CN106526000A - Method for establishing fingerprint of rhizoma cimicifugae radix puerariae decoction composition and fingerprint - Google Patents
Method for establishing fingerprint of rhizoma cimicifugae radix puerariae decoction composition and fingerprint Download PDFInfo
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Abstract
The invention discloses a method for controlling the quality of a rhizoma cimicifugae radix puerariae decoction composition, and belongs to the field of traditional Chinese medicine analysis. The method adopts a high performance liquid chromatographic method for establishing a fingerprint, chromatographic conditions are as follows: an octadecyl silane bonded silica gel chromatography column is used as a chromatography column, flow rate is 0.9ml / min-1.1ml / min, column temperature is 25 to 35 DEG, an UV detector is used as a detection instrument, detection wavelength is 210nm-250nm; theoretical plate number is not less than 4000 on the basis of a puerarin peak; a reference solution is a methanol solution of daidzin; an acetonitrile-0.1% phosphoric acid solution system is used as a moving phase, and gradient elution is performed in the following order shown in the specification. The fingerprint fully reflects the quality information of a rhizoma cimicifugae radix puerariae decoction, so that the purpose of more effective and comprehensive control of the quality of the rhizoma cimicifugae radix puerariae decoction product can be achieved.
Description
Technical field
The invention belongs to important analysis field, and in particular to the finger printing of SHENGMA GEGEN TANG compositionss.
Background technology
SHENGMA GEGEN TANG compositionss are made up of Rhizoma Cimicifugae, Radix Puerariae, the Radix Paeoniae Alba, four taste Chinese medicine of Radix Glycyrrhizae Preparata, with pungent cool expelling pathogenic factors from muscles, rash
Effect of removing toxic substances；Modern clinic except treatment measles in addition to, also for treating the diseases such as herpess, chickenpox, acute dysentery, hepatitis, in addition
Continue to use side of the we as children, the old man felt cold to hyperpyrexia of catching a cold, children's curative effect are favourable.In order to more comprehensively, effectively control
The quality control level of this Chinese medicine composition, and international joint, allow clinical application safety and curative effect to have ensured, it is right to need
This Chinese medicine composition adopts more advanced quality control method.
As Chinese medicine and its compound preparation are multi-component complex system, thus evaluate its quality should be using being adapted therewith
, the detection method of abundant authentication information can be provided, but the method such as existing microscopical identification, physicochemical identification and assay is not
Be enough to solve this problem.Chinese medicine fingerprint refer to some Chinese crude drugs or Chinese medicine cut open agent it is appropriately processed after, using certain
Analysis means, what is obtained can indicate the chromatogram or spectrogram of its chemical feature.Chinese medicine fingerprint be it is a kind of comprehensive, can
The identification of means of quantization, more can comprehensively reflect contained chemistry in Chinese medicine and its preparation, and the species of composition is with quantity and then right
Drug quality carries out whole description and evaluation.So, Chinese medicine fingerprint has become Chinese medicine relatively advanced in the world at present
Quality control method.Refer both in FDA (Food and Drug Adminstration) (FDA) botanical drug product industrial directory, WHO medical herbs evaluation guides
Go out, if the active component of herbal medicinal product can not differentiate, can be proved by finger printing (fingerprint spectrum)
Product quality it is consistent.The European Community also indicates that in herbal quality guide annotation medical herbs and its preparation are using overall as effectively
Material, therefore, the effective ingredient that the quality stability of medical herbs is only known by determining oneself is inadequate, it should shown by finger printing
Various composition contained by which.India herbal medicine allusion quotation, British Herbal Pharmacopoeia, German medicinal plants association, Canadian medicinal and fragrant plant
Association etc., also acceptance ensure the concordance of the unknown herbal products quality of effective ingredient with finger printing.Current national drug
To Chinese medicine, oneself requires that, with related finger printing, the quality to improving Chinese medicine serves pass for Surveillance Authority
Key is acted on, but the still no Compulsory Feature of Chinese medicine to other dosage forms.
Chinese medicine fingerprint includes that Chemistry for Chinese Traditional Medicine finger printing, protein fingerprint spectrum, DNA fingerprinting and biological effect refer to
Stricture of vagina collection of illustrative plates etc..The finger printing generally said refers to chemical fingerprint, is that Chinese crude drug, Chinese medicine extract or Chinese patent medicine Jing suitably locate
After reason, using certain analysis means, what is obtained can indicate the chromatograph or spectrum of the Chinese medicine main chemical compositions characteristic.
Chemistry for Chinese Traditional Medicine finger printing can be divided into Chinese medicine spectrum fingerprint, such as infrared spectrum (IR), ultraviolet spectra (UV),
X ray diffracting spectrum etc.；Chromatographic fingerprinting, such as thin layer chromatography (TLC), gas chromatogram (GC), high performance liquid chromatography (HPLC),
Capillary electrophoresis collection of illustrative plates (CE) etc.；Wave spectrum finger printing, such as mass spectrum (MS), NMR (Nuclear Magnetic Resonance) spectrum (NMR) etc.；DNA fingerprint figure figure
Spectrum；And the multi-dimensional information characteristics spectrum (characteristic obtained using the combination of various modern analytical tool
Fingerprint of muhrdimension andmuhrdata), such as HPLC/MS, HPLC/Ms/MS, GC/MS, CE/MS etc..
Chinese medicine fingerprint research method is although more, but chromatographic processes should be main stream approach, especially TLC, HPLC
With GC chromatographic techniques, the analysis means of three kinds of routines having become recognized by everybody are that current Study of Traditional Chinese Medicine finger printing should
Top-priority method.
Chinese medicine fingerprint has two effects：One is the characteristic by finger printing, can effectively differentiate the true of Chinese crude drug
The pseudo- or place of production：Two is the formulation of the area or ratio by finger printing principal character peak, the quality of energy effective control product, really
Protect the relatively uniform of product quality.
The making and application of Chinese medicine fingerprint often includes：(1) representative traditional Chinese medicine sample is gathered, takes appropriate
Method prepares need testing solution, and from appropriate standard product (object of reference), is analyzed the preferred of method；(2) basis
It is determined that analysis method, enter to have done separation analysis to the traditional Chinese medicine sample of certain batch, the multiple batches of data of gained processed,
Obtain standard finger-print；(3) sample is carried out sample treatment, separated point according to identical method being made with standard finger-print
After analysis, the finger printing of gained and standard finger-print are carried out into similarity-rough set, typically more than 80% is reached to spend
It is qualified to be considered as the sample.The calculating and evaluation methodology of similarity is generally using correlation coefficient and coefficient or the included angle cosine of being harmonious
Method.The units such as Central South University of China, Zhejiang University are carried according to above method, and it is mutually false for completing to have worked out corresponding computer software
Degree is evaluated, wherein with " similarity evaluation " of Chinese Pharmacopoeia Commission's recommendation using more.Cause
This reflects the global feature of compositionss comprehensively using fingerprint pattern technology, and is further associated with its activity, for improving mesh
Front method of quality control, it is ensured that clinical application is safely and effectively very necessary.At present, with regard to SHENGMA GEGEN TANG compositionss
The research of finger printing not yet have document to disclose report to be more not set up the finger printing of SHENGMA GEGEN TANG, from quality control side
Face, does not also report about the standard finger-print of SHENGMA GEGEN TANG compositionss.
The content of the invention
Therefore, the technical problem to be solved in the present invention is to overcome of the prior art without with regard to SHENGMA GEGEN TANG combination
The finger printing of thing and set up SHENGMA GEGEN TANG compositionss finger printing document report defect, so as to provide a kind of liter
The method for building up and finger printing of numb GEGEN TANG compositionss finger printing.
For this purpose, the present invention provides following technical scheme：
The method for building up of the finger printing of SHENGMA GEGEN TANG compositionss includes setting up Rhizoma Cimicifugae Pueraria lobota using high performance liquid chromatography
Root soup compositionss finger printing, chromatographic condition is：
Chromatographic column adopts octadecylsilane chemically bonded silica chromatographic column；
Flow velocity 0.9ml/min～1.1ml/min；Column temperature：25 DEG C～35 DEG C；
Detecting instrument adopts UV-detector, the Detection wavelength 210nm～250nm of finger printing；
Theoretical cam curve is not less than 4000 in terms of puerarin peak；
Methanol solution of the reference solution for daiazi；
It is system that mobile phase is -0.1% phosphoric acid solution of acetonitrile, carries out gradient elution in the following order：
The SHENGMA GEGEN TANG compositionss are prepared by the following two kinds method：
(1) Rhizoma Cimicifugae 2g, Radix Puerariae 3g, Radix Paeoniae Alba 2g, Radix Glycyrrhizae Preparata 2g decoction pieces are weighed respectively, cut into the thick of about 2mm～8mm or so
Granule, is placed in 2L Traditional Chinese medicine decocting earthen pots, plus 300ml water, soaks 30 minutes, adds a cover decoction, and boiling is heated under the voltage of 220V
10 minutes, under 170V voltages keep micro-boiling to medicinal liquid about 100ml, filter medicinal residues, obtain final product the first SHENGMA GEGEN TANG compositionss；Or
(2) following decoction pieces Rhizoma Cimicifugae 666.7g, Radix Puerariae 1000g, Radix Paeoniae Alba 666.7g, Radix Glycyrrhizae Preparata are weighed in parts by weight
666.7g, cuts into the coarse granule of granularity 2mm～8mm, adds water 12 times and measures water, decocts 30min, filtration, then by filtrate decompression
Thick paste is concentrated into, with maltodextrin, is dried, is mixed, stiffened fatty acid magnesium, dry-pressing granule are made 1000g, obtain final product the second Rhizoma Cimicifugae Pueraria lobota
Root soup compositionss.
The finger printing includes 21 total peaks：
No. 1 peak relative retention time RRT is that 8.944, No. 2 peak relative retention time RRT are protected for 14.111, No. 3 peaks are relative
It is that 24.036, No. 5 peak relative retention time RRT are 27.333,6 that time RRT is stayed for 20.583, No. 4 peak relative retention time RRT
It is 37.305, No. 8 peak relative retention time RRT that number peak relative retention time RRT is 32.806, No. 7 peak relative retention time RRT
For 40.061, it is 45.699, No. 11 peak phases that No. 9 peak relative retention time RRT are 42.859, No. 10 peak relative retention time RRT
To retention time RRT for 54.817, S peaks relative retention time RRT for 61.159, No. 13 peak relative retention time RRT it is
67.543, No. 14 peak relative retention time RRT are that 70.776, No. 15 peak relative retention time RRT are relative for 73.526, No. 16 peaks
Retention time RRT for 80.076, No. 17 peak relative retention time RRT for 83.260, No. 18 peak relative retention time RRT is
86.766, No. 19 peak relative retention time RRT are that 91.200, No. 20 peak relative retention time RRT are relative for 98.200, No. 21 peaks
Retention time RRT is 111.034, wherein, No. 12 S peaks are the chromatographic peaks of object of reference.
Need testing solution for high-performance liquid chromatogram determination adopts water as solvent, and reference solution makees molten using methanol
Agent.
The preparation process of reference solution is：Take daiazi reference substance appropriate, plus methanol is configured to 1ml methanol containing daiazi
The reference solution of 200 μ g.
The preparation process of need testing solution is：The first SHENGMA GEGEN TANG compositionss 5ml are taken, is centrifuged, is taken supernatant, obtain final product；
Or the second SHENGMA GEGEN TANG compositionss 1.5g are taken, and to put in 50ml volumetric flasks, dilute, ultrasonic dissolution let cool, and it is fixed to add water
Hold, shake up, take solution, be centrifuged, take supernatant, obtain final product.
Sample size is 10 μ l, and the Detection wavelength of the finger printing is 210nm～250nm.
The finger printing of SHENGMA GEGEN TANG compositionss adopts the high performance liquid chromatography, chromatographic condition to be：
Chromatographic column adopts octadecylsilane chemically bonded silica chromatographic column；
Flow velocity 0.9ml/min～1.1ml/min；Column temperature：25 DEG C～35 DEG C；
Theoretical cam curve is not less than 4000 in terms of puerarin peak；
Methanol solution of the reference solution for daiazi；
It is system that mobile phase is -0.1% phosphoric acid solution of acetonitrile, carries out gradient elution in the following order：
The Detection wavelength is 210nm～250nm.
The finger printing includes 21 total peaks：
No. 1 peak relative retention time RRT is that 8.944, No. 2 peak relative retention time RRT are protected for 14.111, No. 3 peaks are relative
It is that 24.036, No. 5 peak relative retention time RRT are 27.333,6 that time RRT is stayed for 20.583, No. 4 peak relative retention time RRT
It is 37.305, No. 8 peak relative retention time RRT that number peak relative retention time RRT is 32.806, No. 7 peak relative retention time RRT
For 40.061, it is 45.699, No. 11 peak phases that No. 9 peak relative retention time RRT are 42.859, No. 10 peak relative retention time RRT
To retention time RRT for 54.817, S peaks relative retention time RRT for 61.159, No. 13 peak relative retention time RRT it is
67.543, No. 14 peak relative retention time RRT are that 70.776, No. 15 peak relative retention time RRT are relative for 73.526, No. 16 peaks
Retention time RRT for 80.076, No. 17 peak relative retention time RRT for 83.260, No. 18 peak relative retention time RRT is
86.766, No. 19 peak relative retention time RRT are that 91.200, No. 20 peak relative retention time RRT are relative for 98.200, No. 21 peaks
Retention time RRT is 111.034, wherein, No. 12 S peaks are the chromatographic peaks of object of reference.
Technical solution of the present invention, has the advantage that：
1st, the SHENGMA GEGEN TANG compositionss finger printing that the present invention is provided, reflects the matter of SHENGMA GEGEN TANG compositionss comprehensively
Amount information such that it is able to reach the purpose for more fully and effectively controlling SHENGMA GEGEN TANG composite preparation product quality.
2nd, the method for building up of the SHENGMA GEGEN TANG compositionss finger printing that the present invention is provided is carried using Chinese Pharmacopoeia Commission
For similarity evaluation surveyed finger printing is recognized, it is easy to operate, quick；And,
Preparation finger is evaluated with the Xiang Yidu results that this draws, conclusion is more objective, accurate.
3rd, the method for building up of the SHENGMA GEGEN TANG compositionss finger printing that the present invention is provided is with reference to Chinese medicine fingerprint image
The requirement of spectrum, is studied to finger printing according to the present invention, is referred to through the investigation to test sample preparation method and measure
The conditions such as the instrument of stricture of vagina collection of illustrative plates, chromatographic column, mobile phase, Detection wavelength carry out the preferred of system, establish determining fingerprint pattern bar
Part has simultaneously carried out methodological study, on the basis of to many batches of this Chinese medicine composition finger printing testing results, gradually accumulates number
According to, it is proposed that standard finger-print, as this product finger printing standard, so as to reach can more comprehensively, efficiently control preparation
The purpose of quality.
4th, identification of the present invention to measured finger printing provides Chinese medicine chromatographic fingerprint figure using Chinese Pharmacopoeia Commission
Spectrum similarity evaluation system as this Chinese medicine composition fingerprint similarity software for calculation, Jing test of many times researchs, by with
The method for calculating relative retention time and relative peak area compares, and the evaluation conclusion for being drawn is basically identical, using Chinese medicine color
Spectrum fingerprint similarity evaluation system evaluates the similarity of finger printing, easy to operate, quick, the similarity knot drawn with which
Really, preparation finger is evaluated, conclusion is more objective, accurate.
Description of the drawings
In order to be illustrated more clearly that the specific embodiment of the invention or technical scheme of the prior art, below will be to concrete
Needed for embodiment or description of the prior art, accompanying drawing to be used is briefly described, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, can be with according to these other accompanying drawings of accompanying drawings acquisition.
Fig. 1 is multi-wavelength detection result；
Fig. 2 is 200～400nm scanning optical spectrums 3D figures；
Fig. 3 be chromatographic line from the bottom to top：215nm、220nm、225nm、230nm、240nm、250nm；
Fig. 4 is 200～400nm scanning optical spectrums 3D figures；
Fig. 5 is Chromatographic Comparison's figure of two kinds of solvent systems；
Fig. 6 is need testing solution chromatogram；
Fig. 7 is need testing solution chromatogram；
Fig. 8 is that test sample dissolution solvent investigates Chromatographic Comparison's figure；
Fig. 9 is object of reference chromatographic peak and need testing solution chromatographic peak comparison diagram；
200～400nm scanning optical spectrums 3D figures in Figure 10 determining fingerprint patterns；
Daiazi reference solution chromatogram in Figure 11 determining fingerprint patterns
Reference fingerprint in Figure 12 determining fingerprint patterns
The continuous four batches of sample finger printing of Figure 13 contrast chromatogram
Figure 14 is integrity test chromatogram；
Specific embodiment
It is to preferably further understand the present invention to provide following embodiments, it is not limited to the optimal embodiment party
Formula, is not construed as limiting to present disclosure and protection domain, anyone the present invention enlightenment under or by the present invention and its
The feature of his prior art be combined and draw it is any with the present invention it is same or like as product, all fall within the present invention
Within protection domain.
Unreceipted specific experiment step or condition person in embodiment, according to the normal experiment described by document in the art
The operation of step or condition can be carried out.Agents useful for same or the unreceipted production firm person of instrument, be can by city available from
Conventional reagent product.
Reagent of the present invention and instrument：
Reagent：
SHENGMA GEGEN TANG compositionss (make by oneself, and 2) preparation method is shown in embodiment by laboratory；
Hesperetic acid (lot number：111698-201103 China pharmaceutical biological product calibrating academy, purity is 99.2%)；
Peoniflorin (lot number：110736-201136, Chinese pharmaceutical biological product examine and determine academy, and purity is 96.0%)；
Puerarin (lot number：110752-201418, Chinese pharmaceutical biological product examine and determine academy, and purity is 95.5%)；
Ammonium glycyrrhizinate reference substance (lot number：110731-201418, Chinese pharmaceutical biological product examine and determine academy, 93.1%),
Liquirtin reference substance (lot number：111610-201106, Chinese pharmaceutical biological product examine and determine academy, purity
93.7%),
Cimifugin (lot number：111710-200602, Chinese pharmaceutical biological product examine and determine academy),
Daidzein reference substance (lot number：111502-2004021, Chinese pharmaceutical biological product examine and determine academy),
Daiazi reference substance (lot number：111738-201302, Chinese pharmaceutical biological product examine and determine academy),
Lactone glucoside of Radix Paeoniae reference substance (lot number：GR-133-141025, Nanjing Guang Dong biological product company limited, purity
98.0%),
Rhizoma Cimicifugae (lot number：160220, the place of production：Jilin)；
Radix Puerariae (lot number：160222, the place of production：Hubei)；
The Radix Paeoniae Alba (lot number：160223, the place of production：Anhui)；
Radix Glycyrrhizae Preparata (lot number：160221, the place of production：Xinjiang).Reagent is pure for analysis, and water is ultra-pure water.
Instrument：1260 high performance liquid chromatographs of Agilent (1290DAD detectors)
Waters E2695 high performance liquid chromatographs (2998DAD detectors)
Embodiment 1. prepares reference substance solution
Hesperetic acid reference substance solution takes Hesperetic acid reference substance in right amount, accurately weighed, puts in brown measuring bottle, plus 10% second
Alcohol makes solution of every 1ml containing 20 μ g of Hesperetic acid, obtains final product.
Cimifugin reference substance solution takes cimifugin reference substance in right amount, accurately weighed, puts in brown measuring bottle, plus 10% ethanol system
Into solution of every 1ml containing 80 μ g of Hesperetic acid, obtain final product.
Peoniflorin reference substance solution takes peoniflorin reference substance in right amount, accurately weighed, plus methanol is made every 1ml and contains the molten of 60 μ g
Liquid, obtains final product.
Lactone glucoside of Radix Paeoniae reference substance solution takes lactone glucoside of Radix Paeoniae reference substance in right amount, accurately weighed, puts in brown measuring bottle, plus
10% ethanol makes solution of every 1ml containing 50 μ g of Hesperetic acid, obtains final product.
Puerarin reference substance solution takes puerarin reference substance in right amount, accurately weighed, plus 30% ethanol makes every 1ml containing 80 μ g
Solution, obtain final product.
Daidzein reference substance solution takes puerarin reference substance in right amount, accurately weighed, plus methanol is made every 1ml and contains 70 μ g's
Solution, obtains final product.
Daiazi reference substance solution takes daiazi reference substance in right amount, accurately weighed, plus methanol is made every 1ml and contains the molten of 70 μ g
Liquid, obtains final product.
Liquirtin reference substance solution extracting liquorice glycosides reference substance is appropriate, accurately weighed, plus 70% ethanol is respectively prepared every 1ml and contains
The solution of 20 μ g of liquirtin, obtains final product.
Ammonium glycyrrhizinate reference substance solution extracting liquorice acid ammonium reference substance is appropriate, accurately weighed, plus 70% ethanol is respectively prepared often
Solution of the 1ml containing ammonium glycyrrhizinate 0.2mg, obtains final product.
Mixed reference substance solution：Above-mentioned reference substance 1ml is drawn respectively to put in 5ml volumetric flasks, is mixed, is obtained final product.
The preparation of 2. SHENGMA GEGEN TANG compositionss of embodiment
First SHENGMA GEGEN TANG compositionss：Weigh four taste decoction pieces of following weight proportion：Rhizoma Cimicifugae 300g；Radix Puerariae 450g；In vain
Chinese herbaceous peony 300g；Radix Glycyrrhizae Preparata 300g, cuts into the coarse granule of about 2mm～8mm or so, weighs 9g (Rhizoma Cimicifugae 2g, Radix Puerariae 3g, the Radix Paeoniae Alba respectively
2g, Radix Glycyrrhizae Preparata 2g) it is placed in 2L Traditional Chinese medicine decocting earthen pots, plus 300ml water, soak 30 minutes, add a cover decoction, heat (magnitude of voltage about 220V)
To boiling (about 10 minutes), micro-boiling (magnitude of voltage about 170V) is kept to medicinal liquid about 100ml (about 38 minutes), using the medical yarn of two-layer
Cloth filters medicinal residues, obtains final product.
Second SHENGMA GEGEN TANG compositionss：Following decoction pieces Rhizoma Cimicifugae 666.7g, Radix Puerariae 1000g are weighed in parts by weight, in vain
Chinese herbaceous peony 666.7g, Radix Glycyrrhizae Preparata 666.7g, cut into the coarse granule of granularity 2mm～8mm, add water 12 times and measure water, decoct 30min, filter,
Filtrate is obtained, thick paste, plus appropriate maltodextrin is concentrated filtrate to, is dried, mixed, plus appropriate magnesium stearate, dry-pressing
Grain, makes 1000g, obtains final product.2. compositionss are aluminized/polyethylene composite film using polyester, and packing specification is 3.0g/ bags.
Embodiment 3：The selection of chromatographic condition
(1) selection of Detection wavelength
With octadecylsilane chemically bonded silica as filler：With methanol as mobile phase A, 0.1mol/L phosphoric acid is Mobile phase B,
According to following table, gradient elution, 10 μ l of sample size, flow velocity are carried out：1.0ml/min, column temperature：30℃.Gradient program is as follows.
1 gradient elution program 1 of table
Using HPLC multi-wavelength detection technologies, Detection wavelength respectively λ=316, λ=290, λ=270, λ=250, λ=
237, λ=230, to be evaluated with chromatograph peak number, separating degree etc., acquired results are shown in Fig. 1,2.
From 3D spectral scan figures, in SHENGMA GEGEN TANG decocting liquid contained major part material in end absorption (210nm～
250nm), understand with reference to the wavelength investigated by Fig. 1, decocting liquid can be more comprehensively reacted ultraviolet end wavelength is selected as far as possible
Multiple wavelength are carried out further under optimization chromatograph gradient condition in the wave-length coverage of 210nm～250nm by finger print information
Investigate, as a result see Fig. 3,4.
In summary, when Detection wavelength is 220nm, not only baseline is steady, and it is also more that chromatographic peak points out information, therefore selects
The Detection wavelength of finger printing is 220nm.
(2) investigation of flowing phase composition
As compositionss adopt water extraction, often polarity is larger to leach composition, and four taste decoction pieces compositions mostly are flavonoid, glycosides
Class etc., polarity is also larger.With reference to the conventional flowing phase composition that HPLC is adopted, respectively methanol-water, acetonitrile water system are carried out
Investigate.Result of the test is shown in Fig. 5.
Result of the test shows：In chromatographic peak number, it is good compared with methanol water system that chromatographic peak is separated acetonitrile water system,
Therefore acetonitrile water system is selected to carry out chromatograph gradient optimizing research.
(3) optimization of mobile phase ratio
On the basis of acetonitrile water system, in the case of remaining condition all same, gradient elution program is carried out excellent
Change research.Constantly adjustment and optimized by eluent gradient, result of the test is shown in Table 2,3, Fig. 6,7.
Chromatograph optimal conditions one：
2 gradient elution program 2 of table
As seen from the figure, chromatographic peak major part separating degree is preferable, but separates in retention time 37min, 60min or so chromatographic peak
It is poor, it is necessary to further to optimize.
Chromatograph optimal conditions two：
3 gradient elution program 1 of table
Embodiment 4：Test sample solvent is investigated
Solvent, temperature be affect Solubility of Substances key factor, identical material in different solvents dissolubility exist
Difference, the rising of the solubility with temperature of material in same solvent and increase.SHENGMA GEGEN TANG compositionss are 1. in extraction process
Temperature is higher, and the dissolubility of material is of a relatively high, and after room temperature is cooled to, moieties will be separated out because dissolubility is reduced, and be caused
1. compositionss are presented certain precipitation or suspended matter, the part that this precipitation or suspended matter are constituted as compositionss, it should to which
Quality is studied accordingly, while the preparation process of need testing solution should consider the reservation of this moieties.
The first SHENGMA GEGEN TANG compositionss are prepared by preparation method, is cooled between 30 DEG C～35 DEG C, respectively according to series
Mode prepares need testing solution：1. sample introduction (stock solution) is directly centrifuged：Take the first SHENGMA GEGEN TANG compositionss appropriate, 12000r/min
Centrifugation 10min, takes supernatant sample introduction, and 10 μ l of sample size are obtained final product.2. 30% methanol, 50% methanol, 70% methanol as solvent：Point
Precision does not measure 5ml the first SHENGMA GEGEN TANG compositionss and is placed in 10ml volumetric flasks, is separately added into methanol 3ml, 5ml, shakes up, plus
To scale, respectively as 30%, 50% methanol solvate, precision measures 3ml the first SHENGMA GEGEN TANG compositionss and is placed in 10ml appearances water
In measuring bottle, plus methanol dilution is to scale, shakes up, used as 70% methanol solvate；It is appropriate that above-mentioned three kinds of solution is taken respectively,
12000r/min is centrifuged 10min, takes supernatant sample introduction, and 20 μ l of sample size are obtained final product.Result of the test is shown in Fig. 8.
As shown in Figure 8, the test sample that four kinds of modes are processed is separated in chromatographic peak, consistent on peak number, it is contemplated that compositionss
Itself adopt water extraction, water as solvent more can response composite itself composition qualities, therefore select finger printing test sample
The dissolution solvent of solution is water.
The selection of 5. object of reference of embodiment
Object of reference is mainly used in the guide for recognizing and evaluating Fingerprints in finger printing, is not equal to containing measurement
Fixed reference substance.According to《Quality standards in Chinese drugs studies and defines technical requirements》The object of reference of finger printing is typically chosen and is easily obtained
One or more preparations in main active or index components, for investigating the degree of stability and weight of finger printing
Existing property.The active component or index components of the object of reference first-selection monarch drug of compound Chinese medicinal preparation, and object of reference should be retention time and fit
In, peak area size is suitable, separate stable chromatographic peak.
The preparation of reference solution：Take Hesperetic acid, ferulic acid, puerarin, peoniflorin, liquirtin, lactone glucoside of Radix Paeoniae, liter
Numb element, daidzein, daiazi, ammonium glycyrrhizinate reference substance in right amount, plus methanol be prepared into every 1ml contain 74.4 μ g of Hesperetic acid,
72.0 μ g of ferulic acid, 74.6 μ g of puerarin, 77.4 μ g of peoniflorin, 67.5 μ g of liquirtin, 77.6 μ g of lactone glucoside of Radix Paeoniae, cimifugin
81.1 μ g, 73.1 μ g of daiazi, 56 μ g of daidzein, the reference substance solution of 74.0 μ g of ammonium glycyrrhizinate, obtain final product.
As shown in Figure 9：The separation that daiazi, liquirtin, ammonium glycyrrhizinate can be good in need testing solution, wherein Radix Paeoniae
Lactone glycoside, daidzein are relatively low because of experimental cultivar content, and chromatographic peak area is less；Peoniflorin, puerarin do not have in chromatogram
Preferably separated；Cimifugin, Hesperetic acid, ferulic acid three because of the similar retention time in chromatogram of structure relatively,
Therefore separate out of condition, and peak area of the three in need testing solution is less, is not suitable as object of reference.Daiazi
From Radix Puerariae decoction pieces, Radix Puerariae is ministerial drug in side, and daiazi retention time, peak area in chromatogram is moderate, and in front and back without dry
Peak is disturbed, it is final to determine daiazi as finger printing object of reference.
Embodiment 6. has peak determination and the foundation of reference fingerprint.
Using same batch decoction pieces, 1. 10 parts of SHENGMA GEGEN TANG compositionss are prepared, 1. SHENGMA GEGEN TANG compositionss supply respectively
Test sample solution, is determined by determining fingerprint pattern method, records collection of illustrative plates.By analysis, its common characteristic peaks is determined for 21 (see accompanying drawing
10-12), relative retention time deviation RSD of 21 described common characteristic peaks is respectively less than 2%, i.e.,：
No. 1 peak average relative retention time RRT is 0.41% for 7.642, RSD%；
No. 2 peak average relative retention times RRT are 0.49% for 9.853, RSD%；
No. 3 peak average relative retention times RRT are 0.33% for 16.126, RSD%；
No. 4 peak average relative retention times RRT are 0.23% for 22.259, RSD%；
No. 5 peak average relative retention times RRT are 0.37% for 26.222, RSD%；
No. 6 peak average relative retention times RRT are 0.44% for 28.988, RSD%；
No. 7 peak average relative retention times RRT are 0.21% for 35.621, RSD%；
No. 8 peak average relative retention times RRT are 0.38% for 39.980, RSD%；
No. 9 peak average relative retention times RRT are 0.41% for 42.276, RSD%；
No. 10 peak average relative retention times RRT are 0.53% for 45.416, RSD%；
No. 11 peak average relative retention times RRT are 0.51% for 48.500, RSD%；
No. 12 peak average relative retention times RRT are 0.19% for 59.605, RSD%；
No. 13 peak average relative retention times RRT are 0.25% for 65.159, RSD%；
No. 14 peak average relative retention times RRT are 0.18% for 70.071, RSD%；
No. 15 peak average relative retention times RRT are 0.15% for 73.203, RSD%；
No. 16 peak average relative retention times RRT are 0.05% for 75.359, RSD%；
No. 17 peak average relative retention times RRT are 0.10% for 80.548, RSD%；
No. 18 peak average relative retention times RRT are 0.09% for 85.038, RSD%；
No. 19 peak average relative retention times RRT are 0.08% for 92.278, RSD%；
No. 20 peak average relative retention times RRT are 0.06% for 99.758, RSD%；
No. 21 peak average relative retention times RRT are 0.03% for 110.939, RSD%；
Wherein, No. 12 S peaks are the chromatographic peaks of object of reference.
Collection of illustrative plates is processed with chromatographic fingerprints of Chinese materia medica similarity evaluation software (version in 2012), using 160511-1 as ginseng
According to collection of illustrative plates, SHENGMA GEGEN TANG compositionss 1. reference fingerprint is obtained.Sample similarity is calculated using Cosin method, as a result table
It is bright, 10 parts of SHENGMA GEGEN TANG compositionss of decoction 1. similarity between 0.983～1.000, similarity is higher.
4 10 parts of SHENGMA GEGEN TANG compositionss of table 1. similarity evaluation result
Relevant parameter according to given by 10 batches of need testing solution chromatograms, selects with distinctive 21 total peaks, with
No. 12 reference peak-to-peak areas calculate the ratio of other each common characteristic fingerprint peakses peak areas, the results are shown in Table 5 as 1.
1. 5 10 parts of SHENGMA GEGEN TANG compositionss of table have peak area ratio result
The decoction pieces corresponding to the first SHENGMA GEGEN TANG compositionss are taken, continuous the second SHENGMA GEGEN TANG of three batches compositionss are prepared
Sample, is determined by finger print measuring method, and record chromatogram (see accompanying drawing 13) is generated with the first SHENGMA GEGEN TANG compositionss
Reference fingerprint calculates the similarity of 3 batch the second SHENGMA GEGEN TANG compositionss as retinue control collection of illustrative plates.As a result see below
Table.
Second SHENGMA GEGEN TANG compositionss Similarity Measure result
7. fingerprint spectrum method of embodiment is verified.
1 blank assay
To investigate interference of the test sample dissolution solvent to each chromatographic peak in finger printing, enter line blank test.By the side of drafting
Method, 10 μ l deionized waters of sample introduction operation gradient, records chromatogram, as a result shows that blank sample baseline is steady, substantially noiseless.
2 integrity tests
Record after the chromatogram of 120min by condition of drafting, be further continued for running 120min, and record chromatogram (see Figure 14).
It can be seen that chromatographic peak is had no after SHENGMA GEGEN TANG compositionss 120min, determine gradient operation 120min.
3 replica tests
According to need testing solution preparation method, 6 parts of need testing solutions are prepared, respectively 10 μ l of sample introduction, record chromatogram, adopted
Similarity evaluation calculates similarity, and RSD values, acquired results are shown in Table 6.
6 replica test similarity result of table
Result of the test shows：The method repeatability is good.
4 need testing solution stability
Need testing solution is taken, respectively at 0h, 2h, 4h, 8h, 16h, 24h, 36h time point sample introduction, chromatogram is recorded, is calculated
Similarity and RSD values, acquired results are shown in Table 7.
7 test sample stability test similarity result of table
Result of the test shows：Need testing solution relative standard deviation within 36h is less than 3%, has good stability.
Obviously, above-described embodiment is only intended to clearly illustrate example, and the not restriction to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description other multi-forms change or
Change.There is no need to be exhaustive to all of embodiment.And thus it is extended obvious change or
Among changing still in the protection domain of the invention.
Claims (10)
1. the method for building up of the finger printing of SHENGMA GEGEN TANG compositionss, it is characterised in that include adopting high performance liquid chromatography
SHENGMA GEGEN TANG compositionss finger printing is set up, chromatographic condition is：
Chromatographic column adopts octadecylsilane chemically bonded silica chromatographic column；
Flow velocity 0.9ml/min～1.1ml/min；Column temperature：25 DEG C～35 DEG C；
Detecting instrument adopts UV-detector, the Detection wavelength 210nm～250nm of finger printing；
Theoretical cam curve is not less than 4000 in terms of puerarin peak；
Methanol solution of the reference solution for daiazi；
It is system that mobile phase is -0.1% phosphoric acid solution of acetonitrile, carries out gradient elution in the following order：
2. the method for building up of finger printing according to claim 1, the SHENGMA GEGEN TANG compositionss are by following two
The method of kind is prepared：
(1) Rhizoma Cimicifugae 2g, Radix Puerariae 3g, Radix Paeoniae Alba 2g, Radix Glycyrrhizae Preparata 2g decoction pieces are weighed respectively, cut into thick of about 2mm～8mm or so
Grain, is placed in 2L Traditional Chinese medicine decocting earthen pots, plus 300ml water, soaks 30 minutes, adds a cover decoction, boiling 10 is heated under the voltage of 220V
Minute, under 170V voltages keep micro-boiling to medicinal liquid about 100ml, filter medicinal residues, obtain final product the first SHENGMA GEGEN TANG compositionss；Or
(2) following decoction pieces Rhizoma Cimicifugae 666.7g, Radix Puerariae 1000g, Radix Paeoniae Alba 666.7g, Radix Glycyrrhizae Preparata 666.7g are weighed in parts by weight,
The coarse granule of granularity 2mm～8mm is cut into, is added water 12 times and is measured water, decoct 30min, then filtration concentrates filtrate to thick
Cream, with maltodextrin, is dried, mixes, and stiffened fatty acid magnesium, dry-pressing granule make 1000g, obtains final product the combination of the second SHENGMA GEGEN TANG
Thing.
3. the method for building up of finger printing according to claim 2, it is characterised in that the finger printing includes 21 altogether
There is peak：
When No. 1 peak relative retention time RRT is that 8.944, No. 2 peak relative retention time RRT retain for 14.111, No. 3 peaks are relative
Between RRT be 20.583, No. 4 peak relative retention time RRT be 24.036, No. 5 peak relative retention time RRT be 27.333, No. 6 peaks
Relative retention time RRT for 32.806, No. 7 peak relative retention time RRT for 37.305, No. 8 peak relative retention time RRT is
40.061, No. 9 peak relative retention time RRT are that 42.859, No. 10 peak relative retention time RRT are relative for 45.699, No. 11 peaks
It is that 61.159, No. 13 peak relative retention time RRT are 67.543 that retention time RRT is 54.817, S peaks relative retention time RRT,
When No. 14 peak relative retention time RRT are that 70.776, No. 15 peak relative retention time RRT retain for 73.526, No. 16 peaks are relative
Between RRT be 80.076, No. 17 peak relative retention time RRT be 83.260, No. 18 peak relative retention time RRT be 86.766,19
It is 98.200, No. 21 peak relative retention time that number peak relative retention time RRT is 91.200, No. 20 peak relative retention time RRT
RRT is 111.034, wherein, No. 12 S peaks are the chromatographic peaks of object of reference.
4. the method for building up of finger printing according to claim 3, it is characterised in that for high-performance liquid chromatogram determination
Using water as solvent, reference solution adopts methanol as solvent to need testing solution.
5. the method for building up of finger printing according to claim 4, it is characterised in that the preparation process of reference solution
For：Take daiazi reference substance in right amount, plus methanol is configured to reference solution of the 1ml methanol containing 200 μ g of daiazi.
6. the method for building up of finger printing according to claim 5, it is characterised in that the preparation process of need testing solution
For：The first SHENGMA GEGEN TANG compositionss 5ml are taken, is centrifuged, is taken supernatant, obtain final product；Or take the second SHENGMA GEGEN TANG compositionss 1.5g,
Put in 50ml volumetric flasks, dilute, ultrasonic dissolution let cool, and add water constant volume, shake up, take solution, be centrifuged, take supernatant, i.e.,
.
7. the method for building up of finger printing according to claim 6, it is characterised in that sample size is 10 μ l, the fingerprint
The Detection wavelength of collection of illustrative plates is 210nm～250nm.
8. the finger printing of SHENGMA GEGEN TANG compositionss, it is characterised in that adopt the high performance liquid chromatography, chromatographic condition to be：
Chromatographic column adopts octadecylsilane chemically bonded silica chromatographic column；
Flow velocity 0.9ml/min～1.1ml/min；Column temperature：25 DEG C～35 DEG C；
Theoretical cam curve is not less than 4000 in terms of puerarin peak；
Methanol solution of the reference solution for daiazi；
It is system that mobile phase is -0.1% phosphoric acid solution of acetonitrile, carries out gradient elution in the following order：
9. the finger printing of SHENGMA GEGEN TANG compositionss according to claim 8, it is characterised in that the Detection wavelength is
210nm～250nm.
10. the finger printing of SHENGMA GEGEN TANG compositionss according to claim 9, it is characterised in that the finger printing
Including 21 total peaks：
When No. 1 peak relative retention time RRT is that 8.944, No. 2 peak relative retention time RRT retain for 14.111, No. 3 peaks are relative
Between RRT be 20.583, No. 4 peak relative retention time RRT be 24.036, No. 5 peak relative retention time RRT be 27.333, No. 6 peaks
Relative retention time RRT for 32.806, No. 7 peak relative retention time RRT for 37.305, No. 8 peak relative retention time RRT is
40.061, No. 9 peak relative retention time RRT are that 42.859, No. 10 peak relative retention time RRT are relative for 45.699, No. 11 peaks
It is that 61.159, No. 13 peak relative retention time RRT are 67.543 that retention time RRT is 54.817, S peaks relative retention time RRT,
When No. 14 peak relative retention time RRT are that 70.776, No. 15 peak relative retention time RRT retain for 73.526, No. 16 peaks are relative
Between RRT be 80.076, No. 17 peak relative retention time RRT be 83.260, No. 18 peak relative retention time RRT be 86.766,19
It is 98.200, No. 21 peak relative retention time that number peak relative retention time RRT is 91.200, No. 20 peak relative retention time RRT
RRT is 111.034, wherein, No. 12 S peaks are the chromatographic peaks of object of reference.
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Effective date of registration: 20221201 Address after: No.1 Guanqing Road, Guanlan hi tech park, Guanhu street, Longhua District, Shenzhen, Guangdong Province Patentee after: CHINA RESOURCES SANJIU MEDICAL & PHARMACEUTICAL Co.,Ltd. Patentee after: China Resources Sanjiu modern traditional Chinese Medicine Pharmaceutical Co.,Ltd. Address before: No.1 Guanqing Road, Guanlan high tech park, Longhua New District, Shenzhen, Guangdong 518110 Patentee before: CHINA RESOURCES SANJIU MEDICAL & PHARMACEUTICAL Co.,Ltd. |