CN105866289A - Method for quickly extracting and measuring various phenolic acid components in compound radix salviae miltiorrhizae tablets - Google Patents
Method for quickly extracting and measuring various phenolic acid components in compound radix salviae miltiorrhizae tablets Download PDFInfo
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- CN105866289A CN105866289A CN201610303995.8A CN201610303995A CN105866289A CN 105866289 A CN105866289 A CN 105866289A CN 201610303995 A CN201610303995 A CN 201610303995A CN 105866289 A CN105866289 A CN 105866289A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The invention relates to a method for quickly extracting and measuring seven types of phenolic acid components in compound radix salviae miltiorrhizae tablets. The method includes grinding the compound radix salviae miltiorrhizae tablets and preparing to-be-tested substance solution from the ground compound radix salviae miltiorrhizae tablets by hydrous methanol or hydrous ethanol by the aid of microwave-assisted extraction processes; measuring contents by the aid of high-performance liquid chromatography processes, eluting the to-be-tested substance solution in a gradient manner by acetonitrile, phosphoric acid aqueous solution or formic acid aqueous solution or acetic acid aqueous solution, plotting standard curves according to the concentration and peak areas of mixed reference substance solution and quickly computing the contents of the seven components in each compound radix salviae miltiorrhizae tablet. The hydrous methanol or the hydrous ethanol is used as a solvent. The acetonitrile is used as a mobile phase A, and the phosphoric acid aqueous solution or the formic acid aqueous solution or the acetic acid aqueous solution is used as a mobile phase B. The method has the advantages that the seven types of phenolic acid components can be effectively separated from one another by the aid of the high-performance liquid chromatography processes within a short time; the various phenolic acid components in the compound radix salviae miltiorrhizae tablets are measured, accordingly, the quality of medicines can be effectively controlled, and the product quality control level can be upgraded.
Description
Technical field
The present invention relates to the detection field of a kind of Effective Component of Chinese Medicine, particularly in a kind of rapid extraction and mensuration Fufang Danshen Pian
The method of seven kinds of liposoluble ingredients.
Background technology
Fufang Danshen Pian is clinical conventional a kind of Chinese patent drug treating cardiovascular and cerebrovascular disease, and function is promoting blood circulation and removing blood stasis, regulating qi-flowing for relieving pain,
For the obstruction of qi in the chest of caused by energy stagnation and blood stasis, disease sees uncomfortable in chest, pareordia shouting pain；The diseases such as coronary disease and angina pectoris.This product has the red sage root, pseudo-ginseng
Form with borneol.Said preparation now records in the version Pharmacopoeia of the People's Republic of China in 2015.Cardiovascular and cerebrovascular disease in recent years
The incidence of disease increasingly rise, the clinical practice of Fufang Danshen Pian is the most extensive, and the market sales volume constantly increases.Said preparation at present
Description more, manufacturer is also a lot, and for ensureing curative effect and the safety of product, the quality control for medicine seems outstanding
For important.
According to drug matching rule, in Fufang Danshen Pian, the red sage root is monarch drug in a prescription, plays primary treatment effect.Containing root of red-rooted salvia phenolic acid in the red sage root
With tanshinone two class principle active component, according to the preparation technology of Fufang Danshen Pian, this two constituents is also same in Fufang Danshen Pian
Sample contains.Therefore the chemical composition coming solely from the red sage root in Fufang Danshen Pian just has multiple.For compound Danshen Root in " Chinese Pharmacopoeia "
The quality control of sheet only determines root of red-rooted salvia phenolic acid B and tanshinone IIA.The sample treatment measuring root of red-rooted salvia phenolic acid B need ultrasonic
Process 30min and carry out high effective liquid chromatography for measuring again.Measure single component and be difficult to react the feature of Chinese medicine multicomponent comprehensive function.
Root of red-rooted salvia phenolic acid constituents is a class main active in Fufang Danshen Pian, and this constituents includes salviol acid A, B, Rosmarinic acid,
The Multiple components such as alkannic acid, danshensu.Therefore, measure these compositions simultaneously and be conducive to improving the quality control water of Fufang Danshen Pian
Flat.Pharmacopeia and existing patent all use ultrasonic extraction to prepare sample, and the time is long, power consumption height, and this patent uses Microwave-assisted Extraction
Take and prepare sample and can significantly shorten the time, improve efficiency, and the HPLC analytical method optimized can also shorten mensuration
Time, reach the effect of rapid and accurate determination.
Summary of the invention
The present invention provides a kind of rapid extraction and measures the method for seven kinds of liposoluble ingredients in Fufang Danshen Pian, is remarkably improved quality
Analysis efficiency and the quality control level of product, and beneficially energy-conserving and environment-protective.
For achieving the above object, the present invention provides techniques below scheme:
A kind of method of multiple liposoluble ingredient in rapid extraction and mensuration Fufang Danshen Pian, its step is as follows:
(1) preparation is containing danshensu, protocatechualdehyde, caffeic acid, Rosmarinic acid, alkannic acid, salviol acid A and root of red-rooted salvia phenolic acid
The mixing reference substance solution of seven kinds of compositions of B；
(2) microwave-assisted extraction is used to prepare the need testing solution of Fufang Danshen Pian；
(3) reference substance solution analysis: the mixing reference substance solution injecting chromatograph described in preparation variable concentrations step (1),
Chromatogram to the mixing reference substance solution of each concentration；
(4) need testing solution analysis: by step (2) described need testing solution injecting chromatograph, obtain the chromatogram of sample solution
Figure.
(5) cubage: concentration and peak area according to mixing reference substance solution draw calibration curve, by corresponding for test sample composition
Peak area substitute into calibration curve and calculate the content of this composition in need testing solution, calculate the described seven kinds of compositions of step (1) multiple
Content in side's Danshen Tablets.
Described step 2) in microwave-assisted extraction be:
Fufang Danshen Pian is removed coating, finely ground one-tenth Fufang Danshen Pian powder, takes Fufang Danshen Pian powder, Fufang Danshen Pian powder
Adding solvent by mass volume ratio 1:60～150, stir evenly, be placed in microwave radiation exaraction device extraction, extraction conditions is: power
400～1000W, temperature 50～75 DEG C, extract 1～6min, put to room temperature after extraction；Before complementing to extract with described solvent
Fufang Danshen Pian powder and the weight of solvent, shake up, and takes subsequent filtrate as need testing solution；Described solvent is concentration 30～80%
Methanol solution or concentration are 30～80% ethanol solution.
Described step 2) and step 3) in chromatographic condition be: chromatographic column is C18 bonded silica gel post, and flow velocity is 1.00mL/min,
Sampling volume is 10～20 μ L, and detection wavelength is 280～330nm, and column temperature is 20～35 DEG C, gradient elution time 35min,
Condition of gradient elution is as follows:
During 0min, mobile phase A: Mobile phase B=13 87 (volume ratio)
During 15min, mobile phase A: Mobile phase B=23 77 (volume ratio)
During 30min, mobile phase A: Mobile phase B=30 70 (volume ratio)
During 35min, mobile phase A: Mobile phase B=100 0 (volume ratio)
Described mobile phase A is acetonitrile；
Described Mobile phase B is 0.03～the phosphate aqueous solution of 0.06% (volume ratio), 0.05～the formic acid of 0.1% (volume ratio)
The aqueous solution or 0.1～the aqueous acetic acid of 0.5% (volume ratio).
Compared with prior art, the present invention uses microwave auxiliary extraction technology quickly to prepare sample, and energy high efficiency extraction compound simultaneously is red
Joining the root of red-rooted salvia phenolic acid constituents in sheet, the time significantly shortens, and reduces energy resource consumption, beneficially energy-conserving and environment-protective.Set up
Seven kinds of liposoluble ingredients can be efficiently separated within the shorter time by efficient liquid-phase chromatography method, improves efficiency, saves organic examination
The consumption of agent.Measure the multiple liposoluble ingredient in Fufang Danshen Pian simultaneously and can better control over drug quality, improve the matter of product
Amount control level.
Accompanying drawing explanation
Fig. 1 is the liquid chromatogram of seven kinds of standard items mixed solutions.
The material that 1～No. 7 chromatographic peak is corresponding is followed successively by danshensu, protocatechualdehyde, caffeic acid, Rosmarinic acid, alkannic acid, the red sage root
Phenolic acid B, salviol acid A.
Fig. 2 is the high-efficient liquid phase chromatogram of Fufang Danshen Pian need testing solution, and 1～No. 7 chromatographic peak is corresponding to phase in standard items collection of illustrative plates
Answering the chemical composition of retention time, the material that i.e. 1～No. 7 peak is corresponding is danshensu successively, protocatechualdehyde, caffeic acid, rosemary
Acid, alkannic acid, root of red-rooted salvia phenolic acid B, salviol acid A.
Detailed description of the invention
(1) preparation of reference substance solution: precision weighs following reference substance in 10mL volumetric flask: danshensu 0.81mg, former
Catechu aldehyde 0.37mg, caffeic acid 0.49mg, Rosmarinic acid 0.90mg, alkannic acid 1.21mg, salviol acid A 0.68mg,
Root of red-rooted salvia phenolic acid B 7.50mg, adding concentration is that 60% methanol solution dissolves, and is settled to 10mL, is made into mixing reference substance solution.
(2) preparation of need testing solution: take Fufang Danshen Pian 10, removes coating, accurately weighed, finely ground.Precision weighs
Powder 0.200g puts in tool plug round-bottomed flask, and it is the methanol aqueous solution of 60% that precision adds 30mL concentration, tool plug, accurate title
Fixed.Put in microwave radiation exaraction device, microwave power 400W, temperature 60 C, extracts 2min, takes out, put to room temperature, essence
Close weighed, supply the front Fufang Danshen Pian powder of extraction and the gross weight of methanol aqueous solution with the methyl alcohol that concentration is 60%, shake up, use
0.45 μm filtering with microporous membrane, subsequent filtrate is as need testing solution.
(3) chromatographic condition: chromatographic column is C18 bonded silica gel post, mobile phase A is acetonitrile, Mobile phase B be concentration be 0.05%
Phosphate aqueous solution, sampling volume 20 μ L, column temperature 25 DEG C, flow velocity 1mL/min, detect wavelength 280nm, gradient elution
Time 35min, elution requirement is as follows:
During 0min, mobile phase A Mobile phase B=13 87 (volume ratio)
During 15min, mobile phase A Mobile phase B=23 77 (volume ratio)
During 30min, mobile phase A Mobile phase B=30 70 (volume ratio)
During 35min, mobile phase A Mobile phase B=100 0 (volume ratio)
(4) measure: by step 1) the mixing reference substance solution concentration prepared be 70% methanol solution dilute 2 successively, 5,10,
20,50,100 times, obtaining the reference substance solution of six kinds of concentration, precision draws the reference substance solution 20 μ L of variable concentrations respectively
Injecting liquid chromatograph to be analyzed measuring, with the concentration of reference substance as abscissa, corresponding chromatographic peak area is that ordinate is drawn
Calibration curve.Accurate pipette samples solution 20 μ L injects liquid chromatograph again, calculates the peak of each chromatographic peak under identical wavelength
Area, calculates the concentration of each composition in sample solution according to peak area and calibration curve, calculates seven kinds of compositions respectively at compound Danshen Root
Content in sheet.
The above is the preferred embodiments of the present invention, for the staff of the art, may according to circumstances exist
Being suitably modified on the basis of the present invention, without departing from this method, these improvement also should be regarded as protection scope of the present invention.
Claims (2)
1. a rapid extraction and the method for multiple liposoluble ingredient in mensuration Fufang Danshen Pian, it is characterised in that step is as follows:
(1) preparation is containing danshensu, protocatechualdehyde, caffeic acid, Rosmarinic acid, alkannic acid, salviol acid A and root of red-rooted salvia phenolic acid
The mixing reference substance solution of seven kinds of compositions of B；
(2) microwave-assisted extraction is used to prepare the need testing solution of Fufang Danshen Pian；
(3) reference substance solution analysis: preparation variable concentrations step 1) described in mixing reference substance solution injecting chromatograph, obtain
The chromatogram of the mixing reference substance solution of each concentration；
(4) need testing solution analysis: by step 2) described need testing solution injecting chromatograph, obtain the chromatogram of sample solution.
(5) cubage: concentration and peak area according to mixing reference substance solution draw calibration curve, by corresponding for test sample composition
Peak area substitute into calibration curve and calculate the content of this composition in need testing solution, calculate step 1) described seven kinds of compositions are multiple
Content in side's Danshen Tablets；
Described step 2) and step 3) in chromatographic condition be: chromatographic column is C18 bonded silica gel post, and flow velocity is 1.00mL/min,
Sampling volume is 10～20 μ L, and detection wavelength is 280～330nm, and column temperature is 20～35 DEG C, gradient elution time 35min,
Condition of gradient elution is as follows:
During 0min, mobile phase A: Mobile phase B=13 87 volume ratios
During 15min, mobile phase A: Mobile phase B=23 77 volume ratios
During 30min, mobile phase A: Mobile phase B=30 70 volume ratios
During 35min, mobile phase A: Mobile phase B=100 0 volume ratios
Described mobile phase A is acetonitrile；
Described Mobile phase B be concentration be 0.03～the phosphate aqueous solution of 0.06%, concentration be 0.05～the aqueous formic acid of 0.1% or
Concentration is the aqueous acetic acid of 0.1～0.5%.
The method of multiple liposoluble ingredient, its feature in a kind of rapid extraction the most as claimed in claim 1 and mensuration Fufang Danshen Pian
Be: described step 2) in microwave-assisted extraction step be:
Fufang Danshen Pian is removed coating, finely ground one-tenth Fufang Danshen Pian powder, takes Fufang Danshen Pian powder, Fufang Danshen Pian powder
Adding solvent by mass volume ratio 1:60～150, stir evenly, be placed in microwave radiation exaraction device extraction, extraction conditions is: power
400～1000W, temperature 50～75 DEG C, extract 1～6min, put to room temperature after extraction；Before complementing to extract with described solvent
Fufang Danshen Pian powder and the weight of solvent, shake up, and takes subsequent filtrate as need testing solution；Described solvent is concentration 30～80%
Methanol solution or concentration 30～80% ethanol solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107607646A (en) * | 2017-09-14 | 2018-01-19 | 神威药业集团有限公司 | A kind of platelet cell membrane chromatography |
| CN108195972A (en) * | 2018-03-15 | 2018-06-22 | 北京红太阳药业有限公司 | Method that is a kind of while measuring a variety of phenolic acid compound contents in red seven preparations |
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| CN103776908A (en) * | 2012-10-17 | 2014-05-07 | 广州白云山和记黄埔中药有限公司 | Detection method of phenolic acid compounds in compound radix salviae miltiorrhizae tablet |
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| CN103776908A (en) * | 2012-10-17 | 2014-05-07 | 广州白云山和记黄埔中药有限公司 | Detection method of phenolic acid compounds in compound radix salviae miltiorrhizae tablet |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107607646A (en) * | 2017-09-14 | 2018-01-19 | 神威药业集团有限公司 | A kind of platelet cell membrane chromatography |
| CN108195972A (en) * | 2018-03-15 | 2018-06-22 | 北京红太阳药业有限公司 | Method that is a kind of while measuring a variety of phenolic acid compound contents in red seven preparations |
| CN108195972B (en) * | 2018-03-15 | 2020-12-15 | 北京红太阳药业有限公司 | Method for simultaneously determining contents of multiple phenolic acid compounds in salvia miltiorrhiza and notoginseng preparation |
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Application publication date: 20160817 |