CN104689058B - A kind of liriope muscari Baily astragalin composition of platelet aggregation-against - Google Patents
A kind of liriope muscari Baily astragalin composition of platelet aggregation-against Download PDFInfo
- Publication number
- CN104689058B CN104689058B CN201510140415.3A CN201510140415A CN104689058B CN 104689058 B CN104689058 B CN 104689058B CN 201510140415 A CN201510140415 A CN 201510140415A CN 104689058 B CN104689058 B CN 104689058B
- Authority
- CN
- China
- Prior art keywords
- solution
- composition
- water
- liriope muscari
- ophiopogon
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The preparation method of liriope muscari Baily astragalin composition of the present invention includes as follows：Take liriope muscari Baily, soak 6 18h, smash, add water and decoct 0.5 6h, twice, extracting solution filters, after standing cools, centrifugation, discards insoluble matter, supernatant, cross the absorption of 101 resin columns of D, repeat loading once, stand 6 18h, be first negative with water elution to eluent molish reactions, eluted again with 30% ethanol solution, finally eluted with 85% ethanol solution, solvent is separately recovered in eluent, and 30% ethanol position and 85% ethanol position are mixed, it is lyophilized, obtain liriope muscari Baily astragalin composition.Liriope muscari Baily astragalin composition of the present invention possesses antithrombotic, anticoagulation, shortens clotting time, platelet aggregation-against, anti-lung thrombus, the activity for protecting anoxia/reoxygenation cell.
Description
Technical field：
The invention belongs to the field of Chinese medicines, and in particular to a kind of liriope muscari Baily astragalin composition and preparation method thereof, and
Said composition is in antithrombotic, platelet aggregation-against, the application on the extension clotting time.
Background technology：
Thrombotic diseases (thrombotic disease) are the blood as caused by a variety of causes in the blood vessels or in heart,
It is changed into curdled appearance from flow regime, so that a series of synthesis for causing the different degrees of embolism of blood vessel (embolism) and being formed
Sign.Therefore thrombotic diseases are also known as thrombotic disease.Substantial amounts of research data shows that thrombotic disease becomes danger
An important factor for evil population health.Even thromboembolism can be throughout big parteriole, vein, capillary, thus caused cardiac muscle
Infarct, cerebral infarction, pulmonary infarction etc., the death rate is high with disability rate, is first of the various causes of disease.The primary condition of thrombosis：1、
Vascular damaged (predominantly blood vessel endothelium injury) 2, blood flow change (blood stream stasis is with being vortexed) 3, the change (blood coagulation of blood constituent
The factor changes and platelet counts increase or hyperfunction), and impaired vascular endothelium is most important and most common.
Presently used antithrombotic reagent is broadly divided into three classes：When antiplatelet drug, second, anticoagulant, third, molten
Bolt medicine.
Blood platelet is a kind of multi-functional cell, is to be come off to form by the cytoplasm of megacaryocyte ripe in marrow, average
Distribution in blood, has the function of adhesion, aggregation, release and secretory granules content (such as serotonin, ADP).In thrombus
During formation, hematoblastic adhesion plays important initiating effect, excites endogenous and exogenous blood coagulation again therewith
Process.Slow blood flow, vortex are formed, blood coagulability increases and cardiovascular inner membrance is damaged when factors occur, or with certain
When kind factor is main, the blood platelet to keep to the side is then attached on exposed collagenous fibres, and discharges ADP and thromboxane A2, is promoted more
More platelet adhesion reactions, but after coagulation process starts, the fibrinogen transformation fibroblast cells in blood plasma, the latter makes blood small
Plate securely aggegation in impaired intimal surface.Above process is repeated, and blood platelet, which sticks Ji Dui, constantly to be increased, increase, and forms more
A blood platelet hillock, block blood flow, so as to cause thrombus.Therefore Platelet is the important ginseng for indicating thrombosis trend
Number, platelet activation assemble the occurrence and development that can cause or promote these diseases, so, the research for antiplatelet drug
Tool has very important significance.
Clinically, aspirin, clopidogrel and GPIIb/IIIa antagonists are the main bodys of current antiplatelet drug, preceding
Both are orally available, and the latter can only be injected intravenously, and be only applicable to acute phase of disease.But the above two also often cause adverse reaction, such as
Aspirin often causes purple plague purpura, gum blood, hemorrhage of digestive tract or postoperative hemorrhage etc..Clopidogrel often causes Neutrophilic granulocytopenia,
Thrombotic thrombocytopenic purpura and symptom of digestive tract etc..
Anticoagulant, it is currently used to have heparin class, hirudin and its genetic recombination preparation and vitamin K antagon etc..
The above two are intravenous injection administration, and the latter is oral anticoagulation thing.The main adverse reaction of heparin class medicine is bleeding, also may be used
Cause osteoporosis, allergy etc..The adverse reaction of lepirudin 023 ludon well below other anti-coagulants, but due to its effect can not
Doctor is used with caution against property.Common vitamin K antagon have neodicoumarin (warfarin) and acenocoumarol (acencoumarol,
Trade name sintrom) two kinds.Neodicoumarin can cause different degrees of bleeding, and its during Clinical practice influence factor compared with
It is more.
Thrombolytic drug, mainly there is urokinase, streptokinase etc., can be completely dissolved thrombus, but needs injection to apply more, has one
Fixed antigenicity, heavy dose of use can cause systemic bleeding, allergy etc. occur.
Phenomena such as this, the safe and effective antithrombotic reagent of research and development are particularly important.Ophiopogon is Liliaceae
The dried root of plant ophiopogon, its sweet in flavor, slight bitter, is slightly cold, the thoughts of returning home, lung, stomach, has Yin nourishing and lung moistening, reinforcing stomach reg fluid, clears away heart-fire and remove
The effect of tired, be clinically used for the angiocarpy such as diseases associated with inflammation and arrhythmia cordis and myocardial ischemia such as treatment bronchitis, pneumonia
Disease.Ophiopogon contains steroid saponin, homoisoflavone, polysaccharide, amino acid isoreactivity constituents, and modern pharmacology experiment was proved in ophiopogon
Contained monomer component has clear and definite pharmacological activity, as ophiopogonin D has antioxidation activity, Short Mordant Mountain Ophiopogon japonicus C tool
Growth of lung carcinoma cell etc. can be suppressed by playing the role of clear and definite antitumor cell transfer and ophiopogonin B.But monomer constituents
Preparation process is cumbersome, and yield is very low, and cost is higher.Set out so we select the extract for containing each monomer to be used as
Point, optimizes its separation and prepares method of purification, it is therefore an objective to can obtain, with the clear and definite ophiopogon japonicus extract of therapeutic effect, making it can
To be directly used in medical field, and various preparations are made into, its raw material is fully utilized.
201310710650.0 disclose a kind of preparation method of the extract with platelet aggregation inhibitory activity, this method
Including：Using ophiopogon medicinal material as raw material, extracted with organic solvent, extracting solution centrifugation, macroreticular resin separation, is washed with ethanol solution
It is de-, eluent is collected, is drying to obtain extract.
201310710650.0 embodiments 1 disclose a kind of preparation side of the extract with platelet aggregation inhibitory activity
Method, this method include：Ophiopogon medicinal material is taken, adds about 15 times of amount water refluxing extractions of ophiopogon raw material weight.Extracting solution filters, and standing is put
After cool, centrifugation, discards insoluble matter, supernatant, cross D101 type macroreticular resin post separations, first anti-to eluent molish with water elution
It should be negative, then be eluted with 30% ethanol solution, finally be eluted with 85% ethanol solution, eluent recycling design is dry
Up to ophiopogon different elution positions.Experimental data shows that the chief active position of the antiplatelet aggregative activity of ophiopogon is 85%
Alcohol elution, maintains an equal level with positive drug.
Ophiopogon respectively elutes the influence for the Platelet Aggregation in Rabbits rate that position induces ADP
Conclusion：Experimental data shows that the chief active position of the antiplatelet aggregative activity of ophiopogon is 85% ethanol elution
Position, maintains an equal level with positive drug.So preferably going out ophiopogon active site, i.e. ophiopogon japonicus extract according to experiment, and preparation is extracted to it
Technique is further optimized.
It can thus be appreciated that ophiopogon has a good antithrombotic acitivity, and the side effect of ophiopogon or adverse reaction are from there are no report
Road, prompts ophiopogon to be studied as safe and effective antithrombotic reagent, is of great significance.But which is produced actually
The ophiopogon on ground and which components group are its main active sites
For this query, the present invention is using whole animal model and external anticoagulation, platelet aggregation-against and cell
Experiment, screening ophiopogon antithrombotic acitivity position；And the preparation process of active site is optimized, establish reliable and stable quality
Control standard.
Technical solution
The active component and its pharmaceutical applications of ophiopogon of the present invention includes as follows：
The present invention relates to a kind of liriope muscari Baily astragalin composition of antithrombotic.
The present invention relates to a kind of anticoagulant liriope muscari Baily astragalin composition.
The present invention relates to a kind of liriope muscari Baily astragalin composition for shortening clotting time activity.
The present invention relates to a kind of liriope muscari Baily astragalin composition of platelet aggregation-against.
The present invention relates to a kind of liriope muscari Baily astragalin composition for protecting anoxia/reoxygenation cell.
