CN105920096A - Composition with salviae miltiorrhiza and safflower as well as preparation method and application thereof - Google Patents
Composition with salviae miltiorrhiza and safflower as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a composition with salviae miltiorrhiza and safflower. The weight ratio of salviae miltiorrhiza to safflower is (5 to 1) to (5 to 2). Through intensive research on a large number of ancient and modern prescriptions and traditional Chinese medicine application data, active components in salviae miltiorrhiza and safflower are classified according to chemical structure types on the basis of salviae miltiorrhiza and safflower to the existing research; the HPLC-PDA technique is adopted; a chemical substance library is built; a chemical fuzzy recognition strategy is introduced; separated chromatographic peaks of a chromatogram are used for classifying and identifying components and structures according to a characteristic ultraviolet absorption spectrum; dynamic variation rules of different types of chemical components under different preparation methods and different weight ratios are researched by designing symmetry ratios; a weight ratio medicine pair of salviae miltiorrhiza and safflower with optimal weight ratio is screened; the experiment result shows that the preferably-prepared composition with salviae miltiorrhiza and safflower has a good function of resisting cardiovascular and cerebrovascular diseases.
Description
Technical field
The present invention relates to a kind of Chinese medicine composition, be specifically related to the red sage root and the composition of safflower and extracting method thereof and the new application in preparing resisting thrombotic diseases medicine.
Background technology
" medicine to " is the compatibility form that tcm clinical practice drugs to form a prescription is conventional, is the distillation of ancient Chinese medicine doctor accumulation clinical application experience.Medicine, to being to connect the bridge between single medicinal material and some prescriptions, is elite and the core place of the compatibility of medicines in a prescription, and possessed prescription cures mainly function substantially, embodies the overall curative effect of prescription.The red sage root-safflower is famous blood-activating drug pair, and the red sage root leads to blood network, Disperse hepatic depression；Safflower stimulates the menstrual flow network, Removing Blood Stasis.Two medicines all enter the heart, Liver Channel, and two medicines share plays merit promoting blood circulation and removing blood stasis, to promote blood circulation thin network, calming heart and tranquilizing mind altogether.Extract after the red sage root and safflower compatibility is widely used in treating cardiovascular and cerebrovascular disease clinically, has been developed that into more than 20 kind of Chinese patent drug, and wherein the red sage root-Sofflower injection annual sales amount has surpassed 4,000,000,000.
Chemical composition rule is studied and is provided scientific basis for the reasonability disclosing Compatibility Law of TCM Prescriptions and clinical practice thereof by medicine.The red sage root is the dry root and rhizome of Lamiaceae Salvia platymiscium red sage root Salvia miltrorrhiza Bge., and its active ingredient has two classes, the danshinolic acid constituents with tanshin polyphenolic acid B as representative and the tanshinone component with tanshinone IIA as representative.Safflower is the cylinder tubular corolla of feverfew safflower Carthamus tinctorius L., and its active component is mainly the chalcones composition with hydroxyl radical carthamin yellow carthamus A as representative and flavones ingredient.At present, the research of the red sage root-safflower compatibility is essentially consisted in danshinolic acid constituents changes of contents after detection compatibility, and relevant pharmacology and the research of clinical practice, to Change of Chemical Components research shorter mention comprehensive after its compatibility.It is the primary link of bioactive components research to the qualitative and quantitative analysis of chemical composition before and after compatibility to medicine all the time.
Therefore, the red sage root-safflower compatibility chemical composition repercussion study is the important prerequisite disclosing its compatibility mechanism.But, due to complexity and the compatibility application of kinds of traditional Chinese medicines of Chinese medicine material, accurately become a heavy task with complete Identification chinese herbs medicine chemical substance.Although existing scholar is by consulting literatures data and reference substance comparison, identify the chemical composition of some Chinese medicine and compound thereof.The scarcity of reference substance and the indefinability of traditional Chinese medicine ingredients structure annoying researcher the most always.Recently, LC-MS identifies more chemical composition by analysis strategies such as the difference of collision energy, characteristic ion filtration and energy neutral loss, but standard items the most in short supply, testing cost is expensive, these methods all can't be extensively and accurately for the complicated ingredient of Identification chinese herbs medicine.
