CN100462072C - Medicine composition used for injection and its preparing method - Google Patents
Medicine composition used for injection and its preparing method Download PDFInfo
- Publication number
- CN100462072C CN100462072C CNB2007100010535A CN200710001053A CN100462072C CN 100462072 C CN100462072 C CN 100462072C CN B2007100010535 A CNB2007100010535 A CN B2007100010535A CN 200710001053 A CN200710001053 A CN 200710001053A CN 100462072 C CN100462072 C CN 100462072C
- Authority
- CN
- China
- Prior art keywords
- salvianolic acid
- preparation
- add
- radix salviae
- salviae miltiorrhizae
- 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.)
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- 239000007924 injection Substances 0.000 title claims abstract description 111
- 238000002347 injection Methods 0.000 title claims abstract description 111
- 239000000203 mixture Substances 0.000 title claims abstract description 23
- 239000003814 drug Substances 0.000 title claims description 54
- 229940079593 drug Drugs 0.000 title claims description 23
- 238000000034 method Methods 0.000 title claims description 18
- 239000000843 powder Substances 0.000 claims abstract description 35
- 230000008569 process Effects 0.000 claims abstract description 4
- YMGFTDKNIWPMGF-QHCPKHFHSA-N Salvianolic acid A Natural products OC(=O)[C@H](Cc1ccc(O)c(O)c1)OC(=O)C=Cc2ccc(O)c(O)c2C=Cc3ccc(O)c(O)c3 YMGFTDKNIWPMGF-QHCPKHFHSA-N 0.000 claims description 225
- SNKFFCBZYFGCQN-UHFFFAOYSA-N 2-[3-[3-[1-carboxy-2-(3,4-dihydroxyphenyl)ethoxy]carbonyl-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-dihydro-1-benzofuran-4-yl]prop-2-enoyloxy]-3-(3,4-dihydroxyphenyl)propanoic acid Chemical compound C=1C=C(O)C=2OC(C=3C=C(O)C(O)=CC=3)C(C(=O)OC(CC=3C=C(O)C(O)=CC=3)C(O)=O)C=2C=1C=CC(=O)OC(C(=O)O)CC1=CC=C(O)C(O)=C1 SNKFFCBZYFGCQN-UHFFFAOYSA-N 0.000 claims description 224
- YMGFTDKNIWPMGF-AGYDPFETSA-N 3-(3,4-dihydroxyphenyl)-2-[(e)-3-[2-[(e)-2-(3,4-dihydroxyphenyl)ethenyl]-3,4-dihydroxyphenyl]prop-2-enoyl]oxypropanoic acid Chemical compound C=1C=C(O)C(O)=C(\C=C\C=2C=C(O)C(O)=CC=2)C=1/C=C/C(=O)OC(C(=O)O)CC1=CC=C(O)C(O)=C1 YMGFTDKNIWPMGF-AGYDPFETSA-N 0.000 claims description 224
- SNKFFCBZYFGCQN-VWUOOIFGSA-N Lithospermic acid B Natural products C([C@H](C(=O)O)OC(=O)\C=C\C=1C=2[C@H](C(=O)O[C@H](CC=3C=C(O)C(O)=CC=3)C(O)=O)[C@H](OC=2C(O)=CC=1)C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 SNKFFCBZYFGCQN-VWUOOIFGSA-N 0.000 claims description 224
- STCJJTBMWHMRCD-UHFFFAOYSA-N salvianolic acid B Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=O)C=Cc2cc(O)c(O)c3OC(C(C(=O)OC(Cc4ccc(O)c(O)c4)C(=O)O)c23)c5ccc(O)c(O)c5 STCJJTBMWHMRCD-UHFFFAOYSA-N 0.000 claims description 224
- 238000002360 preparation method Methods 0.000 claims description 209
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 199
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 120
- 239000000243 solution Substances 0.000 claims description 91
- WTPPRJKFRFIQKT-UHFFFAOYSA-N 1,6-dimethyl-8,9-dihydronaphtho[1,2-g][1]benzofuran-10,11-dione;1-methyl-6-methylidene-8,9-dihydro-7h-naphtho[1,2-g][1]benzofuran-10,11-dione Chemical compound O=C1C(=O)C2=C3CCCC(=C)C3=CC=C2C2=C1C(C)=CO2.O=C1C(=O)C2=C3CCC=C(C)C3=CC=C2C2=C1C(C)=CO2 WTPPRJKFRFIQKT-UHFFFAOYSA-N 0.000 claims description 79
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- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 60
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- 239000012535 impurity Substances 0.000 claims description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
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- JGSARLDLIJGVTE-UHFFFAOYSA-N 3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid Chemical compound O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-UHFFFAOYSA-N 0.000 claims description 10
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Landscapes
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
A medicinal composition for injection, perfusion, or freeze-dried powder injection contains high-purity danshenolic acid A and high-purity danshenolic acid B. Its preparing process and quality control method are also disclosed.
Description
Technical field
The present invention relates to technical field of traditional Chinese medicine pharmacy, be specifically related to pharmaceutical composition of pharmaceutically active ingredient salvianolic acid A, salvianolic acid B in a kind of containing and preparation method thereof.
Background technology
Middle pharmaceutically active ingredient is the monomeric compound that extracts from Chinese medicine, on the basis that has kept the Chinese medicine function, have clearly characteristics of content height, active strong, action target spot, under the prerequisite of active substance basis and clear mechanism, under the guidance of theory of Chinese medical science, the effective ingredient exploitation is become clinically Chinese medicine preparation safely and effectively, significant aspect modernization of cmm.
The chemical constituent of Radix Salviae Miltiorrhizae mainly can be divided into water soluble ingredient and liposoluble constituent, as everyone knows, traditional application mode of Radix Salviae Miltiorrhizae is a decocting liquid, therefore, its effective ingredient should be based on water-soluble substances, red sage root water soluble ingredient has the phenolic acid structure more, i.e. effective ingredient such as salvianolic acid A, B, C, D, E, and what have fine pharmacologically active is mainly salvianolic acid A and salvianolic acid B.A lot of medical workers wish salvianolic acid A and salvianolic acid B are developed to new Chinese medicine, particularly salvianolic acid A and salvianolic acid B are made up and develop, but exist very big query: (1) salvianolic acid A and salvianolic acid B are the stronger chemical compounds of nature antioxidant activity, be easy to oxidized, unstable in aqueous solution, easier degraded during high temperature sterilize, therefore its stability has become the technology barrier that is difficult to cross over, by the two certain combination, whether can avoid degraded and oxidation, make the two more stable; (2) salvianolic acid A, salvianolic acid B belong to same Effective Components of Chinese Herb, character such as pharmacologically active are close, whether the two combination exists certain internal relation, whether combination can be brought into play bigger effect by certain combination, rather than the combination of simple 1+1=2; (3) salvianolic acid A and salvianolic acid B enter in the body after by certain combination, and whether situations such as its absorption, distribution, metabolism have new variation; Or the like a lot of problems perplexing the medical scientific research person, the two combination and exploitation can not be become safe and effective medicine preparation clinically.
Chinese patent " a kind of method and lyophilized injectable powder thereof that extracts each component of phenolic acid from salviamiltiorrhizabung " (application number 03132382.0) discloses salvianolic acid A and salvianolic acid B combined preparation lyophilized injectable powder, but it mainly is to study how to extract from Radix Salviae Miltiorrhizae to obtain danshen root salvianolic acid A and salvianolic acid B, and its combination has just been carried out superficial research by pharmacological evaluation: many more its pharmacological actions of the consumption of salvianolic acid B are good more in the combination; This research just simply superposes effective component in red sage, to the further investigation of combination of active principles without any practical significance.Document " the different proportionings of salvianolic acid A/salvianolic acid B are to the protective effect of rat myocardial ischemia and reperfusion damage " (" Hebei Chinese medicine journal "; 2006 21 the 2nd phases of volume); disclose salvianolic acid A and salvianolic acid B combination have been studied; its conclusion is a salvianolic acid A: during the weight ratio 2:1 of salvianolic acid B pharmacological action best, also be simple research have been carried out in combination from the drug effect aspect.These two pieces of documents are all studied at salvianolic acid A and salvianolic acid B combination, but too come to the surface, just the two has been carried out simple stack, think that the effective ingredient addition is good, not at salvianolic acid A and the deep research of salvianolic acid B combination carrying out, do not excavate profound thing, the prescription of middle pharmaceutically active ingredient is become a mere formality; Determining of middle pharmaceutically active ingredient compound recipe, be on the basis of analyzing pathogenesis, determine method of treatment, method of treatment guidance under form the prescription meet the state of an illness needs, prescription is made up of medicine, but it is not the rabble of pharmaceutically active ingredient in the random use, but be foundation with the method for treatment, select the appropriate drug effective ingredient, under instruction of Chinese Medicine theory, combine, method of treatment is the theoretical foundation of prescription, prescription is the imbody of method of treatment, and therefore, prescription is a method of treatment, method of treatment is a prescription, the two is indivisible, is used as medicine as Effective Components of Chinese Herb, has These characteristics equally.
Summary of the invention
For these reasons, we find by long term studies, danshen root salvianolic acid A and salvianolic acid B of Radix Salviae Miltiorrhizae are made up according to certain rules: pharmaceutical composition and preparation carry out high effective liquid chromatography for measuring, obtain 2 characteristic peaks, one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A are more than or equal to 0.01 and less than 0.4; Be preferably and obtain 2 characteristic peaks, one is salvianolic acid B, another salvianolic acid A, and the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A are more than or equal to 0.01 and smaller or equal to 0.16; Be preferably and obtain 2 characteristic peaks, one is salvianolic acid B, another salvianolic acid A, and the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A are more than or equal to 0.25 and smaller or equal to 0.38.This combination is not an effective ingredient weight simple addition in the prior art, but pass through high performance liquid chromatography, by the peak area ratio resize ratio, the form of expression (aspects such as stable effective ingredients, pharmacodynamics, pharmacokinetics, toxicology) in echelon in this certain limit that is combined in peak area ratio, less than this scope or greater than this scope, the decline that is in line of its form of expression, this has proved absolutely that salvianolic acid A and salvianolic acid B exist intrinsic relation.
The present invention is achieved through the following technical solutions.
One. the preparation of compositions effective ingredient and formulation preparation
The danshen root salvianolic acid A preparation method:
Radix Salviae Miltiorrhizae water or alcoholic solution extract and obtain aqueous extract or alcohol extract, alcohol extract concentrates ethanol to most, transfer pH value to 7.5-9.0,30-80 ℃ temperature, heating 1-6 hours or transfer pH value to 3.5-6.0,110-130 ℃ temperature, gauge pressure 0.05MPa-0.17MPa pressure, heated 1-6 hours; Solution filters, and filtrate is separated through nonpolar or low pole macroporous resin column chromatography, and first water, 10-30% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 30-70% concentration is collected eluent, reclaims ethanol to most; Concentrated solution separates with sephadex lh-20 or polyamide chromatography post, uses the eluant eluting, collects eluent, and eluent reclaims eluant to most; Concentrated solution is transferred pH value to 2-5, through organic solvent extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
The salvianolic acid B of Radix Salviae Miltiorrhizae preparation method:
Radix Salviae Miltiorrhizae water or alcoholic solution extract and obtain aqueous extract or alcohol extract, and alcohol extract concentrates ethanol to most, and concentrated solution separates through nonpolar or low pole macroporous resin column chromatography, elder generation's water eluting is removed impurity, reuse 10-30% Diluted Alcohol eluting, collect eluent, eluent reclaims ethanol to most; Concentrated solution is transferred pH value to 1-4, through organic solvent extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get salvianolic acid B of Radix Salviae Miltiorrhizae;
Macroporous resin column described in its preparation method is HPD-100, HPD-100A, HPD-300, HPD-400, HPD-400A, HPD-450, D101,1300-I, 1400 or AB-8.
Sephadex lh-20 described in its preparation method or polyamide chromatography post separate, and first water, 20-50% alcoholic solution eluting discard eluent, reuse 50-95% alcoholic solution eluting.
Organic solvent described in its preparation method is selected from a kind of in ethyl acetate, propyl acetate, butyl acetate, n-butyl alcohol, the isopropyl alcohol.
Formulation preparation:
Preparation raw material medicine: danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae;
Danshen root salvianolic acid A purity is more than or equal to 80% and less than 100%, and salvianolic acid B of Radix Salviae Miltiorrhizae purity is more than or equal to 80% and less than 100%; (the check and analysis method is carried out according to the experiment of present specification detection method)
Pharmaceutical composition carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, and the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A are more than or equal to 0.01 and less than 0.4.
Wherein pharmaceutical composition carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, and the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A are more than or equal to 0.01 and smaller or equal to 0.16.
Wherein pharmaceutical composition carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, and the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A are more than or equal to 0.25 and smaller or equal to 0.38.
The preparation of aqueous injection: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively fully, transfer pH value, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters merging filtrate, add vitamin C, filtrate adds injection water, fine straining, embedding, sterilize, obtain the aqueous injection of unit dose;
The preparation of infusion solution: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, add isoosmotic adjusting agent sodium chloride or glucose, fine straining, fill, sterilize, obtain the infusion solution of unit dose;
The preparation of injectable powder: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, aseptic filtration or carry out autoclaving according to the requirement of injection is divided in the cillin bottle lyophilization, gland obtains the lyophilized injectable powder of unit dose.
Preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, and the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A are more than or equal to 0.01 and less than 0.4.
Wherein preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, and the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A are more than or equal to 0.01 and smaller or equal to 0.16.
Wherein preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, and the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A are more than or equal to 0.25 and smaller or equal to 0.38.
Wherein said unit dose is 5-500mg;
Preferred described unit dose is 10-200mg;
Unit dose is unfavorable for clinical practice less than the preparation of 5mg;
Aqueous injection, infusion solution, injectable powder are preparing the application that treats and/or prevents in cardiovascular and cerebrovascular disease, hepatic injury, hepatic fibrosis, pulmonary fibrosis, tumor, the old and feeble medicine.
The invention reside in provides a kind of pharmaceutical composition of recently determining to contain danshen root salvianolic acid A and salvianolic acid B of Radix Salviae Miltiorrhizae with the high effective liquid chromatography for measuring peak area;
The present invention also is to provide a kind of and recently determines to contain aqueous injection, infusion solution, the lyophilized injectable powder that the preparation of pharmaceutical compositions of danshen root salvianolic acid A and salvianolic acid B of Radix Salviae Miltiorrhizae becomes with the high effective liquid chromatography for measuring peak area;
The present invention also is to provide a kind of preparation method that contains the pharmaceutical composition ejection preparation of danshen root salvianolic acid A and salvianolic acid B of Radix Salviae Miltiorrhizae.
Two. detection method
Experimental technique:
Chromatographic column: C
18Reversed phase chromatographic column, NUCLEODUR, 250*4.6mm, ODS;
Chromatographic condition and system suitability experiment: with the octadecylsilane chemically bonded silica is filler; Flow velocity 1.0ml/min; 35 ℃ of column temperatures; Detect wavelength 286nm; Number of theoretical plate should be not less than 60000 by salvianolic acid A; With acetonitrile-0.2% aqueous acetic acid is mobile phase, carries out gradient elution by following condition of gradient elution, moves 90 minutes;
In the time of 0-15 minute, the ratio of acetonitrile reduces to 80% by 10% ratio that rises to 20%, 0.2% aqueous acetic acid by 90%; In the time of 15-55 minute, the ratio of acetonitrile reduces to 70% by 20% ratio that rises to 30%, 0.2% aqueous acetic acid by 80%; In the time of 55-65 minute, the ratio of acetonitrile reduces to 50% by 30% ratio that rises to 50%, 0.2% aqueous acetic acid by 70%; In the time of 65-72 minute, the ratio of acetonitrile reduces to 20% by 50% ratio that rises to 80%, 0.2% aqueous acetic acid by 50%; In the time of 72-77 minute, the ratio 20% of ratio 80%, 0.2% aqueous acetic acid of acetonitrile; In the time of 77-80 minute, the ratio of acetonitrile rises to 90% by 80% ratio of reducing to 10%, 0.2% aqueous acetic acid by 20%; In the time of 80-90 minute, keep acetonitrile-0.2% aqueous acetic acid to carry out eluting with the ratio of 10:90;
The preparation of reference substance solution: precision takes by weighing salvianolic acid A, salvianolic acid B reference substance in volumetric flask, adds dissolve with methanol and shakes up, and be diluted to scale;
The preparation of sample solution: get salvianolic acid A sample, salvianolic acid B sample respectively, add dissolve with methanol and shake up, and be diluted to scale; Or precision measures or takes by weighing preparation, adds dissolve with methanol and shakes up, and be diluted to scale;
Algoscopy: the accurate respectively reference substance solution of drawing, inject chromatograph of liquid, the record chromatogram; Get earlier danshen root salvianolic acid A sample, salvianolic acid B of Radix Salviae Miltiorrhizae sample solution respectively, inject chromatograph of liquid, calculate peak area ratio, contrast peak area ratio scope, adjust the configuration of sample solution, the chromatograph of liquid that reinjects, repetitive operation, the peak area ratio of configuration sample solution is more than or equal to 0.01 and less than in 0.4 scope.
Or the accurate formulation samples solution of drawing, inject chromatograph of liquid, calculate peak area ratio.
Or accurate respectively salvianolic acid A sample, the salvianolic acid B sample solution drawn, injecting chromatograph of liquid, the record chromatogram adopts external standard method with calculated by peak area, promptly.
1. preparation check and analysis experiment
Get aqueous injection, infusion solution, injectable powder that the application's process obtains, experimental technique carries out check and analysis according to the above analysis, obtains the peak area of danshen root salvianolic acid A and salvianolic acid B of Radix Salviae Miltiorrhizae in the preparation, calculates its ratio, the results are shown in Table 1:
Table 1 the application preparation salvianolic acid B and salvianolic acid A peak area ratio
Experiment conclusion: show by above-mentioned experiment, the application's pharmaceutical composition be according to salvianolic acid B and salvianolic acid A high performance liquid chromatogram detect peak area ratio more than or equal to 0.01 and less than 0.4 scope in, be prepared into ejection preparation.
Two. study on the stability
Experimental program:
Scheme 1: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.005 and the ejection preparation of preparation;
Scheme 2: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.008 and the ejection preparation of preparation;
Scheme 3: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.009 and the ejection preparation of preparation;
Scheme 4: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.01 and the ejection preparation of preparation;
Scheme 5: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.05 and the ejection preparation of preparation;
Scheme 6: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.16 and the ejection preparation of preparation;
Scheme 7: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.22 and the ejection preparation of preparation;
Scheme 8: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.25 and the ejection preparation of preparation;
Scheme 9: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.38 and the ejection preparation of preparation;
Scheme 10: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.39 and the ejection preparation of preparation;
Scheme 11: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.40 and the ejection preparation of preparation;
Scheme 12: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.43 and the ejection preparation of preparation;
Scheme 13: salvianolic acid A aqueous solution;
Scheme 14: salvianolic acid B aqueous solution;
Experimental technique: the pharmaceutical composition of getting above-mentioned experimental program respectively, carry out check and analysis according to the application's check and analysis experimental technique, with its peak area and the unit of being set at 1, pharmaceutical composition was placed 3 months, check and analysis, calculate salvianolic acid A and salvianolic acid B peak area and, compare with aforementioned peak area and (unit 1), calculate relative amount; Get the preparation of preparation and place 3 months preparation, according to the said method experiment, scheme 13, scheme 14 are as the criterion with the actual result of check and analysis, and experimental result sees Table 2:
Table 2 different experiments scheme result
Experiment conclusion: show by above-mentioned experiment, the ratio of salvianolic acid A and salvianolic acid B peak area has certain rules to the stability of salvianolic acid A and salvianolic acid B, when ratio less than 0.01 the time, salvianolic acid A and salvianolic acid B just have loss in preparation process, place the more of degraded after 3 months; When ratio more than or equal to 0.01 the time, turnover has taken place, its degraded obviously is in halted state; When more than or equal to 0.4 the time, degradation reaction has obviously taken place again; Its variation is trapezoidal change of state; Consider from salvianolic acid A and salvianolic acid B stability situation, the ratio of its peak area should determine more than or equal to 0.01 and less than 0.4 scope in; We can also determine that salvianolic acid B and salvianolic acid A peak area ratio are more than or equal to 0.01 and better smaller or equal to 0.16 stability from experiment; Preferred salvianolic acid B and salvianolic acid A peak area ratio are more than or equal to 0.25 and smaller or equal to 0.38 good stability.
Three. pharmacokinetics is investigated
1. the investigation of half-life
Experimental program:
Scheme 1: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.009 and the ejection preparation of preparation;
Scheme 2: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.01 and the ejection preparation of preparation;
Scheme 3: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.16 and the ejection preparation of preparation;
Scheme 4: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.25 and the ejection preparation of preparation;
Scheme 5: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.38 and the ejection preparation of preparation;
Scheme 6: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.40 and the ejection preparation of preparation;
Scheme 7: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.43 and the ejection preparation of preparation;
Experimental technique:
Laboratory animal: the SD rat, male, 8 ages in week, SPF level; Assay method: select for use the LC-MS/MS method to detect; The plasma treatment method: precision is measured plasma specimen 100 μ l, adds 100 μ l, 0.1% phosphoric acid, 400 μ l methanol, behind the vortex mixing, and the centrifugal 5min of 10000r/min.After 0.2 μ m filter filtered, supernatant was by the automatic sampler sample introduction; Male SD rat, 6 every group, give salvianolic acid A and the salvianolic acid B pharmaceutical composition and the preparation thereof of different peak area ratios through tail vein single, by physiological saline solution, the administration volume is 1ml/kg.2min, 5min, 10min, 20min, 30min, 45min, 1h, 1.5h, 2h, 3h, 4h, 6h collect blood sample (0.3ml) through rat tail vein before medicine and after the administration, calculate the half-life of the salvianolic acid A and the salvianolic acid B of different peak area ratios, experimental result sees Table 3:
The half-life of different peak area ratio salvianolic acid As of table 3 and salvianolic acid B
2. excretory investigation
The animal that gives medicine is continued to raise 3 days, put to death, dissect kidney, detection scheme 1,6,7 has salvianolic acid A, salvianolic acid B trace to accumulate, and scheme 2-5 does not detect salvianolic acid A, salvianolic acid B, and explanation scheme 2-5 is not accumulated in vivo.
Experiment conclusion: show by above-mentioned experiment, when salvianolic acid B and salvianolic acid A peak area ratio less than 0.01 the time, pharmaceutical composition and preparation half-life are obviously bad; When salvianolic acid B and salvianolic acid A peak area ratio more than or equal to 0.01 and less than 0.4 scope in, pharmaceutical composition and preparation have the good half-life; When salvianolic acid B and salvianolic acid A peak area ratio greater than 0.4 the time, pharmaceutical composition and preparation half-life obviously descend again, prove absolutely that the ratio of peak area is trapezoidal aspect pharmacokinetics; Wherein preferably salvianolic acid B and salvianolic acid A peak area ratio more than or equal to 0.01 and have the good half-life smaller or equal to 0.16; Preferred salvianolic acid B and salvianolic acid A peak area ratio are more than or equal to 0.25 and have the good half-life smaller or equal to 0.38.
Four. pharmacodynamics is investigated
Experimental program:
Scheme 1: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.009 and the ejection preparation of preparation;
Scheme 2: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.01 and the ejection preparation of preparation;
Scheme 3: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.16 and the ejection preparation of preparation;
Scheme 4: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.25 and the ejection preparation of preparation;
Scheme 5: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.38 and the ejection preparation of preparation;
Scheme 6: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.40 and the ejection preparation of preparation;
Scheme 7: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.43 and the ejection preparation of preparation;
Scheme 8: danshen root salvianolic acid A;
Scheme 9: salvianolic acid B of Radix Salviae Miltiorrhizae;
Experiment 1
Protective effect to the anesthetized rat myocardial ischemia reperfusion injury
Experimental technique:
Get the healthy SD rat, body weight 240-260g, random packet: blank group, model group, Radix Salviae Miltiorrhizae for injection polyphenol hydrochlorate matched group, experimental program group (8mg/kg) place the pre-raising of equivalent environment 2 days, free diet.After pre-raising finishes, experimentize, animal is weighed, and 20% urethane is pressed the 0.6ml/100g lumbar injection, after treating that anesthesia is satisfied, lie on the back and be fixed on the Mus plate, tracheal intubation connects respirator, by 10～12ml tidal volume, 70 times/minute frequency is exhaled, and continuous positive pressure breathing is inhaled: exhale than being 1: 1.Adjust respiration parameter according to the respiratory frequency and the degree of depth.Connect electrocardiograph subsequently, survey normal ECG.Cut off front field of operation hair, iodine disinfection, cut off skin, subcutaneous tissue, front muscle and fascia 3～4cm, it is long to separate Intercostal muscle 3cm with the 18# vascular forceps along the 3rd intercostal passivity, open thoracic cavity and pericardium, recording ecg, strut 3,4 ribs, refer to hold thoracic cavity, rat right side with left hand four, the assistant upwards pushes away thymus with the ophthalmology tweezer, finds ligation sign blood vessel great cardiac vein between left auricle and pulmonary conus, 2mm place noinvasive roundlet pin band 6-0 silk thread threading below left auricle, depth of needle is 1～1.5mm, wide 2～3mm, recording ecg behind the threading, give corresponding medicinal liquid through the tail vein, recording ecg behind the administration 10min, and with one the band groove little plastics pipe pad at the ligation position, the ligation thereon of two rear line heads.At once recording ecg after the ligation is cyanosis or the II S-T section back of a bow that leads with left chamber antetheca and upwards raises greater than 0.1mv and be that ligation successfully indicates (it is superseded that the S-T section does not have the changer) more than the lasting 0.5h.10min recording ecg is once more cut off ligature behind the ligation 30min after the ligation, realizes perfusion again, and record pours into electrocardiogram at once again, removes in the thoracic cavity layer-by-layer suture thoracic wall behind the hematocele, removes respirator, animal recovery autonomous respiration, and incision of trachea does not process.Irritate again at once, 10min, 20min, 40min, 1h, 2h, 3h recording ecg respectively.Irritated again 3 hours, through abdominal aortic blood, 4000rpm is centrifugal, and 10min gets serum, adopt automatic clinical chemistry analyzer to detect LDH, CK and CK-MB activity, and adopt the corresponding reagent box to detect SOD in serum, MDA, dissection is cored dirty, the residual blood of ice normal saline flush away, cut off atrium and right ventricle, put into refrigerator and cooled immediately and freeze.With heart after refrigerator and cooled is frozen 10min, from the apex of the heart entad the parallel coronary sulcus direction in the end 5 of equal thickness are cut in left chamber, put into the 1%TTC dye liquor, 37 ℃ of dyeing 10min, the necrotic area is not a kermesinus, the necrotic area is canescence.Digital camera is taken pictures.Weighed respectively in necrotic area and non-necrotic area, calculate the percentage ratio that the necrotic area accounts for left ventricular mass, i.e. infarction size.
The detection index is respectively, myocardial infarct size.Experimental result sees table 4 for details:
The influence of myocardial ischemia myocardial infarct size (%) due to table 4 ligation/logical again rat ramus descendens anterior arteriae coronariae sinistrae (x ± s)
Annotate: compare with model group,
*P＜0.05,
*P＜0.01; Compare #P＜0.05 with Radix Salviae Miltiorrhizae for injection polyphenol hydrochlorate.
To the influence of SOD activity, MDA content in the rat heart muscle ischemia/reperfusion model serum due to the logical rat ramus descendens anterior arteriae coronariae sinistrae of ligation/again, experimental technique sees that the present specification result sees table 5 for details.
The influence of SOD activity, MDA content in the rat heart muscle ischemia/reperfusion model serum due to table 5 pair ligation/logical again rat ramus descendens anterior arteriae coronariae sinistrae
Annotate: compare with model group,
*P＜0.05,
*P＜0.01; Compare #P＜0.05 with Radix Salviae Miltiorrhizae for injection polyphenol hydrochlorate.
Experiment 2
Research to the protective effect of intraluminal middle cerebral artery occlusion in rats ischemical reperfusion injury
Experimental technique:
The animal random packet: blank group, Radix Salviae Miltiorrhizae for injection polyphenol acid group, the application's scheme group, each dosage group successive administration 3 days was made middle cerebral artery occlusion (MCAO) model with improvement line bolt method in 20 minutes behind the 4th day medicine.Behind the rat anesthesia, it is fixing that it is lain on the back.Separate right carotid (CCA), internal carotid artery (ICA) and external carotid artery (ECA), ligation ECA and CCA, after closing the ICA distal end with bulldog clamp folder, make a kerf in ECA and ICA crotch rapidly, insert the nylon wire (diameter is 0.25mm, marks apart from pommel 18mm place, is stained with heparin solution before the insertion) that an end is heated into smooth, spherical and has been coated with 0.1% poly-D-lysine, insertion depth is 18mm, realizes that middle cerebral artery occlusion causes cerebral ischemia.Ligation porch, nylon wire are stayed about 1cm, skin suture outward.Lift extremely slightly resistance of institute's the end of a thread that stays after 2 hours gently, realize that middle cerebral artery pours into again, modeling is finished.At ischemia 2h with pour into the body temperature of keeping rat in the 1h with electric blanket again, body temperature maintains 36.5～37.5 ℃ of anus temperature.The animal inclusion criteria is pressed Longa Pyatyi point system, gets function of nervous system's behavior scoring and be 1,2,3,4 minute animal, (0 minute: the impassivity defective symptom; 1 minute: the offside forelimb can not stretch fully; 2 minutes: to sideway swivel; 3 minutes: topple over to offside; 4 minutes: can not oneself walk or stupor).The cerebral infarction scope is measured, rat model pours into 24h again, after behavioristics's scoring, broken end is got brain, removes olfactory bulb, cerebellum and low brain stem, and remainder is at-20 ℃ of freezing 10min of refrigerator, crownly on ice pan be cut into 6, rapidly the brain sheet is placed the TTC dye liquor, 37 ℃ of lucifuge temperature are incubated 1h, take out to be placed on the 24h that keeps in Dark Place in 10% formalin.The non-ischemic region in dyed back is a rose, and infarct is a white.White organized carefully to dig down weigh, account for full brain weight percentage ratio as the cerebral infarction scope with blocking tissue's weight.
Brain water content is measured: after TTC dyeing is weighed, brain placed oven dry 12h claims dry weight in 120 ℃ of vacuum desiccators.Brain water content=(cutaneous horn weight-brain stem is heavy)/cutaneous horn heavy * 100%.Experimental result sees table 6 for details.
The influence of table 6 pair intraluminal middle cerebral artery occlusion in rats ischemical reperfusion injury rat cerebral infarction scope and brain water content (X ± S)
Annotate: compare with model group,
*P＜0.05,
*P＜0.01; Compare #P＜0.05 with Radix Salviae Miltiorrhizae for injection polyphenol hydrochlorate.
Experiment conclusion: show by above-mentioned experiment, when salvianolic acid B and salvianolic acid A peak area ratio less than 0.01 the time, pharmaceutical composition and preparation pharmacological action are obviously bad; When salvianolic acid B and salvianolic acid A peak area ratio more than or equal to 0.01 and less than 0.4 scope in, pharmaceutical composition and preparation have great pharmacological effects (p＜0.01); When salvianolic acid B and salvianolic acid A peak area ratio greater than 0.4 the time, pharmaceutical composition and preparation pharmacological action have descended again, prove absolutely that the ratio of peak area is trapezoidal aspect pharmacodynamics; Wherein preferably salvianolic acid B and salvianolic acid A peak area ratio more than or equal to 0.01 and have great pharmacological effects smaller or equal to 0.16; Preferred salvianolic acid B and salvianolic acid A peak area ratio are more than or equal to 0.25 and have great pharmacological effects smaller or equal to 0.38; Show that by above-mentioned pharmacological evaluation the application's ejection preparation has great pharmacological effects, prove absolutely that the application's preparation has practical significance.
Annotate: above-mentioned different schemes is carried out defying age experiment, anti-tumor experiment, pulmonary fibrosis resistant experiment, anti-hepatic fibrosis experiment, obtain pharmacological datum, we can draw salvianolic acid B and salvianolic acid A peak area ratio equally more than or equal to 0.01 and have great pharmacological effects less than pharmaceutical composition in 0.4 scope and preparation; Wherein preferably salvianolic acid B and salvianolic acid A peak area ratio more than or equal to 0.01 and have great pharmacological effects smaller or equal to 0.16; Preferred salvianolic acid B and salvianolic acid A peak area ratio are more than or equal to 0.25 and have great pharmacological effects smaller or equal to 0.38.
Five. toxicologic study
Scheme 1: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.009 and the ejection preparation of preparation;
Scheme 2: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.01 and the ejection preparation of preparation;
Scheme 3: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.16 and the ejection preparation of preparation;
Scheme 4: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.25 and the ejection preparation of preparation;
Scheme 5: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.38 and the ejection preparation of preparation;
Scheme 6: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.40 and the ejection preparation of preparation;
Scheme 7: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.43 and the ejection preparation of preparation;
Experimental technique: the pharmaceutical composition of above-mentioned different schemes and preparation, carry out toxicological experiment, measure the LD of mice single intravenously administrable acute toxicity
50, experimental result sees Table 7:
The LD of table 7 different schemes
50Value
Experiment conclusion: we work as LD at experimentation
50Value is only safe during greater than 400mg/kg in clinical application; Show by above-mentioned experiment, when salvianolic acid B and salvianolic acid A peak area ratio more than or equal to 0.01 and less than 0.4 the time, safety is best; The peak area of preferred salvianolic acid B and the peak area ratio of salvianolic acid A are more than or equal to 0.01 and smaller or equal to 0.16; Being preferably the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A is more than or equal to 0.25 and smaller or equal to 0.38.
Compared with the prior art:
The application investigates the internal relation of the two combination by salvianolic acid A and salvianolic acid B compositions and preparation stability, pharmacodynamics, pharmacokinetics aspect, determine that salvianolic acid A, salvianolic acid B its stability, pharmacodynamics, pharmacokinetics, safety under certain relation are good, the present preparation high performance liquid chromatography of the certain relation table aspect of the two be salvianolic acid B with the ratio of salvianolic acid A peak area more than or equal to 0.01 and less than 0.4; Be preferably and obtain 2 characteristic peaks, one is salvianolic acid B, another salvianolic acid A, and the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A are more than or equal to 0.01 and smaller or equal to 0.16; Be preferably and obtain 2 characteristic peaks, one is salvianolic acid B, another salvianolic acid A, and the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A are more than or equal to 0.25 and smaller or equal to 0.38.
Preparation embodiment
Embodiment 1
The danshen root salvianolic acid A preparation method:
Radix Salviae Miltiorrhizae obtains aqueous extract with water extraction, transfers pH value to 7.5,30 ℃ of temperature, heating 1 hour; Solution filters, and filtrate is separated through the HPD-100 macroporous resin column chromatography, and first water, 10% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 30% concentration is collected eluent, reclaims ethanol to most; Concentrated solution separates with the sephadex lh-20 chromatographic column, with first water, 20% alcoholic solution eluting, discards eluent, and reuse 50% alcoholic solution eluting is collected eluent, and eluent reclaims ethanol extremely to the greatest extent; Concentrated solution is transferred pH value to 2, through the organic solvent ethyl acetate extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
The salvianolic acid B of Radix Salviae Miltiorrhizae preparation method:
Radix Salviae Miltiorrhizae extracts with 30% alcoholic solution and obtains alcohol extract, and alcohol extract concentrates ethanol to most, and concentrated solution separates through the HPD-100A macroporous resin column chromatography, and first water eluting is removed impurity, and reuse 10% Diluted Alcohol eluting is collected eluent, and eluent reclaims ethanol to most; Concentrated solution is transferred pH value to 1, through the extraction of organic solvent propyl acetate, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get salvianolic acid B of Radix Salviae Miltiorrhizae;
Formulation preparation:
Preparation raw material medicine: danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae;
Danshen root salvianolic acid A purity 80%, salvianolic acid B of Radix Salviae Miltiorrhizae purity 80.3%, compositions is carried out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.01.
The preparation of aqueous injection: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively fully, transfer pH value, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters merging filtrate, add vitamin C, filtrate adds injection water, fine straining, embedding, sterilization obtains 1000 of unit dose aqueous injection;
The preparation of infusion solution: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, add isoosmotic adjusting agent sodium chloride or glucose, fine straining, fill, sterilization obtains 1000 bottles of the infusion solutions of unit dose;
The preparation of injectable powder: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, aseptic filtration or carry out autoclaving according to the requirement of injection is divided in the cillin bottle lyophilization, gland obtains 1000 of the lyophilized injectable powders of unit dose.
Preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.01;
Clinical applying unit dosage 5mg;
Aqueous injection, infusion solution, injectable powder are preparing the application that treats and/or prevents in cardiovascular and cerebrovascular disease, hepatic injury, hepatic fibrosis, pulmonary fibrosis, tumor, the old and feeble medicine.
Embodiment 2
The danshen root salvianolic acid A preparation method:
Radix Salviae Miltiorrhizae extracts with 70% alcoholic solution and obtains alcohol extract, and alcohol extract concentrates ethanol to the greatest extent, transfers pH value to 9.0,80 ℃ of temperature, heating 6 hours; Solution filters, and filtrate is separated through the HPD-300 macroporous resin column chromatography, and first water, 30% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 70% concentration is collected eluent, reclaims ethanol to most; Concentrated solution separates with polyamide chromatography post, and first water, 50% alcoholic solution eluting discard eluent, and reuse 95% alcoholic solution eluting, eluent reclaim ethanol to most; Concentrated solution is transferred pH value to 5, through the organic solvent n-butyl acetate extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
The salvianolic acid B of Radix Salviae Miltiorrhizae preparation method:
Radix Salviae Miltiorrhizae obtains aqueous extract with water extraction, and concentrated solution separates through the HPD-400 macroporous resin column chromatography, and macroporous resin column chromatography separates, and first water eluting is removed impurity, and reuse 30% Diluted Alcohol eluting is collected eluent, and eluent reclaims ethanol extremely to the greatest extent; Concentrated solution is transferred pH value to 4, through the organic solvent n-butanol extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get salvianolic acid B of Radix Salviae Miltiorrhizae;
Formulation preparation:
Preparation raw material medicine: danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae;
Danshen root salvianolic acid A purity 99.8%, salvianolic acid B of Radix Salviae Miltiorrhizae purity 99.9%, pharmaceutical composition carries out high effective liquid chromatography for measuring, obtain 2 characteristic peaks, one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.393.
The preparation of aqueous injection: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively fully, transfer pH value, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters merging filtrate, add vitamin C, filtrate adds injection water, fine straining, embedding, sterilization obtains 1000 of unit dose aqueous injection;
The preparation of infusion solution: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, add isoosmotic adjusting agent sodium chloride or glucose, fine straining, fill, sterilization obtains 1000 bottles of the infusion solutions of unit dose;
The preparation of injectable powder: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, aseptic filtration or carry out autoclaving according to the requirement of injection is divided in the cillin bottle lyophilization, gland obtains 1000 of the lyophilized injectable powders of unit dose.
Preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.38.
Clinical applying unit dosage 500mg:
Aqueous injection, infusion solution, injectable powder are preparing the application that treats and/or prevents in cardiovascular and cerebrovascular disease, hepatic injury, hepatic fibrosis, pulmonary fibrosis, tumor, the old and feeble medicine.
Embodiment 3
The danshen root salvianolic acid A preparation method:
Radix Salviae Miltiorrhizae obtains aqueous extract with water extraction, transfers pH value to 8.0,55 ℃ of temperature, heating 4 hours; Solution filters, and filtrate is separated through the HPD-400A macroporous resin column chromatography, and first water, 20% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 50% concentration is collected eluent, reclaims ethanol to most; Concentrated solution separates with sephadex lh-20, and first water, 35% alcoholic solution eluting discard eluent, and reuse 70% alcoholic solution eluting, eluent reclaim ethanol to most; Concentrated solution is transferred pH value to 4.0, through the organic solvent n-butanol extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
The salvianolic acid B of Radix Salviae Miltiorrhizae preparation method:
Radix Salviae Miltiorrhizae extracts with 50% alcoholic solution and obtains alcohol extract, and alcohol extract concentrates ethanol to most, and concentrated solution separates through the HPD-400A macroporous resin column chromatography, and first water eluting is removed impurity, and reuse 20% Diluted Alcohol eluting is collected eluent, and eluent reclaims ethanol to most; Concentrated solution is transferred pH value to 3, through the extraction of organic solvent isopropyl alcohol, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get salvianolic acid B of Radix Salviae Miltiorrhizae;
Formulation preparation:
Preparation raw material medicine: danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae;
Compositions danshen root salvianolic acid A purity 90.7%, salvianolic acid B of Radix Salviae Miltiorrhizae purity 91.3%, pharmaceutical composition carries out high effective liquid chromatography for measuring, obtain 2 characteristic peaks, one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.16.
The preparation of aqueous injection: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively fully, transfer pH value, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters merging filtrate, add vitamin C, filtrate adds injection water, fine straining, embedding, sterilization obtains 1000 of unit dose aqueous injection;
The preparation of infusion solution: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, add isoosmotic adjusting agent sodium chloride or glucose, fine straining, fill, sterilization obtains 1000 bottles of the infusion solutions of unit dose;
The preparation of injectable powder: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, aseptic filtration or carry out autoclaving according to the requirement of injection is divided in the cillin bottle lyophilization, gland obtains 1000 of the lyophilized injectable powders of unit dose.
Preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.15.
Clinical applying unit dosage 200mg;
Aqueous injection, infusion solution, injectable powder are preparing the application that treats and/or prevents in cardiovascular and cerebrovascular disease, hepatic injury, hepatic fibrosis, pulmonary fibrosis, tumor, the old and feeble medicine.
Embodiment 4
The danshen root salvianolic acid A preparation method:
Radix Salviae Miltiorrhizae extracts with 45% alcoholic solution and obtains alcohol extract, and alcohol extract concentrates ethanol to the greatest extent, transfers pH value to 3.5,110 ℃ of temperature, gauge pressure 0.05MPa pressure, heats 1 hour; Solution filters, and filtrate is separated through the HPD-450 macroporous resin column chromatography, and first water, 10% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 30% concentration is collected eluent, reclaims ethanol to most; Concentrated solution separates with sephadex lh-20, and first water, 20% alcoholic solution eluting discard eluent, and reuse 50% alcoholic solution eluting, eluent reclaim ethanol to most; Concentrated solution is transferred pH value to 2, through the organic solvent ethyl acetate extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
The salvianolic acid B of Radix Salviae Miltiorrhizae preparation method:
Radix Salviae Miltiorrhizae extracts with 45% alcoholic solution and obtains alcohol extract, and alcohol extract concentrates ethanol to most, and concentrated solution separates through the D101 macroporous resin column chromatography, and first water eluting is removed impurity, and reuse 10% Diluted Alcohol eluting is collected eluent, and eluent reclaims ethanol to most; Concentrated solution is transferred pH value to 1, through the extraction of organic solvent propyl acetate, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get salvianolic acid B of Radix Salviae Miltiorrhizae;
Formulation preparation:
Preparation raw material medicine: danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae;
Danshen root salvianolic acid A purity 81.2%, salvianolic acid B of Radix Salviae Miltiorrhizae purity 80.7%, compositions is carried out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.25.
The preparation of aqueous injection: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively fully, transfer pH value, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters merging filtrate, add vitamin C, filtrate adds injection water, fine straining, embedding, sterilization obtains 1000 of unit dose aqueous injection;
The preparation of infusion solution: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, add isoosmotic adjusting agent sodium chloride or glucose, fine straining, fill, sterilization obtains 1000 bottles of the infusion solutions of unit dose;
The preparation of injectable powder: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, aseptic filtration or carry out autoclaving according to the requirement of injection is divided in the cillin bottle lyophilization, gland obtains 1000 of the lyophilized injectable powders of unit dose.
Preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area of salvianolic acid A 0.23.
Clinical applying unit dosage 10mg;
Aqueous injection, infusion solution, injectable powder are preparing the application that treats and/or prevents in cardiovascular and cerebrovascular disease, hepatic injury, hepatic fibrosis, pulmonary fibrosis, tumor, the old and feeble medicine.
Embodiment 5
The danshen root salvianolic acid A preparation method:
Radix Salviae Miltiorrhizae obtains aqueous extract with water extraction, transfers pH value to 6.0,130 ℃ of temperature, gauge pressure 0.17MPa pressure, heats 6 hours; Solution filters, and filtrate is separated through the 1300-I macroporous resin column chromatography, and first water, 30% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 70% concentration is collected eluent, reclaims ethanol to most; Concentrated solution separates with polyamide chromatography post, and first water, 50% alcoholic solution eluting discard eluent, and reuse 95% alcoholic solution eluting, eluent reclaim ethanol to most; Concentrated solution is transferred pH value to 5, through the extraction of organic solvent propyl acetate, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
The salvianolic acid B of Radix Salviae Miltiorrhizae preparation method:
The Radix Salviae Miltiorrhizae water obtains aqueous extract, and concentrated solution separates through 1400 macroporous resin column chromatographies, and first water eluting is removed impurity, and reuse 30% Diluted Alcohol eluting is collected eluent, and eluent reclaims ethanol to most; Concentrated solution is transferred pH value to 4, through the organic solvent n-butyl acetate extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get salvianolic acid B of Radix Salviae Miltiorrhizae;
Formulation preparation:
Preparation raw material medicine: danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae;
Danshen root salvianolic acid A purity 99.3%, salvianolic acid B of Radix Salviae Miltiorrhizae purity 99.1%, compositions is carried out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.38.
The preparation of aqueous injection: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively fully, transfer pH value, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters merging filtrate, add vitamin C, filtrate adds injection water, fine straining, embedding, sterilization obtains 1000 of unit dose aqueous injection;
The preparation of infusion solution: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, add isoosmotic adjusting agent sodium chloride or glucose, fine straining, fill, sterilization obtains 1000 bottles of the infusion solutions of unit dose;
The preparation of injectable powder: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, aseptic filtration or carry out autoclaving according to the requirement of injection is divided in the cillin bottle lyophilization, gland obtains 1000 of the lyophilized injectable powders of unit dose.
Preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.36.
Clinical applying unit dosage 200mg;
Aqueous injection, infusion solution, injectable powder are preparing the application that treats and/or prevents in cardiovascular and cerebrovascular disease, hepatic injury, hepatic fibrosis, pulmonary fibrosis, tumor, the old and feeble medicine.
Embodiment 6
The danshen root salvianolic acid A preparation method:
Radix Salviae Miltiorrhizae extracts with 80% alcoholic solution and obtains alcohol extract, and alcohol extract concentrates ethanol to the greatest extent, transfers pH value to 5.5,120 ℃ of temperature, gauge pressure 0.12MPa pressure, heats 4 hours; Solution filters, and filtrate is separated through the AB-8 macroporous resin column chromatography, and first water, 20% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 50% concentration is collected eluent, reclaims ethanol to most; Concentrated solution separates with the sephadex lh-20 chromatographic column, and first water, 35% alcoholic solution eluting discard eluent, and reuse 75% alcoholic solution eluting is collected eluent, and eluent reclaims ethanol extremely to the greatest extent; Concentrated solution is transferred pH value to 3.5, through the organic solvent n-butyl acetate extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
The salvianolic acid B of Radix Salviae Miltiorrhizae preparation method:
Radix Salviae Miltiorrhizae extracts with 85% alcoholic solution and obtains alcohol extract, and alcohol extract concentrates ethanol to most, and concentrated solution separates through the HPD-100A macroporous resin column chromatography, and first water eluting is removed impurity, and reuse 20% Diluted Alcohol eluting is collected eluent, and eluent reclaims ethanol to most; Concentrated solution liquid is transferred pH value to 2.5, through the organic solvent n-butanol extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get salvianolic acid B of Radix Salviae Miltiorrhizae;
Formulation preparation:
Preparation raw material medicine: danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae;
Danshen root salvianolic acid A purity 91.2%, salvianolic acid B of Radix Salviae Miltiorrhizae purity 94.3%, composite preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.28.
The preparation of aqueous injection: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively fully, transfer pH value, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters merging filtrate, add vitamin C, filtrate adds injection water, fine straining, embedding, sterilization obtains 1000 of unit dose aqueous injection;
The preparation of infusion solution: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, add isoosmotic adjusting agent sodium chloride or glucose, fine straining, fill, sterilization obtains 1000 bottles of the infusion solutions of unit dose;
The preparation of injectable powder: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, aseptic filtration or carry out autoclaving according to the requirement of injection is divided in the cillin bottle lyophilization, gland obtains 1000 of the lyophilized injectable powders of unit dose.
Preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.25.
Clinical applying unit dosage 100mg;
Aqueous injection, infusion solution, injectable powder are preparing the application that treats and/or prevents in cardiovascular and cerebrovascular disease, hepatic injury, hepatic fibrosis, pulmonary fibrosis, tumor, the old and feeble medicine.
Embodiment 7
The danshen root salvianolic acid A preparation method:
Radix Salviae Miltiorrhizae obtains aqueous extract with water extraction, transfers pH value to 8.0,50 ℃ of temperature, heating 3.5 hours; Solution filters, and filtrate is separated through the D101 macroporous resin column chromatography, and first water, 15% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 65% concentration is collected eluent, reclaims ethanol to most; Concentrated solution separates with polyamide chromatography post, and first water, 25% alcoholic solution eluting discard eluent, and reuse 60% alcoholic solution eluting is collected eluent, and eluent reclaims ethanol extremely to the greatest extent; Concentrated solution is transferred pH value to 4.5, through the extraction of organic solvent isopropyl alcohol, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
The salvianolic acid B of Radix Salviae Miltiorrhizae preparation method:
Radix Salviae Miltiorrhizae extracts with 20% alcoholic solution and obtains alcohol extract, and alcohol extract concentrates ethanol to most, and concentrated solution separates through 1400 macroporous resin column chromatographies, macroporous resin column chromatography separates, and first water eluting is removed impurity, reuse 15% Diluted Alcohol eluting is collected eluent, and eluent reclaims ethanol to most; Concentrated solution is transferred pH value to 3.5, through the organic solvent ethyl acetate extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get salvianolic acid B of Radix Salviae Miltiorrhizae;
Formulation preparation:
Preparation raw material medicine: danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae;
Danshen root salvianolic acid A purity 84.5%, salvianolic acid B of Radix Salviae Miltiorrhizae purity 86.3%, composite preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.17.
The preparation of aqueous injection: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively fully, transfer pH value, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters merging filtrate, add vitamin C, filtrate adds injection water, fine straining, embedding, sterilization obtains 1000 of unit dose aqueous injection;
The preparation of infusion solution: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, add isoosmotic adjusting agent sodium chloride or glucose, fine straining, fill, sterilization obtains 1000 bottles of the infusion solutions of unit dose;
The preparation of injectable powder: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, aseptic filtration or carry out autoclaving according to the requirement of injection is divided in the cillin bottle lyophilization, gland obtains 1000 of the lyophilized injectable powders of unit dose.
Preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.16.
Clinical applying unit dosage 20mg;
Aqueous injection, infusion solution, injectable powder are preparing the application that treats and/or prevents in cardiovascular and cerebrovascular disease, hepatic injury, hepatic fibrosis, pulmonary fibrosis, tumor, the old and feeble medicine.
Embodiment 8
The danshen root salvianolic acid A preparation method:
Radix Salviae Miltiorrhizae extracts with 10% alcoholic solution and obtains alcohol extract, and alcohol extract concentrates ethanol to the greatest extent, transfers pH value to 5.5,125 ℃ of temperature, gauge pressure 0.15MPa pressure, heats 5.5 hours; Solution filters, and filtrate is separated through the D101 macroporous resin column chromatography, and first water, 25% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 45% concentration is collected eluent, reclaims ethanol to most; Concentrated solution separates with the sephadex lh-20 chromatographic column, and first water, 45% alcoholic solution eluting discard eluent, and reuse 90% alcoholic solution eluting is collected eluent, and eluent reclaims ethanol extremely to the greatest extent; Concentrated solution is transferred pH value to 4.5, through the extraction of organic solvent propyl acetate, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
The salvianolic acid B of Radix Salviae Miltiorrhizae preparation method:
Radix Salviae Miltiorrhizae obtains aqueous extract with water extraction, and concentrated solution separates through the HPD-400 macroporous resin column chromatography, and first water eluting is removed impurity, and reuse 25% Diluted Alcohol eluting is collected eluent, and eluent reclaims ethanol to most; Concentrated solution is transferred pH value to 3.5, through the organic solvent n-butyl acetate extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get salvianolic acid B of Radix Salviae Miltiorrhizae;
Formulation preparation:
Preparation raw material medicine: danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae;
Danshen root salvianolic acid A purity 95.2%, salvianolic acid B of Radix Salviae Miltiorrhizae purity 94.7%, composite preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.35.
The preparation of aqueous injection: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively fully, transfer pH value, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters merging filtrate, add vitamin C, filtrate adds injection water, fine straining, embedding, sterilization obtains 1000 of unit dose aqueous injection;
The preparation of infusion solution: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, add isoosmotic adjusting agent sodium chloride or glucose, fine straining, fill, sterilization obtains 1000 bottles of the infusion solutions of unit dose;
The preparation of injectable powder: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, aseptic filtration or carry out autoclaving according to the requirement of injection is divided in the cillin bottle lyophilization, gland obtains 1000 of the lyophilized injectable powders of unit dose.
Preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A 0.34.
Clinical applying unit dosage 350mg;
Aqueous injection, infusion solution, injectable powder are preparing the application that treats and/or prevents in cardiovascular and cerebrovascular disease, hepatic injury, hepatic fibrosis, pulmonary fibrosis, tumor, the old and feeble medicine.
Claims (7)
1. pharmaceutical composition that is used for ejection preparation, compositions contains danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, danshen root salvianolic acid A purity is more than or equal to 80% and less than 100%, salvianolic acid B of Radix Salviae Miltiorrhizae purity is more than or equal to 80% and less than 100%, it is characterized in that pharmaceutical composition carries out high effective liquid chromatography for measuring, obtain 2 characteristic peaks, one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A are greater than 0.25 and smaller or equal to 0.38; Preparation of pharmaceutical compositions becomes aqueous injection, infusion solution, lyophilized injectable powder, preparation carries out high effective liquid chromatography for measuring, obtains 2 characteristic peaks, and one is salvianolic acid B, another salvianolic acid A, the peak area of salvianolic acid B and the peak area ratio of salvianolic acid A are greater than 0.25 and smaller or equal to 0.38; Wherein pharmaceutical composition, preparation high-efficient liquid phase chromatogram process measuring method are:
Chromatographic condition and system suitability experiment: with the octadecylsilane chemically bonded silica is filler; Flow velocity 1.0ml/min; 35 ℃ of column temperatures; Detect wavelength 286nm; Number of theoretical plate should be not less than 60000 by salvianolic acid A; With acetonitrile-0.2% aqueous acetic acid is mobile phase, carries out gradient elution by following condition of gradient elution, moves 90 minutes;
In the time of 0-15 minute, the ratio of acetonitrile reduces to 80% by 10% ratio that rises to 20%, 0.2% aqueous acetic acid by 90%; In the time of 15-55 minute, the ratio of acetonitrile reduces to 70% by 20% ratio that rises to 30%, 0.2% aqueous acetic acid by 80%; In the time of 55-65 minute, the ratio of acetonitrile reduces to 50% by 30% ratio that rises to 50%, 0.2% aqueous acetic acid by 70%; In the time of 65-72 minute, the ratio of acetonitrile reduces to 20% by 50% ratio that rises to 80%, 0.2% aqueous acetic acid by 50%; In the time of 72-77 minute, the ratio 20% of ratio 80%, 0.2% aqueous acetic acid of acetonitrile; In the time of 77-80 minute, the ratio of acetonitrile rises to 90% by 80% ratio of reducing to 10%, 0.2% aqueous acetic acid by 20%; In the time of 80-90 minute, keep acetonitrile-0.2% aqueous acetic acid to carry out eluting with the ratio of 10:90;
The preparation of reference substance solution: precision takes by weighing salvianolic acid A, salvianolic acid B reference substance in volumetric flask, adds dissolve with methanol and shakes up, and be diluted to scale;
The preparation of sample solution: precision takes by weighing or measures pharmaceutical composition, formulation samples, adds dissolve with methanol and shakes up, and be diluted to scale;
Algoscopy: the accurate respectively reference substance solution of drawing, inject chromatograph of liquid, the record chromatogram; Accurate pharmaceutical composition, the formulation samples solution drawn injects chromatograph of liquid, calculates peak area ratio.
2. a kind of preparation of drug combination method that is used for ejection preparation according to claim 1 is:
The danshen root salvianolic acid A preparation method:
Radix Salviae Miltiorrhizae water or alcoholic solution extract and obtain aqueous extract or alcohol extract, alcohol extract concentrates ethanol to most, transfer pH value to 7.5-9.0,30-80 ℃ temperature, heating 1-6 hours or transfer pH value to 3.5-6.0,110-130 ℃ temperature, gauge pressure 0.05MPa-0.17MPa pressure, heated 1-6 hours; Solution filters, and filtrate is separated through nonpolar or low pole macroporous resin column chromatography, and first water, 10-30% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 30-70% concentration is collected eluent, reclaims ethanol to most; Concentrated solution separates with sephadex lh-20 or polyamide chromatography post, uses the eluant eluting, collects eluent, and eluent reclaims eluant to most; Concentrated solution is transferred pH value to 2-5, through organic solvent extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
The salvianolic acid B of Radix Salviae Miltiorrhizae preparation method:
Radix Salviae Miltiorrhizae water or alcoholic solution extract and obtain aqueous extract or alcohol extract, and alcohol extract concentrates ethanol to most, and concentrated solution separates through nonpolar or low pole macroporous resin column chromatography, elder generation's water eluting is removed impurity, reuse 10-30% Diluted Alcohol eluting, collect eluent, eluent reclaims ethanol to most; Concentrated solution is transferred pH value to 1-4, through organic solvent extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get salvianolic acid B of Radix Salviae Miltiorrhizae;
Formulation preparation:
Preparation raw material medicine: danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae;
The preparation of aqueous injection: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively fully, transfer pH value, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters merging filtrate, add vitamin C, filtrate adds injection water, fine straining, embedding, sterilize, obtain the aqueous injection of unit dose;
The preparation of infusion solution: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, add isoosmotic adjusting agent sodium chloride or glucose, fine straining, fill, sterilize, obtain the infusion solution of unit dose;
The preparation of injectable powder: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae respectively and add the dissolving of injection water, add the vitamin C dissolving respectively and transfer pH value fully, add 0.5% active carbon, heating in water bath 30 minutes, the cooling back filters, and merging filtrate adds vitamin C, filtrate adds the injection water, aseptic filtration or carry out autoclaving according to the requirement of injection is divided in the cillin bottle lyophilization, gland obtains the lyophilized injectable powder of unit dose.
3. the preparation method of ejection preparation according to claim 2, wherein said macroporous resin column are HPD-100, HPD-100A, HPD-300, HPD-400, HPD-400A, HPD-450, D101,1300-I, 1400 or AB-8.
4. the preparation method of ejection preparation according to claim 2, wherein said sephadex lh-20 or polyamide chromatography post separate, and first water, 20-50% alcoholic solution eluting discard eluent, reuse 50-95% alcoholic solution eluting.
5. the preparation method of ejection preparation according to claim 2, wherein said organic solvent are selected from a kind of in ethyl acetate, propyl acetate, butyl acetate, n-butyl alcohol, the isopropyl alcohol.
6. the preparation method of ejection preparation according to claim 2, wherein said unit dose is 5-500mg.
7. the preparation method of ejection preparation according to claim 2, wherein said unit dose is 10-200mg.
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| JPH02215713A (en) * | 1989-02-16 | 1990-08-28 | Taisho Pharmaceut Co Ltd | Antiulcer grug |
| CN1513848A (en) * | 2003-08-19 | 2004-07-21 | 江苏扬子江药业集团有限公司 | Method of extracting phenolic components from chinese medicine red sage root and its freeze dried powder injection agent |
| CN1240695C (en) * | 2003-09-29 | 2006-02-08 | 华东理工大学 | Technical method for extracting salvianolic acid B from radix salivae miltiorrhizae |
| CN1788748A (en) * | 2004-12-17 | 2006-06-21 | 雅安三九药业有限公司 | Injection formulation containing raw material herb red sage root and its quality control method |
| CN1830947A (en) * | 2006-04-21 | 2006-09-13 | 王国振 | Method for extracting 'Danfen' phenolic acid-A |
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2007
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Patent Citations (5)
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|---|---|---|---|---|
| JPH02215713A (en) * | 1989-02-16 | 1990-08-28 | Taisho Pharmaceut Co Ltd | Antiulcer grug |
| CN1513848A (en) * | 2003-08-19 | 2004-07-21 | 江苏扬子江药业集团有限公司 | Method of extracting phenolic components from chinese medicine red sage root and its freeze dried powder injection agent |
| CN1240695C (en) * | 2003-09-29 | 2006-02-08 | 华东理工大学 | Technical method for extracting salvianolic acid B from radix salivae miltiorrhizae |
| CN1788748A (en) * | 2004-12-17 | 2006-06-21 | 雅安三九药业有限公司 | Injection formulation containing raw material herb red sage root and its quality control method |
| CN1830947A (en) * | 2006-04-21 | 2006-09-13 | 王国振 | Method for extracting 'Danfen' phenolic acid-A |
Non-Patent Citations (2)
| Title |
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| 丹酚酸A/丹酚酸B不同配比对大鼠心肌缺血再灌注性损伤的保护作用. 王国振，严玉平，朱长福.河北中医药学报，第21卷第2期. 2006 |
| 丹酚酸A/丹酚酸B不同配比对大鼠心肌缺血再灌注性损伤的保护作用. 王国振，严玉平，朱长福.河北中医药学报，第21卷第2期. 2006 * |
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