The present invention relates to a kind of liriope muscari Baily astragalin composition of anti-lung thrombus.
The present invention relates to a kind of extract of the n-butanol of the liriope muscari Baily astragalin composition of antithrombotic.
The present invention relates to a kind of extract of the n-butanol of anticoagulant liriope muscari Baily astragalin composition.
The present invention relates to a kind of extract of the n-butanol for the liriope muscari Baily astragalin composition for shortening clotting time activity.
The present invention relates to a kind of extract of the n-butanol of the liriope muscari Baily astragalin composition of platelet aggregation-against.
The present invention relates to a kind of carrying for n-butanol of the liriope muscari Baily astragalin composition of protection anoxia/reoxygenation cell
Take thing.
The present invention relates to a kind of extract of the n-butanol of the liriope muscari Baily astragalin composition of anti-lung thrombus.
The present invention relates to a kind of hydrolysate of the liriope muscari Baily astragalin composition of antithrombotic.
The present invention relates to a kind of hydrolysate of anticoagulant liriope muscari Baily astragalin composition.
The present invention relates to a kind of hydrolysate for the liriope muscari Baily astragalin composition for shortening clotting time activity.
The present invention relates to a kind of hydrolysate of the liriope muscari Baily astragalin composition of platelet aggregation-against.
The present invention relates to a kind of hydrolysate of the liriope muscari Baily astragalin composition of protection anoxia/reoxygenation cell.
The present invention relates to a kind of hydrolysate of the liriope muscari Baily astragalin composition of anti-lung thrombus.
The species of ophiopogon of the present invention includes：River ophiopogon, liriope muscari Baily, Hubei ophiopogon；
The medicinal material position of ophiopogon of the present invention includes：The root tuber of ophiopogon, the fibrous root of ophiopogon；
The active component of ophiopogon of the present invention includes as follows：Ophiopogonin composition；Ophiopogonin composition is just
The extract of butanol；The hydrolysate of ophiopogonin composition；
The active component of ophiopogon of the present invention includes as follows：River ophiopogonin composition；
The active component of ophiopogon of the present invention includes as follows：Fibre Ophiopogon astragalin composition；
The active component of ophiopogon of the present invention includes as follows：Liriope muscari Baily astragalin composition；Liriope muscari Baily soap
The extract of the n-butanol of glycoside composition；The hydrolysate of liriope muscari Baily astragalin composition；
The active component of ophiopogon of the present invention includes as follows：Liriope muscari Baily fibrous root astragalin composition；Short Roripa mountain wheat
The extract of the n-butanol of winter fibrous root astragalin composition；The hydrolysate of liriope muscari Baily fibrous root astragalin composition；
The active component of ophiopogon of the present invention includes as follows：Hubei ophiopogonin composition；
The preparation method of ophiopogonin composition of the present invention includes as follows：The medicinal material position of ophiopogon is taken, adds water logging
6-18h is steeped, is smashed, adds water and decocts 0.5-6h, twice.Extracting solution filters, and after standing cools, centrifugation, discards insoluble matter, supernatant
Liquid, cross the absorption of D-101 resin columns, repeats loading once, stands 6-18h, first with water elution to eluent molish reactions in the moon
Property, then eluted with 30% ethanol solution, finally eluted with 85% ethanol solution, solvent is separately recovered in eluent, by 30%
Ethanol position and the mixing of 85% ethanol position, freeze, obtain ophiopogonin composition.
The preparation method at the extracting n-butyl alcohol position of ophiopogonin composition of the present invention includes as follows：Ophiopogonin
Composition, is repeatedly extracted using water-saturated n-butanol, collects n-butanol layer and water layer respectively, recycles n-butanol, n-butanol layer, water
Layer is freeze-dried respectively.The weight of freeze-dried powder is recorded, and calculates yield, obtains the extracting n-butyl alcohol portion of ophiopogonin composition
Position.
The preparation method of the hydrolysate of ophiopogonin composition of the present invention includes as follows：Ophiopogonin composition,
Addition aqueous acid, which is placed in boiling water bath, to be hydrolyzed, and is taken out, is put to room temperature, adjusts pH to filter to neutrality, residue is dried to constant weight.
Through petroleum ether surname extraction, extracting solution recycling design, residue is suspended with water, is freeze-dried the hydrolysis up to ophiopogonin composition
Thing.
Analyze and identify method
The present invention relates to a kind of thin-layer chromatography Qualitive test of ophiopogonin,
Take equal amount 1,2,3,4, No. 5 samples, the dissolving of 1ml methanol, SupelcleanTMLC-C18SPE solid phase extraction columns
Absorption, is eluted with water, each 8ml of 40%, 75%, 85% methanol solution respectively, is collected 75% elution position, is volatilized solvent, with less
Methanol dissolving is measured, it is spare.
In order to compare the difference between 5 kinds of sample saponin components, three development systems are selected to be compared.
Development system is as follows：
(1) chloroform-methanol-water (17: 6: 2)
(2) acetone and ethyl acetate-water-acetic acid (5: 5: 1: 0.2)
(3) water-saturated n-butanol system.
Quantitative analysis is carried out to ophiopogonin C the present invention relates to a kind of HPLC-ELSD methods
In order to further compare the difference between different sources dwarf lilyturf tuber total saponins component, HPLC-ELSD is selected to carry out component
Qualitative determination.
Chromatographic column：Agilent ZORBAX SB-C18 (4.6 × 250mm, 5 μm)；
Chromatographic condition：Methanol-water gradient elution.Elution requirement is seen below
Total saposins HPLC-ELSD chromatographic conditions
Assay is carried out to ophiopogonin the present invention relates to a kind of colorimetric method
The preferred process of technical scheme is as follows：
Experimental animal of the present invention includes as follows：ICR mouse, 18~22g of weight, male and female dual-purpose, by Yangzhou University
Comparative medicine center provides；Wistar rats, weight 180-220g, male, is provided by Yangzhou University's comparative medicine center, is permitted
Card number：SUXR (Soviet Union) 2012~0004；Large ear rabbit, male, 2.5~3kg of weight, is provided by Qinglongshan animal center.
Experiment cell of the present invention includes as follows：Human endothelial cells strain EA.hy926 cells, purchased from Chinese Academy of Sciences Shanghai
Cell bank.
Preferably surrounding for technical solution of the present invention is active and expansion：
The preparation method at present oligosaccharides position：
River ophiopogon 100g is weighed, 5 times of amount water immersion 12h is separately added into, smashes, 5 times of amount water, 25 DEG C of temperature extractions is added and takes 2
Secondary, each 1h, is filtered, merging filtrate, is freeze-dried after recycling design to small size up to Fibre Ophiopogon oligosaccharides.
Liriope muscari Baily 100g is weighed, 5 times of amount water immersion 12h is separately added into, smashes, adds 5 times of amount water, 25 DEG C of temperature leachings
Extraction 2 times, each 1h, is filtered, merging filtrate, is freeze-dried after recycling design to small size up to liriope muscari Baily oligosaccharides.
The preparation of glucosides of the present invention
Liriope muscari Baily fibrous root 100g is weighed, 10 times of amounts, 5 times of amount water immersion 12h is separately added into, smashes, then be separately added into
10 times of amounts, 5 times of amount water, 60 DEG C of temperature extractions take 2 times, each 1h, filter, merging filtrate, are freeze-dried after recycling design to small size
Up to liriope muscari Baily fibrous root glucosides.
River ophiopogon 100g is weighed, 10 times of amounts, 5 times of amount water immersion 12h is separately added into, smashes, then be separately added into 10 times of amounts, 5
60 DEG C of temperature extractions of amount water again take 2 times, each 1h, filter, merging filtrate, river wheat to obtain the final product is freeze-dried after recycling design to small size
Winter fibrous root glucosides.
The preparation of ophiopogon water extract
River ophiopogon 100g, 5 times of amount water immersion 4h, then add 5 times of amount water to decoct twice, each 1.5h, filtrate merges, and is concentrated into
2g/ml, it is spare.
In the present invention, ophiopogon water extract can substantially suppress the formation of mouse tail thrombus, its effect is suitable with aspirin.
Ophiopogon water extract can be with the clotting time of the extension mouse of conspicuousness, and anticoagulant effect is better than aspirin, so this result carries
Showing the anti-thrombosis activity of ophiopogon water extract may be realized by influencing blood coagulation system.
The preparation flow of ophiopogonin composition is as follows：
The invention discloses a kind of preparation method of the extract with platelet aggregation inhibitory activity, this method includes：Take
Ophiopogon medicinal material, adds about 5 times of amount water immersion 12h of ophiopogon raw material weight, smashes, next day adds 5 times of amount water to decoct 2h, 8 times of amounts again
1.5h twice.Extracting solution filters, and after standing cools, centrifugation, discards insoluble matter, supernatant, cross the absorption of D-101 resin columns, repeats
Loading once, stands 12h, is first negative with water elution to eluent molish reactions, then is eluted with 30% ethanol solution, washes
De- liquid recycling design, 30% ethanol position freeze, and obtain 30% ethanol position of ophiopogonin.
