CN102539599A - Method for detecting liver-enhancing medicine - Google Patents
Method for detecting liver-enhancing medicine Download PDFInfo
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- CN102539599A CN102539599A CN2011104207869A CN201110420786A CN102539599A CN 102539599 A CN102539599 A CN 102539599A CN 2011104207869 A CN2011104207869 A CN 2011104207869A CN 201110420786 A CN201110420786 A CN 201110420786A CN 102539599 A CN102539599 A CN 102539599A
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Abstract
The invention provides a method for detecting a liver-enhancing medicine. The liver-enhancing medicine is composed of oriental wormwood, isatis root, angelica, white paeony root, danshen root, Radix curcumae, Astragalus mongholicus, Codonopsis pilosula, Rhizoma alismatis, sealwort, rehmannia, yam, hawthorn, large-leaved gentian, liquorice and medicated leaven. The detecting method comprises the step of detecting paeoniflorin, gentiamarin and salvianolic acid B in the liver-enhancing medicine by using a high efficiency liquid chromatography method, wherein conditions are as follows: a chromatographic column takes octadecylsilane chemically bonded silica as a filling material; mobile phases comprise a mobile phase A which is acetonitrile and a mobile phase B which is an acidic water solution, and the mobile phases are subjected to gradient elution; the flow velocity is 1.0mL/min; the column temperature is 30 DEG C; the detection wavelength is 210nm to 400nm; and the number of theoretical plates is calculated according to the paeoniflorin peak and should not be less than 6000. According to the detecting method provided by the invention, the content of active ingredients of the paeoniflorin, the gentiamarin and the salvianolic acid B in the liver-enhancing medicine can be detected at the same time, so that the active ingredients of the liver-enhancing medicine can be comprehensively represented, and the method has the characteristics of high precision, high stability and high repeatability.
Description
Technical field
The invention belongs to the Pharmaceutical Analysis detection range, relate to a kind of detection method of hepatinica thing, particularly relate to the detection method that adopts high performance liquid chromatography (HPLC) to detect the various ingredients of said hepatinica thing simultaneously.
Background technology
Herbal mixture is the principal mode of tcm clinical practice medication, is the characteristic and the marrow of traditional Chinese medicine and pharmacy.Chinese medicine be one by multicomponent, the multifactor complex system that constitutes; The diversity of its chemical constitution and complicacy are the material bases of its curative effect; Foundation meets the modern quality control system of traditional Chinese medicine characteristics; Capture the difficult problem of quality analysis of traditional Chinese medicine and evaluation, improve the problem that the existing method of quality control of Chinese medicine has become people's active research.
Foundation must be based on the characteristic of Chinese medicine to the quality control system of Chinese medicine.The mass action characteristics of herbal mixture have determined Chinese medicine to be different from Western medicine.The method of quality control of Chinese medicine must be controlled whole compositions (organic principle, inorganic constituents and complex compound composition) of onset.Only in this way, the quality control system of being set up could really reach the control traditional Chinese medicine quality, guarantee Chinese medicine purpose safe and effective for medication.It is means that traditional Chinese medicine quality control just turns to the advanced technology from simple single component content mensuration, polycomponent, many indexs Determination on content.
Present most Chinese medicine standard still adopts spectrum or chromatogram means to differentiate and measure a certain or several kinds of effective constituent or index components, and the routine inspection project of pharmacopeia regulation.For chemicals, its effective component is a clear structure simplification compound, and structure-activity relationship is clear and definite, and its content and purity are directly expressed its validity and security.Yet the characteristics of middle medical drugs are compound compatibilities, any single effectively or the content of active component height all can not express its whole curative effect.
Liver-strengthening capsule records in " national drug standards new drug become a full member standard " the 35; Form by oriental wormwood, Radix Isatidis, Radix Angelicae Sinensis, the root of herbaceous peony, the red sage root, root tuber of aromatic turmeric, the Radix Astragali, Radix Codonopsis, rhizoma alismatis, sealwort, glutinous rehmannia, Chinese yam, hawthorn, bark of ash, Radix Glycyrrhizae, Medicated Leaven 16 flavor Chinese medicines, have that clearing heat and promoting diuresis, tonifying spleen nourish blood, beneficial gas is separated strongly fragrant effect.Be used to treat chronic hepatitis, early-phase hepatocirrhosis, fatty liver, toxic hepatitis, liver fibrosis etc.
Liver-strengthening capsule is the big kind of Chinese medicine that east, Shijiazhuang pharmaceutcal corporation, Ltd produces, and proves that after deliberation liver-strengthening capsule has stronger effect of anti hepatic fibrosis, has the protection liver cell, promotes liver cell regeneration, recovering liver function and the effect of inhibition fiberization.Be used to clinically treat liver fibrosis, early-phase hepatocirrhosis, virus hepatitis, toxic hepatic disease, fatty liver etc., its result of treatment has obtained clinical checking.How guaranteeing content of effective in quality and the liver-strengthening capsule of medicine, is the basis of decision liver-strengthening capsule curative effect.Existing liver-strengthening capsule quality standard has been stipulated thin-layer chromatography discrimination method and the Paeoniflorin high performance liquid chromatography content assaying method of oriental wormwood, the root of herbaceous peony.As the big compound of Chinese medicine, only from appearance character, simply differentiate and single component assay equal angles is carried out quality control to liver-strengthening capsule, obviously can not accurately explain its inherent quality can not guarantee its curative effect comprehensively.Control the effect of liver-strengthening capsule, must its material integral body be controlled, characterize the quality of Chinese medicine on the whole effectively, set up safety, effective, stabilized quality standards system according to the characteristics of Chinese medicine polycomponent, many target spots, multipath effect.
The bibliographical information of relevant liver-strengthening capsule quality control is less.Present most detection method all only is confined to measure in the big compound of liver-strengthening capsule one, two kind of index components, but how can the macroscopic quality control method of carrying out of the liver-strengthening capsule quality of the pharmaceutical preparations not appeared in the newspapers from the angle of macroscopic view.Simultaneously; Because the raw material of liver-strengthening capsule comprises between 16 flavor Chinese medicines and the traditional Chinese medicine ingredients and has complex interactions; Cause setting up the effective constituent detection method that reflects liver-strengthening capsule inherent quality and curative effect fully and effectively and have big difficulty; Therefore, this area exists can characterize the demand of the detection method of this medicine of liver-strengthening capsule comprehensively at present.
