CN102539599B - Method for detecting liver-enhancing medicine - Google Patents
Method for detecting liver-enhancing medicine Download PDFInfo
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- CN102539599B CN102539599B CN201110420786.9A CN201110420786A CN102539599B CN 102539599 B CN102539599 B CN 102539599B CN 201110420786 A CN201110420786 A CN 201110420786A CN 102539599 B CN102539599 B CN 102539599B
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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 Pharmaceutical Analysis detection field, relate to a kind of detection method of liver-enhancing medicine, particularly relate to the detection method that adopts high performance liquid chromatography (HPLC) simultaneously to detect the various ingredients of described liver-enhancing medicine.
Background technology
Herbal mixture is the principal mode of tcm clinical practice medication, is characteristic and the marrow of traditional Chinese medicine and pharmacy.Chinese medicine be one by multicomponent, the multifactor complex system forming, the diversity of its chemical composition and complicacy are the material bases of its curative effect, foundation meets the modern quality control system of traditional Chinese medicine feature, capture a difficult problem for quality analysis of traditional Chinese medicine and evaluation, improving the existing method of quality control of Chinese medicine has become the problem of people's active research.
Set up the characteristic that must be based on Chinese medicine to the quality control system of Chinese medicine.The mass action feature of herbal mixture has determined that Chinese medicine is different from Western medicine.The method of quality control of Chinese medicine must be controlled whole compositions of onset (organic principle, inorganic constituents and complex compound composition).Only in this way, the quality control system of setting up could really reach controls traditional Chinese medicine quality, guarantee Chinese medicine object safe and effective for medication.Traditional Chinese medicine quality control is just measured and is turned to taking advanced technology as means from simple single component content, the mensuration of polycomponent, many indexs content.
Current most Chinese medicine standard still adopts spectrum or chromatogram means differentiate and measure a certain or several effective constituent or index components, and the routine inspection project that specifies of pharmacopeia.For chemicals, the single compound that its effective component is clear in structure, structure-activity relationship is clear and definite, and its content and purity are directly expressed its effectiveness and reliability.But the feature of middle medical drugs is compound compatibility, the content height of any single effective or active component all can not be expressed its overall curative effect.
Liver-strengthening capsule records in " national drug standards new drug become a full member standard " the 35, formed 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 taste Chinese medicines, have that clearing heat and promoting diuresis, tonifying spleen nourish blood, the strongly fragrant effect of beneficial gas solution.Be used for the treatment of chronic hepatitis, early-phase hepatocirrhosis, fatty liver, toxic hepatitis, liver fibrosis etc.
Liver-strengthening capsule is the large kind of Chinese medicine that Shijiazhuang Dongfang Pharmaceutical Co., Ltd. produces, and proves after deliberation, and liver-strengthening capsule has stronger effect of anti hepatic fibrosis, has protection liver cell, promotes liver cell regeneration, recovers liver function and suppresses fiberization effect.Be used for the treatment of clinically liver fibrosis, early-phase hepatocirrhosis, virus hepatitis, toxic hepatic disease, fatty liver etc., its result for the treatment of has obtained clinical checking.How ensureing the content of effective constituent in the quality of medicine and liver-strengthening capsule, is the basis that determines liver-strengthening capsule curative effect.Existing liver-strengthening capsule quality standard has specified TLC Identification and the Paeoniflorin high performance liquid chromatography content assaying method of oriental wormwood, the root of herbaceous peony.As the large compound of Chinese medicine, only from appearance character, simply discriminating and single component assay equal angles, liver-strengthening capsule is carried out to quality control, obviously can not accurately illustrate comprehensively and the quality that it is inherent can not ensure its curative effect.Control effect of liver-strengthening capsule, must, according to the feature of Chinese medicine polycomponent, many target spots, multipath effect, its material entirety be controlled, effectively characterize on the whole the quality of Chinese medicine, set up safe, effective, stable quality standard system.
About the bibliographical information of liver-strengthening capsule quality control less.Current most detection method is all only confined to measure in the large 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 be had no to report from macroscopical angle.Simultaneously, because containing between 16 taste Chinese medicines and traditional Chinese medicine ingredients, the raw material packet of liver-strengthening capsule there is complicated interaction, cause setting up and reflect that fully and effectively the effective substance method of liver-strengthening capsule inherent quality and curative effect exists larger 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 object of this invention is to provide a kind of liver-enhancing medicine that detects, as the method for liver-strengthening capsule content, medicine activity component and content thereof that the method can complete detection liver-enhancing medicine, thus characterize and control its inherent quality.
The object of the invention is to be achieved through the following technical solutions:
On the one hand, the invention provides a kind of detection method of liver-enhancing medicine, described liver-enhancing medicine forms (for example liver-strengthening capsule 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), described detection method comprises that employing high performance liquid chromatography detects Paeoniflorin, gentiamarin and the tanshin polyphenolic acid B in described liver-enhancing medicine simultaneously, wherein, the condition of described high performance liquid chromatography comprises:
Chromatographic column: taking octadecylsilane chemically bonded silica as packing material;
Mobile phase: mobile phase A is acetonitrile, Mobile phase B is 0.05% volume fraction phosphate aqueous solution, carries out gradient elution;
Described gradient elution program is as follows, and wherein mobile 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 described high performance liquid chromatography also comprises:
Flow velocity: 1.0mL/min;
Column temperature: 30 DEG C;
Detect wavelength: 210nm～400nm;
Theoretical cam curve is pressed Paeoniflorin peak and is calculated, and should be not less than 6000.
Wherein, described detection method comprises by following steps prepares reference substance solution: get Paeoniflorin, gentiamarin and tanshin polyphenolic acid B reference substance, add methyl alcohol to dissolve and make the solution of every 1ml containing Paeoniflorin 0.06mg, gentiamarin 0.03mg and tanshin polyphenolic acid B 0.05mg, as mixing reference substance solution.
And described detection method also comprises by following steps and prepares need testing solution: get the about 0.3g of described liver-enhancing medicine composition, be placed in conical flask or 25ml volumetric flask, accurately add 70% volume fraction methanol aqueous solution 25mL, weigh, ultrasonic processing or add hot reflux 30min, puts to room temperature, weigh, supply weight with 70% volume fraction methanol aqueous solution again, shake up, miillpore filter filters, discard just filtrate, get subsequent filtrate and get final product.
Preferably, described high performance liquid chromatography comprises the following steps:
1) preparation mixes reference substance solution: get Paeoniflorin reference substance, gentiamarin and tanshin polyphenolic acid B reference substance appropriate, add methyl alcohol to dissolve and be diluted to the mixed solution of every 1ml containing Paeoniflorin 0.06mg, gentiamarin 0.03mg and tanshin polyphenolic acid B 0.05mg, as mixing reference substance solution;
2) prepare need testing solution: get the about 0.3g of described liver-enhancing medicine, precise weighing, is placed in conical flask or 25ml volumetric flask, accurately add 70% volume fraction methanol aqueous solution 25mL, weigh, ultrasonic processing or add hot reflux 30min, puts to room temperature, weigh, supply weight with 70% volume fraction methanol aqueous solution, shake up, miillpore filter filters, discard just filtrate, get subsequent filtrate and get final product;
3) measure: accurate absorption mixed reference substance solution and the each 10 μ l injection high performance liquid chromatographs of need testing solution, measures according to following chromatographic condition:
Chromatographic column: taking octadecylsilane chemically bonded silica as filler;
Mobile phase: mobile phase A is acetonitrile, Mobile phase B is 0.05% volume fraction phosphate aqueous solution, carries out gradient elution;
Flow velocity: 1.0mL/min;
Column temperature: 30 DEG C;
Ultraviolet detects wavelength: 230nm;
Record peak area, adopt external standard method to calculate Paeoniflorin, gentiamarin and tanshin polyphenolic acid B.
Described gradient elution program is as follows, and wherein mobile 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 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 is below detailed description of the present invention.
Chromatographic condition
Compared methanol-water and acetonitrile-water system, result shows, acetonitrile-water system eluting power is stronger, and separation efficiency is higher, and velocity of separation is very fast, therefore select acetonitrile-water system to separate.Meanwhile, survey in index components and contain phenolic acid class water soluble ingredient, the mobile phase of acidifying can suppress liposoluble ingredient and dissociate, and reduces chromatographic peak hangover.Therefore, finally selecting acetonitrile-0.05% volume fraction phosphate aqueous solution is mobile phase.Because the polarity difference of each index components is larger, must adopt the method for gradient elution to carry out compartment analysis.In test, investigated different gradient, finally determined the condition of gradient elution of liver-strengthening capsule: 0～40min, 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 diode array detector (DAD) in 210～400nm wavelength coverage, to carry out full wavelength scanner, and the chromatogram under each wavelength is analyzed relatively.Adopt ultraviolet spectrophotometer respectively gentiamarin, Paeoniflorin, 3 reference substances of tanshin polyphenolic acid B to be scanned, the maximum absorption wavelength of result gentiamarin, Paeoniflorin, tanshin polyphenolic acid B is respectively 271.5nm, 230.5nm and 288.0nm place simultaneously.Adopt DAD detecting device sample to be carried out to the full wavelength scanner of 200～400nm, the chromatogram under each wavelength is analyzed relatively.Consider, under 230nm, each composition characteristics peak is obvious, and peak type is better, suitable many indexs assay.Therefore determine that 230nm is for detecting wavelength.
Mix the preparation of reference substance solution
Respectively accurately weighed gentiamarin, Paeoniflorin, tanshin polyphenolic acid B are appropriate, dissolve and are diluted to scale with methyl alcohol, are mixing reference substance solution.
Need testing solution preparation
Investigate by test repeatedly the preparation method who has determined need testing solution in this method of quality control, test has been compared with 50% volume fraction ethanol water, 70% volume fraction ethanol water, 50% volume fraction methanol aqueous solution, 70% volume fraction methanol aqueous solution, methyl alcohol does and extracts the impact of solvent on assay, determines that to make solvent effect with 70% methanol aqueous solution good, ultrasonic and backflow Different Extraction Method and difference extraction times is compared simultaneously.Result shows to use 70% volume fraction methanol aqueous solution ultrasonic or add hot reflux 30 minutes, and gained chromatographic peak is more, and peak area is larger.Because this product flavour of a drug are many, extraction process is decocting, consider that this product macromolecular substances is more, chromatographic column load is larger, therefore this experiment is also investigated sample removal of impurities disposal route, investigate the extraction efficiency impact on each index components with normal butyl alcohol, ethyl acetate extraction and solution pH value, result is undesirable.Finally be defined as getting liver-strengthening capsule content appropriate, precise weighing, puts in conical flask, adds 70% volume fraction methanol aqueous solution, weighs, ultrasonic processing or add hot reflux, takes out, and puts to room temperature, supplies weight, shakes up, filtering with microporous membrane, discards just filtrate, gets subsequent filtrate, to obtain final product.
The present invention has carried out specificity investigation.Take the about 0.3g of negative control product 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 definite chromatographic condition, measure and mix reference substance solution, need testing solution and negative control product solution injection high performance liquid chromatograph, record HPLC figure, see accompanying drawing 1,2,3.3 chromatograms are analyzed to contrast, in 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 negative control chromatogram, occur without corresponding composition chromatographic peak, prompting the method is noiseless to the assay of gentiamarin, Paeoniflorin, tanshin polyphenolic acid B, proves that many index determinings method of the present invention has specificity.
Adopt described liver-strengthening capsule multi-target ingredient assay method, if the content of gentiamarin, Paeoniflorin, tanshin polyphenolic acid B in the product to be measured of measuring is in following ranges: gentiamarin 0.30～0.70%, Paeoniflorin 0.15～0.32%, tanshin polyphenolic acid B 0.20～0.80% are specification product.
Compared with prior art, liver-enhancing medicine detection method provided by the invention has following good effect:
First, liver-strengthening capsule content involved in the present invention is 16 taste Chinese medicine compositions, the contained complex chemical composition of each medicinal material, during to content detection, each composition causes interference mutually, in the time adopting HPLC to detect, cause other compositions to disturb each index components, make content results poor repeatability, so must strictly control the chromatographic condition of mobile phase, just can make each index components obtain good characteristic peak.Experiment showed, and only have practicable, the specific chromatographic condition that adopts the present invention to obtain through great many of experiments, 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.
Secondly, method of the present invention can be carried out content detection to main effective constituent gentiamarin, Paeoniflorin, the tanshin polyphenolic acid B of for example liver-strengthening capsule of liver-enhancing medicine simultaneously, thereby realize, the maximum possible chemical composition of liver-enhancing medicine is detected, be conducive to the monitoring in all directions to its quality, so that the method for quality control of liver-enhancing medicine is more perfect.
And the present invention has that method is easy, stable, precision is high, favorable reproducibility, be easy to the feature grasped.Under same test condition, realized for example assay of gentiamarin, Paeoniflorin, tanshin polyphenolic acid B in liver-strengthening capsule of liver-enhancing medicine simultaneously.
Brief description of the drawings
Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:
Fig. 1 is the HPLC figure that mixes 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 in embodiment 5;
Fig. 7 adopts national drug standards WS in embodiment 6
3in-133 (Z-133)-2002 (Z), the mobile phase of chromatographic condition detects the HPLC figure that liver-strengthening capsule obtains.
Embodiment
Referring to specific embodiment, the present invention is described.It will be appreciated by those skilled in the art that these embodiment are only for the present invention is described, the scope that it does not limit the present invention in any way.
The reference substance source using in following embodiment is as follows:
Gentiamarin, purchased from Nat'l Pharmaceutical & Biological Products Control Institute, lot number 110770-200712, for assay, content is in 99%; Paeoniflorin, purchased from Nat'l Pharmaceutical & Biological Products Control Institute, lot number 0736-9710; Tanshin polyphenolic acid B, purchased from Tianjin Yi Fang Science and Technology Ltd..
The test sample liver-strengthening capsule using in following embodiment, for the inventor and Shijiazhuang Dongfang Pharmaceutical Co., Ltd. are according to the 35th WS3-133 (Z-133)-2002 (Z) preparation of the national drug standards (new drug become a full member standard), has fully been focused on place of production attribute, collecting season feature, the technology controlling and process feature of each medicinal material in prescription in preparation process.The batch number making sees the following form 1:
The lot number of the liver-strengthening capsule adopting in 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, diode array detector (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 (METTLER TOLEDO company of Switzerland).
Acetonitrile (chromatographically pure) is purchased from Concord, Tianjin Science and Technology Ltd.; Phosphoric acid (top grade is pure) is purchased from Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd.; Normal butyl alcohol (analyzing pure) is purchased from Tianjin Kai Xin chemical industry company limited; 95% ethanol (analyzing pure) is purchased from Tianjin Kai Xin chemical industry company limited; Methyl alcohol (top grade is pure) is purchased 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: accurately weighed gentiamarin 7.55mg respectively, Paeoniflorin 3.76mg, tanshin polyphenolic acid B 5.66mg is respectively to 25ml measuring bottle, and methyl alcohol dissolved dilution, to scale, shakes up, as storing solution.Precision measures gentiamarin, and Paeoniflorin storing solution, in each 2ml to the 10ml measuring bottle of tanshin polyphenolic acid B storing solution, is diluted to scale with methyl alcohol, shakes up, and obtains mixing reference substance solution.
Need testing solution preparation: take the about 0.3g of liver-strengthening capsule content, precise weighing, is placed in conical flask or 25ml volumetric flask, accurately adds 70% volume fraction methanol aqueous solution 25mL, weigh, ultrasonic processing or add hot reflux 30min, puts to room temperature, supplies weight with 70% volume fraction methanol aqueous solution, shake up, miillpore filter filters, and discards just filtrate, gets subsequent filtrate and get final product.
Negative control product solution preparation: take 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, precise weighing, by need testing solution preparation method preparation, obtains negative control solution.
The mensuration of multi-target ingredient: draw above-mentioned mixing reference substance solution and liver-strengthening capsule need testing solution injection liquid chromatography, according to high effective liquid chromatography for measuring, wherein chromatographic determination condition comprises:
Chromatographic column: Luna 5 μ C
18(2) 250 × 4.6mm NO:497626-12;
Detect wavelength: 230nm; Column temperature: 30 DEG C; 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 product Solution H PLC result and see respectively Fig. 1, Fig. 2 and Fig. 3.
need testing solution preparation method's investigation in embodiment 2 multicomponent detection methods
Need testing solution preparation method investigates
1, extracting solvent investigates
The about 0.5g of sample thief, precise weighing, accurately adds respectively methyl alcohol, 70% methanol aqueous solution, 75% ethanol water, the each 25ml of 50% ethanol water is as extracting solvent, close plug, weighs, ultrasonic processing 30 minutes, take out, be placed to room temperature, weigh, supply weight with corresponding solvent, shake up, with 0.45 μ m miillpore filter filtration, get subsequent filtrate and measure by definite chromatographic condition.Test figure sees the following form 3.
Table 3 extracts the selection test figure of solvent
| Extraction solvent | 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 relatively shows, gentiamarin, Paeoniflorin, root of red-rooted salvia phenolic acid B content that 70% methanol aqueous solution is surveyed are the highest; Make extraction solvent with 70% methanol aqueous solution, gentiamarin, Paeoniflorin, three kinds of compositions of root of red-rooted salvia phenolic acid B in sample can be extracted as much as possible, therefore adopt 70% methanol aqueous solution to make extraction solvent.
2. extracting method is investigated
Take 4 parts, sample, make extraction solvent with 70% methanol aqueous solution, close plug, weighs, ultrasonic, reflux each 2 parts, respectively extraction process 30min, takes off, and is placed to room temperature, weighs, and supplies weight with extraction solvent, shake up, filter, filtrate is measured, and test findings is in table 4.
Table 4 extracting method is investigated
| Extracting method | 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 |
Result shows, refluxes and has no notable difference with ultrasonic processing, points out two kinds of extracting method all can adopt.
3. extraction time is investigated
Make solvent with 70% methanol aqueous solution, the ultrasonic processing time is investigated, respectively ultrasonic processing 20min, 30min, 45min, 60min are prepared need testing solution and measured, test figure is in table 5.
Table 5 extraction time is investigated
| 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 |
Result shows, the ultrasonic processing time has no notable difference, therefore selects ultrasonic processing 30min three kinds of compositions can be extracted completely.
4. sample removal of impurities disposal route is investigated
(1) extraction: ethyl acetate extraction 3 times have been investigated in experiment, normal butyl alcohol-chloroform (1: 1) extraction 5 times, water-saturated n-butanol extracts respectively 3,4,5 times, and result shows, that does with normal butyl alcohol that extraction solvent can be more extracts three kinds of compositions, but with extracting n-butyl alcohol 5 times still can not be by gentiamarin, Paeoniflorin, tanshin polyphenolic acid B extracts completely, and adjusting pH with HCl is the 2 rear extracting n-butyl alcohols of using, extract completely not yet, therefore abandon extraction.
(2) resin method: be adjusted into HPD300 macroporous resin column on water extraction liquid, measure washing part and 95% ethanol elution part, result shows, cross post after each chromatographic peak degree of accuracy undesirable, change after chromatographic column still undesirable.
(3) direct extraction method: through series of experiments comparison, reduction sample weighting amount is 0.3g, makes solvent with 70% methanol aqueous solution, after sample introduction, investigate, 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, determine that need testing solution preparation method is for getting the about 0.3g of liver-enhancing medicine, accurately weighed, put in conical flask or 25mL measuring bottle, accurately add 70% methanol aqueous solution 25ml, weigh, ultrasonic processing or refluxing extraction 30 minutes, put to room temperature, weigh, supply weight with 70% methanol aqueous solution, shake up, filter, get subsequent filtrate and get final product.
the methodological study of embodiment 3 multicomponent detection methods
1) precision test
Adopt operation and the condition identical with embodiment 1, the about 0.3g of sample thief, accurately weighed, by need testing solution, preparation method processes, the accurate need testing solution 10 μ L that draw, inject high performance liquid chromatograph, continuous sample introduction 6 times under above-mentioned chromatographic condition, its RSD value is respectively 0.55%, 0.69% and 1.48%.Illustrate that precision is good.
2) study on the stability
Adopt operation and the condition identical with embodiment 1, the about 0.3g of sample thief, extracts by need testing solution preparation, respectively at 0,1,3,6,9,12,24h 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%.Illustrate that need testing solution is placed 24h internal stability under room temperature, air-proof condition good.
3) linear relationship is determined
Draw respectively in three reference substance storing solutions 0.5,1,2,3ml to 10ml measuring bottle, draw in gentiamarin, each 4ml to the 10ml measuring bottle of Paeoniflorin reference substance storing solution, draw in gentiamarin 5ml, tanshin polyphenolic acid B 4ml to 10ml measuring bottle, drawing tanshin polyphenolic acid B 6ml puts in 10ml measuring bottle,, shake up to scale by methanol constant volume; Respectively measure 10 μ L injection liquid chromatographies, under the chromatographic condition of embodiment 1, measure peak area.
Taking reference substance peak area absorption value as ordinate (Y), (X) drawing standard curve taking sample size as horizontal ordinate, calculate regression equation, result gentiamarin is good in 0.151～1.51 μ g scope internal linear relation, 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 operation and the condition identical with embodiment 1, get the about 0.3g of same lot number sample, accurately weighed 6 parts, undertaken after extraction process by need testing solution preparation method, measure each sample solution 10 μ L, inject high performance liquid chromatograph, measure, its RSD value of result is respectively 1.29%, 1.83% and 1.72%, and repeatability is good.
5) recovery test
Get the about 0.15g of sample of known content, accurately weighed 9 parts, add a certain amount of reference substance solution by 80%, 100%, 120% precision of sample determination amount respectively, operate by need testing solution preparation method, under above-mentioned chromatographic condition, measure calculate recovery rate.The average recovery rate % of result gentiamarin is 99.24%, 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 (being provided by Shijiazhuang Dongfang Pharmaceutical Co., Ltd.) content is measured to the content of its gentiamarin, Paeoniflorin, tanshin polyphenolic acid B.
Adopt the preparation of the operation identical with embodiment 1 and condition to mix reference substance solution and need testing solution, accurately measure each 10 μ L injection liquid chromatographies, record peak area, calculate content by external standard method.Result following table 6.
The measurement result of table 6 liver-strengthening capsule content multi-target ingredient
Multicomponent detection method provided by the invention can be for controlling the quality of liver-enhancing medicine.Adopt the content of gentiamarin, Paeoniflorin, tanshin polyphenolic acid B in this detection method detection liver-enhancing medicine product, if its content is specification product within the limits prescribed.
the choice and optimization of condition of gradient elution in embodiment 5 chromatographic detection methods
Because liver-enhancing medicine (liver-strengthening capsule) is large compound, flavour of a drug are many, complicated component, and easily cause interference between composition while detecting.Before determining chromatogram testing conditions of the present invention, carry out repeatedly comparative experiments.Wherein the definite of condition of gradient elution comprises:
Determining using acetonitrile-0.05% volume fraction phosphate aqueous solution after wash-out mobile phase, in other processing and the identical situation of method of the present invention, attempt 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 shown in 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 shown in 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 shown in Fig. 6.
Experimental result shows, under other condition of gradient elution, all can not make gentiamarin, Paeoniflorin, tanshin polyphenolic acid B well be separated simultaneously, as 1,3 composition of gradient elution program all can not reach good separation; Although gradient 2 makes gentiamarin and Paeoniflorin obtain good detached peaks, tanshin polyphenolic acid B has obvious interference separation weak effect; Gradient 3 can make gentiamarin and Paeoniflorin obtain good detached peaks, but wash-out is not out for tanshin polyphenolic acid B chromatographic peak.
Therefore, from a large amount of condition of gradient elution experimental studies, optimize condition of gradient elution of the present invention, can make gentiamarin, Paeoniflorin, tanshin polyphenolic acid B is well separated simultaneously, noiseless with the adjacent chromatographic peak of each composition, and there is a good reappearance, through negative control product, test also shows that elution requirement of the present invention becomes swarming to disturb without other, the condition of gradient elution of the present invention main effective constituent gentiamarin to for example liver-strengthening capsule of liver-enhancing medicine simultaneously, Paeoniflorin, tanshin polyphenolic acid B carries out content detection, effectively characterize on the whole the multifarious inherent quality of traditional Chinese medicine ingredients.
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 (annex VI D of Chinese Pharmacopoeia version in 2000) to measure.
Adopt the mobile phase acetonitrile-water (15: 85) of the chromatographic condition of this national standard to measure liver-strengthening capsule content, obtain chromatogram and see Fig. 7.This figure shows to adopt the condition of national standard only can characterize 1,2,3 three compositions of chromatographic peak, therefore can not effectively characterize on the whole the inherent quality of Chinese medicine liver-strengthening capsule.And under chromatographic condition of the present invention, can obtain gentiamarin, Paeoniflorin, 3 composition chromatographic peaks of tanshin polyphenolic acid B, can better characterize material base diversity and the complicacy of liver-strengthening capsule compound.
In addition, in the national drug standards, test sample preparation method is the ultrasonic processing of methyl alcohol, adjust pH=9～10, after, with normal butyl alcohol-methenyl choloride (1: 1) extraction, the method can make a part of composition as organic acid after adjusting 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 ethyl acetate solvent extraction only can extract fat-soluble strong composition, can extract not exclusively as gentiamarin, Paeoniflorin the large composition of some polarity.And the preparation that adopts need testing solution of the present invention can make 3 compositions of gentiamarin in this product, Paeoniflorin, tanshin polyphenolic acid B extract completely as much as possible, thereby can effectively characterize better on the whole the inherent quality of traditional Chinese medicine ingredients.
the correlation analysis of embodiment 7 multicomponent content and drug effect
Employing quadracycline causes mouse fatty liver, phenixin causes 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.
Silybin Capsules: 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 compound turtle shell): NeiMenggu FuRuiZhong MengYao Science Co., Ltd, 0.5g/ sheet, lot number 20100613.
2) reagent:
Triglyceride Reagent box (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.
Malondialdehyde 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 Bioengineering Research Institute purchased from Nanjing.
HA (hyaluronic acid) kit: lot number 101025, purchased from Tianjin Hao ocean biological products Science and Technology Ltd..
LN (laminin) kit: lot number 101025, purchased 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, Tianjin Kai Xin chemical industry company limited.
3) instrument:
TU-1810 ultraviolet-visible pectrophotometer: Beijing Puxi General Instrument Co., Ltd's product.
Microplate reader: 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 preventive and therapeutic effect of mouse fatty liver to quadracycline
(it is refined etc. that grandson creates reference literature, and degrease pill for treating cirrhosis causes the impact of mouse Models of Fatty Liver liver fat on tetracycline, Yunnan University of Traditional Chinese Medicine's journal, 2002; 25 (1): 8-10) method, selects Kunming mouse, body weight 22-25g.Be divided at random 11 groups by body weight, 10 every group, all male and female half and half.Administration component liver-strengthening capsule (sample S40) 2,4g crude drug/kg dosage group, liver-strengthening capsule (sample S41) 2,4g crude drug/kg dosage group, liver-strengthening capsule (sample S42) 2,4g crude drug/kg dosage group, 4.68/kg of positive drug DONGBAO GANTAI and Silybin Capsules 100mg/kg dosage group, adopt gastric infusion, gavage volume is 0.2ml/10g body weight.The equal gavage of Normal group and model control group waits 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 Normal group of administration, every day 1 time, continuous 3 times.After last gavage and drug administration by injection 1 hour, eye socket was got blood, measured 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, result adopts t-value method to carry out statistical study.Separately get part hepatic tissue, fix with 10% formalin, histopathological examination is carried out in HE dyeing under light microscopic.Hepatic injury classification is as follows:
-hepatic tissue has no work and 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 the visual field, vacuolar degeneration ≯ 1/2
++++swelling of liver cell, endochylema is obviously loose, vacuolar degeneration of hepatic cell > 1/2 visual field
2) preventive and therapeutic effect of liver-strengthening capsule to rat liver fibrosis due to phenixin
Reference literature (Peng little Dong etc., phenixin hypodermic injection builds the research of Rat Liver Fibrosis Model, Jiangxi Medical College's journal, the 45th volume the 2nd phase 5-11 in 2005) method, select male Wistar rat, body weight 100-130g, be divided at random 11 groups by body weight, administration component liver-strengthening capsule (sample S40) 1, 2g crude drug/kg dosage group, liver-strengthening capsule (sample S41) 1, 2g crude drug/kg dosage group, liver-strengthening capsule (sample S42) 1, 2g crude drug/kg dosage group, positive drug FUFANG BIEJIA RUANGAN PIAN 2g medicinal powder/kg and Silymarin Capsule 75mg/kg dosage group, adopt gastric infusion, gavage volume is 1ml/100g body weight.The equal gavage of Normal group and model control group waits capacity 0.5%CMC, once a day, and continuous 8 weeks.Except Normal group, all the other respectively organize rat all in administration, subcutaneous injection of carbon tetrachloride (being made into 50%, 0.3ml/100g body weight with olive oil), 2 times weekly, continuous 8 weeks.After last gastric infusion 1 hour, eye socket is got blood, measure Serum ALT, AST, TBIL, ALB, TP and hydroxyproline content, enzyme is exempted from method and is measured Serum hyaluronic acid (HA), laminin (LN) content, and result adopts t-value method to carry out statistical study.Separately get part hepatic tissue, fix with 10% formalin, histopathologic examination is carried out in HE dyeing under light microscopic.Liver fibrosis classification is as follows:
-hepatic tissue has no work and becomes
+ rarely seen a small amount of vacuolar degeneration of hepatic cell
++ 1/2 of the vacuolar degeneration of hepatic cell < visual field, proliferation of fibrous tissue is not obvious
+++ 1/2 of the vacuolar degeneration of hepatic cell > visual field, the trend that fibr tissue has hyperplasia and has pseudolobuli to form.
+++ the 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 by giving above-mentioned animal model, find that liver-strengthening capsule all has obvious preventive and therapeutic effect to mouse fatty liver, rat liver fibrosis, best with product 1 effect, the results are shown in following table 7 and table 8:
Table 7 liver-strengthening capsule product 1,2 and 3 (4g crude drug amount) causes the impact of mouse fatty liver biochemical indicator on tetracycline
#p < 0.05
##p < 0.01 (with Normal group comparison)
*p < 0.05
*p < 0.01 is (with model control group comparison
The impact of table Final 8's capsule for protecting liver product 1,2 and 3 (2g crude drug amount) on phenixin liver fibrosis
#: P < 0.05
##: P < 0.01 (with Normal group comparison)
*: P < 0.05
*: P < 0.01 (with model control group comparison)
4, many indexs assay of sample for drug efficacy study
Adopt HPLC detection method of the present invention to carry out multicomponent detection to 3 products, the results are shown in Table 9.
Each principal ingredient data analysis of table 9 liver-strengthening capsule content
| Sample title | Product 1 | Product 2 | Product 3 |
| 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, although three kinds of sample crude drug amounts are identical, its component content has certain difference; Between product 1 and product 2, differ not obvious, although drug effect result has certain difference, have no obvious difference; And product 3 is obvious with the component content difference of first two sample, drug effect result also shows obvious otherness.Prompting is arrived when certain limit when multi-target ingredient content in liver-enhancing medicine is low, can affect the drug effect of product, that is to say and can have influence on curative effect of medication, therefore illustrate that 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 to control liver-enhancing medicine.
In sum, detection method provided by the invention can adopt high performance liquid chromatography to detect Paeoniflorin, gentiamarin and the tanshin polyphenolic acid B in liver-enhancing medicine simultaneously, therefore can characterize this product active constituents of medicine content comprehensively, thereby its inherent quality of standard, in ensureing the stable uniform of product, guarantee the safety and effectiveness of product.
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 present invention is only as example.In the situation that not violating purport of the present invention and scope, can carry out various changes and improvements to the present invention.For example, the measurement result possibility that uses different detecting instruments to obtain is different, but as long as use method of quality control of the present invention, all within protection domain of the present invention.
Claims (1)
1. the detection method of a liver-enhancing medicine, described liver-enhancing medicine 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, described detection method comprises that employing high performance liquid chromatography detects Paeoniflorin, gentiamarin and the tanshin polyphenolic acid B in described liver-enhancing medicine simultaneously, wherein, described high performance liquid chromatography comprises the following steps:
1) preparation mixes reference substance solution: get Paeoniflorin reference substance, gentiamarin and tanshin polyphenolic acid B, add methyl alcohol to dissolve and make the solution of every 1ml containing Paeoniflorin 0.06mg, gentiamarin 0.03mg and tanshin polyphenolic acid B 0.05mg, as mixing reference substance solution;
2) prepare need testing solution: get described liver-enhancing medicine 0.3g, be placed in conical flask or 25ml volumetric flask, accurately add 70% volume fraction methanol aqueous solution 25mL, weigh, ultrasonic processing or add hot reflux 30min, put to room temperature, weigh, supply weight with 70% volume fraction methanol aqueous solution, shake up, miillpore filter filters, and discards just filtrate, gets subsequent filtrate and get final product;
3) measure: accurate absorption mixed reference substance solution and the each 10 μ l injection high performance liquid chromatographs of need testing solution, measures according to following chromatographic condition:
Chromatographic column: taking octadecylsilane chemically bonded silica as packing material;
Mobile phase: mobile phase A is acetonitrile, Mobile phase B is 0.05% volume fraction phosphate aqueous solution, carries out gradient elution;
Described gradient elution program is as follows, and wherein mobile 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%;
And the condition of described high performance liquid chromatography also comprises:
Flow velocity: 1.0mL/min;
Column temperature: 30 DEG C;
Detect wavelength: 230nm;
Theoretical cam curve is pressed Paeoniflorin peak and is calculated, and should be not less than 6000;
Record peak area, adopt external standard method to calculate Paeoniflorin, gentiamarin and tanshin polyphenolic acid B.
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| CN113804771B (en) * | 2020-06-17 | 2024-03-29 | 浙江维康药业股份有限公司 | Method for simultaneously measuring contents of various components of traditional Chinese medicine for treating liver diseases |
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