The invention discloses a kind of preparation method of the extract with platelet aggregation inhibitory activity, this method includes：Take
Ophiopogon medicinal material, adds about 5 times of amount water immersion 12h of ophiopogon raw material weight, smashes, next day adds 5 times of amount water to decoct 2h, 8 times of amounts again
1.5h twice.Extracting solution filters, and after standing cools, centrifugation, discards insoluble matter, supernatant, cross the absorption of D-101 resin columns, repeats
Loading once, stands 12h, is first negative with water elution to eluent molish reactions, then is eluted with 85% ethanol solution, washes
De- liquid recycling design, 85% ethanol position freeze, and obtain 85% ethanol position of ophiopogonin.
The invention discloses a kind of preparation method of the extract with platelet aggregation inhibitory activity, this method includes：Take
Ophiopogon medicinal material, adds about 5 times of amount water immersion 12h of ophiopogon raw material weight, smashes, next day adds 5 times of amount water to decoct 2h, 8 times of amounts again
1.5h twice.Extracting solution filters, and after standing cools, centrifugation, discards insoluble matter, supernatant, cross the absorption of D-101 resin columns, repeats
Loading once, stands 12h, is first negative with water elution to eluent molish reactions, then is eluted with 30% ethanol solution, most
Eluted afterwards with 85% ethanol solution, solvent is separately recovered in eluent, and 30% ethanol position and 85% ethanol position are mixed, and freezes
It is dry, obtain ophiopogonin composition.
Ophiopogonin composition no corresponding table
The yield of ophiopogonin composition
9 lyophilized samples such as ophiopogonin composition of preparation, and the weight of freeze-dried powder is recorded, sample is calculated respectively to be obtained
Rate, as a result following 1-9 samples freeze-dried powder yield
Ophiopogon glucosides is numbered：OJ-oc：River ophiopogon glucosides, LM-oc：Liriope muscari Baily glucosides
Ophiopogon water extract is numbered：River ophiopogon water extract OJ-ae
The ophiopogonin composition of the present invention is with 201310710650.0 the special of embodiment 1,2,5：
1. the preparation process of ophiopogonin composition of the present invention takes ophiopogon medicinal material, add about 5 times of amount water of ophiopogon raw material weight
Soak 12h........It is this to belong to the mode soaked with water temperature, mainly obtain oligosaccharides position.
2. the ophiopogonin composition of the present invention is the freeze-dried powder of the mixture at 30% ethanol position and 85% ethanol position.
The special-effect of the ophiopogonin composition of the present invention is special preparation process based on more than.It is specific point below
Analysis：
The preparation method at the extracting n-butyl alcohol position of ophiopogonin composition：
No. 4 samples are further purified, obtain No. 4 sample 100mg, using 6 extractions of water-saturated n-butanol, are collected respectively
N-butanol layer and water layer, recycle n-butanol, n-butanol layer, water layer are freeze-dried respectively.The weight of freeze-dried powder is recorded, and is calculated
Rate, as a result for：N-butanol layer 89.49%, water layer 7.04%.
ICR mouse 40, are randomly divided into control group, No. 4 sample sets (50mg/kg), extracting n-butyl alcohol position group
(44.75mg/kg) and water position group (3.52mg/kg).Experimental result is compared with blank group, extracting n-butyl alcohol position and No. 4 samples
Product significantly extend the clotting time of mouse, and as the increase of saponin content, anticoagulant active also further increase, so
Illustrate the content of total saposins has vital influence to the anticoagulant active of ophiopogon.Water position influences compared with blank group
Less, compared with extracting n-butyl alcohol position, there is the difference (P ＜ 0.05) of conspicuousness.
The preparation method of the hydrolysate of ophiopogonin composition：
Weigh No. 4 sample 1.11g, add 2% aqueous sulfuric acids of 130ml and be placed in boiling water bath and hydrolyzes 5h, take out, put to
After room temperature, with 16% sodium hydroxide solution tune pH to neutrality, filter, residue is dried to constant weight together with filter paper at 75 DEG C.Through stone
Oily ether surname extraction 5h, extracting solution recycling design, residue are suspended with water, are freeze-dried the hydrolysis up to ophiopogonin composition
Thing, it is 25.72% to calculate yield.
ICR mouse 40, are randomly divided into control group, the hydrolysate of ophiopogonin composition is high, neutralizes low dose group
(22mg/kg、11mg/kg、5.5mg/kg).Test result indicates that (convert during 11mg/kg from astragalin composition) to mouse
Though there is extension effect in the clotting time, the indifference compared with blank group.5.5mg/kg and 22mg/kg dosages can be extremely notable
Extension mouse clotting time, so, select low dosage administration.
ICR mouse, are randomly divided into the hydrolysate group (5.5mg/ of control group, Aspirin groups, ophiopogonin composition by 50
Kg), OJ-oc (5.31g/kg) and LM-oc (5.31g/kg) group.Test result indicates that：The hydrolysate portion of ophiopogonin composition
Position extremely can significantly extend clotting time of mice (P ＜ 0.01) in 5.5mg/kg dosages compared with blank group.Oligosaccharides group
Experimental result it is identical with prerun, under the dosage of 5.31g/kg, oligosaccharides position can shorten the clotting time of mouse,
OJ-oc and blank group relatively have a significant difference, and LM-oc indifferences.The oligosaccharides position of two separate sources compares, OJ-
The blood coagulation enhancing effect of oc is stronger compared with LM-oc.
The pharmacological testing of the present invention includes as follows：
The antithrombotic of ophiopogon water extract, the pharmacological testing for extending the clotting time
Ophiopogon water extract Carrageenan causes the influence of mouse tail thrombus
ICR male mices 50,18-22g, are randomly divided into 5 groups, i.e. blank control group, aspirin group (33.6mg/
Kg), the high, medium and low dosage group of river ophiopogon water extract (1g/kg, 2g/kg, 4g/kg).1% carrageenan is subcutaneously injected in mouse back
(50mg/kg), observes 24h, 36h, 48h, 72h mouse tail thrombosis situation.
After carrageenan stimulation 24h is subcutaneously injected, has all there is kermesinus thrombus area in model group afterbody front end, but is administered
Group only has small part to occur.After stimulating 36h, model group thrombus area is progressively longer, and thrombus area also occurs substantially in administration group.During 48h
Model group reaches maximum with administration group thrombus area, starts slowly to be hardened after 72h, is dried, some are started shedding off.Tied more than
Fruit understands that ophiopogon water extract can substantially suppress the formation of mouse tail thrombus with aspirin.
The influence of tail thrombus caused by ophiopogon water extract Carrageenan
Statistical analysis：Experimental result is represented with mean ± SD, using the conspicuousness of each group difference of t check analyses.
Influence of the ophiopogon water extract to clotting time of mice
ICR male mices 50,18-22g, are randomly divided into 5 groups, i.e. blank control group, aspirin group (33.6mg/
Kg), the high, medium and low dosage group of ophiopogon water extract (1g/kg, 2g/kg, 4g/kg).It is administered once a day, successive administration three days, end
Fasting (can't help water) 12h before secondary administration, 1h after administration in the 3rd day, the mouse intraocular corner of the eyes is inserted into the capillary glass tube of internal diameter 0.5mm
Ball rear vein beard, autoblood, which pours, starts timing in pipe, is taken out after filling, and fractures capillary about 0.5cm every 30s, and to the left
The right side is slowly pulled open, and whether the observation place of fractureing has blood clotting silk, and untill blood clotting silk occurs, the time undergone is the clotting time
(CT)。
After successive administration 3 days, ophiopogon water extract extremely can significantly extend mouse under 1g/kg, 2g/kg dosage
Clotting time, the clotting time of mouse can be obviously prolonged under 4g/kg dosages, but in, compared with low dosage
Compared with effect is weaker.
Influence of the ophiopogon water extract to clotting time of mice
Ophiopogon water extract can substantially suppress the formation of mouse tail thrombus, its effect is suitable with aspirin.Ophiopogon water extract
Can be with the clotting time of the extension mouse of conspicuousness, and anticoagulant effect is better than aspirin, this result prompting ophiopogon water extract
Anti-thrombosis activity may be realized by influencing blood coagulation system.
Ophiopogonin composition, the hydrolysate of ophiopogonin composition, ophiopogon glucosides, to rabbit extracorporeal platelet aggregation
Influence
Large ear rabbit, male, 2~2.5Kg, etherization, fixation of facing upward, arteria carotis intubation take blood, and blood is with 3.8%
1: 9 anti-freezing of sodium citrate, 1000r/min centrifugation 18min, prepare platelet rich plasma (platelet-rich plasma,
PRP), remainder then with 3000r/min centrifuge 15min, prepare platelet poor plasma (platelet-poor plasma,
PPP).Aggregation inducing agent is with adenosine diphosphate (ADP) (ADP) (5 μM/ml of final concentration).Various concentrations are often added in 275 μ l PRP of pipe
Added in medicine 15 μ l, control group PRP after 15 μ l, PRP of physiological saline are incubated 3min altogether with medicine and add 10 μ lADP, measured
Hematoblastic maximum aggregation rate in 6min, calculates inhibiting rate, formula is as follows according to formula：
Ophiopogonin composition, the hydrolysate of ophiopogonin composition, ophiopogon glucosides are to rabbit extracorporeal platelet aggregation rate
Influence
Ophiopogonin composition, the hydrolysate of ophiopogonin composition, ophiopogon glucosides are to rabbit extracorporeal platelet aggregation rate
Influence
Influence to clotting time of mice
Influence of the ophiopogonin composition to clotting time of mice
The selection of ophiopogonin composition dosage
Arbitrarily take the sample that No. 4 samples are groped as wheat ophiopogonin composition dosage.
