CN103601789B - Neotigogenin compounds as well as preparation method and application thereof - Google Patents
Neotigogenin compounds as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to neotigogenin compounds with structural general formulas of (I) and (II). The neotigogenin compounds are extracted from a whole herb of C.tuberosum and are {neotigogenin,3-O-beta-D-glucose-(1->2)-[beta-D-glucose-(1->4)]-beta-D-glucose-(1->4)-beta-D-galactopyranoside} (I), and {neotigogenin,3-O-beta-D-glucose-(1->2)-[beta-D-glucose-(1->3)]-beta-D-glucose-(1->4)-beta-D-galactopyranoside (II). The neotigogenin compounds have platelet aggregation induction activity and can be further applied to preparation of in vivo hemostatic drugs, so that the neotigogenin compounds have an important clinical application value.
Description
Technical field
The present invention relates to a kind of Liliaceae bracketplant genus plants extract and preparation method thereof and application, especially new for accusing saponin compound 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 the generation reducing cardiovascular and cerebrovascular adverse events.The antiplatelet aggregative activity of steroidal saponin be its as treatment diseases of cardiovascular and cerebrovascular systems medicine, one of mechanism of action of illness such as treatment atherosclerosis, myocardial infarction etc.
Steroidal saponin is the activeconstituents that in plant, a class is important, and the medicine as treatment diseases of cardiovascular and cerebrovascular systems is widely used clinically, and its indication comprises the sequela etc. of coronary heart disease, myocardial ischemia, cerebral arteriosclerosis and cerebral thrombosis.The medicine 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 successfully got permission European Union's registration listing in 2012.This is the therapeutic pharmaceuticals that China successfully enters that first of EU market has independent intellectual property right, obtains first plant amedica of the market access beyond Ye Shi EU member country.
It is Liliaceae (Liliacea) per nnial herb that bracketplant belongs to (Chlorophytum), and the whole world about has 215 kinds.Bracketplant platymiscium has anti-diabetic, regulates the multiple pharmacologically active such as immunity of organisms, reducing blood-fat, anti-oxidant, antifatigue, antalgic and inflammation relieving, and 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 between florescence or fruiting period, India and Africa mainly as nourishing drug use, have the effectiveness such as enhancing body immunizing power, cardiac stimulant, sexual desire promoting, treatment dysentery, lactagogue.But 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 new for accusing saponin compound.
Another technical problem to be solved by this invention is to provide a kind of new for accusing saponin mixture.
Another technical problem to be solved by this invention is to provide the above-mentioned new preparation method for accusing saponin mixture.
Another technical problem to be solved by this invention is to provide the above-mentioned new application for accusing saponin mixture.
For solving the problems of the technologies described above, technical scheme of the present invention is:
A kind of new for accusing saponin compound ﹛ NSC 232021,3-O-β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 4)]-β-D-Glucose-(1 → 4)-β-D-gala pyrans Tang Gan ﹜, has following structural formula (I) formula:
A kind of new for accusing saponin mixture, by above-mentioned new for accusing saponin compound (the first compound) ﹛ NSC 232021,3-O-β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 4)]-β-D-Glucose-(1 → 4)-β-D-gala pyrans Tang Gan ﹜ (I) and the second compound NSC 232021 3-O-β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 3)]-β-D-Glucose-(1 → 4)-β-D-synthesis (II) form, and have following structural formula (I) formula and (II) formula respectively:
Preferably, above-mentioned new physical chemistry and Spectroscopic Properties for accusing saponin mixture: white powder (methyl alcohol), in various development system, is single spot, and 10% sulfuric acid ethanol shows purplish grey spot; Nuclear magnetic data is:
1h-NMR (C
5d
5n, 400MHz) δ 1.10 (3H, d, J=6.6Hz, Me-21), 1.04 (3H, d, J=7.2Hz, Me-27), 0.76 (3H), 0.65 (3H), 3.33 (1H, br d, J=10.2Hz, H-26
ax), 5.53 (1H, d, J=7.2Hz), 5.46 (1H, d, J=7.8Hz), 5.25 (1H, d, J=7.8Hz), 5.16 (1H, d, J=7.8Hz), 5.12 (1H, d, J=7.8Hz), 5.08 (1H, d, J=7.8Hz), 4.89 (1H, d, J=7.2Hz), 4.87 (1H, d, J=7.8Hz); Carbon modal data is shown in embodiment part table 1.
Preferably, above-mentioned new for accusing saponin mixture, 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;
(3) by 70% ethanol elution thing through ODS column chromatography, water-100% acetonitrile, collect 50% acetonitrile washing separation of flow part, obtained component, through preparation liquid phase, acetonitrile, silicagel column elution, obtains new for accusing saponin mixture.
Preferably, above-mentioned new for accusing saponin mixture, obtained by following method:
(1) C.tuberosum herb 8 times amount 50% alcohol reflux 3 times, 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 things;
(3) by 70% ethanol elution thing through ODS column chromatography, water-100% acetonitrile, collect 50% acetonitrile washing separation of flow part, obtained component is through preparation liquid phase, acetonitrile, 40-63 μm silicagel column elution, eluting solvent is methylene dichloride: methyl alcohol: water=6.2:2.8:1(v/v/v), obtain this newly for accusing saponin mixture.
The above-mentioned new preparation method for accusing saponin mixture, 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, collect 50% acetonitrile washing separation of flow part, obtained component, through preparation liquid phase, acetonitrile, silicagel column elution, obtains new for accusing saponin mixture (mixture of the first compound and the second compound).
Preferably, the above-mentioned new preparation method for accusing saponin mixture, concrete steps are as follows:
(1) C.tuberosum herb 8 times amount 50% alcohol reflux 3 times, 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 things;
(3) by 70% ethanol elution thing through ODS column chromatography, water-100% acetonitrile, collect 50% acetonitrile washing separation of flow part, obtained component is through preparation liquid phase, acetonitrile, 40-63 μm silicagel column elution, eluting solvent is methylene dichloride: methyl alcohol: water=6.2:2.8:1(v/v/v), obtain new for accusing saponin mixture (mixture of the first compound and the second compound).
The invention discloses above-mentioned new for accusing the application of saponin mixture in induced platelet aggregation is active.
Preferably, the invention discloses the above-mentioned new announcement saponin mixture that replaces and prepare the application in blood-clotting agent.
There is the above-mentioned new pharmaceutical composition for accusing saponin mixture, comprising and treating and/or preventing the new for accusing saponin mixture and the acceptable vehicle of optional pharmacy of significant quantity.
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 depend on the administering mode or disease type and state that are used for the treatment of particular patient, for the preparation method of the said synthetic processes of specific administration pattern completely in the ken of pharmaceutical field technician.Such as, the thinner of pharmaceutical field routine, carrier, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier and lubricant etc. can be comprised as the acceptable vehicle of pharmacy, if desired, flavouring agent, preservative and sweetener etc. can also be added in pharmaceutical composition.
Above-mentioned composition can make 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 formulation all can be prepared according to the ordinary method of pharmaceutical field.
The invention has the beneficial effects as follows:
The new announcement saponin compound that replaces of the present invention obtains by extracting in the herb of C.tuberosum, has induced platelet aggregation activity, can be applied to the preparation of haemostatic medicament in body further, have important clinical value.
Accompanying drawing explanation
Fig. 1 is that compound is newly for the HMBC correlogram of accusing saponin(e (I);
Fig. 2 is that compound is newly for the HMBC correlogram of accusing saponin(e (II)
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 (Bruker company of Switzerland, AVIII type 400MHz and 600MHz); Developer: 10% sulfuric acid ethanol; A reagent (the aubepine ethanolic soln of 1%); E reagent (the paradimethy laminobenzaldehyde methanol solution of 1%); Aniline-pentanoic-phosphoric acid solution.
Embodiment 1
Activeconstituents is newly for the preparation (extraction and isolation flow process) of accusing saponin mixture:
The herb of C.tuberosum is provided by International School of Tianjin University Of Traditional Chinese Medicine Africa foreign student Abdulai JawoBah, is taken back, about 1.5kg by Africa, with 8 times amount 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, through macroporous resin column chromatography after filtration, 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% acetonihile gradient elution), wherein component Fr27-31 is through preparation liquid phase (acetonitrile), silica gel column chromatography (eluting solvent: methylene dichloride: methyl alcohol: water=6.2:2.8:1), obtain sample 1(S1 respectively), namely the blend sample of the first compound (I) and the second compound (II) is about 50mg, and the first compound is the new compound having no bibliographical information; And sample 2(S2), namely { NSC 232021 3-O-β-D-Glucose-(1 → 2)-[β-D-wood sugar-(1 → 3)]-β-D-Glucose-(1 → 4)-β-D-synthesis } and the mixture of isomers thereof are about 10mg.
Above-mentioned new for accusing saponin mixture (I and II formula mixture) sample, white powder (methyl alcohol), in various development system, is single spot.10% sulfuric acid ethanol shows purplish grey spot, and A reagent colour development is yellow spotting, and E reagent does not develop the color, and supposition may be spirostanol saponin.There is quasi-molecular ion peak m/z:1081.88 [M+H] in ESI-MS
+with 1125.73 [M+HCOO]
-, infer that its molecular weight is 1080.In positive ion first mass spectrometric, main fragmention is m/z:431.16 [M+H-162-162-162-162]
+, 595.54 [M+H-162-162-162]
+, 757.01 [M+H-162-162]
+, 919.24 [M+H-162-162]
+; In negative ion second order ms (with 1079 for parent ion), main fragmention is m/z:1079.54 [M-H]
-, 917.48 [M-H-162]
-, 593.37 [M-H-162-162-162]
-, according to ESI-MS
2mass-spectrometric data, infers in molecular structure containing four hexoses.
1h-NMR (C
5d
5n, 400MHz) compose existing eight sugared terminal hydrogen signal δ 5.53 (1H, d, J=7.2Hz), 5.46 (1H, d, J=7.8Hz), 5.25 (1H, d, J=7.8Hz), 5.16 (1H, d, J=7.8Hz), 5.12 (1H, d, J=7.8Hz), 5.08 (1H, d, J=7.8Hz), 4.89 (1H, d, J=7.2Hz), 4.87 (1H, d, J=7.8Hz), the fragmention information provided with ESI-MS produces contradiction, and therefore we infer may be a mixture.In hydrogen spectrum, except the hydrogen signal on sugar chain, two mixture aglycon part hydrogen signals overlap better, such as the signal of two methyl 21 and 27 overlaps completely, and chemical shift is δ 1.10 (3H, d, J=6.6Hz, Me-21) and 1.04 (3H, d, J=7.2Hz, Me-27), 18 and 19 displacement studies that methylate are very close, are respectively δ 0.77/0.76(3H, s), 0.65/0.64(3H, s).Therefore infer that two compound aglycon parts may be identical, and have fine distinction at sugar chain portion.
13c-NMR (C
5d
5n, 100MHz) spectrum gives eight sugared end group carbon signal δ 105.1,104.9,104.6,104.4,104.3,104.2,103.0,102.9, the further above-mentioned supposition of confirmation.Between chemical shift δ 60-90, have 36 carbon signals, the carbon signal of deduction aglycon part, its quantity is much smaller than eight due carbon signal numbers of hexose, and some carbon signal is apparently higher than other carbon signals, infers that sugar chain also overlaps thus.Examine carbon to compose us and find that two compound aglycon carbon signals overlap substantially, the carbon signal do not overlapped is respectively δ 84.0/83.9 (C-3), 44.48/44.40 (C-5), 33.96/33.89(C-4), be sugar chain and change the chemical shift difference caused, and numerical value is very close.
In carbon spectrum, δ 109.6 is characteristic signals of spirostane alcohol compound 22 spiro atoms; δ 81.1 and 64.9 is respectively spirostane alcohol 16 and 26 carbon signals; Carbon-13 nmr spectra high field region shows four methyl carbon signals and is respectively δ 16.5,13.3,14.7,16.2, above data and NSC 232021 nuclear magnetic data basically identical, in conjunction with the fragment ion peak of m/z433.17 in ESI-MS, infer that the aglycon part of compound (I) and (II) is NSC 232021.
As depicted in figs. 1 and 2, the order of connection of sugar is inferred according to HMBC spectrum, the terminal hydrogen signal δ 4.89 (1H of semi-lactosi, d, J=7.2Hz) and 4.87 (1H, d, J=7.8Hz) exist long-range relevant to aglycon C-3 position carbon signal δ 84.0/83.9, prove that semi-lactosi is directly connected on aglycon 3.The terminal hydrogen signal δ 5.12 (1H, d, J=7.8Hz) of glucose, 5.08 (1H, d, J=7.8Hz) exist long-range relevant to the C-4' position δ 79.6 of semi-lactosi, prove that this glucose is connected on the 4' position of semi-lactosi.Glucose terminal hydrogen signal δ 5.53 (1H, d, J=7.2Hz), 5.46 (1H, d, J=7.8Hz) exist long-range relevant to glucose 2'' position, inner side carbon signal δ 81.0, therefore infer that it is connected in glucose C-2'' position, inner side.In compound 5, the terminal hydrogen signal δ 5.25 (1H, d, J=7.8Hz) of glucose exists long-range relevant to glucose 3'' position, inner side carbon signal δ 88.4 outside another; In compound 3, the terminal hydrogen signal δ 5.16 (1H, d, J=7.8Hz) of glucose exists long-range relevant to glucose 4'' position, inner side carbon signal δ 87.3 outside another.Sugar terminal hydrogen signal coupling constant is all about 8.0Hz, infers that sugar is β-D configuration.
Can infer that the sugar chain order of connection of compound (I) is β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 4)]-β-D-Glucose-(1 → 4)-β-D-semi-lactosi in sum, the sugar chain order of connection of compound (II) is β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 3)]-β-D-Glucose-(1 → 4)-β-D-semi-lactosi.
In order to confirm the structure of aglycon part in compound (I), (II) and sugared kind, carried out acid hydrolysis (acid hydrolytic reaction is as previously described) to it, spectral data is as follows:
1h-NMR (C
5d
5n, 400MHz) δ 1.10 (3H, d, J=6.6Hz, Me-21), 1.04 (3H, d, J=7.2Hz, Me-27), 0.76 (3H), 0.65 (3H), 3.33 (1H, br d, J=10.2Hz, H-26
ax), 5.53 (1H, d, J=7.2Hz), 5.46 (1H, d, J=7.8Hz), 5.25 (1H, d, J=7.8Hz), 5.16 (1H, d, J=7.8Hz), 5.12 (1H, d, J=7.8Hz), 5.08 (1H, d, J=7.8Hz), 4.89 (1H, d, J=7.2Hz), 4.87 (1H, d, J=7.8Hz); Carbon modal data is in table 1.
ESI-MS m/z：1081.88[M+H]
+,1125.73[M+HCOO]
-；ESI-MS
2m/z：431.16[M+H-162-162-162-162]
+,595.54[M+H-162-162-162]
+,757.01[M+H-162-162]
+，919.24[M+H-162-162]
+;1079.54[M-H]
-,917.48[M-H-162]
-,593.37[M-H-162-162-162]
-。
Its aglycon of the results show is NSC 232021, and in sugar chain, sugar consists of β-D glucose and β-D semi-lactosi.Comprehensively
1h-NMR,
13c-NMR, HMBC, HSQC, TOCSY, LC-MS also compare with document carbon modal data, are speculated as the mixture of NSC 232021 3-O-β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 4)]-β-D-Glucose-(1 → 4)-β-D-synthesis (Compound I) and NSC 232021 3-O-β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 3)]-β-D-Glucose-(1 → 4)-β-D-synthesis (Compound II per).
Carbon modal data (the C of table 1 compound (I), (II)
5d
5n, 100MHz)
Judge that its structure is as follows according to new physico-chemical property and Spectral Characteristic for accusing saponin(e:
Wherein new is the new compound having no bibliographical information for accusing saponin(e (I) formula.
Embodiment 2
Compound of the present invention is newly for accusing saponin(e (C.tuberosum) to the impact of platelet aggregation
Utilize extracorporeal platelet aggregation active testing system to test C.borivianum50% ethanol extraction and be separated the component and part of compounds that obtain to the impact of platelet aggregation through macroporous resin column, result shows that 70% ethanol elution thing has the activity of induced platelet aggregation, and 95% ethanol elution thing has inhibit activities to the platelet aggregation that ADP induces.
1 laboratory apparatus and reagent
Laboratory apparatus:
Flexstation3 microplate reader (Molecular Device); Supercentrifuge (Thermo ElectronCorporation company of the U.S., Thermo-RC6
+); 96 hole enzyme plates, ten thousand/balance (METTLERTOLEDO instrument Shanghai company limited, AL204), thrombocyte mirror; Oscillating agitator (Medical Instruments factory of Jintan City, XH-C); Liquid-transfering gun (1000 μ l, 200 μ l, 20 μ l, 10 μ l, 2.5 μ l, Eppendorf company of the U.S.); 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, sigma company of the U.S.); Trisodium Citrate, glucose, acetylsalicylic acid, NaCl, NaHCO
3, CaCl
2, KH
2pO
4, Tris-base, MgCl
2be sigma company of the U.S. to produce.
Preparation of reagents method:
1 × ACD prepares: 38mM citric acid, 75mM Trisodium Citrate, 124mM glucose;
1 × BufferA(PH7.4) preparation: 130mM NaCl, 10mM Trisodium Citrate, 9mMNaHCO
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 value is about 1; Anticoagulant 50 μ l; ADP50 μ L(20 μM, it act as induced platelet aggregation); Hatch for 37 DEG C.
2 sample preparations
Sample preparation: by each for C.tuberosum component DMSO or water dissolution, component preparation 10mg/ml, monomeric compound is mixed with 20mM, then is diluted to the concentration needed for experiment 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 weight, with 10% chloral hydrate anesthesia (0.5mL/100g rat), carries skin near anus place, directly opens 1-shaped open, cut off integumentary musculature, expose abdominal cavity, and the frictional force by cotton peels off intestinal tube.Clamp the right artery of ilium with small size mosquito forceps, use the 10ml syringe (ACD accounts for 15% of whole blood) through ACD rinse, with centripetal end 1 ~ 3mm place of abdominal aortic bifurcation place for optimum puncturing point, blood is got in puncture.The desirable blood 10mL of 200g rat.
3.2 thrombocytes extract
Whole blood is through centrifugal (200 × g, 10min, room temperature), being separated the supernatant liquor obtained is platelet rich plasma (Platelet-rich plasma, PRP), 2 μ LPGE1(500 × PGE1 are added in every milliliter of platelet rich plasma) make the final concentration of prostaglandin(PG) be 1 μM; Centrifugal (800 × g, 2650rpm, 10min, room temperature).Precipitation uses Buffer A washing and resuspendedly namely obtains thrombocyte suspension, counts, be adjusted to about 5 × 10 under thrombocyte mirror
8individual/mL.
3.3 preliminary experiment
As there is aggreation in thrombocyte, the turbidity of corresponding thrombocyte system and the OD value of sample can decline, therefore can characterize the situation (i.e. turbidimetry) of platelet aggregation with the change of OD value.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).Flexstation3 microplate reader (Molecular Device) is used to carry out platelet aggregation test.Open Flexstation3 microplate reader, detect the OD value of 405nm place sample, if necessary, adjustment sample concentration makes OD about 1 further.After adding 50 μ L positive drug (acetylsalicylic acid) respectively, hatch 10min and continuous vibration in 37 DEG C.Add 50 μ L ADP (final concentration 20 μm of ol/L), microplate reader is set in 37 DEG C of every 45s readings once, intermittent phase continuous vibration, stop experiment when no longer changing to OD value, finally obtain 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).Flexstation3 microplate reader (Molecular Device) is used to carry out platelet aggregation test.Open Flexstation3 microplate reader, detect the OD value of 405nm place sample, if necessary, adjustment sample concentration makes OD about 1 further.After adding 50 μ L BufferA respectively, hatch 10min and continuous vibration in 37 DEG C.Add 50 μ L ADP (final concentration 20 μm of ol/L), microplate reader is set in 37 DEG C of every 45s readings once, intermittent phase continuous vibration, stop experiment when no longer changing to OD value, finally obtain 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 affect experimental result).Flexstation3 microplate reader (Molecular Device) is used to carry out platelet aggregation-against test.Open Flexstation3 microplate reader, detect the OD value of 405nm place sample, if necessary, adjustment sample concentration makes OD about 1 further.After adding 50 μ L BufferA respectively, hatch 10min and continuous vibration in 37 DEG C.Add 50 μ LBufferA again, microplate reader is set in 37 DEG C of every 45s readings once, intermittent phase continuous vibration, stop experiment when no longer changing to OD value, finally obtain the time dependent kinetic curve of OD value.
Background: 200 μ L buffer A
3.4 formally test
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 affect experimental result).Flexstation3 microplate reader (MolecularDevice) is used to carry out platelet aggregation test.Open Flexstation3 microplate reader, detect the OD value of 405nm place sample, if necessary, adjustment sample concentration makes OD about 1 further.After adding 50 μ L samples respectively, hatch 10min and continuous vibration in 37 DEG C.Add 50 μ L ADP (final concentration 20 μm of ol/L), microplate reader is set in 37 DEG C of every 45s readings once, intermittent phase continuous vibration, stop experiment when no longer changing to OD value, finally obtain 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 medicine to be measured provides contrast), negative group (for detecting the calculating of thrombocyte the stress function and Drug inhibition rate), Control group (monitoring spontaneous platelet aggregation extent), experimental group and background (i.e. background color group).
The grouping of table 2-1 platelet aggregation test is arranged
Note: this experiment adopts ADP to be that agonist carrys out induced platelet aggregation, and positive drug is acetylsalicylic acid.
3.5 platelet aggregation rates and medicine are to the calculating assembling inhibiting rate
The OD(of each each time point of hole of Office Excel software analysis is used to deduct background) Value Data, adopt following formula:
Platelet aggregation rate
t=(OD
t=0-OD
t)/OD
t=0× 100%
Convert the platelet aggregation rate of each time point, finally obtains the time dependent kinetic curve of platelet aggregation rate.The aggregation rate in each hole, compared with the aggregation rate of negative control, draws the inhibiting rate of each time point of thrombocyte according to formulae discovery, adopt inhibiting rate that the MA in this hole is corresponding to be the final platelet aggregation-against inhibiting rate of medicine to be measured.Formula is:
Medicine is to the inhibiting rate %=(feminine gender group MA-corresponding experimental group aggregation rate of platelet aggregation)/negative group MA × 100%.
Calculate the inhibiting rate of different medicine, different concns experimental group.Take drug concentration as X-coordinate, with the inhibiting rate of medicine for ordinate zou, with the matched curve of Origin mapping software, draw the dose effect curve of medicine, input the concentration of certain medicine, according to this beneficial effect curve, namely obtain the inhibiting rate of medicine under this concentration.
4 experimental results
C.tuberosum50% ethanol extraction and five components obtained 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 the solution that concentration is about 10mg/ml respectively, except 95% ethanol elution thing is with except DMSO dissolving, each component all adopts water-soluble, be diluted to final concentration 100 μ g/ml (concentration in 200 μ L reaction systems) with buffer A when testing, the inhibiting rate of the platelet aggregation that each component is induced for ADP is as shown in table 2-2.
Table 2-2C.tuberosum gross sample and each component platelet aggregation-against inhibiting rate
Note: 95% ethanol elution thing solvability is poor, the concentration therefore prepared is 50 μ g/ml.
As can be seen from table 2-2, compared 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 more weak anticoagulant, and the platelet aggregation that 50% ethanol elution thing induce ADP is almost without affecting.
Find in an experiment, after adding macroporous resin column 70% ethanol elution thing in test system, do not add ADP induction, mix OD value in the process of hatching namely occur downward trend at sample with thrombocyte, this phenomenon shows that 70% ethanol elution thing may contain the chemical composition of induced platelet aggregation.For this reason, to general extractive and 5 components when not adding ADP, the activity of its induced platelet aggregation is tested.Experimental result is as shown in table 2-3.
Table 2-3C.tuberosum50% ethanol total extract and each component platelet aggregation rate
Table 2-3 confirms that 70% ethanol elution thing has the activity of induced platelet aggregation, and all the other each components are active without induced platelet aggregation.
Spirostanol saponin is 70% ethanol elution thing main component, and this experiment is separated the sample of two spirostanol saponins obtained when not adding ADP to 70% ethanol elution thing, test the activity of its induced platelet aggregation.Experimental result as shown in Table 2-4.
Wherein, sample 1(S1) be the mixture of compound (I), (II), first compound (I) is NSC 232021 3-O-β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 4)]-β-D-Glucose-(1 → 4)-β-D-synthesis, and the second compound (II) is NSC 232021 3-O-β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 3)]-β-D-Glucose-(1 → 4)-β-D-synthesis; Sample 2(S2) be the mixture of compound { NSC 232021 3-O-β-D-Glucose-(1 → 2)-[β-D-wood sugar-(1 → 3)]-β-D-Glucose-(1 → 4)-β-D-synthesis } and isomers thereof.
Table 2-4S1 and S2 platelet aggregation test result
Experimental result shows, it is active that S1, S2 have stronger induced platelet aggregation.
The above-mentioned detailed description newly replacing announcement saponin compound and preparation method thereof and application to carry out to this with reference to embodiment; illustrative instead of determinate; several embodiments can be listed according to institute's limited range; therefore in the change do not departed under general plotting of the present invention and amendment, should belong within protection scope of the present invention.
Claims (9)
1. one kind is replaced announcement saponin compound, it is characterized in that: ﹛ Tigogenin, 3-O-β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 4)]-β-D-Glucose-(1 → 4)-β-D-gala pyrans Tang Gan ﹜, has following structural formula (I) formula:
2. one kind is replaced announcement saponin mixture, soap glycoside compound ﹛ Tigogenin is accused by replacing described in claim 1,3-O-β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 4)]-β-D-Glucose-(1 → 4)-β-D-gala pyrans Tang Gan ﹜ (I) and Tigogenin 3-O-β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 3)]-β-D-Glucose-(1 → 4)-β-D-synthesis (II) form, and have following structural formula (I) formula and (II) formula respectively:
3. according to claim 2 for accusing saponin mixture, it is characterized in that: physical chemistry and Spectroscopic Properties: white powder, in various development system, be single spot, 10% sulfuric acid ethanol shows purplish grey spot; Nuclear magnetic data is:
1h-NMR (C
5d
5n, 400MHz) δ 1.10 (3H, d, J=6.6Hz, Me-21), 1.04 (3H, d, J=7.2Hz, Me-27), 0.76 (3H), 0.65 (3H), 3.33 (1H, br d, J=10.2Hz, H-26
ax), 5.53 (1H, d, J=7.2Hz), 5.46 (1H, d, J=7.8Hz), 5.25 (1H, d, J=7.8Hz), 5.16 (1H, d, J=7.8Hz), (5.12 1H, d, J=7.8Hz), 5.08 (1H, d, J=7.8Hz), 4.89 (1H, d, J=7.2Hz), 4.87 (1H, d, J=7.8Hz); Carbon modal data is embodiment part table 1.
4. according to claim 2 for accusing saponin mixture, it is characterized in that: 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;
(3) by 70% ethanol elution thing through ODS column chromatography, water-100% acetonitrile, collect 50% acetonitrile washing separation of flow part, obtained component, through preparation liquid phase, acetonitrile, silicagel column elution, obtains for accusing saponin mixture.
5. replacing according to claim 2 or 4 accuses saponin mixture, it is characterized in that: obtained by following method:
(1) C.tuberosum herb 8 times amount 50% alcohol reflux 3 times, 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 things;
(3) by 70% ethanol elution thing through ODS column chromatography, water-100% acetonitrile, collect 50% acetonitrile washing separation of flow part, obtained component is through preparation liquid phase, acetonitrile, 40-63 μm silicagel column elution, eluting solvent is methylene dichloride: methyl alcohol: water=6.2:2.8:1 (v/v/v), obtains this for accusing saponin mixture.
6. the preparation method for accusing saponin mixture according to claim 2, 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, collect 50% acetonitrile washing separation of flow part, obtained component, through preparation liquid phase, acetonitrile, silicagel column elution, obtains for accusing saponin mixture.
7. the preparation method for accusing saponin mixture according to claim 6, is characterized in that: concrete steps are as follows:
(1) C.tuberosum herb 8 times amount 50% alcohol reflux 3 times, 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 things;
(3) by 70% ethanol elution thing through ODS column chromatography, water-100% acetonitrile, collect 50% acetonitrile washing separation of flow part, obtained component is through preparation liquid phase, acetonitrile, 40-63 μm silicagel column elution, eluting solvent is methylene dichloride: methyl alcohol: water=6.2:2.8:1 (v/v/v), obtains this for accusing saponin mixture.
8. the announcement saponin mixture that replaces according to claim 2 is preparing the application in blood-clotting agent.
9. to have described in claim 2 for accusing the pharmaceutical composition of saponin mixture, comprise treat and/or prevent significant quantity for accusing saponin mixture and the acceptable vehicle of optional pharmacy.
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| 蒺藜果中两种新甾体皂苷的分离和鉴定;徐雅娟等;《药学学报》;20011231;第36卷(第10期);750-753 * |
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