Summary of the invention
Goal of the invention: the technical problem to be solved is to overcome the deficiencies in the prior art, on the basis of motherland's traditional Chinese medicine theory, at the red sage root and safflower medicine on Research foundation, the red sage root is classified according to chemical structural type with the active component in safflower, use HPLC-PDA technology, by setting up chemical substance storehouse, and introduce chemical fog recognition strategy, the chromatographic peak that chromatogram separates is carried out constituent structure taxonomic identification according to feature ultraviolet absorption spectrum.The present invention is by symmetrical proportion design, study under its different preparation method, different ratio, the dynamic rule of dissimilar chemical composition, for exploring the scientific meaning offer scientific basis of the red sage root and the application of safflower optimal clinical compatibility, also the effective substance for drug matching is studied and is provided new thinking and method.
Technical scheme: in order to realize object above, the technical scheme that the present invention takes is:
A kind of Chinese medicine composition, it is made up of the red sage root and safflower.
Preferably, above-described Chinese medicine composition, the weight ratio of the red sage root and safflower is 5:1～5:2.
As more preferably scheme, above-described Chinese medicine composition, the weight ratio of the red sage root and safflower is 5:2.
Preferably, the extracting method of above-described Chinese medicine composition, the red sage root and safflower composition are added water, methyl alcohol or volumetric concentration are that 50% methanol aqueous solution extracts.
Preferably, the extracting method of above-described Chinese medicine composition, take the red sage root and safflower composition, add volumetric concentration 50% methanol aqueous solution, refluxing extraction 1～3 times, each 1～2h, merge extract, concentrate, both.
The extracting method of Chinese medicine composition of the present invention, the extract of the described red sage root and safflower composition be mainly composed of danshinolic acid class, tanshinone, quinoid chalcones or flavone compound.
The Chinese medicine composition of the present invention application in preparing medicament against cardiovascular disease.
Beneficial effect: the red sage root and safflower Chinese medicine composition that the present invention provides have the advantage that
The present invention is using the red sage root-safflower as research object, the red sage root-safflower proportioning composition by great many of experiments screening Different Weight proportioning, chemically based material storehouse, chemical fog recognition strategy is used to carry out textural classification ownership chemical composition in sample, filter out the red sage root and the safflower composition of optimal weight proportion, thus the reasonability for tcm clinical practice medication provides reference frame with scientific.
Test result indicate that, when to use the weight ratio of the red sage root that preferably obtains of the present invention and safflower be 5:1～5:2, the composition antithrombotic efficacy of the red sage root and safflower is substantially better than the composition of other weight ratio, achieves extraordinary unforeseeable technique effect.
Accompanying drawing explanation
Fig. 1 is the chromatogram of each reference substance.
Fig. 2 is the red sage root and safflower compatibility Aqueous extracts (A), methanol-water extract (B), the chromatogram of methyl alcohol extract (C).
Fig. 3 is the relative stripping quantity of four compounds in the red sage root-safflower under different preparation method, different ratio-compare change curve.
Detailed description of the invention
According to following embodiment, the present invention be may be better understood.But, as it will be easily appreciated by one skilled in the art that concrete material proportion, process conditions and result thereof described by embodiment are merely to illustrate the present invention, and should be also without limitation on the present invention described in detail in claims.
Embodiment 1
1, instrument and reagent
U.S. Waters 2695 highly effective liquid phase chromatographic system, Waters 2998PDA detector, Empower data management system,16 desk centrifuges (Beckman Coulter company of the U.S.), the omnipotent centrifuge of FW80 type high speed (Tianjin Stettlen Instrument Ltd.), YRE-301 Rotary Evaporators (Yuhua Instrument Co., Ltd., Gongyi City), BT125 electronic balance (Germany Sai Duolisi scientific instrument Co., Ltd), EPED ultrapure water system (Nanjing Yi Pudayi development in science and technology Co., Ltd).
Acetonitrile (Scharlab company of Spain) is chromatographically pure reagent, and methyl alcohol (Nanjing Chemistry Reagent Co., Ltd.), that formic acid (Germany Merck company) is analysis is pure, and ultra-pure water (is made by oneself).Reference substance: tanshin polyphenolic acid B (DFB, lot number 111562), tanshinone IIA (DST, lot number 110766) are purchased from Nat'l Pharmaceutical & Biological Products Control Institute；Hydroxyl radical carthamin yellow carthamus A (HSYA, lot number 140827) is purchased from Sichuan Wei Keqi；6-hydroxyl Kaempferol, 6,7-tri--O-β-D-Glucose glycosides (HTG, laboratory is made by oneself), four compounds are through normalized, and purity is all more than 98%.The structure of four reference substances is shown in Fig. 1.The red sage root is the dry root and rhizome of labiate red sage root Salvia miltrorrhizaBge., originates from Shandong District (lot number 141201)；Safflower is the cylinder tubular corolla of feverfew safflower Carthamus tinctorius L., originates from Xinjiang region (lot number 121220)
2 experimental techniques and result
The preparation of 2.1 reference substance solution
It is accurate respectively that to weigh the reference substance being dried to constant weight appropriate, add 80% methyl alcohol to make concentration and be respectively tanshin polyphenolic acid B (172.20 μ g/mL), tanshinone IIA (42.75 μ g/mL), hydroxyl radical carthamin yellow carthamus A (102.52 μ g/mL), 6-hydroxyl Kaempferol, the mixing reference substance storing solution of 6,7-tri--O-β-D-Glucose glycosides (85.51 μ g/mL), in this, as No. 1 mixing reference substance solution.Accurate draw No. 1 mixing reference substance solution 1mL and put in 2mL measuring bottle, add 80% methyl alcohol to scale, shake up and to obtain No. 2 mixing reference substance solution, use stepwise dilution legal system to obtain 3～No. 8 mixing reference substance solution with method.Each reference substance solution is centrifuged 10min through 12000r/min before sample introduction.All of reference substance solution is all stored under the conditions of 4 DEG C.
The preparation of 2.2 need testing solutions
Precision weighs the red sage root-flos carthami (1:0,5:1,5:2,2:1,1:1,1:2,2:5,1:5,0:1, g/g) dried powder (the crossing 40 mesh sieves) 1.0g of Different Weight ratio.To different ratio mixing medicinal material, the red sage root-safflower medicine is carried out hot recirculation water extraction respectively, heat backflow 50% methyl alcohol extracts and the extraction of hot methanol at reflux.Add 30mL Extraction solvent, refluxing extraction 1h, let cool, supply weight.Precision takes supernatant 20mL in round-bottomed flask, is concentrated into 10mL.Take concentrate, be centrifuged 10min through 12000r/min, take the supernatant after being centrifuged as need testing solution.The parallel preparation of each need testing solution 6 parts, obtains each test liquid A series.Owing in the red sage root-safflower, same class component content difference is very big, consider based on linear, therefore by each test liquid A serial dilution 10 times, obtain each test liquid B series, to ensure that it is in the range of linear.
2.3 chromatographic condition
Use Apollo C18Chromatographic column (250mm × 4.6mm, 5 μm)；Flowing is acetonitrile (A)-0.5% aqueous formic acid (B) mutually；Flow velocity is 1mL/min；Column temperature is 35 DEG C；The detection wavelength of danshinolic acid class, tanshinone, chalcones and flavones ingredient is followed successively by 280,270,350,403nm；Condition of gradient elution is: 0～15min, 2%～10%A；15～20 min, 10%～17%A；20～52min, 17%～28%A；52～58min, 28%～30%A；58～63min, 30%～40%A；63～70min, 40%～90%A；70～78min, 90%A；78～80min, 90%～2%A.Sample size is 10 μ L.
The selection at 2.4 peaks processes with data
In this experiment, have the danshinolic acid class of certain response intensity, tanshinone, quinoid chalcones, the peak of flavones ingredient are selected as detecting object for detecting and analysis of compounds interphase interaction.In the red sage root, the peak area at the peak of detected compound should be greater than 33000；In safflower, the response intensity at the peak of detected compound should be greater than 26000.
2.5 chemical fog recognition strategies
Chemical fog recognition strategy of the present invention includes three steps.First, the quinoid chalcones set up in the red sage root in danshinolic acid class, tanshinone and safflower according to pertinent literature, the chemical substance storehouse of flavonoids.This chemical substance storehouse inclusion compound title, structural formula, molecular formula, molecular weight and UV information.In this experiment, the chemical substance storehouse of four class materials in the red sage root and safflower is established respectively.Secondly, select the red sage root, safflower simple and geometric ratio compatibility herbal extract sample introduction, in full wavelength scanner collection of illustrative plates, find reference substance peak according to UV information corresponding under retention time.The selection of reference substance is according to its corresponding content in medicinal material, selects the known compound that in medicinal material, in dissimilar compound, content is higher as reference substance.Owing to the ultraviolet spectrum characteristic of same class material is similar, the classification for other compound is provided foundation by the UV information of these reference substances.3rd, according to the red sage root set up, safflower chemical libraries, according to retention time in full wavelength scanner collection of illustrative plates, the UV information of compound is compared with to the UV information of reference substance, the basic parent nucleus of compound rather than definite compound structure can be determined, and it being classified as inhomogeneity compound, each class all has identical parent nucleus.
2.6 quantitative analysis
In the red sage root, safflower, the quantitative analysis of four compounds selects HPLC-PDA method for combined use, uses UV-quantitative analysis.Doing calibration curve with respective type compound control product, the quantitative of same type compound all carries out quantitative analysis with the calibration curve of same standard items.
2.7 methodological study
2.7.1 linear relationship, detection limit (LOD) are investigated with quantitative limit (LOQ)
Preparation DFB, DST, HSYA, HTG concentration is respectively the hybrid standard product test solution of 172.24,42.75,102.52,85.51 μ g/mL, and stepwise dilution is 2,4,8,16,32,64,128 times, is configured to serial standards solution.Precision draws each hybrid standard product test solution 10 μ L injection HPLC respectively, measures respective peaks area by chromatographic condition under " 2.3 " item.With peak area as ordinate (Y), with constituent concentration to be measured as abscissa (X), carry out linear regression, obtain the calibration curve of four standard items, the chromatogram of four standard items is shown in that (in figure, No. 1 peak is 6-hydroxyl Kaempferol to Fig. 1,6,7-tri--O-β-D-Glucose glycosides；No. 2 peaks are hydroxyl radical carthamin yellow carthamus A；No. 3 peaks are tanshin polyphenolic acid B；No. 4 peaks are tanshinone IIA).LOD and LOQ value measures when signal is 3 and 10 with noise values respectively, the results are shown in Table 1.
2.7.2 precision test
Take No. 1 mixing reference substance solution, by chromatographic condition detection under " 2.3 " item, in one day, repeat sample introduction 6 times and in continuous 3 days, repeat sample introduction 3 times, each sample introduction 10 μ L, measuring the peak area of each reference substance, with each calculated by peak area in a few days and day to day precision.The results are shown in Table 2.
2.7.3 repeated experiment
Take the red sage root-safflower (1:1) sample and prepare the need testing solution of 50% methanol-water according to 6 parts of test samples of parallel preparation under " 2.2 " restraining Preparation Method, sample introduction 10 μ L respectively, detect under identical chromatographic condition, calculate its repeatability with the content of reference substance composition each in sample.The results are shown in Table 2.
2.7.4 stability test
The water taking the red sage root-safflower 1:1 carries, and 50% methanol-water carries, and the test solution that methyl alcohol carries is respectively at 0h, 2h, 4h, 6h, 8h, 12h, sample introduction when 16h, 20h, investigates its stability with the peak area of four standard items, represents that it makes a variation with its relative standard deviation (RSD).The stability result of the test solution of different preparation methods is shown in Table 2.
2.7.5 average recovery test
Taking the sample powder 9 parts of the red sage root-safflower (1:1) of known content, about 1g, accurately weighed, respectively by the 50%, 100% of known content, 150%3 levels add DFB, DST, HSYA, HTG reference substance.Operating by under " 2.2 " item, under " 2.3 " item, chromatographic condition sample introduction measures, and calculates its rate of recovery.Result shows: the rate of recovery of 4 kinds of reference substances, between 94.89～98.01%, the results are shown in Table 2.Result confirms that the method set up has the higher degree of accuracy for measuring four constituents in the red sage root, safflower.
The equation of linear regression of 1 four standard items of table and LOD and LOQ measurement result
Table 2 precision, repeatability, stability and average recovery experiment
2.8 sample determination
Accurate pipette samples solution 10 μ L, injects high performance liquid chromatograph, in conjunction with the feature ultraviolet spectra of four compounds, uses chemical fog recognition strategy that four constituents in the red sage root-safflower are carried out semi-quantitative analysis.
2.8.1 the relative dissolution rate of the different proportion red sage root-Safflower extracts
Owing to the red sage root is after water carries, tanshinone component response is the lowest, therefore in the water extract of the red sage root, the dissolution rate of tanshinone component is not considered.As Fig. 2-A testing result shows, in the red sage root-Safflower extracts, identify 47 compositions altogether.Wherein, danshinolic acid constituents 15, quinoid chalcones composition 20, flavones ingredient 12.Can be obtained by the proportioning stripping quantity curve of the red sage root-saffron aqueous solution, the order of the relative dissolution rate of danshinolic acid class, quinoid chalcones and flavones ingredient is followed successively by: 5:1 ＞ 5:2 ＞ 2:1 ＞ 1:0 ＞ 2:5 ＞ 1:1 ＞ 1:5 ＞ 1:2；1:5 ＞ 5:2 ＞ 0:1 ＞ 2:5 ＞ 2:1 ＞ 5:1 ＞ 1:2 ＞ 1:1；5:2 ＞ 1:5 ＞ 0:1 ＞ 2:5 ＞ 1:2 ＞ 1:1 ＞ 2:1 ＞ 5:1, three constituents are followed successively by 5:1,1:5 and 5:2 relative to the best proportion of dissolution rate.The ratio being surveyed the total relative dissolution rate of three class materials the highest is 5:2, and the minimum ratio of total relative dissolution rate is 1:2.
2.8.2 the relative dissolution rate of the different proportion red sage root-safflower 50% methanol-water extract
The red sage root-safflower is after 50% methanol-water carries, and the four class materials surveyed all have preferable response on HPLC-PDA.As Fig. 2-B testing result shows, in the red sage root-safflower 50% methanol-water extract, identify 57 compositions altogether.Wherein, danshinolic acid constituents 16, tanshinone component 8, quinoid chalcones composition 22, flavones ingredient 11.Can be obtained by the proportioning stripping quantity curve of the red sage root-safflower 50% methanol-water extract, danshinolic acid class, tanshinone, quinoid chalcones dissolution rate relative with flavones ingredient order is followed successively by: 5:1 ＞ 5:2 ＞ 1:0 ＞ 1:1 ＞ 2:1 ＞ 2:5 ＞ 1:5 ＞ 1:2；5:1 ＞ 1:0 ＞ 5:2 ＞ 2:5 ＞ 2:1 ＞ 1:5 ＞ 1:1 ＞ 1:2；5:2 ＞ 1:5 ＞ 0:1 ＞ 2:5 ＞ 2:1 ＞ 5:1 ＞ 1:1 ＞ 1:2；5:2 ＞ 1:5 ＞ 0:1 ＞ 2:5 ＞ 5:1 ＞ 2:1 ≈ 1:2 ＞ 1:1, four constituents are followed successively by 5:1,5:1,5:2 and 5:2 relative to the best proportion of dissolution rate.The ratio being surveyed the total relative dissolution rate of four class materials the highest is 5:2, and the minimum ratio of total relative dissolution rate is 1:2.
2.8.3 the relative dissolution rate of the different proportion red sage root-safflower methyl alcohol extract
Owing to methyl alcohol has preferable solvability, four class materials have higher response in analysis liquid phase.As Fig. 2-C testing result shows, in the methanolic extract of the red sage root-safflower, identify 43 compositions altogether.Wherein, danshinolic acid constituents 12, tanshinone component 8, quinoid chalcones composition 14, flavones ingredient 9.Can be obtained by the proportioning stripping quantity curve of the red sage root-safflower methanol extract liquid, danshinolic acid class, tanshinone, quinoid chalcones dissolution rate relative with flavones ingredient order is followed successively by: 5:1 ＞ 5:2 ≈ 1:0 ＞ 2:1 ＞ 2:5 ＞ 1:1 ＞ 1:5 ＞ 1:2；1:2 ＞ 5:1 ＞ 1:0 ＞ 2:5 ＞ 5:2 ≈ 1:1 ＞ 2:1 ＞ 1:5；1:5 ＞ 0:1 ＞ 2:5 ＞ 1:1 ＞ 5:2 ≈ 1:2 ＞ 2:1 ＞ 5:1；1:5 ＞ 0:1 ＞ 2:5 ＞ 1:2 ＞ 1:1 ＞ 5:2 ＞ 2:1 ＞ 5:1, four constituents are followed successively by 5:1,1:2,1:5 and 1:5 relative to the best proportion of dissolution rate.The ratio being surveyed the total relative dissolution rate of four class materials the highest is 5:2, and the ratio that dissolution rate is minimum relatively is 1:2.
2.8.4 the comparison of the relative dissolution rate of the red sage root-safflower different proportion difference preparation method extract
Under same ratio, danshinolic acid class, tanshinone, quinoid chalcones, the flavones ingredient relative dissolution rate order in different preparation methods is respectively as follows: 50% methanol-water and carries ＞ water and carry ＞ methyl alcohol and carry；Methyl alcohol carries ＞ 50% methanol-water and carries；50% methanol-water carries ＞ water and carries ＞ methyl alcohol and carry；50% methanol-water carries ＞ water and carries ＞ methyl alcohol and carry, and under different preparation methods, the relative dissolution rate figure of four constituents is shown in Fig. 3 (A-pressure differential self B-tanshinone compound C-chalcone compounds D-flavone compound).Under the red sage root-safflower difference preparation method, except tanshinone component, other three constituents relative dissolution rate under 50% methanol-water preparation method, carry, obviously higher than water under the identical red sage root-safflower ratio, the relative dissolution rate carried with methyl alcohol.In the red sage root-saffron aqueous solution, the red sage root-safflower 50% methanol-water extract and methyl alcohol extract, the optimization range of the relative dissolution rate that surveyed composition is total is all 5:1～5:2, and the highest rate of total relative dissolution rate is all 5:2.
Embodiment 2 antithrombotic contrast experiment
The red sage root of Different Weight ratio and the safflower impact on the clotting time
At present, the screening conventional method of antithrombotic compound is the activity investigating compound suppression platelet aggregation and the impact on the clotting time, and the impact in clotting time is investigated each compound antithrombotic acitivity by measuring compound by the present invention.
Concrete grammar: take healthy male rabbit, the yellow Jackets normal saline solution auricular vein injecting anesthetic of 30mg/kg rabbit body weight, operation separates arteria carotis communis and takes blood, is collected in plastic centrifuge tube, 3.8% sodium citrate aqueous solution anti-freezing (blood and anti-coagulants volume ratio are 9:1).800r/min is centrifuged 10min, prepares platelet rich plasma (Platelet-rich plasma, PRP), and 3000r/min is centrifuged 10min, prepares platelet poor plasma (Platelet-poor plasma, PPP).
Take the red sage root-flos carthami by different weight ratio (1:0,5:1,5:2,2:1,1:1,1:2,2:5,1:5,0:1, g/g) dried powder 100 grams (cross 40 mesh sieves), is separately added into 50% methanol eddy of medicinal material 10 times amount and extracts 2 times, each 2 hours, merge extract, concentrate, obtain the red sage root and the extract of safflower composition of Different Weight ratio, during use, be diluted with water into 10mg/ml.
The mensuration of PT (prothrombin time):
Principle: APTT and calcium ion mixture can make prothombin be fibrin ferment, fibrin ferment makes fibrinogen be changed into fibrin clot, and the time that grumeleuse is formed is negative correlation with the extrinsic coagulation factor content in blood plasma.
Method: add solvent or given the test agent 10 μ L, PPP 50 μ L, pre-temperature 3min in 37 DEG C of pre-temperature holes in test cup, test cup proceeds to TCH test channel, adds the derivant PT reagent 100 μ L of 37 DEG C of pre-temperature, the time of record PPP solidification.
The mensuration of APTT (activated partial thromboplastin time):
Principle: test plasma adds activated partial thromboplastin solution, fibrinogen is changed into fibrin, measures the time needed for solidification, is test plasma activated partial thromboplastin time (APTT).If intrinsic pathway is defective, setting time i.e. extends, and the degree lacked to single-factor is directly proportional.The most also lack to the accumulation of the factor needed for intrinsic pathway and be directly proportional.
Method: solvent or given the test agent 10 μ L, addition PPP 50 μ L and the APTT reagent 50 μ L of pre-temperature, pre-temperature 5min in 37 DEG C of pre-temperature holes in test cup, proceeds to test cup TCH test channel, adds the derivant CaCl of 37 DEG C of pre-temperature2Reagent 50 μ L, the time of record PPP solidification.
The mensuration of TT (thrombin time):
Principle: test plasma adds the thrombin solution demarcated, fibrinogen is changed into fibrin, measures the time needed for solidification, is test plasma thrombin time (TT).
Method: add solvent or given the test agent 10 μ L, PPP 50 μ L, pre-temperature 3min in 37 DEG C of pre-temperature holes in test cup, test cup proceeds to TCH test channel, adds the derivant TT reagent 50 μ L of room temperature, the time of record PPP solidification.
The mensuration of FIB (fibrinogen):
Principle: quantitative determination fibrinogen is the classical way commonly used, this method is to measure the aggegation time of diluting plasma after adding fibrin ferment.
Method: 1.: the preparation of calibration curve: the definite value blood plasma after redissolving is respectively prepared the diluting plasma of 1:5,1:10,1:15,1:20,1:30.Take each 200 μ L of diluting plasma of variable concentrations, 37 DEG C of pre-temperature 3 minutes, it is then respectively adding FIB reagent 100 μ L, measures setting time, coagulo meter automatically generate curve and preserve.2.: test cup adds solvent or given the test agent 10 μ L, PPP 50 μ L, pre-temperature 3min in 37 DEG C of pre-temperature holes, test cup is proceeded to TCH test channel, fibrin ferment (FIB) the 50 μ L of room temperature, the time of record PPP solidification or concentration are added.
All above experimental data withRepresenting, between group, mean compares employing t inspection, and specific experiment result is as shown in table 1.
The red sage root of table 1 Different Weight proportioning and safflower affect situation to thrombin time
As can be seen from the above data, the red sage root of Different Weight proportioning is different on the impact of thrombin time with safflower.When wherein the red sage root and safflower weight ratio are 5:1 and 5:2, effect is optimum, hence it is evident that is better than the composition of other weight ratio, and compares with the single red sage root or the safflower of same dose, the red sage root and safflower, by weight during for 5:1 and 5:2, serve the effect of good Synergistic.
Embodiment of above is only for technology design and the feature of the explanation present invention; its object is to allow person skilled in the art understand present invention and to be carried out; can not limit the scope of the invention with this; all equivalence changes done according to spirit of the invention or modification, all should contain in protection scope of the present invention.
Claims (7)
1. a Chinese medicine composition, it is characterised in that it is made up of the red sage root and safflower.
Chinese medicine composition the most according to claim 1, it is characterised in that returning the weight ratio with safflower is 5:1～5:2.
Chinese medicine composition the most according to claim 2, it is characterised in that returning the weight ratio with safflower is 5:2.
4. the extracting method of the Chinese medicine composition described in any one of claims 1 to 3, it is characterised in that by the red sage root and safflower group
Compound adds water, methyl alcohol or volumetric concentration are that 50% methanol aqueous solution extracts.
The extracting method of Chinese medicine composition the most according to claim 4, it is characterised in that take the red sage root and safflower composition,
Add volumetric concentration 50% methanol aqueous solution, refluxing extraction 1～3 times, each 1～2h, merge extract, concentrate, both.
The extracting method of Chinese medicine composition the most according to claim 4, it is characterised in that the described red sage root and safflower combination
The extract of thing be mainly composed of danshinolic acid class, tanshinone, quinoid chalcones or flavone compound.
7. the application in preparing medicament against cardiovascular disease of the Chinese medicine composition described in any one of claims 1 to 3.
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