CN100569219C - A kind of pharmaceutical composition that is used for oral formulations and preparation method thereof - Google Patents
A kind of pharmaceutical composition that is used for oral formulations and preparation method thereof Download PDFInfo
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- CN100569219C CN100569219C CNB200710001054XA CN200710001054A CN100569219C CN 100569219 C CN100569219 C CN 100569219C CN B200710001054X A CNB200710001054X A CN B200710001054XA CN 200710001054 A CN200710001054 A CN 200710001054A CN 100569219 C CN100569219 C CN 100569219C
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- China
- Prior art keywords
- salvianolic acid
- preparation
- radix salviae
- salviae miltiorrhizae
- peak area
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- 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 abstract description 265
- 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 abstract description 265
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Landscapes
- Medicinal Preparation (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a kind of pharmaceutical composition that is used for oral formulations, wherein 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%, be prepared into tablet, capsule, granule, pellet, drop pill, soft capsule, oral liquid, it is characterized in that pharmaceutical composition and preparation carry out high effective liquid chromatography for measuring, obtaining 2 characteristic peaks, is the peak area of object of reference with the salvianolic acid B standard substance and be that the peak area ratio of object of reference is more than or equal to 0.01 and less than 0.4 with the salvianolic acid A standard substance; By the scope that study on the stability, pharmacokinetics are investigated, pharmacodynamics is investigated, toxicology is investigated definite above-mentioned peak area ratio is science; Pharmacological evaluation shows that pharmaceutical composition and preparation in the application's peak area ratio scope have great pharmacological effects.
Description
Technical field
The present invention relates to technical field of traditional Chinese medicine pharmacy, be specifically related to the pharmaceutical composition and the oral formulations preparation method thereof of pharmaceutically active ingredient salvianolic acid A, salvianolic acid B in a kind of containing.
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: the pharmacological action in 2: 1 o'clock of the weight ratio of salvianolic acid B is best, also is from the drug effect aspect simple research have been carried out in combination.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 less than 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 oral formulations 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, adjust pH to 7.5-9.0,30-80 ℃ temperature, heating 1-6 hour or adjust pH to 3.5-6.0,110-130 ℃ temperature, gauge pressure (gauge pressure is the temperature, pressure table) 0.05MPa-0.17MPa pressure, heated 1-6 hour; 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; The concentrated solution adjust pH through organic solvent extraction, separates the organic solvent phase to 2-5, 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; The concentrated solution adjust pH through organic solvent extraction, separates the organic solvent phase to 1-4, 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 50% and less than 100%, and salvianolic acid B of Radix Salviae Miltiorrhizae purity is more than or equal to 50% 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 04.
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 less than 0.38.
Preparation tablets: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to the conventional tablet that requires to be prepared into unit dose of tablet pharmaceutics;
Capsule preparation: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to the conventional capsule that requires to be prepared into unit dose of capsule pharmaceutics;
Granule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to the conventional granule that requires to be prepared into unit dose of capsule pharmaceutics;
Soft capsule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to the conventional soft capsule that requires to be prepared into unit dose of soft capsule pharmaceutics;
Pellet: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to the conventional pellet that requires to be prepared into unit dose of pellet pharmaceutics;
Drop pill: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to the conventional drop pill that requires to be prepared into unit dose of drop pill pharmaceutics;
Oral liquid: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, learn the conventional oral liquid that requires to be prepared into unit dose according to oral liquid medicine.
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 less than 0.38.
Wherein said unit dose is 10-1000mg;
Preferred described unit dose is 20-500mg;
Unit dose is unfavorable for clinical practice less than the preparation of 5mg;
Tablet, capsule, granule, soft capsule, pellet, drop pill, oral liquid 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.
In the experiment of the application's oral solid formulation dissolution, in the time of 45 minutes, the salvianolic acid A dissolution is more than 80%, and the salvianolic acid B dissolution is more than 80%.
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 tablet, capsule, granule, soft capsule, pellet, drop pill, the oral liquid 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 oral preparation of Chinese traditional medicinal 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 10: 90 ratio;
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; Or precision measures or takes by weighing preparation, adds methanol, filters, and is 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 tablet, capsule, granule, soft capsule, pellet, drop pill, oral liquid that the application's process obtains, experimental technique carries out check and analysis according to the above analysis, obtain the peak area of danshen root salvianolic acid A and salvianolic acid B of Radix Salviae Miltiorrhizae in the preparation, calculate 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 oral formulations.
Two. study on the stability
Experimental program:
Scheme 1: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.004 and the oral formulations of preparation;
Scheme 2: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.007 and the oral formulations of preparation;
Scheme 3: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.008 and the oral formulations of preparation;
Scheme 4: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.01 and the oral formulations of preparation;
Scheme 5: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.10 and the oral formulations of preparation;
Scheme 6: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.16 and the oral formulations of preparation;
Scheme 7: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.24 and the oral formulations of preparation;
Scheme 8: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.25 and the oral formulations of preparation;
Scheme 9: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.38 and the oral formulations of preparation;
Scheme 10: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.39 and the oral formulations of preparation;
Scheme 11: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.40 and the oral formulations of preparation;
Scheme 12: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.43 and the oral formulations of preparation;
Scheme 13: salvianolic acid A;
Scheme 14: salvianolic acid B;
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 6 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 6 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 less than 0.38 good stability.
Three. pharmacokinetics is investigated
1. the investigation of bioavailability
Experimental program:
Scheme 1: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.009 and the oral formulations of preparation;
Scheme 2: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.01 and the oral formulations of preparation;
Scheme 3: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.16 and the oral formulations of preparation;
Scheme 4: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.25 and the oral formulations of preparation;
Scheme 5: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.38 and the oral formulations of preparation;
Scheme 6: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.40 and the oral formulations of preparation;
Scheme 7: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.43 and the oral formulations 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, before the gastric infusion medicine, get blank blood, in taking medicine back 15min, 30min, 45min, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h and 12h extracting vein blood 0.3ml in anticoagulant tube, separated plasma, calculate the salvianolic acid A of different peak area ratios and salvianolic acid B pharmaceutical composition, preparation bioavailability, experimental result sees Table 3:
The bioavailability of different peak area ratio salvianolic acid As of table 3 and salvianolic acid B
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 bioavailability 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 good bioavailability; When salvianolic acid B and salvianolic acid A peak area ratio greater than 0.4 the time, pharmaceutical composition and preparation bioavailability 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 good bioavailability 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 good bioavailability less than 0.38.
Four. pharmacodynamics is investigated
Experimental program:
Scheme 1: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.007 and the oral formulations of preparation;
Scheme 2: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.01 and the oral formulations of preparation;
Scheme 3: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.16 and the oral formulations of preparation;
Scheme 4: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.25 and the oral formulations of preparation;
Scheme 5: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.38 and the oral formulations of preparation;
Scheme 6: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.40 and the oral formulations of preparation;
Scheme 7: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.43 and the oral formulations 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, FUFANG DANSHEN DIWAN matched group, experimental program group (32mg/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, irritate stomach and give corresponding medicinal liquid, recording ecg behind the administration 30min, 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 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 Composite Salvia Dropping Pill group.
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 is seen in this please the description result see 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 Composite Salvia Dropping Pill group.
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, Composite Salvia Dropping Pill group, the application's scheme group, each dosage group was irritated stomach successive administration 3 days, 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, place 120 ℃ of vacuum to dry 12h in dry device in brain and claim dry weight.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 Composite Salvia Dropping Pill group.
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 less than 0.38; Show that by above-mentioned pharmacological evaluation the tasty and refreshing formulation in this Shen 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 less than 0.38.
Five. toxicologic study
Scheme 1: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.007 and the oral formulations of preparation;
Scheme 2: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.01 and the oral formulations of preparation;
Scheme 3: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.16 and the oral formulations of preparation;
Scheme 4: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.25 and the oral formulations of preparation;
Scheme 5: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.38 and the oral formulations of preparation;
Scheme 6: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.40 and the oral formulations of preparation;
Scheme 7: the pharmaceutical composition of salvianolic acid B and salvianolic acid A peak area ratio 0.43 and the oral formulations of preparation;
Experimental technique: the pharmaceutical composition of above-mentioned different schemes and preparation, carry out toxicological experiment, measure the LD of mice single oral administration 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 912mg/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 less than 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 less than 0.38.
Preparation embodiment
Embodiment 1
The danshen root salvianolic acid A preparation method:
Radix Salviae Miltiorrhizae obtains aqueous extract with water extraction, adjust pH 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 adjust pH 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 70% 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 adjust pH 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 50.2%, salvianolic acid B of Radix Salviae Miltiorrhizae purity 50.0%, 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.
Preparation tablets: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 in the conventional tablet that requires to be prepared into unit dose of tablet pharmaceutics; Clinical applying unit dosage 10mg;
Capsule preparation: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional capsules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 10mg;
Granule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 bags of the conventional granules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 10mg;
Soft capsule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional soft capsules that requires to be prepared into unit dose of soft capsule pharmaceutics; Clinical applying unit dosage 10mg;
Pellet: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional pellet 10000 balls that require to be prepared into unit dose of pellet pharmaceutics; Clinical applying unit dosage 10mg;
Drop pill: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional drop pill 10000 balls that require to be prepared into unit dose of drop pill pharmaceutics; Clinical applying unit dosage 10mg;
Oral liquid: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, learn 1000 bottles of the conventional oral liquids that requires to be prepared into unit dose according to oral liquid medicine.Clinical applying unit dosage 10mg;
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;
Tablet, capsule, granule, pellet, drop pill, soft capsule, oral liquid 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 50% alcoholic solution and obtains alcohol extract, and alcohol extract concentrates ethanol to the greatest extent, adjust pH 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 adjust pH 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 adjust pH 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.
Preparation tablets: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 in the conventional tablet that requires to be prepared into unit dose of tablet pharmaceutics; Clinical applying unit dosage 100mg;
Capsule preparation: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional capsules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 1000mg;
Granule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 bags of the conventional granules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 1000mg;
Soft capsule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional soft capsules that requires to be prepared into unit dose of soft capsule pharmaceutics; Clinical applying unit dosage 1000mg;
Pellet: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional pellet 10000 balls that require to be prepared into unit dose of pellet pharmaceutics; Clinical applying unit dosage 1000mg;
Drop pill: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional drop pill 10000 balls that require to be prepared into unit dose of drop pill pharmaceutics; Clinical applying unit dosage 1000mg;
Oral liquid: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, learn 1000 bottles of the conventional oral liquids that requires to be prepared into unit dose according to oral liquid medicine.Clinical applying unit dosage 1000mg;
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.
Tablet, capsule, granule, pellet, drop pill, soft capsule, oral liquid 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, adjust pH 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 adjust pH 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 85% 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 adjust pH 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 62.3%, salvianolic acid B of Radix Salviae Miltiorrhizae purity 68.0%, 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.
Preparation tablets: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 in the conventional tablet that requires to be prepared into unit dose of tablet pharmaceutics; Clinical applying unit dosage 500mg;
Capsule preparation: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional capsules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 500mg;
Granule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 bags of the conventional granules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 500mg;
Soft capsule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional soft capsules that requires to be prepared into unit dose of soft capsule pharmaceutics; Clinical applying unit dosage 500mg;
Pellet: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional pellet 10000 balls that require to be prepared into unit dose of pellet pharmaceutics; Clinical applying unit dosage 500mg;
Drop pill: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional drop pill 10000 balls that require to be prepared into unit dose of drop pill pharmaceutics; Clinical applying unit dosage 500mg;
Oral liquid: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, learn 1000 bottles of the conventional oral liquids that requires to be prepared into unit dose according to oral liquid medicine.Clinical applying unit dosage 500mg;
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 0.14.
Tablet, capsule, granule, pellet, drop pill, soft capsule, oral liquid 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 80% alcoholic solution and obtains alcohol extract, and alcohol extract concentrates ethanol to the greatest extent, and adjust pH to 3.5,110 ℃ of temperature, gauge pressure 0.05MPa pressure heated 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 adjust pH 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 adjust pH 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 85.1%, salvianolic acid B of Radix Salviae Miltiorrhizae purity 86.7%, 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.
Preparation tablets: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 in the conventional tablet that requires to be prepared into unit dose of tablet pharmaceutics; Clinical applying unit dosage 780mg;
Capsule preparation: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional capsules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 780mg;
Granule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 bags of the conventional granules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 780mg;
Soft capsule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional soft capsules that requires to be prepared into unit dose of soft capsule pharmaceutics; Clinical applying unit dosage 780mg;
Pellet: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional pellet 10000 balls that require to be prepared into unit dose of pellet pharmaceutics; Clinical applying unit dosage 780mg;
Drop pill: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional drop pill 10000 balls that require to be prepared into unit dose of drop pill pharmaceutics; Clinical applying unit dosage 780mg;
Oral liquid: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, learn 1000 bottles of the conventional oral liquids that requires to be prepared into unit dose according to oral liquid medicine.Clinical applying unit dosage 780mg;
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 0.22.
Tablet, capsule, granule, pellet, drop pill, soft capsule, oral liquid 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, and adjust pH to 6.0,130 ℃ of temperature, gauge pressure 0.17MPa pressure heated 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 adjust pH 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 adjust pH 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 75.0%, salvianolic acid B of Radix Salviae Miltiorrhizae purity 74.1%, 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 0.28.
Preparation tablets: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 in the conventional tablet that requires to be prepared into unit dose of tablet pharmaceutics; Clinical applying unit dosage 20mg;
Capsule preparation: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional capsules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 20mg;
Granule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 bags of the conventional granules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 20mg;
Soft capsule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional soft capsules that requires to be prepared into unit dose of soft capsule pharmaceutics; Clinical applying unit dosage 20mg;
Pellet: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional pellet 10000 balls that require to be prepared into unit dose of pellet pharmaceutics; Clinical applying unit dosage 20mg;
Drop pill: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional drop pill 10000 balls that require to be prepared into unit dose of drop pill pharmaceutics; Clinical applying unit dosage 20mg;
Oral liquid: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, learn 1000 bottles of the conventional oral liquids that requires to be prepared into unit dose according to oral liquid medicine.Clinical applying unit dosage 20mg;
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.
Tablet, capsule, granule, pellet, drop pill, soft capsule, oral liquid 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 10% alcoholic solution and obtains alcohol extract, and alcohol extract concentrates ethanol to the greatest extent, and adjust pH to 5.5,120 ℃ of temperature, gauge pressure 0.12MPa pressure heated 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 adjust pH 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 20% 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 adjust pH 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 95.7%, salvianolic acid B of Radix Salviae Miltiorrhizae purity 94.0%, 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 0.38.
Preparation tablets: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 in the conventional tablet that requires to be prepared into unit dose of tablet pharmaceutics; Clinical applying unit dosage 120mg;
Capsule preparation: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional capsules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 120mg;
Granule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 bags of the conventional granules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 120mg;
Soft capsule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional soft capsules that requires to be prepared into unit dose of soft capsule pharmaceutics; Clinical applying unit dosage 120mg;
Pellet: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional pellet 10000 balls that require to be prepared into unit dose of pellet pharmaceutics; Clinical applying unit dosage 120mg;
Drop pill: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional drop pill 10000 balls that require to be prepared into unit dose of drop pill pharmaceutics; Clinical applying unit dosage 120mg;
Oral liquid: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, learn 1000 bottles of the conventional oral liquids that requires to be prepared into unit dose according to oral liquid medicine.Clinical applying unit dosage 120mg;
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 0.37.
Tablet, capsule, granule, pellet, drop pill, soft capsule, oral liquid 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, adjust pH 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 adjust pH 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 60% 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 adjust pH 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 65.9%, salvianolic acid B of Radix Salviae Miltiorrhizae purity 72.3%, 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 0.18.
Preparation tablets: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 in the conventional tablet that requires to be prepared into unit dose of tablet pharmaceutics; Clinical applying unit dosage 300mg;
Capsule preparation: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional capsules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 300mg;
Granule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 bags of the conventional granules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 300mg;
Soft capsule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional soft capsules that requires to be prepared into unit dose of soft capsule pharmaceutics; Clinical applying unit dosage 300mg;
Pellet: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional pellet 10000 balls that require to be prepared into unit dose of pellet pharmaceutics; Clinical applying unit dosage 300mg;
Drop pill: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional drop pill 10000 balls that require to be prepared into unit dose of drop pill pharmaceutics; Clinical applying unit dosage 300mg;
Oral liquid: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, learn 1000 bottles of the conventional oral liquids that requires to be prepared into unit dose according to oral liquid medicine.Clinical applying unit dosage 300mg;
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.
Tablet, capsule, granule, pellet, drop pill, soft capsule, oral liquid 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 40% alcoholic solution and obtains alcohol extract, and alcohol extract concentrates ethanol to the greatest extent, and adjust pH to 5.5,125 ℃ of temperature, gauge pressure 0.15MPa pressure heated 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 adjust pH 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 adjust pH 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 0.34.
Preparation tablets: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 in the conventional tablet that requires to be prepared into unit dose of tablet pharmaceutics; Clinical applying unit dosage 80mg;
Capsule preparation: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional capsules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 80mg;
Granule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 bags of the conventional granules that requires to be prepared into unit dose of capsule pharmaceutics; Clinical applying unit dosage 80mg;
Soft capsule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to 1000 of the conventional soft capsules that requires to be prepared into unit dose of soft capsule pharmaceutics; Clinical applying unit dosage 80mg;
Pellet: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional pellet 10000 balls that require to be prepared into unit dose of pellet pharmaceutics; Clinical applying unit dosage 80mg;
Drop pill: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to conventional drop pill 10000 balls that require to be prepared into unit dose of drop pill pharmaceutics; Clinical applying unit dosage 80mg;
Oral liquid: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, learn 1000 bottles of the conventional oral liquids that requires to be prepared into unit dose according to oral liquid medicine.Clinical applying unit dosage 80mg;
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 0.33.
Tablet, capsule, granule, pellet, drop pill, soft capsule, oral liquid 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 (6)
1. pharmaceutical composition that is used for oral formulations, wherein compositions contains danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, and wherein danshen root salvianolic acid A purity is more than or equal to 50% and less than 100%, and salvianolic acid B of Radix Salviae Miltiorrhizae purity is more than or equal to 50% 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, 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 less than 0.38; Wherein preparation of pharmaceutical compositions becomes tablet, capsule, granule, pellet, soft capsule, oral liquid, preparation 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 more than or equal to 0.25 and less than 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 10: 90 ratio;
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;
Wherein the preparation method of preparation 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, adjust pH to 7.5-9.0,30-80 ℃ temperature, heating 1-6 hour or adjust pH to 3.5--6.0,110-130 ℃ temperature, gauge pressure 0.05MPa-0.17MPa pressure, heated 1-6 hour; 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 liquid adjust pH through organic solvent extraction, separates the organic solvent phase to 2-5, 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; The concentrated solution adjust pH through organic solvent extraction, separates the organic solvent phase to 1-4, 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;
Preparation tablets: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to the conventional tablet that requires to be prepared into unit dose of tablet pharmaceutics;
Capsule preparation: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to the conventional capsule that requires to be prepared into unit dose of capsule pharmaceutics;
Granule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to the conventional granule that requires to be prepared into unit dose of capsule pharmaceutics;
Soft capsule: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to the conventional soft capsule that requires to be prepared into unit dose of soft capsule pharmaceutics;
Pellet: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to the conventional pellet that requires to be prepared into unit dose of pellet pharmaceutics;
Drop pill: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, according to the conventional drop pill that requires to be prepared into unit dose of drop pill pharmaceutics;
Oral liquid: get danshen root salvianolic acid A, salvianolic acid B of Radix Salviae Miltiorrhizae, learn the conventional oral liquid that requires to be prepared into unit dose according to oral liquid medicine.
2. a kind of pharmaceutical composition that is used for oral formulations according to claim 1, macroporous resin column described in its preparation method are HPD-100, HPD-100A, HPD-300, HPD-400, HPD-400A, HPD-450, D101,1300-I, 1400 or AB-8.
3. a kind of pharmaceutical composition that is used for oral formulations according to claim 1, sephadex lh-20 described in its preparation method or polyamide chromatography post separate, elder generation's water, 20-50% alcoholic solution eluting discard eluent, reuse 50-95% alcoholic solution eluting.
4. a kind of pharmaceutical composition that is used for oral formulations according to claim 1, the organic solvent described in its preparation method are selected from a kind of in ethyl acetate, propyl acetate, butyl acetate, n-butyl alcohol, the isopropyl alcohol.
5. a kind of pharmaceutical composition that is used for oral formulations according to claim 1, the unit dose described in its preparation method is 10-1000mg.
6. a kind of pharmaceutical composition that is used for oral formulations according to claim 1, the unit dose described in its preparation method is 20-500mg.
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| CN103446082A (en) * | 2012-06-01 | 2013-12-18 | 天士力制药集团股份有限公司 | Application of salvianolic acid B to preparation of medicament for prevention and / or treatment of pulmonary microcirculation disorder or lung injury |
| CN102772487A (en) * | 2012-08-01 | 2012-11-14 | 辽宁盛生医药集团有限公司 | Preparation method of Salvia miltiorrhiza soft capsule |
| CN103083303B (en) * | 2012-11-20 | 2016-01-20 | 江西青峰药业有限公司 | Salvianolic acid A compositions is for the preparation of the purposes improving the function of nervous system's symptom medicine after cerebral ischemia |
| CN103083301B (en) * | 2012-11-20 | 2016-01-20 | 江西青峰药业有限公司 | Salvianolic acid A compositions is for the preparation of prevention and or the purposes for the treatment of cerebral thrombosis medicine |
| CN103083300B (en) * | 2012-11-20 | 2016-04-27 | 江西青峰药业有限公司 | Salvianolic acid A compositions is for the preparation of the purposes of protection cerebrovascular endothelial cell medicine |
| CN103083306B (en) * | 2012-11-20 | 2016-01-20 | 江西青峰药业有限公司 | Salvianolic acid A compositions is for the preparation of the purposes suppressing brain neuron damage or dead medicine |
| CN103044251B (en) * | 2012-11-20 | 2015-01-07 | 江西青峰药业有限公司 | Method for preparing salvianolic acid A |
| CN103083302B (en) * | 2012-11-20 | 2016-01-20 | 江西青峰药业有限公司 | Salvianolic acid A compositions is for the preparation of the purposes of protection ischemic tissue of brain damage medicine |
| CN103083294B (en) * | 2012-11-20 | 2016-01-06 | 江西青峰药业有限公司 | Salvianolic acid A freeze-dried powder is for the preparation of the purposes of protection ischemic tissue of brain damage medicine |
| CN103083304B (en) * | 2012-11-20 | 2016-01-20 | 江西青峰药业有限公司 | Salvianolic acid A compositions is for the preparation of the purposes preventing and/or treating cerebral thrombosis medicine |
| CN103083305B (en) * | 2012-11-20 | 2016-04-27 | 江西青峰药业有限公司 | A kind of salvianolic acid A compositions and prepare medicinal usage |
| CN102993015B (en) * | 2012-11-20 | 2015-04-08 | 江西青峰药业有限公司 | Method for purifying salvianolic acid A |
| CN110702707A (en) * | 2019-10-16 | 2020-01-17 | 四川轻化工大学 | Method for obtaining nuclear waste barrel chromatography gamma scanning image |
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