ICR mouse 32, are randomly divided into control group, No. 4 high, medium and low dosage groups of sample (100,50,25mg/kg), by reality
Test the results show that ophiopogonin composition dosage is obviously prolonged the clotting time of mouse in 50mg/kg, 100mg/kg,
Relatively there is significant difference with blank group.In order to compare the anticoagulant active of separate sources ophiopogonin composition, we select
Dosage of the 50mg/kg as medicine efficacy screening.
The selection of ophiopogonin composition dosage
Note：* represents that P ＜ 0.01, * represent P ＜ 0.05
Influence of the ophiopogonin composition to clotting time of mice
Glass-tube method：ICR mouse, half male and half female, random packet.It is administered once a day, successive administration three days, before last dose
Fasting (can't help water) 12h, 1h after administration in the 3rd day, with vein after the capillary glass tube insertion mouse intraocular corner of the eyes ball of internal diameter 0.5mm
Capillary is taken out in clump, the timing since being begun to flow into pipe blood immediately after filling, interval 30s fractures capillary about from both ends
Whether 0.5cm, and slowly pulling open to the left and right, the observation place of fractureing have the appearance of blood clotting silk, and blood clotting silk appearance the undergone time is
Clotting time (CT).
Influence of the ophiopogonin composition to clotting time of mice
In order to compare the antithrombotic acitivity of separate sources ophiopogonin composition, we select single dose to carry out pharmacodynamics
Comparative studies.ICR mouse 70, are randomly divided into control group, 1,2,3,4, No. 5 sample sets (50mg/kg) and Aspirin groups
(33.6mg/kg).Test result indicates that：The ophiopogonin composition of different sources and medicinal part gives medicament 50mg/kg's
In the amount lower clotting time (P ＜ 0.01) that can significantly extend mouse, prompt ophiopogonin composition that there is anticoagulant active.Into one
Walk and discovery is compared to the anticoagulant active of each sample, the anticoagulating active of No. 2 and No. 4 samples is apparently higher than other each groups, with 1
Number, No. 3 samples compare, anticoagulant active has significant difference (P ＜ 0.05).No. 2, No. 4 sample anti-freezings compared with Aspirin
Blood effect is slightly weak, but there was no significant difference.
Influence of the ophiopogonin composition to clotting time of mice
Note：* represents that P ＜ 0.01, * represent P ＜ 0.05
Influence of the ophiopogonin composition extracting n-butyl alcohol position to clotting time of mice
No. 4 samples are further purified, obtain No. 4 sample 100mg, using 6 extractions of water-saturated n-butanol, are collected respectively
N-butanol layer and water layer, recycle n-butanol, n-butanol layer, water layer are freeze-dried respectively.The weight of freeze-dried powder is recorded, and is calculated
Rate, as a result for：N-butanol layer 89.49%, water layer 7.04%.
ICR mouse, half male and half female, random packet.It is administered once a day, successive administration three days, fasting is (no before last dose
Prohibit water) 12h, 1h after administration in the 3rd day, are inserted into mouse intraocular corner of the eyes ball rear vein beard, from blood with the capillary glass tube of internal diameter 0.5mm
Liquid, which is begun to flow into, starts timing in pipe, takes out capillary after filling immediately, and interval 30s wrecks disconnected capillary about 0.5cm from two, and
Slowly pull open to the left and right, whether the observation place of fractureing has the appearance of blood clotting silk, and blood clotting silk appearance the undergone time is the clotting time
(CT)。
Experimental result is as follows：
ICR mouse 40, are randomly divided into control group, No. 4 sample sets (50mg/kg), extracting n-butyl alcohol position group
(44.75mg/kg) and water position group (3.52mg/kg).Experimental result is as shown in table 2.2.3, compared with blank group, n-butanol extraction
Take position and No. 4 samples significantly to extend the clotting time of mouse, and with the increase of saponin content, anticoagulant active also into
One step increase, so the content of explanation total saposins has vital influence to the anticoagulant active of ophiopogon.Water position and sky
White group compares, and influences less, compared with extracting n-butyl alcohol position, there is the difference (P ＜ 0.05) of conspicuousness.
Ophiopogonin composition extracting n-butyl alcohol position and the comparison of ophiopogonin composition
Influence of the hydrolysate of ophiopogon glucosides and ophiopogonin composition to clotting time of mice
ICR mouse 40, half male and half female, random packet.It is administered once a day, successive administration three days, prohibits before last dose
(can't help water) 12h, 1h after administration in the 3rd day are eaten, mouse intraocular corner of the eyes ball rear vein beard is inserted into the capillary glass tube of internal diameter 0.5mm,
The timing since being begun to flow into pipe blood, takes out capillary immediately after filling, interval 30s wrecks disconnected capillary about from two
Whether 0.5cm, and slowly pulling open to the left and right, the observation place of fractureing have the appearance of blood clotting silk, and blood clotting silk appearance the undergone time is
Clotting time (CT).
The selection of ophiopogon glucosides dosage
In addition to steroid saponin, other most of materials are mainly polysaccharide for ophiopogon water extract part.Arbitrarily OJ-oc is taken to make
For the sample of glucosides dosage selection.ICR mouse 40, are randomly divided into river ophiopogon glucosides basic, normal, high 1 and high 2 dosage group
(0.4g/kg、1.2g/kg、2.4g/kg、5.31g/kg).Ophiopogon, glucosides was in 0.4-2.4g/kg it can be seen from experimental result
When, though extending the clotting time of mouse, compared with blank group, do not have significant difference (P ＞ 0.05), and when administration
When dosage is 5.31g/kg, the effect of ophiopogon glucosides reverses completely, extremely significantly shortens the clotting time of mouse.Since glucosides
Influence is had no on the clotting time of mouse, so the dosage of this experimental selection 5.31g/kg compares.
The selection of ophiopogon glucosides dosage
Note：* represents that P ＜ 0.01, * represent P ＜ 0.05
The selection of the hydrolysate dosage of ophiopogonin composition
ICR mouse 40, are randomly divided into control group, the hydrolysate of ophiopogonin composition is high, neutralizes low dose group 22mg/
kg、11mg/kg、5.5mg/kg).Mouse is coagulated test result indicates that (converting during 11mg/kg from ophiopogonin composition)
Though there is extension effect in the blood time, the indifference compared with blank group.5.5mg/kg and 22mg/kg dosages can be extremely significant
Extend the clotting time of mouse, so, select low dosage administration.
The selection of the hydrolysate dosage of ophiopogonin composition
Note：* represents that P ＜ 0.01, * represent P ＜ 0.05
Influence of the hydrolysate of ophiopogon glucosides and ophiopogonin composition to clotting time of mice
ICR mouse, are randomly divided into the hydrolysate group (5.5mg/ of control group, Aspirin groups, ophiopogonin composition by 50
Kg), OJ-oc (5.31g/kg) and LM-oc (5.31g/kg) group.Test result indicates that：The hydrolysate portion of ophiopogonin composition
Position extremely can significantly extend clotting time of mice (P ＜ 0.01), glucosides group in 5.5mg/kg dosages compared with blank group
Experimental result it is identical with prerun, under the dosage of 5.31g/kg, glucosides position can shorten the clotting time of mouse,
OJ-oc and blank group relatively have a significant difference, and LM-oc indifferences.The oligosaccharides position of two separate sources compares, OJ-
The blood coagulation enhancing effect of oc is stronger compared with LM-oc.
Influence of the hydrolysate of glucosides and ophiopogonin composition to clotting time of mice
Note：* represents that P ＜ 0.01, * represent P ＜ 0.05
Influence of the ophiopogon each position to anoxia/reoxygenation human endothelial cells
Cell：Human endothelial cells strain EA.hy926 cells, purchased from Chinese Academy of Sciences's Shanghai cell bank.
The protective effect of ECV304 cell hypoxia reoxygenation models
12800 nutrient solutions of DMEM：Culture medium dry powder (grams) Milli-Q water 300mL dissolves, magnetic agitation 30min,
Fully 1.5g NaHCO are added after dissolving3, 0.06g penicillin (100U/mL) and 0.10g streptomysins (100U/mL), add
Milli-Q water makes it fully dissolve, mix to 1000mL, magnetic agitation at least 4h.Then it is equipped with two layers through what is sterilized in advance
The Suction filtration device filtration sterilization of 0.22 μm of miillpore filter, and dispense into 5 vials through autoclaved 250mL, 4 DEG C are cold
Hide.In use, plus such as 10% Vicente hyclone, as 12800 culture mediums of 10%DMEM.
0.1M phosphate buffers (PBS)：8g NaCl, 2.08g Na are weighed respectively2HPO4, 0.2g KCl and 0.2g
KH2PO4Into 1000ml triangular flasks, 1000mL Milli-Q water is added, is allowed to be completely dissolved under magnetic agitation, it is micro- through 0.22 μm
After the membrane filtration of hole, packing.Again through high pressure steam sterilization 30min, 4 DEG C of refrigerations after room temperature are placed.
0.25% trypsin solution：250mg trypsase is weighed, is dissolved in the 0.1M PBS bufferings after 100mL sterilizings
In liquid, magnetic agitation is to being completely dissolved, through 0.22 μm of filtering with microporous membrane, 4 DEG C of refrigerations.
MTT storing liquids：5mg/ml MTT are prepared with PBS, after 0.22 μm of membrane filtration is degerming, 4 DEG C are kept in dark place.
MTT working solutions：Used after diluting MTT storing liquids according to 1: 10 with serum free medium.
The protective effect of medicine Human Umbilical Vein Endothelial Cells strain anoxia/reoxygenation
Endothelial cell is suspended with 12800 nutrient solutions of 10%DMEM, with 1 × 105The cell density of cell/mL is inoculated in 96
On well culture plate (100 μ L/ holes), 37 DEG C, 5%CO2Cultivate 24h.In the exponential phase of endothelial cell strain, different medicines are added
Thing, 37 DEG C, 5%CO212h is co-cultured with cell.Culture medium is changed into sugar-free culture-medium after 12h, 37 DEG C, 5%CO2, 1%O2Bar
Anoxic 4h in anoxic case under part, taking-up change the culture medium containing 0.2% serum, 37 DEG C, 5%CO into2Reoxygenation 2h, finally uses
Mtt assay measures cell viability.
This experiment carries out research discovery, No. 2 and No. 4 samples using human endothelial cells EA.hy926 cell lines Hypoxia-reoxygenation model
Product can reach more than 50% to the protective rate of EA.hy926 cell hypoxia reoxygenations.Prompt the antithrombotic work of No. 2 and No. 4 samples
Property is stronger.
Under the conditions of hypoxia-reoxygenation, human endothelial cells strain EA.hy926 cell viabilities are decreased obviously, and are added before anoxic treatment each
Sample solution, saponin(e, glucosides, ophiopogonin composition hydrolysate final concentration of 1 μ g/ml successively, each saponin(e and effect
12h, reoxygenation 12h, sample sets can significantly raise damaged cell vigor, calculate each sample to human endothelial cells strain EA.hy926 cells
The effect of the protection of hypoxia-reoxygenation, 2,4 and No. 5 protective effects of the sample to anoxia/reoxygenation cell are preferable, can reach
55.16%th, 51.18% and 48.36%.
Adrenaline adds cold stimulation rat to cause lung thrombus model
Modeling method：Reference literature method modeling, and improved.Wistar male rats 80, are randomly divided into 10 groups,
That is model control group, blank control group, No. 1 sample sets, No. 2 sample sets, No. 3 sample sets, No. 4 sample sets, No. 5 sample sets, OJ-
Oc groups, GY groups and Aspirin groups.Every group is pressed 70mg/kg, 70mg/kg, 70mg/kg, 70mg/kg, 70mg/kg, 36.8g/ respectively
The dosage gastric infusion of kg, 15.4mg/kg and 23.1mg/kg.Each group medicine is made into suspension solution, daily gavage with distilled water
It is administered once, successive administration 7d.Model control group, blank control group are then administered with distilled water by weight.In addition to blank control group,
Each group 1h after administration in the 6th day, is subcutaneously injected again after 1% adrenalin hydrochloride solution 0.08ml/100g, 4h is subcutaneously injected
1% adrenalin hydrochloride 0.08ml/100g, double injection are spaced two hours, and rat is placed in 0~5 DEG C of frozen water went swimming
5min, fasting after taking-up, free water.Physiological saline, but not ice bath is subcutaneously injected in blank group 0.1ml/100g.It is administered within 7th day
After 30min, heparin is used with 10% chloraldurate 0.4ml/100g intraperitoneal injection of anesthesia rats, abdominal aortic blood, 4ml blood
Anti-freezing, it is spare.Residual blood uses 3.8% sodium citrate, 1: 9 anti-freezing.
Detection method：2mL is taken out from the 4mL whole bloods added with heparin and adds LBY-N6B rotary blood viscosimeters, respectively
Measure undercut (10/s), in cut (40/s) and height cuts whole blood viscosity under (120/s) 3 shear rates, remaining anticoagulant heparin blood is adopted
Hematocrit value is measured with micro-capillary tubes method.Added with the blood of sodium citrate, under the conditions of 4 DEG C, 3000r/min centrifugations
10min, takes blood plasma, and PT, APTT and FIB are measured by kit requirement.Lung tissue segment is taken, is homogenized with electric homogenizer, is obtained
10% homogenate, measure protein content, NO contents and MPO vigor are required according to kit.
Protective effect to mouse lung thrombus
ICR mouse, male, 50,18~22g, is randomly divided into 5 groups, is respectively control group, aspirin group (93.4mg/
Kg), the high, medium and low dosage group (400,200,100mg/kg) of ophiopogonin composition, every group 10, continuous gavage administration 5
My god, 1h after the last administration, mouse tail vein injection 100 μ l collagens (22 μ g)-adrenaline (5 μ g) mixed liquor, observation
The survival number of mouse in 3min.
Adrenaline adds cold stimulation rat to cause lung thrombus model
Influence to hemorheology
From experimental result, the whole blood viscosity of model group illustrates mould apparently higher than blank group (P ＜ 0.01, P ＜ 0.05)
Type is successful.For administration group compared with model group, No. 4 samples have the blood viscosity of rat reduction effect, but and model group
Compare and no significant difference (P ＞ 0.05), the blood viscosity of other groups prompt it for blood viscosity base compared with model group height
Without influence on this.Modeling there is no hematocrit value influence with administration, not have difference more with blank group.
Influence of the ophiopogon each position to cold stimulation hemorheology of rat
Note：Compared with blank group：## represents that P ＜ 0.01, # represent P ＜ 0.05；Compared with model group：* represents P ＜
0.01, * represents P ＜ 0.05.
Influence to blood plasma PT, APTT, FIB
Test result indicates that：Compared with blank group, prothrombin time (PT) and the activated partial thromboplastin of model group
Time (APTT) extremely significantly shortens (P ＜ 0.01), and fibrinogen (FIB) content extremely significantly increases (P ＜ 0.01), explanation
Modelling success.For this index of PT, the hydrolysate group of ophiopogonin composition substantially without influencing (P ＞ 0.05), other
Each sample can significantly extend the PT times (P ＜ 0.01 or P ＜ 0.05)；For this index of APTT, (the P ＞ in addition to No. 4 samples
0.05), other each sample can significantly extend the APTT times (P ＜ 0.01 or P ＜ 0.05) of rat's blood stasis model；The equal energy of total saposins group
Significantly reduce the content (P ＜ 0.01 or P ＜ 0.05) of FIB, hydrolysate and the river ophiopogon glucosides position pair of ophiopogonin composition
This index does not influence (P ＞ 0.05).Next being compared each sample group, influence of No. 4 samples for PT is extremely normal
Significantly, compared with other each groups difference extremely significantly (P ＜ 0.01), the influence for FIB, except with addition to 1, No. 2 sample indifference
(P ＞ 0.05), has significant difference (P ＜ 0.05) compared with other groups, illustrates influence of No. 4 samples to coagulation indexes PT, FIB
It is very big.
But its on APTT substantially without influence.Influence of the ophiopogon each position to cold stimulation P of Rats T, APTT, FIB
Note：Compared with blank group：## represents that P ＜ 0.01, # represent P ＜ 0.05；Compared with model group：* represents P ＜
0.01, * represents P ＜ 0.05.
Influence to lung tissue NO, MPO
Lung tissue segment is taken, after weighing, 10% tissue homogenate is prepared into physiological saline, requires to carry out according to kit
Measure.
Experimental result is shown：Compared with blank group, model group NO contents significantly reduce, and MPO vigor significantly raises (P ＜
0.05), hints model is successful.For NO, each administration group can significantly reduce NO contents, with the more variant (P of model group
＜ 0.05.P ＜ 0.01)；For MPO, 1,2,3, No. 4 sample sets can significantly raise MPO vigor in lung tissue.
Influence of the ophiopogon each position to NO, MPO in cold stimulation lung tissue of rats
Note：Compared with blank group：## represents that P ＜ 0.01, # represent P ＜ 0.05；Compared with model group：* represents P ＜
0.01, * represents P ＜ 0.05.
Protective effect to mouse lung thrombus
Experimental result is shown：Ophiopogonin composition has certain guarantor to the lung thrombus mouse of collagen and adrenaline induction
Shield acts on, and high dose is that survival rate can reach 60%.
Protective effect of the liriope muscari Baily fibrous root astragalin composition to mouse lung thrombus
The ophiopogon anticoagulating active at each position studies brief summary
Ophiopogon water extract substantially suppresses the mouse tail blood of carrageenan induction in 1~4g/kg (crude drug amount) dosage range
Bolt is formed, and extends clotting time of mice, and this chapter investigates the two big main component antithrombotic acitivities at its water extract position.
Influence of the ophiopogon each position to normal clotting time of mice is primarily looked at.
