CN111948316B - Establishment method and detection method of Zhuang medicine Jinmu granule fingerprint spectrum - Google Patents
Establishment method and detection method of Zhuang medicine Jinmu granule fingerprint spectrum Download PDFInfo
- Publication number
- CN111948316B CN111948316B CN202010815171.5A CN202010815171A CN111948316B CN 111948316 B CN111948316 B CN 111948316B CN 202010815171 A CN202010815171 A CN 202010815171A CN 111948316 B CN111948316 B CN 111948316B
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- volume percentage
- changed
- jinmu
- zhuang medicine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to the field of drug analysis, in particular to a construction method of Zhuang medicine Jinmu particle fingerprint spectrum, which comprises the following steps: (1) preparing Zhuang medicine Jinmu granules into a test solution; (2) high performance liquid chromatography determination: and (3) injecting the test solution into a high performance liquid chromatograph for determination to obtain the Zhuang medicine Jinmu granule fingerprint spectrum with the common characteristic peak. The high performance liquid chromatography method has the advantages of high precision, good reproducibility, short analysis time and certain specificity; the separation effect of each characteristic peak in the obtained characteristic map is better.
Description
Technical Field
The invention relates to the field of drug analysis, in particular to a construction method and a detection method of Zhuang medicine Jinmu granule fingerprint.
Background
The prescription of Zhuang medicine Jinmu granule is optimized on the basis of the prescription of patent Zhuang medicine Fuyajing granule. Mainly comprises strong medicinal materials such as Gaeguinhhgang (Zhuang medicine name) Jingang (Chinese medicine name), Gaeumei (Zhuang medicine name) Huochun (Chinese medicine name), Gaebengzlaz (Zhuang medicine name) sargentg (Chinese medicine name), Godiengganggh (Zhuang medicine name) Huzhang (Chinese medicine name), Gaeulonghauh (Zhuang medicine name) glabrous greenbrier rhizome (Chinese medicine name), Godungzhau (Zhuang medicine name) white back root of the leaf (Chinese medicine name) and the like. Has effects in clearing away heat and toxic materials, eliminating dampness, and stopping leukorrhagia, and can be used for treating leukorrhagia, lower abdomen pain, lumbosacral pain, pudendal pruritus, and dysmenorrhea caused by downward flow of damp-heat toxin.
The establishment of the quality standard of the preparation is an indispensable part for declaring new drugs, and finally, the safety and the effectiveness of the used drugs are ensured.
At present, no report about a construction method and a detection method of Zhuang medicine Jinmu granule fingerprint spectrum is found.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a construction method and a detection method of Zhuang medicine Jinmu particle fingerprint spectrum.
The purpose of the invention can be realized by the following technical scheme:
a construction method of Zhuang medicine Jinmu granule fingerprint spectrum comprises the following steps:
(1) preparing Zhuang medicine Jinmu particles into a test solution;
(2) high performance liquid chromatography determination: and (3) taking the test solution, injecting the test solution into a high performance liquid chromatograph for determination, and obtaining the Zhuang medicine Jinmu particle fingerprint spectrum with the common characteristic peak.
In one embodiment, in step (2), the chromatographic conditions are as follows: bonding a silica gel chromatographic column by using C18; the column temperature is 25-35 ℃, acetonitrile is used as a mobile phase A, 0.05-0.3% (V/V) phosphoric acid aqueous solution is used as a mobile phase B, gradient elution is adopted, the flow rate of the mobile phase is 0.5-1.5 mL/min, and the detection wavelength is 280-300 nm.
In one embodiment, the mobile phase change in the gradient elution of step (2) is set as follows: changing the volume percentage of the mobile phase A from 5% to 8% and the volume percentage of the mobile phase B from 95% to 92% in 0-40 min; the volume percentage of the mobile phase A is changed from 8% to 14% and the volume percentage of the mobile phase B is changed from 92% to 86% in 40-60 min; 60-95 min, changing the volume percentage of the mobile phase A from 14% to 19%, and changing the volume percentage of the mobile phase B from 86% to 81%; 95-110 min, wherein the volume percentage of the mobile phase A is changed from 19% to 30%, and the volume percentage of the mobile phase B is changed from 81% to 70%; 110-135 min, wherein the volume percentage of the mobile phase A is changed from 30% to 60%, and the volume percentage of the mobile phase B is changed from 70% to 40%; 135-145 min, wherein the volume percentage of the mobile phase A is changed from 60% to 95%, and the volume percentage of the mobile phase B is changed from 40% to 5%; 145-150 min, the volume percentage of the mobile phase A is changed from 95% to 5%, and the volume percentage of the mobile phase B is changed from 5% to 95%.
Preferably, the construction method of the Zhuang medicine Jinmu granule fingerprint comprises the following steps: in the step (1), 2g of Zhuang medicine Jinmu granules are precisely weighed and placed in a conical flask with a plug of 150ml, 25ml of 60% ethanol is precisely added, the mixture is tightly plugged and weighed, the mixture is subjected to ultrasonic treatment for 30min, the mixture is cooled, the lost mass is compensated by 60% ethanol, and the mixture is filtered to obtain a subsequent filtrate, namely a sample solution; in the step (2), 10 mu l of sample solution is precisely absorbed and injected into a high performance liquid chromatograph for determination, and the Zhuang medicine Jinmu granule fingerprint spectrum with common characteristic peaks is obtained.
In one embodiment, the chromatographic column used in the step (2) is a Symmetry C18 column with the following dimensions: 5 μm, 4.6mm × 250mm, the flow rate of the mobile phase is 1ml/min, the column temperature is 30 ℃, the detection wavelength is 291nm, and the concentration of the phosphoric acid aqueous solution is 0.3% (V/V).
The invention also provides a detection method of Zhuang medicine Jinmu granule fingerprint spectrum, which comprises the following steps:
(1) preparing Zhuang medicine Jinmu granules into a test solution;
(2) high performance liquid chromatography determination: and (3) taking the test solution, and injecting the test solution into a high performance liquid chromatograph for measurement to obtain the test solution.
In one embodiment, the chromatogram of the test solution obtained by the detection in the step (2) is compared with the fingerprint obtained by the construction method, the similarity is calculated, and the quality of Zhuang medicine Jinmu particles is evaluated.
In one example, in step (2), the chromatographic conditions are as follows: and (3) chromatographic column: symmetry C18 column, size 5 μm, 4.6mm × 250 mm; the column temperature is 30 ℃, acetonitrile is taken as a mobile phase A, 0.3% (V/V) phosphoric acid aqueous solution is taken as a mobile phase B, the flow rate of the mobile phase is 1mL/min, and the detection wavelength is 291 nm; with gradient elution, the mobile phase changes were set as follows: changing the volume percentage of the mobile phase A from 5% to 8% and the volume percentage of the mobile phase B from 95% to 92% in 0-40 min; the volume percentage of the mobile phase A is changed from 8% to 14% and the volume percentage of the mobile phase B is changed from 92% to 86% in 40-60 min; 60-95 min, changing the volume percentage of the mobile phase A from 14% to 19%, and changing the volume percentage of the mobile phase B from 86% to 81%; 95-110 min, wherein the volume percentage of the mobile phase A is changed from 19% to 30%, and the volume percentage of the mobile phase B is changed from 81% to 70%; 110-135 min, the volume percentage of the mobile phase A is changed from 30% to 60%, and the volume percentage of the mobile phase B is changed from 70% to 40%; 135-145 min, wherein the volume percentage of the mobile phase A is changed from 60% to 95%, and the volume percentage of the mobile phase B is changed from 40% to 5%; 145-150 min, the volume percentage of the mobile phase A is changed from 95% to 5%, and the volume percentage of the mobile phase B is changed from 5% to 95%.
Regarding the selection of the detection wavelength, the inventor carries out full-wavelength scanning on the Zhuang medicine golden mother particle test sample solution in the wavelength range of 190-600 nm, and finds that the test sample solution has larger absorption at 265nm, 291nm, 306nm and 345 nm. Therefore, in a preliminary experiment, the chromatograms are respectively compared under the wavelengths, and the result shows that the information of the chromatographic peak at 265nm is less, the separation degree at 345nm is poorer, the base line of the later elution section at 306nm is shifted, the separation degree of the chromatographic peak at 291nm is good, the information is rich, and the characteristic is stronger. In general terms, 291nm was chosen as the detection wavelength.
Regarding the selection of the mobile phase, the inventors investigated different mobile phase systems such as acetonitrile-water, acetonitrile-phosphoric acid aqueous solution, methanol-water, methanol-phosphoric acid aqueous solution, and the like, and as a result, it was found that when methanol is used as the organic phase, the chromatographic peak separation degree is poor and the peak appearance time is late, and when pure water is used as the aqueous phase, the peak information is small and the peak shape is poor, so that it was finally determined that when acetonitrile-0.3% phosphoric acid aqueous solution is used as the elution system, the chromatographic peak information is rich, the retention time is moderate, the peak shape is good, and the separation degree is good.
Regarding the selection of index components, in order to comprehensively control the internal quality of the Zhuang medicine Jinmu granules, components which are greatly related to the Zhuang medicine Jinmu granules for treating sequelae of pelvic inflammatory diseases are selected as the index components, and the inventor finds that gallic acid, polydatin, rutin, kaempferol and astilbin all have certain anti-inflammatory effects by searching related documents.
The invention also aims to provide a fingerprint of Zhuang medicine Jinmu granules, which is constructed by the above fingerprint construction method and has 22 common peaks: the characteristic peaks of the glabrous greenbrier rhizome comprise No. 11, No. 15 and No. 16 peaks; the characteristic peaks of the radix sophorae flavescentis comprise No. 20 and No. 21 peaks; the characteristic peaks of the phellodendron include No. 4, No. 7 and No. 8 peaks; the characteristic peaks of the giant knotweed comprise No. 9, No. 17, No. 18, No. 19 and No. 22 peaks; the characteristic peaks of the coltsfoot violet comprise a No. 12 peak; the characteristic peaks of the fire carbon mother include No. 1, No. 2, No. 5 and No. 10 peaks; peak 3 is the common peak of sargentgloryvine stem, bock greenbrier rhizome and phellodendron bark; 6. the 13 th peak is the common peak of the adamantine thorn and the glabrous greenbrier rhizome; the 14 th peak is the common peak of the coltsfoot root and the bock greenbrier rhizome.
Compared with the prior art, the method has the following advantages:
1. the high performance liquid chromatography method has the advantages of high precision, good reproducibility, short analysis time and certain specificity; the separation effect of each characteristic peak in the obtained characteristic map is better.
2. The characteristic spectrum established by the invention has 22 common peaks, has larger information quantity, completely reserves the effective components in the test solution, and defines 6 medicinal materials corresponding to 18 common characteristic peaks.
Drawings
FIG. 1 shows HPLC finger prints of 10 Zhuang medicine Jinmu granules.
FIG. 2 is an HPLC chart of a mixed control (upper panel) and Zhuang medicine Jinmu granule test sample (lower panel); wherein the icons are 2 gallic acid, 9 polydatin, 11 rutin, 13 astilbin and 20 kaempferol respectively.
FIG. 3 is the common peak belonging map in Zhuang medicine Jinmu granule fingerprint.
FIG. 4 is a graph of Zhuang medicine 'jin mother's granule fingerprint clustering analysis.
FIG. 5 is a graph showing the results of selective investigation of detection wavelengths; wherein the icons are respectively A.2458nm, B.291nm, C.306nm and D.354nm.
FIG. 6 is a diagram showing the results of a selective examination of mobile phases; wherein the icons are respectively A. acetonitrile-0.1% phosphoric acid, B. acetonitrile-pure water, C. methanol-pure water, and D. methanol-0.1% phosphoric acid.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 establishment of HPLC fingerprint of Zhuang medicine Jinmu granule
First, experimental material
1.1 instrumentation
Waterse model 2695 high performance liquid chromatograph, Waters corporation, USA; KQ3200B ultrasonic cleaner, ultrasonic instruments ltd, kunshan; HWS-26 model electric heating constant temperature water bath, Shanghai Qixin scientific instruments, Inc.; JM-A5002 electronic balance, Supersizer devices, Inc., of Zhu corporation; DGG-9426A type electric heating constant temperature blast drying oven, Shanghai Qixin scientific instruments Co., Ltd; SQP type electronic balance, sydows scientific instruments (beijing) ltd.
1.2 reagents and reagents
Anhydrous ethanol (batch No. 2019022802) and methanol (batch No. 202019060502) were purchased from Kyoto Kolon Chemicals, Inc. Chromatographic grade acetonitrile (batch number: 172563), chromatographic grade methanol (batch number: 178500) were purchased from Saimer Feishel technologies, Inc. Gallic acid (purity: 91.5%, batch number: 110831-. Diamond (Smilax Scobinicaulis Radix, lot number: Y18080604), Chinese knotweed (Polygonum Chinense Herba, lot number: 17070202), Smilax glabra (Smilacis Glabrae Rhizoma, lot number: 18031901), Polygonum cuspidatum (Polygonum Cuspidifolia Rhizoma et Radix, lot number: 18051501), Absidia (Violae Herba, lot number: Y18080604), Phellodendron amurense (Phellodenron Chinense Cortex, lot number: 17102601), Sophora flavescens (Sophora flavescens Radix, lot number: 18071702), Sargentodoxa cuneata (Phellodendride Cortex, lot number: 17042504), and Malloti Apeltae Rhizoma root (Malloti Rhizoma, lot number: 19060401) were purchased from Raphiao Zhengchi Radix pharmaceutical industries, Ltd.
1.3 samples
Zhuang Yao jin mu Ke Li (a medicine of Guangxi university of traditional Chinese medicine, developed by the pharmaceutical engineering center, lot numbers 20190601-20190610, recorded as S1-S10 in turn).
Second, Experimental methods
2.1 preparation of Zhuang medicine Jinmu granule
According to table 1, 9 medicinal materials are put into a multifunctional extraction pot, heated and refluxed with 60% ethanol for two times, each time for 45 minutes, 60% ethanol is added according to the material-liquid ratio of 1: 10 for the first time, and 60% ethanol is added according to the material-liquid ratio of 1: 8 for the second time. Mixing extractive solutions, filtering, recovering ethanol from the filtrate until no ethanol smell exists, concentrating the filtrate to obtain concentrated solution with relative density of 1.08(60 deg.C), weighing and recording the concentrated solution, extracting appropriate amount of concentrated solution to determine solid content, calculating total solid content in the concentrated solution according to formula "total solid content is solid content multiplied by total weight of the concentrated solution", adding adjuvants into the concentrated solution according to the ratio of "total solid content to dextrin is 1: 1", mixing, adding prescribed amount of steviosin, spray drying, and collecting spray dried powder. Weighing, calculating the paste adding precision according to the weight of 1000 g-spraying dry powder, adding auxiliary materials, uniformly mixing with the spraying dry powder, tabletting by using a dry granulating machine to obtain Zhuang medicine Jinmu granules, and granulating into 1000 g.
2.2 preparation of control solutions
Accurately weighing appropriate amount of gallic acid, polydatin, rutin, astilbin, and kaempferol as reference substances, and adding methanol to obtain mixed reference substance solutions with mass concentrations of 0.067, 0.572, 0.635, 0.522, and 0.668mg/ml respectively.
2.3 preparation of test solutions
Weighing 2g of Zhuang medicine rhizoma anemarrhenae granules, precisely weighing, placing in a conical flask with a plug of 150ml, precisely adding 25ml of 60% ethanol, sealing the plug, weighing for mass, performing ultrasonic treatment for 30min, cooling, supplementing 60% ethanol to the loss, filtering, and taking the subsequent filtrate.
2.4 preparation of solutions of Individual herbs
As shown in Table 1, the concentrated solutions of the ingredients of Jingangci, herb Polygoni chinensis, Smilax glabra, Phellodendri cortex, Polygonum cuspidatum, etc. were prepared according to item "2.1" and the test solutions of the individual ingredients were prepared according to item "2.3".
2.5 chromatographic conditions
The chromatographic column is a Symmetry C18 column (4.6mm × 250mm, 5 μm); the mobile phase is acetonitrile-0.3% phosphoric acid, and the gradient elution is as follows: 0-40 min, 5-8% acetonitrile; 40-60 min, 8-14% acetonitrile; 60-95 min, 14% -19% acetonitrile; 95-110 min, 19% -30% acetonitrile; 110-135 min, 30-60% acetonitrile; 135-145 min, 60% -95% acetonitrile; 145-150 min, 95% -5% acetonitrile; flow rate: 1 ml/min; column temperature: 30 ℃; detection wavelength: 291 nm; sample introduction amount: 10 μ l.
Third, experimental results
3.1 determination of common peaks
Taking 10 batches of Zhuang medicine Jinmu granule samples, preparing a sample solution according to the method under item 2.3, injecting samples according to the chromatographic condition under item 2.5, performing chromatographic peak matching by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and selecting 22 chromatographic peaks with good separation degree and high content as fingerprint common characteristic peaks of the Zhuang medicine Jinmu granules.
3.2 evaluation of similarity
The fingerprint spectrums of 10 batches of Zhuang medicine Jinmu particle samples are led into a traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system (2012 edition), a median method is adopted, 0.1min is taken as time width, S1 is taken as a reference spectrum, automatic matching is carried out after chromatogram multipoint correction, 10 batches of Zhuang medicine Jinmu particle fingerprint spectrums (figure 1) are generated, wherein, 11 th peak (rutin) is taken as the reference peak and the similarity is calculated, and the similarity of the chromatogram of a test sample measured by S1-S10 and the similarity of a reference fingerprint spectrum are respectively 0.997, 0.976, 0.991, 0.987, 0.985, 0.990, 0.962, 0.993, 0.978 and 0.985. The results show that the similarity among 10 batches of samples is good, which reflects the stability of the Zhuang medicine Jinmu granule preparation process to a certain extent. Taking the No. 11 peak (rutin) as a reference peak and calculating the relative retention time and the relative peak area of the fingerprint of 10 batches of the Zhuang medicine mother-of-gold particles, wherein the RSD of the 22 common peaks in the 10 batches of the Zhuang medicine mother-of-gold particles is less than 0.7 percent, which shows that the peak emergence time of the 22 common peaks is relatively stable, and the RSD of the relative peak area is less than 3 percent, which shows that the component contents in different batches of particles are slightly different, and the results are shown in tables 2 and 3.
3.3 chromatographic Peak identification
By comparing the fingerprints of the mixed reference substance and the Zhuang medicine rhizoma polygonati particle and comparing the retention time of the two, the peaks 2, 9, 11, 13 and 20 in the fingerprint of the Zhuang medicine rhizoma polygonati particle can be identified as gallic acid, polydatin, rutin, astilbin and kaempferol respectively. See fig. 2.
3.4 correlation study of common peaks with herbs
Comparing the chromatogram of each single medicinal material solution of Zhuang medicine-Jinmu granules with a reference fingerprint (figure 3), finding that the chromatographic peak of each medicinal material corresponds to the reference chromatographic peak of the Zhuang medicine-Jinmu granules, tracing the common peaks of 10 batches of granule fingerprints to each single medicinal material, and confirming that the peaks 1, 2 (gallic acid) and 5 and 10 are the exclusive peaks of the herb of Bingchun; 4. peaks 7 and 8 are exclusive peaks of phellodendron; peaks 9 (polydatin), 17, 18, 19 and 22 are the exclusive peaks of the giant knotweed; peaks 11 (rutin), 15 and 16 are exclusive peaks of rhizoma smilacis glabrae; no. 12 peak is the exclusive peak of the coltsfoot weevil; 20 (kaempferol) and 21 # peak are the exclusive peak of kuh-seng; peak 3 is the common peak of sargentgloryvine stem, bock greenbrier rhizome and phellodendron bark; 6. the 13 th peak is the common peak of the adamantine thorn and the glabrous greenbrier rhizome; the 14 peak is the common peak of Absidia and Smilax Bockii, and the results are shown in Table 4.
3.5 Cluster analysis
SPSS21.0 statistical software is used, the relative peak area data of 22 common peaks are used as variables, the Ward connection method is used as a clustering model identification method, the squared Euclidean distance is used as a measurement standard, clustering analysis is carried out on the samples, and the result is shown in figure 4. The results show that when the euclidean distance is 5, the samples can be classified into 3 types, wherein the samples of S1, S3 and S4 are grouped into one type, the samples of S5, S6 and S9 are grouped into one type, and the samples of S2, S7, S8 and S10 are grouped into one type. Because the preparation process of the particles is consistent, the samples gathered into a class can reflect the similarity of the used medicinal materials on the expression of the common peak.
3.6 principal Components analysis
By using SPSS21.0 statistical software and taking 22 common peak relative peak area data as variables and a characteristic value greater than 1 as an extraction principle, main component analysis is carried out to obtain 2 main components, the cumulative variance contribution rate is 88.778%, the two main components can express 88.778% of chemical information in the Zhuang medicine Jinmu granule fingerprint, and the result is shown in Table 5. According to a load matrix (table 6), determining that PC1 mainly reflects information of chromatographic peaks 1, 4, 5, 7-11, 13, 16-18, 21 and 22, wherein the peak 9 is polydatin, the peak 11 is rutin, and the peak 13 is astilbin; PC2 mainly reflects information of chromatographic peak numbers 2, 6, 15 and 20, wherein peak number 2 is gallic acid and peak number 20 is kaempferol. Taking the proportion of the variance contribution rates corresponding to the two principal components in the accumulated variance contribution rate of the extracted principal components as a weight coefficient, taking peak areas of 22 common peaks as variables (after Z standardization), establishing a model, calculating the score of the principal components, wherein the model is Y0.9329 × PC1+0.0671 × PC2, calculating the comprehensive score of each sample through the model, sequencing the samples according to the score, and displaying the result that the samples can be divided into 3 types, wherein the samples of S2, S7, S8 and S10 are one type; samples S5, S6 and S9 are one type; samples No. S1, S3 and S4 are a class; among them, the S2 sample had better overall quality, and the results are shown in Table 7.
Example 2 precision test
The same sample solution (S1) was sampled and repeatedly injected 6 times, and the sample was measured under the chromatography condition of "2.5 chromatography condition" in example 1, and the relative retention time and relative peak area of 22 common peaks were calculated with reference to peak No. 11 (rutin), and the results are shown in table 8. The result shows that the relative retention time of each common peak and the RSD of the relative peak area are both less than 3.00 percent, which indicates that the precision of the instrument is good.
Example 3 stability test
The same sample solution (S1) was sampled at 0, 3, 6, 9, 12, and 24h under the chromatographic condition of "chromatographic condition 2.5" in example 1, and the relative retention time and relative peak area of 22 common peaks were calculated with reference to peak No. 11 (rutin), and the results are shown in Table 8. The result shows that the relative retention time of each common peak and the RSD of the relative peak area are both less than 3.00 percent, which indicates that the stability of the test solution is good within 24 hours.
Example 4 repeatability experiments
Taking 6 parts of Zhuang medicine-rhizoma-lonicerae-japonicas particles (S1), preparing a test solution according to the method under the item of '2.3 preparation of the test solution' in example 1, injecting samples according to the chromatographic condition under the item of '2.5 chromatographic condition' in example 1, and calculating the relative retention time and the relative peak area of 22 common peaks by taking the No. 11 peak (rutin) as a reference, wherein the results are shown in Table 8. The result shows that the relative retention time of all the shared peaks and the RSD of the relative peak area are both less than 3.00 percent, which indicates that the method has good repeatability.
TABLE 1 Zhuang medicine Jinmu granule formulation
| Name of Chinese medicine/name of auxiliary Material | Zhuang medicine name | Dosage (g) |
| Diamond thorn | Gaeuginhgangh (tone: diamond hook) | 555 |
| Charcoal nut | Gaeumei (Sound: colluding berry) | 555 |
| Rhizoma Smilacis Glabrae | Gaeulonghauh (tone: caraway) | 555 |
| Root of white back blade | Godungzhau (sound: the understand luxury) | 555 |
| Caulis Sargentodoxae | Gaebengzlaz (tone: hook handle loudspeaker) | 444 |
| Giant knotweed rhizome | Godiengganggh (tone: Tiangang) | 370 |
| Sophora flavescens ait | Caemhgumh (Sound: hook ) | 333 |
| Huang Bai | 333 | |
| Herba Violae Mundae | 333 | |
| Dextrin | Proper amount of | |
| |
10 |
TABLE 210 batch Zhuang medicine-Jinmu granule HPLC fingerprint chromatogram common peak relative retention time
Table 310 Zhuang medicine mother gold particles HPLC fingerprint chromatogram common peak relative peak area
TABLE 4 peak assignment results
TABLE 510 analysis results of fingerprint spectra common peak of Zhuang medicine 'Jinmu' granules
| Principal component | Characteristic value | Variance contribution rate/%) | Cumulative variance contribution/%) |
| PC1 | 18.220 | 82.817 | 82.817 |
| PC2 | 1.311 | 5.960 | 88.778 |
| PC3 | 0.901 | 4.097 | 92.874 |
| PC4 | 0.525 | 2.388 | 95.263 |
TABLE 610 Zhuang medicine Jinmu granule fingerprint chromatogram common peak principal component load matrix
| Peak number | PC1 | PC2 | Peak number | PC1 | PC2 |
| 1 | 0.778 | -0.617 | 12 | 0.845 | 0.375 |
| 2 | 0.214 | 0.926 | 13 | 0.946 | -0.037 |
| 3 | 0.783 | 0.418 | 14 | 0.877 | 0.298 |
| 4 | 0.797 | -0.455 | 15 | -0.355 | 0.891 |
| 5 | 0.823 | 0.141 | 16 | 0.983 | -0.041 |
| 6 | 0.056 | 0.956 | 17 | 0.986 | -0.018 |
| 7 | 0.969 | -0.045 | 18 | 0.946 | 0.204 |
| 8 | 0.957 | 0.004 | 19 | 0.802 | 0.339 |
| 9 | 0.882 | -0.254 | 20 | -0.350 | 0.991 |
| 10 | 0.904 | 0.059 | 21 | 0.992 | -0.020 |
| 11 | 0.996 | -0.015 | 22 | 0.960 | -0.246 |
TABLE 710 Substratescores and comprehensive score orderings of Zhuang medicine-Jinmu granule samples
TABLE 8 methodological observations
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto without departing from the scope of the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (4)
1. A construction method of Zhuang medicine Jinmu granule fingerprint spectrum is characterized by comprising the following steps: (1) preparing Zhuang medicine Jinmu granules into a test solution; (2) high performance liquid chromatography determination: injecting the test solution into a high performance liquid chromatograph for determination to obtain Zhuang medicine Jinmu granule fingerprint with common characteristic peaks;
the chromatographic conditions were as follows: bonding a silica gel chromatographic column by using C18; the column temperature is 25-35 ℃, acetonitrile is used as a mobile phase A, phosphoric acid aqueous solution with volume percentage concentration of 0.05-0.3% is used as a mobile phase B, gradient elution is adopted, the flow rate of the mobile phase is 0.5-1.5 mL/min, and the detection wavelength is 280-300 nm;
the mobile phase change was set as follows: changing the volume percentage of the mobile phase A from 5% to 8% and the volume percentage of the mobile phase B from 95% to 92% in 0-40 min; the volume percentage of the mobile phase A is changed from 8% to 14% and the volume percentage of the mobile phase B is changed from 92% to 86% in 40-60 min; changing the volume percentage of the mobile phase A from 14% to 19% and the volume percentage of the mobile phase B from 86% to 81% within 60-95 min; 95-110 min, wherein the volume percentage of the mobile phase A is changed from 19% to 30%, and the volume percentage of the mobile phase B is changed from 81% to 70%; 110-135 min, the volume percentage of the mobile phase A is changed from 30% to 60%, and the volume percentage of the mobile phase B is changed from 70% to 40%; 135-145 min, wherein the volume percentage of the mobile phase A is changed from 60% to 95%, and the volume percentage of the mobile phase B is changed from 40% to 5%; 145-150 min, wherein the volume percentage of the mobile phase A is changed from 95% to 5%, and the volume percentage of the mobile phase B is changed from 5% to 95%.
2. The construction method according to claim 1, wherein in the step (1), 2g of Zhuang medicine Jinmu granules are taken, precisely weighed, placed in a 150ml conical flask with a plug, precisely added with 25ml of 60% ethanol, tightly plugged, weighed, ultrasonically treated for 30min, cooled, supplemented with 60% ethanol to reduce the mass, filtered, and the subsequent filtrate is taken to obtain the test solution; in the step (2), the adopted chromatographic column is a Symmetry C18 column, and the size is as follows: 5 mu m and 4.6mm multiplied by 250mm, the flow rate of the mobile phase is 1ml/min, the column temperature is 30 ℃, the detection wavelength is 291nm, the volume percentage concentration of the phosphoric acid aqueous solution is 0.3%, 10 mu l of the test sample solution is precisely absorbed and injected into a high performance liquid chromatograph for determination, and the Zhuang medicine Jinmu granule fingerprint spectrum with the common characteristic peak is obtained.
3. A detection method of Zhuang medicine Jinmu granule fingerprint spectrum is characterized by comprising the following steps: (1) preparing Zhuang medicine Jinmu particles into a test solution; (2) high performance liquid chromatography determination: injecting the sample solution into a high performance liquid chromatograph for determination to obtain a chromatogram of the sample solution; (3) comparing the chromatogram of the test solution obtained by the detection in the step (2) with the fingerprint obtained by the construction method of any one of claims 1 or 2, calculating the similarity, and evaluating the quality of Zhuang medicine Jinmu granules;
in the step (2), the chromatographic conditions are as follows: a chromatographic column: symmetry C18 column, size 5 μm, 4.6mm × 250 mm; the column temperature is 30 ℃, acetonitrile is used as a mobile phase A, phosphoric acid aqueous solution with volume percentage concentration of 0.3% is used as a mobile phase B, the flow rate of the mobile phase is 1mL/min, and the detection wavelength is 291 nm; with gradient elution, the mobile phase changes were set as follows: changing the volume percentage of the mobile phase A from 5% to 8% and the volume percentage of the mobile phase B from 95% to 92% in 0-40 min; the volume percentage of the mobile phase A is changed from 8% to 14% and the volume percentage of the mobile phase B is changed from 92% to 86% in 40-60 min; 60-95 min, changing the volume percentage of the mobile phase A from 14% to 19%, and changing the volume percentage of the mobile phase B from 86% to 81%; 95-110 min, wherein the volume percentage of the mobile phase A is changed from 19% to 30%, and the volume percentage of the mobile phase B is changed from 81% to 70%; 110-135 min, wherein the volume percentage of the mobile phase A is changed from 30% to 60%, and the volume percentage of the mobile phase B is changed from 70% to 40%; 135-145 min, wherein the volume percentage of the mobile phase A is changed from 60% to 95%, and the volume percentage of the mobile phase B is changed from 40% to 5%; 145-150 min, the volume percentage of the mobile phase A is changed from 95% to 5%, and the volume percentage of the mobile phase B is changed from 5% to 95%.
4. The detection method according to claim 3, wherein in the step (1), 2g of Zhuang medicine Veronica mother granules are taken, precisely weighed, placed in a 150ml conical flask with a plug, 25ml of 60% ethanol is precisely added, the plug is sealed, the quality is weighed, ultrasonic treatment is carried out for 30min, cooling is carried out, the 60% ethanol is used for supplementing the loss quality, filtering is carried out, and the subsequent filtrate is taken, so that the test solution is obtained.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010815171.5A CN111948316B (en) | 2020-08-13 | 2020-08-13 | Establishment method and detection method of Zhuang medicine Jinmu granule fingerprint spectrum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010815171.5A CN111948316B (en) | 2020-08-13 | 2020-08-13 | Establishment method and detection method of Zhuang medicine Jinmu granule fingerprint spectrum |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111948316A CN111948316A (en) | 2020-11-17 |
| CN111948316B true CN111948316B (en) | 2022-09-13 |
Family
ID=73343049
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010815171.5A Active CN111948316B (en) | 2020-08-13 | 2020-08-13 | Establishment method and detection method of Zhuang medicine Jinmu granule fingerprint spectrum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111948316B (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104764828B (en) * | 2015-04-20 | 2017-01-11 | 广州白云山星群(药业)股份有限公司 | Construction method of fingerprint of Ilex rotunda thunb medicinal material and detection method of Ilex rotunda thunb medicinal material |
-
2020
- 2020-08-13 CN CN202010815171.5A patent/CN111948316B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN111948316A (en) | 2020-11-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109752478B (en) | Traditional Chinese medicine fingerprint detection method of compound garden balsam stem preparation | |
| CN106932509B (en) | Radix astragali and radix puerariae particle fingerprint spectrum and construction method thereof | |
| CN112444579B (en) | Method for establishing UPLC fingerprint spectrum of Chaihingye oral liquid | |
| CN114152705B (en) | HPLC fingerprint quality evaluation method for rhizoma atractylodis stem and leaf | |
| CN110297060B (en) | Fingerprint detection method and fingerprint thereof for ixeris sonchifolia medicinal materials | |
| CN107402265B (en) | Detection method of Kangyun granule fingerprint | |
| CN111175429B (en) | Method for establishing fingerprint spectrum of bactericidal and antipruritic lotion | |
| CN107561192A (en) | R accuses the method accused according to spring and S according to spring content in a kind of measure chromatogram of Radix Isatidis and its product | |
| CN108459129B (en) | Quality control method of radix Stephaniae Tetrandrae and Poria decoction composition | |
| CN1969953A (en) | Quality control method of honeysuckle, scutellarta root and extract thereof and formulation containing the extract | |
| CN111948316B (en) | Establishment method and detection method of Zhuang medicine Jinmu granule fingerprint spectrum | |
| CN115902050A (en) | Method for establishing high performance liquid phase fingerprint spectrum of Chinese herbal medicine compound preparation and application | |
| CN110297047B (en) | Establishment method of HPLC fingerprint of lysimachia capillipes hemsl medicinal material and fingerprint thereof | |
| CN113917009A (en) | Construction method and application of bupleurum chinense non-saponin component HPLC fingerprint | |
| CN108845040B (en) | Method for identifying whether Laijin pills contain pseudo-cynanchum atratum and old melon heads | |
| CN113092655A (en) | Method for detecting effective components in aconite decoction by high performance liquid chromatography | |
| CN115343377A (en) | Fingerprint spectrum of stomach-clearing coptis tablet and construction method and application thereof | |
| CN111751472A (en) | Quality evaluation method of sophora and scutellaria ointment | |
| CN112034085A (en) | HPLC fingerprint detection method for liver-soothing, stomach-harmonizing and pain-relieving traditional Chinese medicine | |
| CN111693630B (en) | Method for constructing HPLC standard fingerprint of Zhining tablet and application thereof | |
| CN114414685B (en) | Fingerprint detection method for traditional Chinese medicine capsules | |
| CN109856262B (en) | Method for qualitative and quantitative analysis of main components of Erding preparation simultaneously | |
| CN115541745B (en) | Quality detection method for hypericum perforatum standard decoction and formula particles | |
| CN113640434B (en) | Construction method and application of high performance liquid chromatography fingerprint of Zilongjin tablets | |
| CN116183751A (en) | Detection method of corydalis saxicola bunting fingerprint |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |