CN103588851B - 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 PDFInfo
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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
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 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 Xin Haike saponin compound.
Another technical problem to be solved by this invention is to provide the extract containing above-mentioned Xin Haike saponin compound.
Another technical problem to be solved by this invention is the preparation method providing above-mentioned Xin Haike saponin compound.
Another technical problem to be solved by this invention is the preparation method of the extract provided containing above-mentioned Xin Haike saponin compound.
Another technical problem to be solved by this invention is the application of the extract provided containing above-mentioned Xin Haike saponin compound.
For solving the problems of the technologies described above, technical scheme of the present invention is:
A kind of Xin Haike 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-Ban lactose Gan ﹜, has following structural formula (I):
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 modal data is 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, collect 50% acetonitrile washing separation of flow part, obtained component is through silica gel column chromatography, and 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) C.tuberosum herb 8 times amount 50%(v/v) 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 through 40-63 μm of 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 containing 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. Objective extraction thing.
Preferably, the preparation method of said extracted thing, concrete steps are as follows:
(1) C.tuberosum herb 8 times amount 50%(v/v) 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 70% ethanol elution thing, i.e. Objective extraction thing.
The invention discloses the application of extract in induced platelet aggregation is active containing above-mentioned Xin Haike saponin compound.
Preferably, the extract that the invention discloses containing above-mentioned Xin Haike saponin compound is preparing the application in blood-clotting agent.
There is the pharmaceutical composition of said extracted thing, comprise the extract and the acceptable vehicle of optional pharmacy that treat and/or prevent 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:
Xin Haike saponin compound of the present invention obtains by extracting in the herb of C.tuberosum, and its 70% ethanol elution thing has induced platelet aggregation activity, can be applied to the preparation of haemostatic medicament in body further, 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 (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
The preparation (extraction and isolation flow process) of activeconstituents monomer Xin Haike saponin(e:
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 Fr23-25 obtains above-mentioned Xin Haike saponin compound (chemical compounds I) about 7mg through silica gel column chromatography (eluting solvent: methylene dichloride: methyl alcohol: water=6.2:2.8:1) again, and 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 molecular weight is 1078 thus.In negative ion second order ms (taking 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 (taking 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 in this compound containing 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 spirostane alcohol 17 tertiary carbons; δ 3.36 (1H, br d, J=10.8Hz, H-26
ax) be the upright hydrogen signal on 26, spirostane alcohol F ring.Due to 12 carbonyl substituted, 11 carbon are made to 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 modal data and new hecogenin hydrogen modal data basically identical, infer that its aglycon parent nucleus is new hecogenin.Hydrogen spectrum gives four sugared terminal hydrogen signals 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 provide 51 carbon signals.δ 109.7 is characteristic signals of spirostane alcohol compound 22 spiral shell carbon atoms; Low place δ 212.7 shows in molecular structure carbonyl substituted; δ 79.7 and 65.1 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 16.2,11.6,13.7,16.0, and above data, in conjunction with ESI-MS
2the fragment ion peak of middle m/z431.19, infers that aglycon parent nucleus is Xin Haike saponin(e further.Showing δ 102.3,104.5,104.8,105.0 in carbon spectrum is four sugared end group carbon signals, is all about 8Hz, infers that four sugar may be β-D configuration in conjunction with four sugared terminal hydrogen signal coupling constants.
As shown in Figure 1, infer the order of connection of sugar further according to HMBC spectrum, the terminal hydrogen signal δ 4.86 of semi-lactosi exists long-range relevant to aglycon C-3 position carbon signal δ 76.9, proves that semi-lactosi is directly connected on aglycon 3.The terminal hydrogen signal δ 5.15 of glucose exists long-range relevant to the C-4' position δ 80.2 of semi-lactosi, proves that this glucose is connected on the 4' position of semi-lactosi.Glucose terminal hydrogen signal δ 5.30 exists long-range relevant to glucose 3'' position, inner side carbon signal δ 88.5, therefore infer that it is connected in glucose C-3'' position, inner side, outside another, the terminal hydrogen signal δ 5.59 of glucose exists long-range relevant to glucose 2'' position, inner side carbon signal δ 81.4, infer that the order of connection of sugar chain is β-D-Glucose-(1 → 2)-[β-D-Glucose-(1 → 3)]-β-D-Glucose-(1 → 4)-β-D-gala pyranose accordingly, (25R)-3 β-[(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 datas of this sugar chain portion NMR data and bibliographical information are basically identical, thus, sugar chain portion structure is determined substantially.
In order to confirm the structure of aglycon part in this compound and sugared kind, acid hydrolysis has been carried out to it, described acid hydrolytic reaction is: the new hecogenin 1mg of Weigh Compound is dissolved in 10ml trifluoroacetic acid aqueous solution (2M), 95 DEG C of reflux 3 hours, after cooling, extract three times with methylene dichloride 10ml, methylene dichloride is reclaimed after merging, obtain this compound aglycon part, with corresponding aglycon thin layer altogether, determine the structure of aglycon.After water layer reclaims, do paper chromatography experiment with 18cm circular filter paper, determine the kind of sugar with standard sugar contrast.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 modal data is 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) in document-[β-D-Glucose-(1 → 3)]-β-D-Glucose-(1 → 4)-β-D-synthesis, and carbon modal data is basically identical.According to
1h-NMR,
13c-NMR, HSQC, HMBC data, belong to its carbon, hydrogen signal, as shown in table 1 below.Its physico-chemical property comprehensive, NMR and ESI-MS data determine that its structure is for (25S)-3 beta-hydroxy-5 α-spiral shell steroid-12-ketone 3-O-β-D-Glucose-(1 → 2)-[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)
Judge that its structure is as follows according to above-mentioned physico-chemical property and Spectral Characteristic:
This compound has no bibliographical information, obtains for being separated first.
Embodiment 2
Xin Haike saponin compound of the present invention and C.tuberosum extract are on the impact of platelet aggregation
Utilize extracorporeal platelet aggregation active testing system to test C.tuberosum50% 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 3-1.
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 3-2.
Table 2-3C.tuberosum50% ethanol total extract and each component platelet aggregation rate
Table 2-3 confirms that the 70% ethanol elution thing containing Xin Haike saponin compound has the activity of induced platelet aggregation, and all the other each components are active without induced platelet aggregation.
Above-mentioned detailed description of this Xin Haike saponin compound and extract and preparation method thereof being carried out with application 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. extra large Ke's saponin compound, it is characterized in that: ﹛ hecogenin, 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):
2. extra large Ke's saponin compound according to claim 1, it is characterized in that: physical chemistry and Spectroscopic Properties: white powder, 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 modal data is embodiment part table 1.
3. the preparation method of extra large Ke's saponin compound according to 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, collect 50% acetonitrile washing separation of flow part, obtained component is through silica gel column chromatography, and methylene chloride-methanol-water elution, obtains extra large Ke's saponin compound.
4. the preparation method of extra large Ke's saponin compound according to claim 3, is characterized in that: concrete steps are as follows:
(1) C.tuberosum herb 8 times amount 50% (v/v) 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 40-63 μm of silica gel column chromatography, and methylene chloride-methanol-water 6.2:2.8:1 (v/v/v) wash-out, obtains this extra large Ke's saponin compound.
5. the extract containing extra large Ke's saponin compound described in claim 1, 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.
6. the preparation method of extract according to 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. Objective extraction thing.
7. the preparation method of extract according to claim 6, is characterized in that: concrete steps are as follows:
(1) C.tuberosum herb 8 times amount 50% (v/v) 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 70% ethanol elution thing, i.e. Objective extraction thing.
8. the extract containing extra large Ke's saponin compound according to claim 5 is preparing the application in blood-clotting agent.
9. there is the pharmaceutical composition of extract described in claim 5, it is characterized in that: comprise the extract and the acceptable vehicle of optional pharmacy that treat and/or prevent significant quantity.
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