Originally experimental studies have found that, dwarf lilyturf tuber total saponins 50mg/kg extremely can significantly extend the clotting time of mouse, and and root tuber
Compare, fibrous root acts on cruor time extending stronger.The hydrolysate 5.5mg/kg of ophiopogonin composition can also be extremely significant
Extend the clotting time of mouse, and glucosides has facilitation to blood coagulation.Prompting, the anticoagulant active position of ophiopogon for saponin(e with
The hydrolysate of ophiopogonin composition.Under 70mg/kg dosages, ophiopogonin can significantly extend rat's blood stasis model plasma A PTT
With the PT times, fibrinogen (FIB) content is reduced, glucosides also can significantly extend APTT the and PT times, but to FIB contents without shadow
Ring.So understanding, ophiopogonin influences blood coagulation system maximum.Prompting dwarf lilyturf tuber total saponins may regulate and control endogenous and external source at the same time
Property blood coagulation system and play anticoagulant active.It can relatively be drawn by data, the anticoagulation of liriope muscari Baily fibrous root total saposins
Active best, its anticoagulation is suitable with Aspirin.
The ophiopogon platelet aggregation inhibitory activity at each position is studied.
Blood platelet is the key component in normal coagulation mechanism in body, has adhesion, aggregation, release, thromboplastic work(
Energy.And aggregation capability is a hematoblastic important physiological property, refer to sticking between blood platelet and blood platelet, display activation
Blood platelet interact pockets of feature, be that blood platelet participates inHemostasisWithThrombusOne of an important factor for forming process.Two phosphorus
Adenosine monophosphate (ADP) is one of common derivant in current antiplatelet drug research.This experiment is after clotting assay, further
Study the platelet aggregation inhibitory activity at ophiopogon each position, it is found that 27 μ g/ml of dwarf lilyturf tuber total saponins can significantly inhibit the external blood of rabbit
The aggregation of platelet, it is suitable with positive drug Aspirin effects.During low dosage, the hydrolysate of glucosides and ophiopogonin composition is all not
Platelet aggregation inhibitory activity is shown, when dosage doubles, the blood coagulation activity of ophiopogonin composition is also to improve very
More times, the hydrolysate of glucosides and ophiopogonin composition also shows significant platelet aggregation inhibitory activity in high dose,
But effect is not as good as total saposins.In all saponin(es, the total saposins activity from fibrous root is better than root tuber, illustrates that saponin content is got over
Height, platelet aggregation inhibitory activity are stronger.Above research finds that ophiopogonin composition can be obviously prolonged the clotting time of mouse,
So prompting ophiopogonin composition may be by suppressing hematoblastic aggregation so as to play anticoagulant activity.Also into one
Walk and provide foundation for the antithrombotic acitivity of dwarf lilyturf tuber total saponins.
Influence of the ophiopogon each position to anoxia/reoxygenation human endothelial cells
Hypoxia-reoxygenation model is the common modeling mode of Cell culture invitro, is widely used for histoorgan ischemia/reperfusion
Note damage and ischemic preconditioning/adapt to the research on cellular and molecular level afterwards.For ischemic disease, recover as early as possible blood flow,
It is best treatment method to shorten Ischemia Time, but while recovery blood flow, the damage of histoorgan often further adds
Weight, here it is ischemia/reperfusion injury, Hypoxia-reoxygenation model is point for body ischemia/reperfusion and ischemic preconditioning/adapt to afterwards
Research in sub- level, important in inhibiting.This experiment using human endothelial cells EA.hy926 cell lines Hypoxia-reoxygenation model into
Row research finds that No. 2 can reach more than 50% with No. 4 samples to the protective rate of EA.hy926 cell hypoxia reoxygenations.Prompting 2
It is number stronger with the antithrombotic acitivity of No. 4 samples.
Influence of the ophiopogon each position to lung thrombus
Above research shows, ophiopogon water extract can significantly extend clotting time of mice with ophiopogonin composition, suppresses ADP
The platelet aggregation of induction, in order to further compare the antithrombotic of ophiopogon different parts, selects lung thrombus as research index.
This experiment by making its excited blood vessel α acceptor be dominant to rat skin lower injection high-dose epinephrine, and and beta receptor
With reference to vasorelaxation relative reduction, as a result make vessel retraction.Frozen water immersion simulation cold air invasion and attack are imposed again, and blood is coagulated because cold.
Both share, and can make hemorheology that glutinous, dense, solidifying, poly- general character be presented, the blood perfusion of microcirculation be influenced, so as to cause micro-
Dyshaemia, causes topically or systemically disease.This experimental result shows that No. 4 samples can not only reduce whole blood viscosity, suppresses red
Cell aggregation, and plasma F IB contents can be reduced.When total saposins constituents can significantly extend rat's blood stasis model plasma A PTT and PT
Between, lung tissue NO contents are reduced, improve MPO vigor.Prompt in dwarf lilyturf tuber total saponins No. 4 samples for main antithrombotic acitivity into
Point.
In order to further verify above-mentioned conclusion, the lung that collagen and adrenalin tail vein injection induces has been selected in this experiment again
Thrombus model, protective effect of No. 4 samples of verification to lung tissue.It was found that No. 4 samples can be reduced due to pulmonary embolism respiratory failure
Caused by the death rate.
In summary, it can be deduced that a conclusion, the antithrombotic acitivity of Rootlet Ophiopogonis are better than Tubers of Ophiopogon japonicus, contain with its saponin(e
Amount height has direct relation.Fibre Ophiopogon total saposins are compared with the effect of liriope muscari Baily fibrous root total saposins, liriope muscari Baily soap
The total saponin content of glycosides is with antithrombotic acitivity portion than more prominent.
Assay and HPLC-ELSD methods are carried out to main to the total saposins of short Roripa mountain wheat the present invention relates to a kind of colorimetric method
Component Short Mordant Mountain Ophiopogon japonicus C carries out quantitative analysis, and experimental result is that total saponin content is 71.1% and Short Mordant Mountain Ophiopogon japonicus C
Content be 5.49%.
The present invention relates to a kind of thin-layer chromatography Qualitive test of liriope muscari Baily fibrous root total saposins,
Take equal amount 1,2,3,4, No. 5 samples, the dissolving of 1ml methanol, SupelcleanTMLC-C18SPE solid phase extraction columns
Absorption, is eluted with water, each 8ml of 40%, 75%, 85% methanol solution respectively, is collected 75% elution position, is volatilized solvent, with less
Methanol dissolving is measured, it is spare.
In order to compare the difference between 5 kinds of sample saponin components, three development systems are selected to be compared.Thin-layer chromatography
Figure is shown in Fig. 1,2 and 3.
Development system is as follows：
(4) chloroform-methanol-water (17: 6: 2)
(5) acetone and ethyl acetate-water-acetic acid (5: 5: 1: 0.2)
(6) water-saturated n-butanol system
By the comparison of thin-layer chromatography, do not have substantially in the total saposins component species of identical place of production difference medicinal part
Change, but the content of component of the same race has certain difference.Different sources total saposins, no matter from species or congruent content
From the point of view of difference it is all larger.
1.1HPLC-ELSD chromatographies compare
In order to further compare the difference between different sources dwarf lilyturf tuber total saponins component, HPLC-ELSD is selected to carry out component
Qualitative determination.
Chromatographic column：Agilent ZORBAX SB-C18 (4.6 × 250mm, 5 μm)；
Chromatographic condition：Methanol-water gradient elution.Elution requirement see the table below.The result is shown in Fig. 4；
Total saposins HPLC-ELSD chromatographic conditions
The apparent component difference for legibly having shown each place of production ophiopogon of HPLC-ELSD collection of illustrative plates, each spectrogram have one altogether
There is peak, it is higher to cut content.No. 1 with No. 2 sample sources in river ophiopogon, No. 3, No. 4 sample sources in liriope muscari Baily, between any two
Do not change substantially in component species, but the sample of separate sources, gap is larger in species.No. 5 sample sources in Hubei ophiopogon,
Compared with the above two, difference is larger in component species.
The assay of 1.2 saponin(es
1.2.1 the preparation of standard items and test sample
The preparation of standard solution：Precision weighs the hydrolysate 3.40mg of ruscogenin, puts in 25ml volumetric flasks, first
Alcohol dissolves, and constant volume, shakes up up to the standard solution that concentration is 0.136mg/ml, spare.
The preparation of test solution：It is appropriate that precision weighs 1,2,3,4, No. 5 sample, puts in 10ml volumetric flasks, methanol dissolving
And constant volume, it is spare.
1.2.2 the selection of maximum absorption wavelength
Precision measures standard solution, No. 4 test solutions and each 0.5ml of methanol into 10ml tool plug test tubes, in water-bath
Volatilize solvent.Taking-up is put to room temperature, sequentially adds 5% vanillic aldehyde-glacial acetic acid solution 0.4mL, and perchloric acid 0.6ml, shakes up, put to
30min is reacted in 80 DEG C of water-baths, is taken out, ice-water bath 3min, then acetic acid 8ml on the rocks, concussion is uniform, is cooled to room temperature, in 400
It is scanned at~760nm wavelength, chooses maximum absorption wavelength 533nm as measure wavelength.The result is shown in Fig. 5.
1.2.3 the preparation of standard curve
It is accurate respectively to measure 0.136mg/ml standard solutions 0.3,0.4,0.5,0.6,0.7,0.8,1.0ml to 10ml tools
Fill in test tube, volatilize solvent.Put to room temperature, develop the color according to 2.4.2 lower methods, extinction is measured under 533nm wavelength
Degree, and draw standard curve.Acquired results are shown in Fig. 6.
Calibration curve equation measured by experimental result is：Y=5.726X-0.110, r=0.9998 (n=3), wherein Y
Reference axis is absorbance (A), and X-coordinate axle is concentration (mg/ml).The range of linearity is the μ g/ml of 40.8 μ g/ml~136.0.Standard is bent
Line is shown in Fig. 6.
Sample measures
Precision measures each test solution 1ml into 10ml tool plug test tubes, volatilizes solvent, puts to room temperature, according to
2.4.2 method develops the color under item, and absorbance is measured under 533nm wavelength, and calculates each sample saponin content.Acquired results are such as
Following table：
The ophiopogon medicinal material of separate sources, prepares two batches according to the method under 2.1.1 respectively.From experimental result, no
It is smaller with total saponin content difference between preparing batch, illustrate that the preparation method under 2.1.1 is stable, reliable.The total soap of every batch of sample
Glycosides content can reach more than 50%.
Total saponin content in each dwarf lilyturf tuber total saponins freeze-dried powders of table 2.1.3
2.4 determination of polysaccharide
2.5.1 the preparation of standard items and test sample
The preparation of standard items：Precision weighs the 80 DEG C of fructose to dry to constant weight 8.30mg, puts in 100ml volumetric flasks, uses methanol
Dissolving, and constant volume, obtain 0.083mg/ml standard solution, shake up, spare.
The preparation of test sample：It is appropriate that precision weighs 1,2,3,4, No. 5 sample, puts into 10ml volumetric flasks, and methanol dissolves simultaneously
Constant volume, shakes up, spare.
2.5.2 the selection of maximum absorption wavelength
Precision measures standard solution 7ml, with methanol dilution and is settled to 10ml.Take test solution 2.5ml to 10ml
In volumetric flask, with methanol dilution and constant volume.It is accurate respectively to measure methanol, solution after standard items dilution, solution after test sample dilution
2ml is each to add 5% phenol solution 1ml into tool plug test tube, then adds the 5ml concentrated sulfuric acids, 40 DEG C of water-baths along tube wall rapidly
15min, ice-water bath 3min terminate reaction, put to room temperature, are scanned at 400~760nm of wavelength, select maximum absorption wave a length of
490nm.The result is shown in Fig. 7.
2.5.3 the preparation of standard curve
Precision measures fructose standard solution 1,2,3,5,7ml into 10ml volumetric flasks, and methanol dilution and constant volume, are made
A series of standard of concentration of 0.0083mg/ml, 0.0166mg/ml, 0.0249mg/ml, 0.0415mg/ml, 0.0581mg/ml
Product solution.The accurate 2ml that measures into tool plug test tube, develops the color according to the method under 2.5.2 from each standard solution respectively, with
Retinue blank solution is reference solution, and absorbance is measured under wavelength 490nm.With concentration (mg/ml) for abscissa, absorbance
(A) standard curve is drawn for ordinate, standard curve is：Y=13.38x-0.002, r=0.9998 (n=3), fructose is in 8.3 μ
It is linear good in the range of the μ g/ml of g/ml~58.1.The result is shown in Fig. 8.
2.5.4 sample measures
Precision measures 1,2,3,4, No. 5 test solution 2.5ml into 10ml volumetric flasks, methanol dilution and constant volume.It is accurate
Test solution develops the color, with molten with line blank into 10ml tool plug test tubes according to the method under 2.5.2 after measuring 2ml dilutions
Liquid is reference solution, and absorbance is measured under wavelength 490nm, the polyoses content of each sample is calculated according to standard curve, as a result
It is shown in Table.
Polyoses content in each dwarf lilyturf tuber total saponins freeze-dried powder
By the polyoses content in phend-sulphuric acid determination sample, No. 3 with No. 4 be respectively liriope muscari Baily root tuber with
Fibrous root total saposins, experimental data show that the polyoses content of fibrous root will be less than root tuber.
Brief description of the drawings
Fig. 1 chloroform-methanol system thin-layer chromatograms
Fig. 2 acetone and ethyl acetate system thin-layer chromatograms
Fig. 3 water-saturated n-butanol system thin-layer chromatograms
The comparison of Fig. 4 ophiopogonin composition HPLC-ELSD spectrograms
The selection of Fig. 5 ophiopogonin composition maximum absorption wavelengths
Fig. 6 ruscogenin standard curves
The selection of Fig. 7 polysaccharide maximum absorption wavelengths
Fig. 8 fructose standard curves
Specific embodiment
Embodiment 1
Liriope muscari Baily fibrous root 54.1g is weighed, 10 times of amounts, 5 times of amount water immersion 12h is separately added into, smashes, then be separately added into
10 times of amounts, 5 times of amount water, 60 DEG C of temperature extractions take 2 times, each 1h, filter, merging filtrate, are freeze-dried after recycling design to small size
Up to liriope muscari Baily fibrous root glucosides.
Embodiment 2
River ophiopogon 54.1g is weighed, 10 times of amounts, 5 times of amount water immersion 12h is separately added into, smashes, then be separately added into 10 times of amounts, 5
60 DEG C of temperature extractions of amount water again take 2 times, each 1h, filter, merging filtrate, river wheat to obtain the final product is freeze-dried after recycling design to small size
Winter fibrous root glucosides.
Embodiment 3
Liriope muscari Baily 100g, 5 times of amount water immersion 4h, then add 5 times of amount water to decoct twice, each 1.5h, filtrate merges, dense
2g/ml is reduced to, it is spare, obtain liriope muscari Baily water extract.
Embodiment 4
River ophiopogon 100g, 5 times of amount water immersion 4h, then add 5 times of amount water to decoct twice, each 1.5h, filtrate merges, and is concentrated into
2g/ml, it is spare, obtain river ophiopogon water extract.
Embodiment 5
100 grams of liriope muscari Baily is taken, adds about 5 times of amount water immersion 12h of liriope muscari Baily raw material weight, smashes, next day is again
5 times of amount water are added to decoct 2h, 8 times of amount 1.5h are twice.Extracting solution filters, and after standing cools, centrifugation, discards insoluble matter, supernatant, mistake
D-101 resin columns adsorb, and repeat loading once, stand 12h, are first negative with water elution to eluent molish reactions, then with
30% ethanol solution elution, is finally eluted, solvent is separately recovered in eluent, by 30% ethanol position with 85% ethanol solution
Mixed with 85% ethanol position, freeze, obtain liriope muscari Baily astragalin composition.
Embodiment 6
100 grams of liriope muscari Baily fibrous root is taken, adds about 5 times of amount water immersion 12h of liriope muscari Baily fibrous root raw material weight, beats
Broken, next day adds 5 times of amount water to decoct 2h again, and 8 times of amount 1.5h are twice.Extracting solution filters, and after standing cools, centrifugation, discards insoluble matter,
Supernatant, cross the absorption of D-101 resin columns, repeats loading once, stands 12h, is in water elution to eluent molish reactions first
Feminine gender, then eluted with 30% ethanol solution, finally eluted with 85% ethanol solution, solvent is separately recovered in eluent, will
30% ethanol position and the mixing of 85% ethanol position, freeze, obtain liriope muscari Baily fibrous root astragalin composition.
Embodiment 7
100 grams of river ophiopogon is taken, adds about 5 times of amount water immersion 12h of river ophiopogon raw material weight, smashes, next day adds 5 times of amount water again
2h is decocted, 8 times of amount 1.5h are twice.Extracting solution filters, and after standing cools, centrifugation, discards insoluble matter, supernatant, cross D-101 resins
Column adsorbs, and repeats loading once, stands 12h, is first negative with water elution to eluent molish reactions, then with 30% ethanol
Solution elutes, and is finally eluted with 85% ethanol solution, solvent is separately recovered in eluent, by 30% ethanol position and 85% ethanol
Position mixes, and freezes, obtains river ophiopogonin composition.
Embodiment 8
100 grams of Fibre Ophiopogon is taken, adds about 5 times of amount water immersion 12h of Fibre Ophiopogon raw material weight, smashes, next day is again
5 times of amount water are added to decoct 2h, 8 times of amount 1.5h are twice.Extracting solution filters, and after standing cools, centrifugation, discards insoluble matter, supernatant, mistake
D-101 resin columns adsorb, and repeat loading once, stand 12h, are first negative with water elution to eluent molish reactions, then with
30% ethanol solution elution, is finally eluted, solvent is separately recovered in eluent, by 30% ethanol position with 85% ethanol solution
Mixed with 85% ethanol position, freeze, obtain Fibre Ophiopogon astragalin composition.
Embodiment 9
100 grams of Hubei ophiopogon is taken, adds about 5 times of amount water immersion 12h of Hubei ophiopogon raw material weight, smashes, next day adds 5 times again
Measure water and decoct 2h, 8 times of amount 1.5h are twice.Extracting solution filters, and after standing cools, centrifugation, discards insoluble matter, supernatant, cross D-101
Resin column adsorbs, and repeats loading once, stands 12h, is first negative with water elution to eluent molish reactions, then with 30%
Ethanol solution elutes, and is finally eluted with 85% ethanol solution, solvent is separately recovered in eluent, by 30% ethanol position and 85%
Ethanol position mixes, and freezes, obtains Hubei astragalin composition.
