregation extent), experimental group and background (being background color group).
The grouping of table 2-1 platelet aggregation test arranges
Note: it is that agonist comes induced platelet to assemble that this experiment adopts ADP, and positive drug is acetylsalicylic acid.
3.5 platelet aggregation rates and medicine are to assembling the calculating of inhibiting rate
Use the OD(deduction background of each each time point of hole of Office Excel software analysis) Value Data, adopt following formula:
Platelet aggregation rate t=(OD t=0-OD t)/OD t=0* 100%
The convert platelet aggregation rate of each time point, finally obtains the time dependent kinetic curve of platelet aggregation rate.The aggregation rate in each hole is compared with the aggregation rate of negative control, calculates the inhibiting rate of each time point of thrombocyte according to formula, the final platelet aggregation-against inhibiting rate that inhibiting rate corresponding to MA that adopts this hole is medicine to be measured.Formula is:
Medicine is to the negative group of the inhibiting rate %=(of platelet aggregation MA-corresponding experimental group aggregation rate)/negative group MA * 100%.
Calculate the inhibiting rate of different medicines, different concns experimental group.Take drug concentration as X-coordinate, the inhibiting rate of medicine of take be the matched curve of ordinate zou ，Yong Origin mapping software, draw the dose effect curve of medicine, input the concentration of certain medicine, according to this beneficial effect curve, obtain the inhibiting rate of medicine under this concentration.
4 experimental results
C.tuberosum50% ethanol extraction and five components that obtain through macroporous resin column chromatography: water elution thing, 30% ethanol elution thing, 50% ethanol elution thing, 70% ethanol elution thing, 95% ethanol elution thing, be made into respectively the solution of the about 10mg/ml of concentration, except 95% ethanol elution thing dissolves with DMSO, each component all adopts water-soluble, while testing, with buffer A, be diluted to final concentration 100 μ g/ml (concentration in 200 μ L reaction systems), the inhibiting rate of the platelet aggregation that each component is induced for ADP is as shown in table 3-1.
Table 2-2C.tuberosum gross sample and each component platelet aggregation-against inhibiting rate
Note: 95% ethanol elution thing solvability is poor, therefore the concentration of preparation is 50 μ g/ml.
From table 2-2, can find out, compare with acetylsalicylic acid, it is active that C.tuberosum95% ethanol elution thing has good anticoagulant, the strongest to the platelet aggregation inhibitory activity of ADP induction.It is active that 30% ethanol elution thing has weak anticoagulant, and the platelet aggregation that 50% ethanol elution thing is induced ADP is almost without impact.
In experiment, find, in test system, add after macroporous resin column 70% ethanol elution thing, do not add ADP induction, mix OD value in the process of hatching occur downward trend at sample with thrombocyte, this phenomenon shows that 70% ethanol elution thing may contain the chemical composition that induced platelet is assembled.For this reason, general extractive and 5 components, in the situation that not adding ADP, are tested to the activity that its induced platelet is assembled.Experimental result is as shown in table 3-2.
Table 2-3C.tuberosum50% total ethanol extract and each component platelet aggregation rate
Figure BDA0000413434670000141
Table 2-3 confirms the activity that the 70% ethanol elution thing that contains Xin Haike saponin compound has induced platelet to assemble, and all the other each components are without induced platelet aggregation activity.
Above-mentioned detailed description of this Xin Haike saponin compound and extract and preparation method thereof and application being carried out with reference to embodiment; illustrative rather than determinate; can list several embodiment according to institute's limited range; therefore in the variation and the modification that do not depart under general plotting of the present invention, within should belonging to protection scope of the present invention.

Claims (10)

1. Yi Zhong Xin Haike saponin compound, it is characterized in that the new hecogenin of: ﹛, 3-O-β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 3)]-β-D-Glucose-(1 → 4)-β-D-Ban lactose Gan ﹜, has following structural formula (I):
Figure FDA0000413434660000011
2. Xin Haike saponin compound according to claim 1, is characterized in that: physical chemistry and Spectroscopic Properties: white powder (methyl alcohol), and nuclear magnetic data is:
1h-NMR (C 5d 5n, 400MHz) δ 0.64 (3H, s, Me-19), 1.06 (3H, s, Me-18), 1.06 (3H, d, J=4.4Hz, Me-27), 1.36 (3H, d, J=6.8Hz, Me-21), 2.21 (1H, dd, J 1=14.4, J 2=4.4Hz, H-11 eq), 2.35 (1H, t, J=13.6Hz, H-11 ax), 2.73 (1H, t, J=6.8Hz, H-17), 3.36 (1H, br d, J=10.8Hz, H-26 ax), 4.86 (1H, J=7.6Hz, Gal-1'), 5.15 (1H, J=8Hz, Glc-1 "), 5.30 (1H, J=8Hz, Glc-1''''), 5.59 (1H, J=7.6Hz, Glc-1'''); Carbon spectrum data are embodiment part table 1.
3. the preparation method of Xin Haike saponin compound claimed in claim 1, is characterized in that: concrete steps are as follows:
(1) after C.tuberosum herb alcohol reflux, extracting solution simmer down to crude extract medicinal extract;
(2) dilution of gained crude extract medicinal extract adding distil water disperses and filters to obtain settled solution, and through macroporous resin column chromatography, water-ethanol gradient elution, obtains 70% ethanol elution thing;
(3) by 70% ethanol elution thing through ODS column chromatography, water-100% acetonitrile wash-out, collect 50% acetonitrile washing separation of flow part, obtained component is through silica gel column chromatography, methylene chloride-methanol-water elution, obtains Xin Haike saponin compound.
4. the preparation method of Xin Haike saponin compound according to claim 3, is characterized in that: concrete steps are as follows:
(1) 8 times of amount 50%(v/v for C.tuberosum herb) alcohol reflux is 3 times, and each 2 hours, united extraction liquid, decompression recycling ethanol, to without alcohol taste, obtains crude extract medicinal extract;
(2) dilution of gained crude extract medicinal extract adding distil water is dispersed to 4L, and after filter paper filters, settled solution is through D101 macroporous resin column chromatography, and water-95% ethanol (v/v) gradient elution, obtains 30%, 50%, 70%, 95%4 ethanol elution thing;
(3) by 70% ethanol elution thing through ODS column chromatography, water-100% acetonitrile wash-out, collect 50% acetonitrile washing separation of flow part, obtained component is through 40-63 μ m silica gel column chromatography, methylene chloride-methanol-water 6.2:2.8:1(v/v/v) wash-out, obtain this Xin Haike saponin compound.
5. an extract that contains Xin Haike saponin compound described in claim 1, is characterized in that: by following method, obtained:
(1) after C.tuberosum herb alcohol reflux, extracting solution simmer down to crude extract medicinal extract;
(2) dilution of gained crude extract medicinal extract adding distil water disperses and filters to obtain settled solution, and through macroporous resin column chromatography, water-ethanol gradient elution, obtains 70% ethanol elution thing.
6. the preparation method of extract claimed in claim 5, is characterized in that: concrete steps are as follows:
(1) after C.tuberosum herb alcohol reflux, extracting solution simmer down to crude extract medicinal extract;
(2) dilution of gained crude extract medicinal extract adding distil water disperses and filters to obtain settled solution, and through macroporous resin column chromatography, water-ethanol gradient elution, obtains 70% ethanol elution thing, i.e. target extract.
7. the preparation method of extract according to claim 6, is characterized in that: concrete steps are as follows:
(1) 8 times of amount 50%(v/v for C.tuberosum herb) alcohol reflux is 3 times, and each 2 hours, united extraction liquid, decompression recycling ethanol, to without alcohol taste, obtains crude extract medicinal extract;
(2) dilution of gained crude extract medicinal extract adding distil water is dispersed to 4L, and after filter paper filters, settled solution is through D101 macroporous resin column chromatography, and water-95% ethanol (v/v) gradient elution, obtains 70% ethanol elution thing, i.e. target extract.
8. the application of the extract that contains Xin Haike saponin compound claimed in claim 5 aspect induced platelet aggregation activity.
9. the application of the extract of the Xin Haike of containing saponin compound claimed in claim 5 in preparing blood-clotting agent.
10. the pharmaceutical composition with extract described in claim 5, is characterized in that: comprise and treat and/or prevent the extract of significant quantity and the optional acceptable vehicle of pharmacy.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106596432A (en) * 2016-12-26 2017-04-26 天津中医药大学 Method for determining activity contribution degrees of compounds in traditional Chinese medicine for promoting blood circulation to remove blood stasis

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HIROMICHI MATSUURA ET AL.: "A Furostanol Glycoside from Garlic,Bulbs of Allium sativum L.", 《CHEM.PHARM.BULL.》 *
徐雅娟等: "蒺藜果中两种新甾体皂苷的分离和鉴定", 《药学学报》 *
杨维力等: "南川鹭鸶草的化学成分", 《药学学报》 *
邹传纯: "特征结构甾体多糖皂甙的汇集式合成研究", 《中国博士论文全文数据库》 *
陈延镛等: "龙舌兰属植物中菌族皂戒元的研究", 《化学学报》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106596432A (en) * 2016-12-26 2017-04-26 天津中医药大学 Method for determining activity contribution degrees of compounds in traditional Chinese medicine for promoting blood circulation to remove blood stasis

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