Summary of the invention
In order to address the above problem, the purpose of this invention is to provide a kind of detection hepatinica thing, like the method for liver-strengthening capsule content, medicine activity component and content thereof that this method can complete detection hepatinica thing, thus characterize and control its inherent quality.
The objective of the invention is to realize through following technical scheme:
On the one hand; The invention provides a kind of detection method of hepatinica thing; Said hepatinica thing is formed (liver-strengthening capsule for example by oriental wormwood, Radix Isatidis, Radix Angelicae Sinensis, the root of herbaceous peony, the red sage root, root tuber of aromatic turmeric, the Radix Astragali, Radix Codonopsis, rhizoma alismatis, sealwort, glutinous rehmannia, Chinese yam, hawthorn, bark of ash, Radix Glycyrrhizae, Medicated Leaven; Record in " national drug standards new drug become a full member standard " the 35); Said detection method comprises that the employing high performance liquid chromatography detects Paeoniflorin, gentiamarin and tanshin polyphenolic acid B in the said hepatinica thing simultaneously, and wherein, the condition of said high performance liquid chromatography comprises:
Chromatographic column: with the octadecylsilane chemically bonded silica is packing material;
Moving phase: mobile phase A is an acetonitrile, and Mobile phase B is 0.05% volume fraction phosphate aqueous solution, carries out gradient elution;
Said gradient elution program is following, and wherein the moving phase ratio is percent by volume:
0～40min, mobile phase A is 10～35%, Mobile phase B is 90%～65%;
40～50min, mobile phase A is 35%～100%, Mobile phase B is 65%～0%;
50～51min, mobile phase A is 100%～10%, Mobile phase B is 0%～90%;
51～55min, mobile phase A is 10%～10%, Mobile phase B is 90%～90%.
Preferably, the condition of said high performance liquid chromatography also comprises:
Flow velocity: 1.0mL/min;
Column temperature: 30 ℃;
Detect wavelength: 210nm～400nm;
Theoretical cam curve is pressed the Paeoniflorin peak and is calculated, and should be not less than 6000.
Wherein, Said detection method comprises through following steps and prepares reference substance solution: get Paeoniflorin, gentiamarin and tanshin polyphenolic acid B reference substance; Add dissolve with methanol and process the solution that every 1ml contains Paeoniflorin 0.06mg, gentiamarin 0.03mg and tanshin polyphenolic acid B 0.05mg, as mixing reference substance solution.
And said detection method also comprises through following steps and prepares need testing solution: get the about 0.3g of said hepatinica thing composition, place conical flask or 25ml volumetric flask, accurately add 70% volume fraction methanol aqueous solution 25mL; Weigh, sonicated or reflux 30min are put to room temperature; Weigh, supply weight with 70% volume fraction methanol aqueous solution again, shake up; Miillpore filter filters, and discards filtrating just, gets subsequent filtrate and promptly gets.
Preferably, said high performance liquid chromatography may further comprise the steps:
1) preparation mixes reference substance solution: it is an amount of to get Paeoniflorin reference substance, gentiamarin and tanshin polyphenolic acid B reference substance; Add dissolve with methanol and be diluted to the mixed solution that every 1ml contains Paeoniflorin 0.06mg, gentiamarin 0.03mg and tanshin polyphenolic acid B 0.05mg, as mixing reference substance solution;
2) preparation need testing solution: get the about 0.3g of said hepatinica thing, precision is weighed, and places conical flask or 25ml volumetric flask, accurately adds 70% volume fraction methanol aqueous solution 25mL; Weigh, sonicated or reflux 30min are put to room temperature; Weigh, supply weight, shake up with 70% volume fraction methanol aqueous solution; Miillpore filter filters, and discards filtrating just, gets subsequent filtrate and promptly gets;
3) measure: accurate absorption mixes reference substance solution and each 10 μ l of need testing solution inject high performance liquid chromatograph, measures according to following chromatographic condition:
Chromatographic column: with the octadecylsilane chemically bonded silica is filler;
Moving phase: mobile phase A is an acetonitrile, and Mobile phase B is 0.05% volume fraction phosphate aqueous solution, carries out gradient elution;
Flow velocity: 1.0mL/min;
Column temperature: 30 ℃;
Ultraviolet detection wavelength: 230nm;
The record peak area adopts external standard method to calculate Paeoniflorin, gentiamarin and tanshin polyphenolic acid B.
Said gradient elution program is following, and wherein the moving phase ratio is percent by volume:
0～40min, mobile phase A is 10～35%, Mobile phase B is 90%～65%;
40～50min, mobile phase A is 35%～100%, Mobile phase B is 65%～0%;
50～51min, mobile phase A is 100%～10%, Mobile phase B is 0%～90%;
51～55min, mobile phase A is 10%～10%, Mobile phase B is 90%～90%.
Adopt said method to measure the multi-target ingredient in the liver-strengthening capsule sample to be measured, the gentiamarin of product to be measured, Paeoniflorin, content of danshinolic acid B scope should be following ranges: gentiamarin 0.30～0.70%, Paeoniflorin 0.15～0.32%, tanshin polyphenolic acid B 0.20～0.80%.
It below is detailed description of the present invention.
Chromatographic condition
Compared methanol-water and acetonitrile-water system, the result shows that acetonitrile-water system eluting power is stronger, and separation efficiency is higher, and velocity of separation is very fast, so select the acetonitrile-water system to separate.Simultaneously, survey and contain the phenolic acids water soluble ingredient in the index components, the moving phase of acidifying can suppress liposoluble ingredient dissociates, and reduces the chromatographic peak hangover.Therefore, finally selecting acetonitrile-0.05% volume fraction phosphate aqueous solution is moving phase.Because the polarity difference of each index components is bigger, must adopt the method for gradient elution to carry out compartment analysis.In the test, investigated different gradient, confirmed the condition of gradient elution of liver-strengthening capsule: 0～40min at last, mobile phase A is 10～35%, and Mobile phase B is 90%～65%; 40～50min, mobile phase A is 35%～100%, Mobile phase B is 65%～0%; 50～51min, mobile phase A is 100%～10%, Mobile phase B is 0%～90%; 51～55min, mobile phase A is 10%～10%, Mobile phase B is 90%～90%.
Need testing solution adopts PDAD (DAD) in 210～400nm wavelength coverage, to carry out full wavelength scanner, and the chromatogram under each wavelength is analyzed comparison.Adopt ultraviolet spectrophotometer respectively gentiamarin, Paeoniflorin, 3 reference substances of tanshin polyphenolic acid B to be scanned simultaneously, the maximum absorption wavelength of gentiamarin, Paeoniflorin, tanshin polyphenolic acid B is respectively 271.5nm, 230.5nm and 288.0nm place as a result.Adopt the DAD detecting device that sample is carried out the full wavelength scanner of 200～400nm, the chromatogram under each wavelength is analyzed comparison.Take all factors into consideration, under the 230nm, each composition characteristics peak is obvious, and the peak type is better, suitable many indexs assay.So confirm that 230nm is for detecting wavelength.
Mix the preparation of reference substance solution
Respectively accurate to claim to decide gentiamarin, Paeoniflorin, tanshin polyphenolic acid B an amount of, with dissolve with methanol and be diluted to scale, is the mixing reference substance solution.
The need testing solution preparation
The preparation method who has confirmed need testing solution in this method of quality control is investigated in test through repeatedly; 70% volume fraction ethanol water, 50% volume fraction methanol aqueous solution have been compared with 50% volume fraction ethanol water in test; 70% volume fraction methanol aqueous solution; Methyl alcohol is done and is extracted the influence of solvent to assay, and it is good to confirm to make the solvent effect with 70% methanol aqueous solution, compares with backflow Different Extraction Method and different extraction times ultrasonic simultaneously.The result shows and used the ultrasonic or reflux of 70% volume fraction methanol aqueous solution 30 minutes, and the gained chromatographic peak is more, and peak area is bigger.Because these article flavour of a drug are many; Extraction process is a decocting; These article of considering macromolecular substances is more, and the chromatographic column load is bigger, so this experiment is also investigated sample removal of impurities disposal route; Investigate with the extraction efficiency influence to each index components of normal butyl alcohol, ethyl acetate extraction and solution pH value, the result is undesirable.Confirm as finally that to get the liver-strengthening capsule content an amount of, precision is weighed, and puts in the conical flask, adds 70% volume fraction methanol aqueous solution, weighs; Sonicated or reflux are taken out, and put to room temperature, supply weight, shake up; Filtering with microporous membrane discards filtrating just, gets subsequent filtrate, promptly gets.
The present invention has carried out the specificity investigation.Take by weighing the about 0.3g of negative control article that lacks bark of ash, the root of herbaceous peony, red rooted salvia by liver-strengthening capsule prescription and extraction process preparation; Press need testing solution preparation method preparation; Then under the chromatographic condition of confirming; Measure and mix reference substance solution, need testing solution and negative control article solution injection high performance liquid chromatograph, record HPLC figure sees accompanying drawing 1,2,3.3 chromatograms are analyzed contrast; In the test sample chromatogram, on the position of gentiamarin, Paeoniflorin, the corresponding retention time of tanshin polyphenolic acid B reference substance chromatographic peak, there is corresponding composition chromatographic peak to occur; And in the negative control chromatogram; Do not have corresponding composition chromatographic peak and occur, point out this method noiseless, prove that many index determinings method of the present invention has specificity the assay of gentiamarin, Paeoniflorin, tanshin polyphenolic acid B.
Adopt described liver-strengthening capsule multi-target ingredient assay method; Like the content of gentiamarin, Paeoniflorin, tanshin polyphenolic acid B in the product of measuring to be measured in following ranges: gentiamarin 0.30～0.70%, Paeoniflorin 0.15～0.32%, tanshin polyphenolic acid B 0.20～0.80% then are specification product.
Compared with prior art, hepatinica object detecting method provided by the invention has following good effect:
At first, liver-strengthening capsule content involved in the present invention is that 16 flavor Chinese medicines are formed the contained complex chemical composition of each medicinal material; Each composition causes interference each other during to content detection; When adopting HPLC to detect, cause other compositions that each index components is disturbed, make the content results poor repeatability; So the chromatographic condition of necessary strict control moving phase just can make each index components obtain good characteristic peak.Experiment showed, to have only and adopt the present invention, just can obtain effective constituent gentiamarin, the Paeoniflorin of this medicine, the characteristic peak of tanshin polyphenolic acid B, thereby realized effective separation of characteristic peak through resulting practicable, the specific chromatographic conditions of a large amount of experiments.
Secondly; Method of the present invention can for example main effective constituent gentiamarin, Paeoniflorin, the tanshin polyphenolic acid B of liver-strengthening capsule carry out content detection to the hepatinica thing simultaneously; Thereby realize the maximum possible chemical constitution of hepatinica thing is detected; Help monitoring in all directions, so that the method for quality control of hepatinica thing is more perfect to its quality.
And the present invention has that method is easy, stable, precision is high, favorable reproducibility, the characteristics that are easy to grasp.Under same test condition, realized the for example assay of gentiamarin, Paeoniflorin, tanshin polyphenolic acid B in the liver-strengthening capsule of hepatinica thing simultaneously.
Description of drawings
Below, specify embodiment of the present invention in conjunction with accompanying drawing, wherein:
Fig. 1 is for mixing the HPLC figure of reference substance, and wherein 1 is gentiamarin, and 2 is Paeoniflorin, and 3 is tanshin polyphenolic acid B;
Fig. 2 is the HPLC figure of liver-strengthening capsule content, and wherein 1 is gentiamarin, and 2 is Paeoniflorin, and 3 is tanshin polyphenolic acid B;
The negative reference substance HPLC figure of Fig. 3;
Fig. 4 to 6 is respectively the HPLC figure of the condition of gradient elution 1 to 3 of test among the embodiment 5;
Fig. 7 adopts national drug standards WS among the embodiment 6
3The moving phase of chromatographic condition detects the HPLC figure that liver-strengthening capsule obtains in-133 (Z-133)-2002 (Z).
Embodiment
The concrete embodiment of following reference explains the present invention.It will be appreciated by those skilled in the art that these embodiment only are used to explain the present invention, the scope that it does not limit the present invention in any way.
Below employed reference substance source is as follows among each embodiment:
Gentiamarin, available from Nat'l Pharmaceutical & Biological Products Control Institute, lot number 110770-200712 supplies assay usefulness, and content is in 99%; Paeoniflorin, available from Nat'l Pharmaceutical & Biological Products Control Institute, lot number 0736-9710; Tanshin polyphenolic acid B is available from Tianjin one side Science and Technology Ltd..
Below among each embodiment employed test sample liver-strengthening capsule for the inventor and east, Shijiazhuang pharmaceutcal corporation, Ltd according to the 35th WS3-133 (Z-133)-2002 (Z) preparation of the national drug standards (new drug become a full member standard), place of production attribute, collecting season characteristic, the technology controlling and process characteristic of each medicinal material in fully having paid attention in the preparation process writing out a prescription.The batch number that makes sees the following form 1:
The lot number of the liver-strengthening capsule that adopts among table 1 embodiment
| Numbering | Lot number |
| S1 | 20090301 |
| S2 | 20090302 |
| S3 | 20090303 |
| S4 | 20090304 |
| S5 | 20090305 |
| S6 | 20090306 |
| S7 | 20090307 |
| S8 | 20090308 |
| S9 | 20090309 |
| S10 | 20090310 |
| S11 | 20090401 |
| S12 | 20090402 |
| S13 | 20090403 |
| S14 | 20090404 |
| S15 | 20090405 |
| S16 | 20090406 |
| S17 | 20090407 |
| S18 | 20090408 |
| S19 | 20090409 |
| S20 | 20090410 |
| S21 | 20090501 |
| S22 | 20090502 |
| S23 | 20090503 |
| S24 | 20090504 |
| S25 | 20090505 |
| S26 | 20090506 |
| S27 | 20090507 |
| S28 | 20090508 |
| S29 | 20090509 |
| S30 | 20090510 |
| S31 | 20090601 |
| S32 | 20090602 |
| S33 | 20090603 |
| S34 | 20090604 |
| S35 | 20090605 |
| S36 | 20090606 |
| S37 | 20090607 |
| S38 | 20090608 |
| S39 | 20090609 |
| S40 | 20090610 |
| S41 | 20090701 |
| S42 | 20090702 |
Other Instruments and reagent:
Adopt Agilent 1100 liquid chromatographs, comprise quaternary pump, online degasser, automatic sampler, PDAD (DAD), column oven, Chemstation workstation; AB204-N electronic balance (METTLER TOLEDO); Ultrasound Instrument: Autoscience AS3120; Electric-heated thermostatic water bath (Medical Apparatus & Instruments Factory Jiangsu Prov.'s production); Rotary Evaporators (Switzerland BUCHi); METTLER TOLEDO PB303-N electronic balance (Switzerland METTLER TOLEDO company).
Acetonitrile (chromatographically pure) is available from Concord, Tianjin Science and Technology Ltd.; Phosphoric acid (top grade is pure) is available from Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd.; Normal butyl alcohol (analyzing pure) is available from the triumphant letter chemical industry in Tianjin company limited; 95% ethanol (analyzing pure) is available from the triumphant letter chemical industry in Tianjin company limited; Methyl alcohol (top grade is pure) is available from Concord, Tianjin Science and Technology Ltd.; It magnetic pure water.
Embodiment 1 adopts HPLC to set up the multicomponent assay method of liver-strengthening capsule content
Mix the preparation of reference substance solution: gentiamarin 7.55mg decided in accurate respectively title, Paeoniflorin 3.76mg, and to the 25ml measuring bottle, dissolve with methanol is diluted to scale to tanshin polyphenolic acid B 5.66mg respectively, shakes up, as storing solution.Precision is measured gentiamarin, and the Paeoniflorin storing solution in each 2ml to 10ml measuring bottle of tanshin polyphenolic acid B storing solution, is diluted to scale with methyl alcohol, shakes up, and promptly gets and mixes reference substance solution.
The need testing solution preparation: take by weighing the about 0.3g of liver-strengthening capsule content, precision is weighed, and places conical flask or 25ml volumetric flask; Accurately add 70% volume fraction methanol aqueous solution 25mL, weigh sonicated or reflux 30min; Put to room temperature, supply weight, shake up with 70% volume fraction methanol aqueous solution; Miillpore filter filters, and discards filtrating just, gets subsequent filtrate and promptly gets.
Negative control article formulations prepared from solutions: take by weighing the about 0.3g of negative control sample that lacks bark of ash, the root of herbaceous peony, the red sage root by liver-strengthening capsule prescription and extraction process preparation, precision is weighed, and presses need testing solution preparation method preparation, promptly gets negative control solution.
The mensuration of multi-target ingredient: draw above-mentioned mixing reference substance solution and liver-strengthening capsule need testing solution and inject liquid chromatograph, according to high effective liquid chromatography for measuring, wherein the chromatographic determination condition comprises:
Chromatographic column: Luna 5 μ C
18(2) 250 * 4.6mm NO:497626-12;
Detect wavelength: 230nm; Column temperature: 30 ℃; Flow velocity: 1.0ml/min; Sample size 10 μ L;
Acetonitrile-0.05% phosphate aqueous solution gradient elution, condition of gradient elution sees the following form 2:
Table 2 assay gradient
Mix reference substance solution, need testing solution and negative control article Solution H PLC result and see Fig. 1, Fig. 2 and Fig. 3 respectively.
Need testing solution preparation method's investigation in the embodiment 2 multicomponent detection methods
The need testing solution preparation method investigates
1, extracting solvent investigates
The about 0.5g of sample thief, precision is weighed, and accurately adds methyl alcohol, 70% methanol aqueous solution respectively; 75% ethanol water, each 25ml of 50% ethanol water is as extracting solvent, and close plug is weighed; Sonicated 30 minutes is taken out, and is placed to room temperature, weighs; Supply weight with corresponding solvent, shake up, filter, get subsequent filtrate and measure by the chromatographic condition of confirming with 0.45 μ m miillpore filter.Test figure sees the following form 3.
Table 3 extracts the choice of Solvent test figure
| Solvent is used in extraction | Gentiamarin (area/g) | Paeoniflorin (area/g) | Root of red-rooted salvia phenolic acid B (area/g) |
| 70% methanol aqueous solution | 1468.9 | 1161.9 | 3279.2 |
| Methyl alcohol | 1309.5 | 1140.8 | 2186.9 |
| 50% ethanol water | 1484.3 | 838.4 | 2735.3 |
| 70% ethanol water | 1004.8 | 970.2 | 2658.5 |
The above results comparison shows that, the gentiamarin that 70% methanol aqueous solution is surveyed, Paeoniflorin, root of red-rooted salvia phenolic acid B content are the highest; Make the extraction solvent with 70% methanol aqueous solution, can gentiamarin, Paeoniflorin, three kinds of compositions of root of red-rooted salvia phenolic acid B in the sample be extracted as much as possible, therefore adopt 70% methanol aqueous solution to make the extraction solvent.
2. method for distilling is investigated
Take by weighing 4 parts in sample, make the extraction solvent with 70% methanol aqueous solution, close plug is weighed, and is ultrasonic, reflux each 2 parts; Extract respectively and handle 30min, take off, be placed to room temperature, weigh, supply weight with the extraction solvent; Shake up, filter, filtrating is measured, and test findings is seen table 4.
Table 4 method for distilling is investigated
| Method for distilling | Gentiamarin (mg/g) | Paeoniflorin (mg/g) | Root of red-rooted salvia phenolic acid B (mg/g) |
| Ultrasonic | 4.50 | 2.08 | 5.86 |
| Reflux | 4.43 | 2.09 | 5.93 |
The result shows that backflow is not seen notable difference with sonicated, points out two kinds of method for distilling all can adopt.
3. extraction time is investigated
Make solvent with 70% methanol aqueous solution, the sonicated time is investigated, respectively sonicated 20min, 30min, 45min, 60min are prepared need testing solution and measure, test figure is seen table 5.
Table 5 extraction time investigation
| Time (min) | Gentiamarin (mg/g) | Paeoniflorin (mg/g) | Root of red-rooted salvia phenolic acid B (mg/g) |
| ?20 | 3.53 | 2.03 | 4.55 |
| ?30 | 3.56 | 2.03 | 5.03 |
| ?45 | 3.45 | 2.03 | 5.08 |
| ?60 | 3.47 | 2.01 | 5.04 |
The result shows that the sonicated time is not seen notable difference, therefore selects sonicated 30min can three kinds of compositions be extracted fully.
4. sample removal of impurities disposal route is investigated
(1) extraction: The effects ethyl acetate extraction 3 times, normal butyl alcohol-chloroform (1: 1) extraction 5 times, water-saturated n-butanol extracts respectively 3,4,5 times; The result shows, with what normal butyl alcohol did that the extraction solvent can be more three kinds of compositions extracted, but still can not be with extracting n-butyl alcohol 5 times with gentiamarin; Paeoniflorin, tanshin polyphenolic acid B extracts fully, and using HCl to transfer pH is that extracting n-butyl alcohol is used in 2 backs; Do not extract fully yet, therefore abandon extraction.
(2) resin method: be adjusted into HPD300 macroporous resin column on the water extract, measure washing part and 95% ethanol elution part, the result shows, cross post after each chromatographic peak degree of accuracy undesirable, change after the chromatographic column still undesirable.
(3) direct extraction method: through a series of experiments relatively, the reduction sample weighting amount is 0.3g, makes solvent with 70% methanol aqueous solution; Investigate behind the sample introduction; The degree of separation of each chromatographic peak and chromatogram sample indistinction after treatment, and free of losses, each composition chromatographic peak precision meets the requirements.
To sum up, confirm the need testing solution preparation method for getting the about 0.3g of hepatinica thing, the accurate title, decide, and puts in conical flask or the 25mL measuring bottle; Accurately add 70% methanol aqueous solution 25ml, weigh sonicated or refluxing extraction 30 minutes; Put to room temperature, weigh, supply weight with 70% methanol aqueous solution; Shake up, filter, get subsequent filtrate and promptly get.
The methodological study of embodiment 3 multicomponent detection methods
1) precision test
Adopt and embodiment 1 identical operations and condition; The about 0.3g of sample thief, the accurate title, decide, and the preparation method handles by need testing solution; The accurate need testing solution 10 μ L that draw; Inject high performance liquid chromatograph, continuous sample introduction is 6 times under above-mentioned chromatographic condition, and its RSD value is respectively 0.55%, 0.69% and 1.48%.Explain that precision is good.
2) study on the stability
Adopt and embodiment 1 identical operations and condition, the about 0.3g of sample thief presses need testing solution and prepares extraction, respectively at 0,1,3,6,9,12, the 24h sample introduction, under above-mentioned chromatographic condition, measures.As a result, gentiamarin RSD is 0.77%, and Paeoniflorin RSD is 1.09%, and tanshin polyphenolic acid B RSD is 0.75%.Explain that need testing solution is good at room temperature, air-proof condition held 24h internal stability.
3) linear relationship is confirmed
Draw respectively in three reference substance storing solutions 0.5,1,2,3ml to the 10ml measuring bottle; Draw in gentiamarin, each 4ml to 10ml measuring bottle of Paeoniflorin reference substance storing solution; Draw in gentiamarin 5ml, tanshin polyphenolic acid B 4ml to the 10ml measuring bottle; Draw tanshin polyphenolic acid B 6ml and put in the 10ml measuring bottle, to scale, shake up with methanol constant volume; Respectively measure 10 μ L and inject liquid chromatograph, under the chromatographic condition of embodiment 1, measure peak area.
With reference substance peak area absorption value is ordinate (Y); With the sample size is horizontal ordinate (X) drawing standard curve, calculates regression equation, and gentiamarin is good in 0.151～1.51 μ g scope internal linear relation as a result; Regression equation is y=662.29x+2.0293, r=0.999 (n=6); Paeoniflorin is good in 0.0752～0.752 μ g scope internal linear relation, and regression equation is y=1184.8x-0.5474, r=0.999 (n=6); Tanshin polyphenolic acid B is good in 0.1132～1.3584 μ g scope internal linear relation, and regression equation is y=1475.4x-26.466, r=0.999 (n=5).
4) replica test
Adopt and embodiment 1 identical operations and condition, get the about 0.3g of same lot number sample, the accurate title, decided 6 parts; After extracting processing by the need testing solution preparation method; Measure each sample solution 10 μ L, inject high performance liquid chromatograph, measure; Its RSD value is respectively 1.29%, 1.83% and 1.72% as a result, and repeatability is good.
5) recovery test
Get the about 0.15g of sample of known content; The accurate title, decided 9 parts, presses a certain amount of reference substance solution of 80%, 100%, 120% accurate adding of sample determination amount respectively, presses need testing solution preparation method operation; Under above-mentioned chromatographic condition, measure calculate recovery rate.The average recovery rate % of gentiamarin is 99.24% as a result, and its RSD value is 0.85%; The average recovery rate % of Paeoniflorin is 101.9%, and its RSD value is 1.81%; The average recovery rate % of tanshin polyphenolic acid B is 102.7%, and its RSD value is 1.89%.
The multicomponent content detection of embodiment semi-finals capsule for protecting liver content
14 batches of liver-strengthening capsules (pharmaceutcal corporation, Ltd provides by east, Shijiazhuang) content is measured the content of its gentiamarin, Paeoniflorin, tanshin polyphenolic acid B.
Adopt and embodiment 1 identical operations and condition preparation mixing reference substance solution and need testing solution, accurately measure each 10 μ L and inject liquid chromatograph, the record peak area calculates content with external standard method.Following table 6 as a result.
The mensuration result of table 6 liver-strengthening capsule content multi-target ingredient
Multicomponent detection method provided by the invention can be used to control the quality of hepatinica thing.Adopting this detection method to detect the content of gentiamarin, Paeoniflorin, tanshin polyphenolic acid B in the hepatinica produce article, then is specification product like its content in the scope of regulation.
The selection and the optimization of condition of gradient elution in embodiment 5 chromatographic detection methods
Because hepatinica thing (liver-strengthening capsule) be big compound, flavour of a drug are many, complicated component, and be prone to cause interference between the composition during detection.Before confirming chromatogram testing conditions of the present invention, carried out repeatedly comparative experiments.Wherein condition of gradient elution confirm comprise:
Confirm with acetonitrile-0.05% volume fraction phosphate aqueous solution as wash-out moving phase after, other handle and the identical situation of method of the present invention under, attempted multiple different condition of gradient elution, for example:
Condition of gradient elution 1: acetonitrile (A)-0.05% phosphate aqueous solution (B), 0～9min, 10%A～25%A; 9～10min, 25%A～35%A; 10～20min, 35%～35%A; 20～23min, 35%～60%A; 23～45min, 60%～85%A; 45～46min, 85%～10%A; Chromatogram is seen Fig. 4;
Condition of gradient elution 2: acetonitrile (A)-0.05% phosphate aqueous solution (B), 0～10min, 15%～20%A; 10～20min, 20%～25%A; 20～25min, 25%～40%A; 25～40min, 40%～60%A; 40～45min, 60%～100%A; 45～50min, 100%～15%A; Chromatogram is seen Fig. 5;
Condition of gradient elution 3: acetonitrile (A)-0.05% phosphate aqueous solution (B), 0～30min, 10%～30%A; 30～35min, 30%～10%A; 35～40min, 10%～10%A; Chromatogram is seen Fig. 6.
Experimental result shows, under other condition of gradient elution, gentiamarin, Paeoniflorin, tanshin polyphenolic acid B is well separated simultaneously, all can not reach good separation like 1,3 composition of gradient elution program; Though gradient 2 makes gentiamarin and Paeoniflorin obtain good detached peaks, tanshin polyphenolic acid B has tangible interference separation weak effect; Gradient 3 can make gentiamarin and Paeoniflorin obtain good detached peaks, but the tanshin polyphenolic acid B chromatographic peak not wash-out come out.
Therefore; From a large amount of condition of gradient elution experimental studies; Optimize condition of gradient elution of the present invention, gentiamarin, Paeoniflorin, tanshin polyphenolic acid B are well separated simultaneously, noiseless with the adjacent chromatographic peak of each composition; And reappearance preferably arranged; Test shows that also elution requirement of the present invention does not have other and becomes swarmings interference through negative control article, condition of gradient elution of the present invention can be simultaneously to the hepatinica thing for example main effective constituent gentiamarin, Paeoniflorin, the tanshin polyphenolic acid B of liver-strengthening capsule carry out content detection, characterize the multifarious inherent quality of traditional Chinese medicine ingredients on the whole effectively.
The comparison of embodiment 6 and liver-strengthening capsule national drug standards detection method
The national drug standards WS of state food and Drug Administration
3The content assaying method to liver-strengthening capsule has been announced in-133 (Z-133)-2002 (Z), wherein for adopting high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D) to measure.
Adopt the moving phase acetonitrile-water (15: 85) of the chromatographic condition of this national standard that the liver-strengthening capsule content is measured, obtain chromatogram and see Fig. 7.This figure shows that the condition that adopts national standard only can characterize 1,2,3 three compositions of chromatographic peak, therefore can not characterize the inherent quality of Chinese medicine liver-strengthening capsule on the whole effectively.And under chromatographic condition of the present invention, can obtain gentiamarin, Paeoniflorin, 3 composition chromatographic peaks of tanshin polyphenolic acid B, can better characterize the material base diversity and the complicacy of liver-strengthening capsule compound.
In addition, the test sample preparation method is the methyl alcohol sonicated in the national drug standards, adjust pH=9～10; After; With normal butyl alcohol-methenyl choloride (1: 1) extraction, this method can make a part of composition such as organic acid after transferring pH, and flavones ingredient, root of red-rooted salvia phenolic acid constituents change; Soluble in water, be unfavorable for the extraction of organic solvent; Adopt the ethyl acetate solvent extraction only can extract fat-soluble strong composition, can extract not exclusively the big composition of some polarity such as gentiamarin, Paeoniflorin.And the preparation of adopting need testing solution of the present invention can make gentiamarin in these article, Paeoniflorin, 3 compositions of tanshin polyphenolic acid B extract fully as much as possible, thereby can characterize the inherent quality of traditional Chinese medicine ingredients better on the whole effectively.
The correlation analysis of embodiment 7 multicomponent content and drug effect
The employing quadracycline causes the mouse fatty liver, phenixin causes the rat liver fibrosis empirical model, carries out biochemical measurement and histopathological examination.
1 experiment material
1) medicine:
Liver-strengthening capsule product 1: brown ceramic powder, sample S40.
Liver-strengthening capsule product 2: brown ceramic powder, sample S41.
Liver-strengthening capsule product 3: brown ceramic powder, sample S42.
Quadracycline: Tianjin recovery fine chemistry industry research institute, lot number 20100710.
Legalon capsule: 35mg/ grain, lot number 100511, Tianjin Tasly Pharmaceutical Co., Ltd.
Methionine compound choline sheet (DONGBAO GANTAI): lot number 100110, Tonghua Dongbao Pharmaceutical Co., Ltd.
Silymarin capsule (Legalon): Madaus AG, 140mg/ grain, lot number B0901894.
FUFANG BIEJIA RUANGAN PIAN (being called for short the compound turtle shell): NeiMenggu FuRuiZhong MengYao Science Co., Ltd, 0.5g/ sheet, lot number 20100613.
2) reagent:
Triglyceride kit (enzyme process): the safe clinical reagent of Beijing northization company limited, lot number 20100728.
T-CHOL kit (enzyme process): the safe clinical reagent of Beijing northization company limited, lot number 20100907.
Superoxide dismutase (SOD) kit: lot number 20101230.
MDA kit: lot number 20101230.
Glutamic-pyruvic transaminase (ALT/GPT) kit: lot number 20101215.
Glutamic-oxalacetic transaminease (AST/GOT) kit: lot number 20101213.
Total bilirubin kit: lot number 20101217.
Protein quantification kit (biuret method): lot number 20101110.
Albumin (bromcresol green colourimetry) kit: lot number 20101208.
Hydroxyproline kit: lot number 20101216.
Above kit all builds up bio-engineering research institute available from Nanjing.
HA (hyaluronic acid) kit: lot number 101025, available from Tianjin Hao ocean biological products Science and Technology Ltd..
LN (laminin) kit: lot number 101025, available from Tianjin Hao ocean biological products Science and Technology Ltd..
Olive oil: chemical pure, lot number F20100512, Chemical Reagent Co., Ltd., Sinopharm Group.
Phenixin: analyze pure, lot number 20100804, the triumphant letter chemical industry in Tianjin company limited.
3) instrument:
TU-1810 ultraviolet-visible pectrophotometer: Beijing Puxi General Instrument Co., Ltd's product.
ELIASA: B10-RAD 550 model USA.
4) animal:
Kunming mouse, Wistar kind rat: licence numbering: scxk (capital) 2006-0009 is provided by Beijing Vital River Experimental Animals Technology Co., Ltd..
2, test method
1) liver-strengthening capsule causes the effect of mouse Prevention and Management of Fatty Liver to quadracycline
(it is refined etc. that grandson creates, and the degrease pill for treating cirrhosis causes the influence of mouse fatty liver model liver fat, Yunnan University of Traditional Chinese Medicine's journal, 2002 to tetracycline for reference literature; 25 (1): 8-10) method, select Kunming mouse for use, body weight 22-25g.Be divided into 11 groups at random by body weight, 10 every group, equal male and female half and half.Administration component liver-strengthening capsule (sample S40) 2,4g crude drug/kg dose groups, liver-strengthening capsule (sample S41) 2,4g crude drug/kg dose groups, liver-strengthening capsule (sample S42) 2,4g crude drug/kg dose groups, 4.68/kg of positive drug DONGBAO GANTAI and legalon capsule 100mg/kg dose groups; Adopt gastric infusion, irritate the stomach volume and be the 0.2ml/10g body weight.Normal control group and model control group are all irritated stomach and are waited capacity 0.5%CMC, once a day, and continuous 10 days.The 8th day equal lumbar injection quadracycline (213mg/kg, 0.2ml/10g body weight) except that the normal control group of administration, every day 1 time, continuous 3 times.Last was irritated behind stomach and the drug administration by injection 1 hour, and eye socket is got blood, measures serum total cholesterol (TC) and triglyceride (TG).Dislocation is put to death, and gets liver and weighs, and calculates liver coefficient (liver weight/10g body weight).Get the about 200mg of hepatic tissue, use ethanol: acetone (1: 1) mixed liquor 2ml homogenate, measure total cholesterol of liver (TC) and triglyceride (TG) content, the result adopts t-value method to carry out statistical study.Other gets the part hepatic tissue, uses 10% formalin fixed, and histopathological examination is carried out in HE dyeing under light microscopic.The hepatic injury classification is following:
-hepatic tissue does not see that work becomes
+ swelling of liver cell, endochylema is loose, and indivedual liver cells see vacuolar degeneration
++ swelling of liver cell, endochylema is loose, and a small amount of liver cell sees vacuolar degeneration
+++swelling of liver cell, endochylema is loose, and liver cell sees vacuolar degeneration ≯ 1/2 visual field
++ ++ swelling of liver cell, endochylema is obviously loose, vacuolar degeneration of hepatic cell＞1/2 visual field
2) liver-strengthening capsule is to the preventive and therapeutic effect of rat liver fibrosis due to the phenixin
Reference literature (Peng Xiaodong etc.; The phenixin hypodermic injection makes up the rat liver fibrosis Study of model, Jiangxi Medical College's journal, 2005 the 45th volume the 2nd phase 5-11) method; Select male Wistar rat for use; Body weight 100-130g is divided into 11 groups at random by body weight, administration component liver-strengthening capsule (sample S40) 1,2g crude drug/kg dose groups, liver-strengthening capsule (sample S41) 1,2g crude drug/kg dose groups, liver-strengthening capsule (sample S42) 1,2g crude drug/kg dose groups, positive drug FUFANG BIEJIA RUANGAN PIAN 2g medicinal powder/kg and silymarin capsule 75mg/kg dose groups; Adopt gastric infusion, irritate the stomach volume and be the 1ml/100g body weight.Normal control group and model control group are all irritated stomach and are waited capacity 0.5%CMC, once a day, and continuous 8 weeks.Except that the normal control group, all the other respectively organize rat all in administration, subcutaneous injection of carbon tetrachloride (being made into 50% with olive oil, the 0.3ml/100g body weight), 2 times weekly, continuous 8 weeks.Behind the last gastric infusion 1 hour, eye socket was got blood, measured Serum ALT, AST, TBIL, ALB, TP and hydroxyproline content, and enzyme is exempted from method and measured Serum hyaluronic acid (HA), laminin (LN) content, and the result adopts t-value method to carry out statistical study.Other gets the part hepatic tissue, uses 10% formalin fixed, and histopathologic examination is carried out in HE dyeing under light microscopic.Liver fibrosis classification is following:
-hepatic tissue does not see that work becomes
+ rarely seen a small amount of vacuolar degeneration of hepatic cell
++ 1/2 of vacuolar degeneration of hepatic cell＜visual field, proliferation of fibrous tissue is not obvious
++ the trend that 1/2 of+vacuolar degeneration of hepatic cell＞visual field, group of fibers are woven with hyperplasia and have pseudolobuli to form.
++ ++ 2/3 of vacuolar degeneration of hepatic cell＞visual field, proliferation of fibrous tissue is obvious and have pseudolobuli to form.
3, test of pesticide effectiveness result
Use product 1 (sample S40), product 2 (sample S41), the product 3 (sample S42) of test sample liver-strengthening capsule through giving above-mentioned animal model; Find that liver-strengthening capsule all has tangible preventive and therapeutic effect to mouse fatty liver, rat liver fibrosis; Best with product 1 effect, the result see the following form 7 with table 8:
Table 7 liver-strengthening capsule product 1,2 and 3 (4g crude drug amount) causes the influence of mouse fatty liver biochemical indicator to tetracycline
#P＜0.05
##P＜0.01 (comparing) with the normal control group
*P＜0.05
*(compare with model control group P＜0.01
Table Final 8's capsule for protecting liver product 1,2 and 3 (2g crude drug amount) are to the influence of phenixin liver fibrosis
#: P＜0.05
##: P＜0.01 (comparing) with the normal control group
*: P＜0.05
*: P＜0.01 (comparing) with model control group
4, drug efficacy study is with many indexs assay of sample
Adopt HPLC detection method of the present invention that 3 products are carried out multicomponent and detect, the result sees table 9.
Each principal ingredient data analysis of table 9 liver-strengthening capsule content
| The sample title | Product 1 | Product 2 | |
| Gentiamarin (%) | 0.52 | 0.46 | 0.24 |
| Paeoniflorin (%) | 0.25 | 0.29 | 0.18 |
| Tanshin polyphenolic acid B (%) | 0.88 | 0.51 | 0.35 |
The composition detection of three kinds of samples, though three kinds of sample crude drug amounts are identical, its component content has certain difference; Differ not obvious between product 1 and the product 2,, do not see evident difference though the drug effect result has certain difference; And the component content obvious difference of product 3 and preceding two kinds of samples, drug effect result also shows evident difference property.When prompting is hanged down to certain limit when multi-target ingredient content in the hepatinica thing; Then can influence the drug effect of product; That is to say to have influence on curative effect of medication, explain that therefore the HPLC detection method that the present invention is directed to gentiamarin, Paeoniflorin and tanshin polyphenolic acid B can be used in the quality that helps control hepatinica thing.
In sum; Detection method provided by the invention can adopt high performance liquid chromatography to detect Paeoniflorin, gentiamarin and tanshin polyphenolic acid B in the hepatinica thing simultaneously; Therefore can characterize these article active constituents of medicine content comprehensively; Thereby its inherent quality of standard in the stable uniform that guarantees product, is guaranteed safety of products validity.
To those skilled in the art, technology contents disclosed according to the present invention, those skilled in the art will very clear other embodiment of the present invention, and the embodiment of the invention is only as an example.Under the situation of not violating purport of the present invention and scope, can carry out various changes and improvement to the present invention.For example, the mensuration the possibility of result that uses the different detection instrument to be obtained is different, but as long as use method of quality control of the present invention, all within protection domain of the present invention.
Claims (6)
1. the detection method of a hepatinica thing; Said hepatinica thing is made up of oriental wormwood, Radix Isatidis, Radix Angelicae Sinensis, the root of herbaceous peony, the red sage root, root tuber of aromatic turmeric, the Radix Astragali, Radix Codonopsis, rhizoma alismatis, sealwort, glutinous rehmannia, Chinese yam, hawthorn, bark of ash, Radix Glycyrrhizae, Medicated Leaven; Said detection method comprises that the employing high performance liquid chromatography detects Paeoniflorin, gentiamarin and tanshin polyphenolic acid B in the said hepatinica thing simultaneously; Wherein, the condition of said high performance liquid chromatography comprises:
Chromatographic column: with the octadecylsilane chemically bonded silica is packing material;
Moving phase: mobile phase A is an acetonitrile, and Mobile phase B is 0.05% volume fraction phosphate aqueous solution, carries out gradient elution;
Said gradient elution program is following, and wherein the moving phase ratio is percent by volume:
0～40min, mobile phase A is 10～35%, Mobile phase B is 90%～65%;
40～50min, mobile phase A is 35%～100%, Mobile phase B is 65%～0%;
50～51min, mobile phase A is 100%～10%, Mobile phase B is 0%～90%;
51～55min, mobile phase A is 10%～10%, Mobile phase B is 90%～90%.
2. detection method according to claim 1 is characterized in that, the condition of said high performance liquid chromatography also comprises:
Flow velocity: 1.0mL/min;
Column temperature: 30 ℃;
Detect wavelength: 210nm～400nm;
Theoretical cam curve is pressed the Paeoniflorin peak and is calculated, and should be not less than 6000.
3. detection method according to claim 1 and 2; It is characterized in that; Said detection method also comprises through following steps and prepares reference substance solution: get Paeoniflorin reference substance, gentiamarin and tanshin polyphenolic acid B; Add dissolve with methanol and process the solution that every 1ml contains Paeoniflorin 0.06mg, gentiamarin 0.03mg and tanshin polyphenolic acid B 0.05mg, as mixing reference substance solution.
4. according to each described detection method in the claim 1 to 3, it is characterized in that said method for building up also comprises through following steps and prepares need testing solution: get the about 0.3g of said hepatinica thing composition; Place conical flask or 25ml volumetric flask, accurately add 70% volume fraction methanol aqueous solution 25mL, weigh; Sonicated or reflux 30min are put to room temperature, weigh; Supply weight with 70% volume fraction methanol aqueous solution again, shake up, miillpore filter filters; Discard filtrating just, get subsequent filtrate and promptly get.
5. according to each described detection method in the claim 1 to 4, it is characterized in that said high performance liquid chromatography may further comprise the steps:
1) preparation mixes reference substance solution: get Paeoniflorin reference substance, gentiamarin and tanshin polyphenolic acid B, add dissolve with methanol and process the solution that every 1ml contains Paeoniflorin 0.06mg, gentiamarin 0.03mg and tanshin polyphenolic acid B 0.05mg, as mixing reference substance solution;
2) preparation need testing solution: get the about 0.3g of said hepatinica thing, place conical flask or 25ml volumetric flask, accurately add 70% volume fraction methanol aqueous solution 25mL; Weigh, sonicated or reflux 30min are put to room temperature; Weigh, supply weight, shake up with 70% volume fraction methanol aqueous solution; Miillpore filter filters, and discards filtrating just, gets subsequent filtrate and promptly gets;
3) measure: accurate absorption mixes reference substance solution and each 10 μ l of need testing solution inject high performance liquid chromatograph, measures according to following chromatographic condition:
Chromatographic column: with the octadecylsilane chemically bonded silica is packing material;
Moving phase: mobile phase A is an acetonitrile, and Mobile phase B is 0.05% phosphate aqueous solution, carries out gradient elution;
Flow velocity: 1.0mL/min;
Column temperature: 30 ℃;
Ultraviolet detection wavelength: 230nm;
The record peak area adopts external standard method to calculate Paeoniflorin, gentiamarin and tanshin polyphenolic acid B.
6. detection method according to claim 5 is characterized in that, said gradient elution program is following, and wherein the moving phase ratio is percent by volume:
0～40min, mobile phase A is 10～35%, Mobile phase B is 90%～65%;
40～50min, mobile phase A is 35%～100%, Mobile phase B is 65%～0%;
50～51min, mobile phase A is 100%～10%, Mobile phase B is 0%～90%;
51～55min, mobile phase A is 10%～10%, Mobile phase B is 90%～90%.
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