Embodiment 9
Liriope muscari Baily fibrous root astragalin composition 100mg, using 6 extractions of water-saturated n-butanol, collects n-butanol respectively
Layer and water layer, recycle n-butanol, n-butanol layer, water layer are freeze-dried respectively.The weight of freeze-dried powder is recorded, and calculates yield, is tied
Fruit is：N-butanol layer 89.49%, water layer 7.04%.
Embodiment 10
Liriope muscari Baily fibrous root saponin(e astragalin composition 1.11g, adds 2% aqueous sulfuric acids of 130ml and is placed in boiling water bath
5h is hydrolyzed, takes out, puts to room temperature, with 16% sodium hydroxide solution tune pH to neutrality, is filtered, by residue 75 DEG C together with filter paper
Under dry to constant weight.It is suspended, is freeze-dried up to ophiopogon soap with water through petroleum ether surname extraction 5h, extracting solution recycling design, residue
The hydrolysate of glycoside composition, it is 24.36% to calculate yield.
Claims (1)
1. a kind of liriope muscari Baily astragalin composition of platelet aggregation-against analyzes and identifies method, it is characterised in that：
The preparation method of liriope muscari Baily astragalin composition is as follows：
Liriope muscari Baily is taken, soaks 6-18h, is smashed, adds water and decocts 0.5-6h, twice, extracting solution filtering, standing cools
Afterwards, centrifuge, discard insoluble matter, supernatant, cross the absorption of D-101 resin columns, repeats loading once, 6-18h is stood, first with water elution
It is negative to eluent molish reactions, then is eluted with 30% ethanol solution, is finally eluted with 85% ethanol solution, elution
Solvent is separately recovered in liquid, and 30% ethanol position and 85% ethanol position are mixed, and freezes, obtains liriope muscari Baily astragalin composition；
The thin-layer chromatography Qualitive test of liriope muscari Baily astragalin composition
Take liriope muscari Baily astragalin composition, the dissolving of 1ml methanol, the absorption of LC-C18SPE solid phase extraction columns, respectively with water,
40%th, each 8ml elutions of 75%, 85% methanol solution, collect 75% elution position, volatilize solvent, dissolved with a small amount of methanol, standby
With；
Development system is as follows：
Acetone and ethyl acetate-water-acetic acid, 5: 5: 1: 0.2；
The HPLC-ELSD chromatographies of liriope muscari Baily astragalin composition,
Component qualitative determination is carried out with HPLC-ELSD；
Chromatographic column：Agilent ZORBAX SB-C18,4.6 × 250mm, 5 μm；
Chromatographic condition：Methanol-water gradient elution, elution requirement；
Total saposins HPLC-ELSD chromatographic conditions
Liriope muscari Baily Methods in Determination of Polysaccaride Content,
The preparation of standard items and test sample
The preparation of standard items：Precision weighs the 80 DEG C of fructose to dry to constant weight 8.30mg, puts in 100ml volumetric flasks, is dissolved with methanol,
And constant volume, 0.083mg/ml standard solution is obtained, is shaken up, it is spare；
The preparation of test sample：It is appropriate that precision weighs liriope muscari Baily astragalin composition sample, puts into 10ml volumetric flasks, methanol is molten
Simultaneously constant volume is solved, is shaken up, it is spare；
The selection of maximum absorption wavelength
Precision measures standard solution 7ml, with methanol dilution and is settled to 10ml；Test solution 2.5ml is taken to 10ml capacity
In bottle, with methanol dilution and constant volume；Accurate respectively to measure methanol, solution after standard items dilution, solution 2ml is extremely after test sample dilution
It is each to add 5% phenol solution 1ml in tool plug test tube, then rapidly the 5ml concentrated sulfuric acids are added along tube wall, 40 DEG C of water-bath 15min,
Ice-water bath 3min terminates reaction, puts to room temperature, is scanned at 400~760nm of wavelength, selects a length of 490nm of maximum absorption wave；
The preparation of standard curve
Precision measures fructose standard solution 1,2,3,5,7ml into 10ml volumetric flasks, and methanol dilution and constant volume, are made
A series of standard of concentration of 0.0083mg/ml, 0.0166mg/ml, 0.0249mg/ml, 0.0415mg/ml, 0.0581mg/ml
Product solution, the accurate 2ml that measures is into tool plug test tube from each standard solution respectively, according to the above-mentioned " choosing of maximum absorption wavelength
Select " method colour developing under item, using blank solution of accompanying as reference solution, absorbance is measured under wavelength 490nm, with concentration mg/
Ml is abscissa, and absorbance A draws standard curve for ordinate, and standard curve is：Y=13.38x-0.002, r=0.9998, n
=3, fructose is linear good in the range of the μ g/ml of 8.3 μ g/ml~58.1；
Sample measures
Precision measures liriope muscari Baily astragalin composition test solution 2.5ml into 10ml volumetric flasks, methanol dilution and constant volume；
Test solution is into 10ml tool plug test tubes after precision measures 2ml dilutions, colour developing, using blank solution of accompanying as reference solution,
Absorbance is measured under wavelength 490nm, the polyoses content of each sample is calculated according to standard curve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510140415.3A CN104689058B (en) | 2015-03-25 | 2015-03-25 | A kind of liriope muscari Baily astragalin composition of platelet aggregation-against |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510140415.3A CN104689058B (en) | 2015-03-25 | 2015-03-25 | A kind of liriope muscari Baily astragalin composition of platelet aggregation-against |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104689058A CN104689058A (en) | 2015-06-10 |
| CN104689058B true CN104689058B (en) | 2018-05-08 |
Family
ID=53336882
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510140415.3A Active CN104689058B (en) | 2015-03-25 | 2015-03-25 | A kind of liriope muscari Baily astragalin composition of platelet aggregation-against |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104689058B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1596900A (en) * | 2004-07-23 | 2005-03-23 | 中国药科大学 | Rusco saponin element and application of its saponin in preparation of medicine preventing and treating embolus disease |
| CN101810769A (en) * | 2010-04-01 | 2010-08-25 | 哈尔滨泰华药业股份有限公司 | Method for preparing dwarf lilyturf tuber total saponins |
| CN103638295A (en) * | 2013-12-23 | 2014-03-19 | 中国药科大学 | Extractive with anti-platelet aggregation activity and preparation method thereof |
-
2015
- 2015-03-25 CN CN201510140415.3A patent/CN104689058B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1596900A (en) * | 2004-07-23 | 2005-03-23 | 中国药科大学 | Rusco saponin element and application of its saponin in preparation of medicine preventing and treating embolus disease |
| CN101810769A (en) * | 2010-04-01 | 2010-08-25 | 哈尔滨泰华药业股份有限公司 | Method for preparing dwarf lilyturf tuber total saponins |
| CN103638295A (en) * | 2013-12-23 | 2014-03-19 | 中国药科大学 | Extractive with anti-platelet aggregation activity and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104689058A (en) | 2015-06-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101433553B (en) | Method for preparing deproteinized extract of calf blood and freeze-dried powder thereof | |
| CN107898782A (en) | A kind of ginkgo diterpenoid-lactone composition | |
| CN105031206B (en) | A kind of river ophiopogonin composition of platelet aggregation-against | |
| CN104689057B (en) | A kind of Hubei ophiopogonin composition of platelet aggregation-against | |
| CN104873746B (en) | A kind of ophiopogonin composition of antithrombotic | |
| CN1977885B (en) | Antihepatitis medicinal composition | |
| CN104689058B (en) | A kind of liriope muscari Baily astragalin composition of platelet aggregation-against | |
| CN100496527C (en) | Preparation method and application of injection containing Erigeron breviscapus | |
| CN104721483B (en) | A kind of liriope muscari Baily fibrous root astragalin composition of platelet aggregation-against | |
| CN101152233B (en) | Pharmaceutical composition of snakegourd fruit and folium ginkgo | |
| CN103405501B (en) | Preparation method of three-component blood-activating and stasis-dissolving capsules | |
| CN103393708B (en) | Ginsenoside-ophiopogonin D composition for treating cardiovascular and cerebrovascular diseases | |
| CN100563647C (en) | The Pharmaceutical composition of Herba Erigerontis and sodium tanshinon IIA silate injection | |
| CN101049355B (en) | Composition of medication prepared from safflower and leaves of hawthorn | |
| WO2019113861A1 (en) | Ginkgo biloba extract medicinal raw material and preparation method therefor | |
| CN101152285A (en) | Pharmaceutical composition of snakegourd fruit and whitethorn leaf | |
| CN1931216B (en) | Medicine composition of safflower and rhodiola root | |
| CN111514212A (en) | Preparation and application of daphne giraldii nitsche anti-liver cancer active total flavone | |
| CN1899352B (en) | Chinese medicine effective part composition for supplementing qi and recovering pulse | |
| CN1977886A (en) | Medicinal composition of oxymatrine, ganoderma lucidum and astragalus | |
| CN101564423A (en) | Medicament for treating cardiovascular and cerebrovascular diseases and preparation method thereof | |
| CN1931214B (en) | Medicine composition of rhodiola root and puerarin | |
| CN115990178B (en) | Application of compound in preparation of anticoagulant drugs | |
| CN101085226B (en) | 'Mailuoning' powder injection and its preparation method | |
| CN103417565A (en) | Application of depolymerized holothurian glycosaminolycan in preparing drugs for curing and preventing thrombotic diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |