CN111398504A - Method for determining content of caffeoylquinic acid components in caulis et folium Periplocae Forrestii and cluster analysis - Google Patents
Method for determining content of caffeoylquinic acid components in caulis et folium Periplocae Forrestii and cluster analysis Download PDFInfo
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Abstract
The invention discloses a method for determining the content of caffeoyl quinic acid components in the ethyl acetate part of caulis et folium piperis nigri by an HP L C-UV method, and comprehensively evaluating the quality of caulis et folium nigri medicinal materials by combining the content determination of the caffeoyl quinic acid components with clustering analysis, principal component analysis and partial least square discriminant analysis.
Description
Technical Field
The invention relates to a content determination and cluster analysis method, in particular to a content determination and cluster analysis method for caffeoylquinic acid components in caulis et folium Periplocae Forrestii.
Background
The caulis et folium Periplocae Forrestii is dry root or whole plant of Periploca sepium (Periploca) Schltr of Periploca of Asclepiadaceae, is used as medicine for minority people in Guizhou province, and is collected in Chinese materia Medica (Miao medicine roll). Is bitter and pungent in taste, has the effects of dispelling pathogenic wind, removing dampness, promoting blood circulation and resolving carbuncle, and can be used for treating rheumatic arthralgia, traumatic injury, and menoxenia. The previous literature research shows that the periploca forrestii schltr has the effects of resisting Rheumatoid Arthritis (RA), strengthening heart, resisting tumors, suppressing immunity and the like, and mainly contains chemical components such as quinones, flavonoids, phenylpropanoids, terpenoids, C21 steroids and the like. The study in the earlier stage of the subject group finds that the periploca forrestii ethyl acetate part has good in-vivo anti-RA activity and is an anti-RA effective part of the periploca forrestii. Research shows that caffeoylquinic acid components in the caulis et folium Periplocae Forrestii have good anti-RA activity. The quality control method of the caulis et folium piperis nigri is mainly based on the quality standard of traditional Chinese medicinal materials and national medicinal materials in Guizhou province (2003 edition), but the detection of the caulis et folium piperis nigri in the standard is only limited to character, microscopic and partial physicochemical identification tests, and the quality of the caulis et folium piperis nigri cannot be well controlled. The functional components are used as index components for measuring the content of the RA-resistant effective part of the periploca forrestii schltr, and are combined with a chemometrics method to be used for the fresh report of quality evaluation of the RA-resistant effect of the periploca forrestii schltr.
Therefore, the method for determining the content of 6 caffeoylquinic acid components at the ethyl acetate part of the periploca forrestii by the HP L C-UV method, and comprehensively evaluating the quality of the periploca forrestii medicinal material by combining cluster analysis, principal component analysis and partial least square discriminant analysis has more important significance for guiding the reasonable harvesting, the production area processing and the resource development of the periploca forrestii medicinal material.
Disclosure of Invention
The invention aims to provide a method for measuring the content of caffeoylquinic acid components in caulis et folium piperis nigri and performing cluster analysis. The method has the characteristics that the content determination of 6 caffeoyl quinic acid components is combined with a stoichiometric method, the effect quality of the caulis et folium piperis nigri for resisting rheumatoid arthritis can be further evaluated, and a reference basis is provided for the quality control of the caulis et folium piperis nigri.
The technical scheme includes that the content of caffeoyl quinic acid components in the periploca forrestii is determined by an HP L C-UV method, and the content of the caffeoyl quinic acid components at the ethyl acetate part of the periploca forrestii is determined by the HP L C-UV method, and the quality of the periploca forrestii is comprehensively evaluated by the aid of the main component analysis and the partial least square discriminant analysis.
In the foregoing method for determining content of caffeoyl quinic acid components in caulis et folium piperis nigri and performing cluster analysis, the chromatographic conditions of the HP L C-UV method are as follows:
Xtimate C18(4.6mm × 250mm,5 mu m) chromatographic column, 0.1 percent of formic acid aqueous solution (A) -acetonitrile (B) as a mobile phase, and gradient elution, wherein the elution procedure comprises 0-16 min, 8-9 percent of B, 16-21 min, 9-11 percent of B, 21-25 min, 11-13 percent of B, 25-60 min, 13-16 percent of B, 60-85 min, 16-16 percent of B, 85-90 min, 16-17 percent of B, 90-98 min, 17-20 percent of B, 98-104 min, 20-21 percent of B, 104-115 min, 21-22 percent of B, sample introduction amount of 10 mu L, flow rate of 1m L/min, column temperature of 25 ℃ and detection wavelength of 327 nm;
preparation of mixed control solution:
precisely weighing 5.02, 5.00, 5.02, 5.03, 5.04 and 5.01mg of reference substances of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C respectively, placing the reference substances in a 5m L volumetric flask, adding methanol to a constant volume to reach a scale to prepare a single reference substance stock solution with corresponding concentration, precisely sucking a proper amount of each reference substance stock solution, and adding methanol to prepare a mixed reference substance solution with mass concentrations of 0.02, 0.10, 0.02 and 0.10mg/m L respectively;
preparation of a test solution:
precisely weighing 14.0-18.0g of medicinal material sample powder, adding 65-75% ethanol solution 350-450m L according to a material-liquid ratio of 1:23-27, heating, refluxing and extracting for 2-4 times, each time for 0.8-1.2h, combining extracting solutions, evaporating to dryness in a 65-75 ℃ water bath, adding 350-450m L water to form a suspension, extracting with equal amount of petroleum ether, chloroform, ethyl acetate and n-butyl alcohol respectively and sequentially for 3 times, combining 3 times of ethyl acetate extracting solutions, concentrating under reduced pressure, evaporating to dryness in a water bath, vacuum drying, grinding to powder to obtain an HGT-C part, precisely weighing 23-27mg of powder of the HGT-C part to a 10m L volumetric flask, adding methanol 2m L, carrying out ultrasonic treatment for 1-3min, cooling, adding methanol to a constant volume to scale, shaking uniformly, filtering through a 0.20-0.24 mu m micropore filter membrane, and taking a subsequent filtrate to obtain a test solution of a test sample solution.
In the method for measuring the content of caffeoyl quinic acid components in the periploca forrestii schltr and performing cluster analysis, the preparation of the test solution comprises the steps of precisely weighing 16.0g of medicinal material sample powder, adding 400m L of 70% ethanol solution according to the material-to-liquid ratio of 1:25, heating and refluxing for 3 times, extracting for 1 hour each time, combining extracting solutions, evaporating to dryness in a 70 ℃ water bath, adding 400m L water to obtain a suspension, extracting with equal amounts of petroleum ether, chloroform, ethyl acetate and n-butyl alcohol respectively and sequentially for 3 times, combining 3 times of ethyl acetate extracting solutions, concentrating under reduced pressure, evaporating to dryness in a water bath, performing vacuum drying, grinding to obtain a powder, obtaining an HGT-C (periploca forrestii schltr ethyl acetate) part, precisely weighing 25mg to 10m L volumetric flask of the powder of the HGT-C part, adding 2m L of methanol, performing ultrasonic treatment for 2min, cooling, adding methanol to scale, shaking uniformly, performing microporous filtration, and obtaining a secondary filtrate, thus obtaining the.
In the method for determining the content of caffeoyl quinic acid components in the caulis et folium Periplocae Forrestii and performing cluster analysis, the caffeoyl quinic acid components comprise neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C.
In the method for determining the content of the caffeoyl quinic acid components in the caulis et folium piperis nigri and the cluster analysis, the cluster analysis takes the content of the caffeoyl quinic acid components as a variable, SPSS24.0 statistical software is used, an inter-group mean number coupling method is used, and the distance formula of the similarity of samples is taken as the squared Euclidean distance, so that the caulis et folium piperis nigri medicinal material samples are subjected to cluster analysis.
In the method for determining the content of caffeoyl quinic acid components in the caulis et folium piperis nigri and performing cluster analysis, the principal component analysis and the partial least squares discriminant analysis are performed by using a multivariate statistical software SIMCA14.1 to obtain a P L S-DA score map, a 3D map and a VIP map of the caulis et folium piperis nigri.
According to the invention, the chemical components of 6 caffeoyl quinic acids such as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C and the like in the effective parts of the Miao medicine caulis Periplocae Forrestii with different sources are measured, and the quality of the effective parts is comprehensively analyzed by combining chemometrics, so that a basis is provided for reasonably and effectively evaluating the effect of the Miao medicine caulis Periplocae Forrestii on rheumatoid arthritis.
The method adopts HP L C-UV method for simultaneous determination, and the chromatographic column is XtimateC18(4.6mm × 250mm,5 μm), the mobile phase is 0.1% formic acid water-acetonitrile, gradient elution is carried out, the sample injection amount is 10 μ L, the flow rate is 1m L/min, the column temperature is 25 ℃, the detection wavelength is 327nm, and the quality of the periploca forrestii medicinal material is comprehensively analyzed and evaluated by adopting SPSS24.0 and SIMCA14.1 software.
As a result: carrying out content determination on 21 batches of periploca forrestii samples from different sources; classifying the 21 batches of samples into III classes through clustering analysis, wherein the II class and the III class have better quality; the results of the principal component analysis and partial least square discriminant analysis are consistent with the clustering results, and chlorogenic acid and isochlorogenic acid C obtained by screening are markers causing quality difference of caulis et folium Periplocae Forrestii from different sources.
And (4) conclusion: by combining the content determination of 6 caffeoylquinic acid components with a stoichiometric method, the effect quality of the caulis et folium piperis nigri for resisting rheumatoid arthritis can be further evaluated, and a reference basis is provided for the quality control of the caulis et folium piperis nigri.
The inventors conducted a number of experiments, and the following are partial experimental studies
Experimental example:
1 instruments and materials
1.1 instruments
L C-2030CN HPLC, which comprises an automatic sample feeding system, a column oven, a diode array detector and a chromatographic workstation (Shimadzu corporation, Japan), an XS205 type one-hundred-ten-thousandth balance (Medt corporation, Switzerland), an FA2204B type electronic balance (Shanghai Tianmei balance instruments Co., Ltd.), and a DRHH-2 type digital display constant temperature water bath (Shanghai Shujie experiment equipment Co., Ltd.).
1.2 reagent
Chlorogenic acid reference substances (batch number: CHB190121), neochlorogenic acid reference substances (batch number: CHB190217), cryptochlorogenic acid reference substances (batch number: CHB180905), isochlorogenic acid A reference substances (batch number: CHB180921), isochlorogenic acid B reference substances (batch number: CHB180923) and isochlorogenic acid C reference substances (batch number: CHB180925) are purchased from Dorkomae Biotechnology Co., Ltd, the mass fractions are all more than 98%, acetonitrile is chromatographically pure (America Tiandi Co., Ltd.), other reagents are analytically pure, and HP L C is purified water from Waaha Ha Co., Ltd.
21 batches of the medicinal materials are different from the medicinal materials of the Periploca forrestii, are identified as dried rhizomes of the Periploca plant of the genus Periploca (Periploca forrestii Schltr.) of the family asclepiadaceae by professor Liuyuchen of Guizhou traditional Chinese medicine university, and the specific information is shown in table 1.
TABLE 1 sample Source information
Table1 Source information of samples
2 methods and results
2.1 chromatographic conditions
Xtimate C18(4.6mm × 250mm,5 mu m) chromatographic column, 0.1 percent of formic acid aqueous solution (A) -acetonitrile (B) as a mobile phase, and gradient elution, wherein the elution procedure comprises 0-16 min, 8-9 percent of B, 16-21 min, 9-11 percent of B, 21-25 min, 11-13 percent of B, 25-60 min, 13-16 percent of B, 60-85 min, 16-16 percent of B, 85-90 min, 16-17 percent of B, 90-98 min, 17-20 percent of B, 98-104 min, 20-21 percent of B, 104-115 min, 21-22 percent of B, sample introduction amount of 10 mu L, flow rate of 1m L/min, column temperature of 25 ℃ and detection wavelength of 327 nm.
2.2 preparation of Mixed control solutions
Respectively and precisely weighing 5.02, 5.00, 5.02, 5.03, 5.04 and 5.01mg of reference substances of neochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C, placing the reference substances in a 5m L volumetric flask, adding methanol to a constant volume to reach a scale to prepare a single reference substance stock solution with corresponding concentration, precisely sucking a proper amount of each reference substance stock solution, and adding methanol to prepare a mixed reference substance solution with mass concentrations of 0.02, 0.10, 0.02 and 0.10mg/m L.
2.3 preparation of test solutions
Precisely weighing 16.0g of medicinal material sample powder, adding 400m L of 70% ethanol solution according to a material-liquid ratio of 1:25, heating and refluxing for 3 times, each time for 1h, combining extracting solutions, evaporating to dryness in a 70 ℃ water bath, adding 400m L of water to form suspension, respectively extracting with equal amount of petroleum ether, chloroform, ethyl acetate and n-butanol for 3 times, combining 3 times of ethyl acetate extracting solutions, concentrating under reduced pressure, evaporating to dryness in a water bath, vacuum drying, grinding to powder, obtaining HGT-C part, precisely weighing 25mg to 10m L volumetric flasks of the HGT-C part powder, adding 2m L of methanol, carrying out ultrasonic treatment for 2min, cooling, adding methanol to a constant volume, shaking uniformly, filtering through a 0.22 mu m microporous membrane, and taking a continuous filtrate to obtain a sample solution, and mixing HP L C pictures of a reference substance (A) and a sample (B) in pictures of figures 1-neochlorogenic acid, 2-chlorogenic acid, 3-chlorogenic acid, 4-isochlorogenic acid B, 5-isochlorogenic acid A and 6-isochlorogenic acid C.
2.4 Linear relationship investigation
Precisely measuring the single reference substance stock solution in volumetric flasks of 1m L-10 m L respectively, adding methanol to fix the volume to obtain a mixed standard substance 1, precisely sucking the mixed standard substance 1-100 m L respectively, adding methanol to fix the volume to obtain a mixed standard substance 2, respectively injecting 1, 5, 10, 15 and 20 mu L into the mixed standard substance 1 under the chromatographic condition of '2.1', respectively injecting 1 and 10 mu L into the mixed standard substance 2, measuring the peak area of each component, drawing a standard curve by taking the injection amount (mu g) of each component as an abscissa (X) and the peak area as an ordinate (Y), and obtaining a linear regression equation, a correlation coefficient (r) and a linear range of each component, taking the reference substance solution concentration of which the signal-to-noise ratio (S/N) is about 3:1 as a detection limit (L OD), and taking the reference substance solution concentration of which the signal-to-noise ratio (S/N) is about 10:1 as a quantitative limit (L OQ), wherein the results are shown in Table 2.
TABLE 26 Linear relationship of caffeoylquinic acid constituents
Table2 Linearity correlations of 6 coffee acyl quinines analytes
2.5 precision test
Precisely sucking the mixed reference substance solution under the item of 2.2, continuously sampling for 6 times under the chromatographic condition under the item of 2.1, and recording the peak area of each component. The results show that the peak areas RSD of the neochlorogenic acid, the chlorogenic acid, the cryptochlorogenic acid, the isochlorogenic acid B, the isochlorogenic acid A and the isochlorogenic acid C are respectively 0.24%, 0.32%, 0.36%, 0.59%, 0.99% and 0.47%, which indicates that the precision of the instrument is good.
2.6 stability test
The test solutions were prepared according to the method under item "2.3", placed at room temperature for 0, 2.5, 5, 10, 20, 40h, measured according to the chromatographic conditions under item 2.1 ", and the peak areas of the components were recorded. The results show that the peak areas RSD of the neochlorogenic acid, the chlorogenic acid, the cryptochlorogenic acid, the isochlorogenic acid B, the isochlorogenic acid A and the isochlorogenic acid C are respectively 2.13%, 2.32%, 2.46%, 2.45% and 2.08%, which indicates that the test solution is stable within 40 h.
2.7 repeatability test
Taking the same batch of periploca forrestii sample (S3) HGT-C part test sample, preparing 6 parts of test sample solution according to the method under the item '2.3', carrying out sample injection measurement according to the chromatographic condition under the item '2.1', recording peak areas of all components, and calculating the mass fraction RSD. The results show that the mass fractions RSD of the neochlorogenic acid, the chlorogenic acid, the cryptochlorogenic acid, the isochlorogenic acid B, the isochlorogenic acid A and the isochlorogenic acid C are respectively 1.77%, 1.29%, 1.08%, 0.76%, 1.14% and 1.28%, which indicates that the method has good repeatability.
2.8 sample recovery test
Taking 12.5mg of a test sample powder at the HGT-C part of the same batch of periploca forrestii sample (S3), precisely weighing, placing in a 10m L volumetric flask, precisely adding different concentrations of neochlorogenic acid (0.01278mg/m L), chlorogenic acid (0.47240mg/m L), cryptochlorogenic acid (0.04608mg/m L), isochlorogenic acid B (0.02912mg/m L), isochlorogenic acid A (0.11860mg/m L) and isochlorogenic acid C (0.42480mg/m L) reference substances respectively at 1m L, carrying out ultrasonic treatment for 2min, adding methanol to fix the volume to the scales, shaking up, preparing 6 parts in parallel, carrying out sample injection measurement according to the chromatographic condition under the item '2.1', recording peak areas of the components, calculating the recovery rates of the components respectively at 102.37%, 100.07%, 98.27%, 101.03%, 100.83% and 96.26%, and calculating the RSD values respectively at 1.06%, 1.67%, 1.51%, 2.88%, 0.77% and 0.95%, and establishing a good measurement result table.
TABLE 3 sample recovery test results (n ═ 6)
Table3 Results of recovery rates(n＝6)
2.9 measurement of sample content
Taking 21 batches of periploca forrestii schltr medicinal material samples, preparing a test solution according to the method under the item 2.3, carrying out sample injection measurement according to the chromatographic condition under the item 2.1, recording peak areas of all components, and calculating the mass fractions of all the components in the samples, wherein the results are shown in a table 4.
Table 421 sample test results
Table4 Determination results of 21 batches of samples
3 chemometric analysis
3.1 Cluster Analysis (CA)
The method comprises the steps of taking the content of 6 caffeoyl quinic acid components in 21 batches of caulis et folium piperis nigri medicinal materials as a variable, applying SPSS24.0 statistical software, adopting a group mean number coupling method, and taking the squared Euclidean distance as a distance formula of sample similarity to perform cluster analysis on medicinal material samples. The results are shown in FIG. 3. The results show that 21 samples are clustered into 3 types, and S10, S14, S16, S5, S20, S9, S18, S19, S11, S12, S13, S2 and S15 are I types; s1, S4, S3, S7, S6, S21 and S17 are type II; s8 is solely group iii.
The clustering analysis result shows that the content of 6 components in the class I sample is lower, 6 samples do not contain neochlorogenic acid, 11 samples do not contain isochlorogenic acid B, wherein the content of chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C in 3 medicinal materials collected in Guangxi province is lower, and 2 samples collected in Liuzhou province do not contain neochlorogenic acid, cryptochlorogenic acid and isochlorogenic acid B; the II type sample has high content of chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C, and other components can be basically detected; in the self-contained S8 sample, the chlorogenic acid content was the highest among the 21 samples, but no isochlorogenic acid B was detected.
2.5.2 Principal Components Analysis (PCA) and partial least squares discriminant analysis (P L S-DA)
The method comprises the steps of obtaining a PCA score map and a 3D map thereof by adopting multivariate variable statistical software SIMCA14.1, and a P L S-DA score map, a 3D map and a VIP map of 21 samples, wherein the results are shown in figures 4, 5 and 6, the results show that the 21 samples are also aggregated into 3 types, are more remarkably separated and are consistent with the CA result, and 2 different markers with statistical significance are obtained by screening by combining a Variable Importance Projection (VIP) method by taking the VIP value greater than 1 as a standard, wherein the influence degrees of the 2 different markers are respectively chlorogenic acid, isochlorogenic acid C, isochlorogenic acid A, cryptogenic acid, isochlorogenic acid B and new chlorogenic acid, isochlorogenic acid C and the 2 components are markers causing the change of the periploca nigra in different places, so that a certain basis is provided for the quality control of the periploca.
Discussion of 3
In order to reflect the component information in the periploca forrestii schltr as much as possible, enable the chromatographic peak of each detected component to have higher response value and achieve better separation, the experiment is carried out in the early stage by groping mobile phase, detection wavelength, column temperature, flow rate, chromatographic column and gradient elution conditions. The mobile phase researches acetonitrile-0.1 percent of phosphoric acid water, acetonitrile-0.1 percent of formic acid water and acetonitrile-0.1 percent of acetic acidThe result of elution conditions of water, acetonitrile-0.2% formic acid water and acetonitrile-0.05% formic acid water shows that the separation effect of the chemical components of the periploca forrestii is better under the elution conditions of the acetonitrile-0.1% formic acid water, so the system is selected, a DAD detector is adopted to carry out 190-450 nm full-wavelength scanning, the result shows that the chromatographic peak separation is better under the wavelength of 327nm, the response value is more complete, the column temperature researches at 20 ℃, 25 ℃ and 30 ℃ show that the chromatographic peak separation is better under the temperature of 25 ℃, the flow rate researches at 0.8m L/min, 1.0m L/min and 1.2m L/min show that the chromatographic peak separation is better under the temperature of 1.0m L/min, and CAPCE LL PAKC is selected18MGⅡ、Xtimate C18And Agilent C 183 kinds of chromatographic column, the result shows that when the chromatographic column is Xitinate C18The separation effect of each measured component is better.
The method of quantitative analysis and multivariate statistical analysis established by the invention can be used for quality control and quality standard improvement of the periploca forrestii schltr, and provides reference for quality evaluation research among batches of different producing areas of the traditional Chinese medicine.
In conclusion, the method has the beneficial effects that the effect quality of the caulis et folium piperis nigri for resisting rheumatoid arthritis can be further evaluated by combining the content determination of 6 caffeoyl quinic acid components with a chemometric method, and a reference basis is provided for the quality control of the caulis et folium piperis nigri.
Drawings
FIG. 1 is a graph of HP L C of a mixed control (A) in which 1-neochlorogenic acid, 2-chlorogenic acid, 3-cryptochlorogenic acid, 4-isochlorogenic acid B, 5-isochlorogenic acid A, and 6-isochlorogenic acid C;
FIG. 2 is a graph of HP L C of sample (B) in the present invention, wherein 1-neochlorogenic acid, 2-chlorogenic acid, 3-cryptochlorogenic acid, 4-isochlorogenic acid B, 5-isochlorogenic acid A, and 6-isochlorogenic acid C;
FIG. 3 is a dendrogram of 21 sample clusters in the cluster analysis of the present invention;
FIG. 4 is a plot of the P L S-DA scores of 21 samples in the principal component analysis and partial least squares discriminant analysis of the present invention;
FIG. 5 is a 3D plot of 21 samples in principal component analysis and partial least squares discriminant analysis according to the present invention;
figure 6 is a VIP plot of 21 samples in the principal component analysis and partial least squares discriminant analysis of the present invention.
Detailed Description
The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.
The embodiment relates to a method for measuring the content of caffeoyl quinic acid components in caulis et folium kadsurae, which is used for measuring the content of 6 caffeoyl quinic acid chemical components such as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C and the like in an ethyl acetate part of the caulis et folium kadsurae by an HP L C-UV method, and comprehensively evaluating the quality of a caulis et folium kadsurae medicinal material by combining cluster analysis, main component analysis and partial least square discriminant analysis, and specifically comprises the following steps:
the chromatographic conditions of the HP L C-UV method are as follows:
Xtimate C18(4.6mm × 250mm,5 mu m) chromatographic column, 0.1 percent of formic acid aqueous solution (A) -acetonitrile (B) as a mobile phase, and gradient elution, wherein the elution procedure comprises 0-16 min, 8-9 percent of B, 16-21 min, 9-11 percent of B, 21-25 min, 11-13 percent of B, 25-60 min, 13-16 percent of B, 60-85 min, 16-16 percent of B, 85-90 min, 16-17 percent of B, 90-98 min, 17-20 percent of B, 98-104 min, 20-21 percent of B, 104-115 min, 21-22 percent of B, sample introduction amount of 10 mu L, flow rate of 1m L/min, column temperature of 25 ℃ and detection wavelength of 327 nm;
preparation of mixed control solution:
precisely weighing 5.02, 5.00, 5.02, 5.03, 5.04 and 5.01mg of reference substances of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C respectively, placing the reference substances in a 5m L volumetric flask, adding methanol to a constant volume to reach a scale to prepare a single reference substance stock solution with corresponding concentration, precisely sucking a proper amount of each reference substance stock solution, and adding methanol to prepare a mixed reference substance solution with mass concentrations of 0.02, 0.10, 0.02 and 0.10mg/m L respectively;
preparation of a test solution:
precisely weighing 16.0g of medicinal material sample powder, adding 400m L of 70% ethanol solution according to a material-liquid ratio of 1:25, heating and refluxing for 3 times, each time for 1h, combining extracting solutions, evaporating to dryness in a 70 ℃ water bath, adding 400m L of water to form a suspension, sequentially extracting with equal amounts of petroleum ether, chloroform, ethyl acetate and n-butanol for 3 times, respectively, combining 3 times of ethyl acetate extracting solutions, concentrating under reduced pressure, evaporating to dryness in a water bath, vacuum drying, grinding to powder, obtaining an HGT-C part, precisely weighing 25mg to 10m L volumetric flask of the powder of the HGT-C part, adding 2m L of methanol, carrying out ultrasonic treatment for 2min, cooling, adding methanol to a constant volume, shaking uniformly, filtering through a 0.22 mu m microporous membrane, and taking a continuous filtrate to obtain a sample solution.
The cluster analysis is to use the content of caffeoyl quinic acid as variable, use SPSS24.0 statistical software, use the inter-group mean number coupling method, use the squared Euclidean distance as the distance formula of sample similarity, and carry out cluster analysis on the medicinal material sample, and classify the caulis et folium piperis nigri.
The principal component analysis and partial least square analysis respectively adopt multivariate statistical software SIMCA14.1 to obtain a P L S-DA score map, a 3D map and a VIP map of the periploca forrestii schltr.
As a result: carrying out content determination on 21 batches of periploca forrestii samples from different sources; classifying the 21 batches of samples into III classes through clustering analysis, wherein the II class and the III class have better quality; the results of the principal component analysis and partial least square discriminant analysis are consistent with the clustering results, and chlorogenic acid and isochlorogenic acid C obtained by screening are markers causing quality difference of caulis et folium Periplocae Forrestii from different sources.
And (4) conclusion: by combining the content determination of 6 caffeoylquinic acid components with a stoichiometric method, the effect quality of the caulis et folium piperis nigri for resisting rheumatoid arthritis can be further evaluated, and a reference basis is provided for the quality control of the caulis et folium piperis nigri.
Claims (6)
1. The method for measuring the content of caffeoyl quinic acid components in the caulis et folium piperis nigri is characterized in that the content of the caffeoyl quinic acid components at the ethyl acetate part of the caulis et folium piperis nigri is measured by an HP L C-UV method, and the quality of the caulis et folium piperis nigri medicinal material is comprehensively evaluated by combining cluster analysis, main component analysis and partial least square discriminant analysis.
2. The method for content determination and cluster analysis of caffeoyl quinic acid components in caulis et folium Periplocae Forrestii of claim 1, wherein the HP L C-UV method has the following chromatographic conditions:
Xtimate C18a chromatographic column with the thickness of 4.6mm and the thickness of × 250mm and the thickness of 5 mu m, a mobile phase of 0.1 percent formic acid aqueous solution A-acetonitrile B, gradient elution, wherein the elution procedure comprises 0-16 min, 8-9 percent B, 16-21 min, 9-11 percent B, 21-25 min, 11-13 percent B, 25-60 min, 13-16 percent B, 60-85 min, 16-16 percent B, 85-90 min, 16-17 percent B, 90-98 min, 17-20 percent B, 98-104 min, 20-21 percent B, 104-115 min, 21-22 percent B, the sample introduction amount of 10 mu L, the flow rate of 1m L/min, the column temperature of 25 ℃ and the detection wavelength of 327 nm;
preparation of mixed control solution:
precisely weighing 5.02, 5.00, 5.02, 5.03, 5.04 and 5.01mg of reference substances of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C respectively, placing the reference substances in a 5m L volumetric flask, adding methanol to a constant volume to reach a scale to prepare a single reference substance stock solution with corresponding concentration, precisely sucking a proper amount of each reference substance stock solution, and adding methanol to prepare a mixed reference substance solution with mass concentrations of 0.02, 0.10, 0.02 and 0.10mg/m L respectively;
preparation of a test solution:
accurately weighing 14.0-18.0g of medicinal material sample powder, adding 65-75% ethanol solution 350-450m L according to a material-liquid ratio of 1:23-27, heating, refluxing and extracting for 2-4 times, each time for 0.8-1.2h, combining extracting solutions, evaporating to dryness in a 65-75 ℃ water bath, adding 350-450m L water to obtain a suspension, extracting with equal amounts of petroleum ether, chloroform, ethyl acetate and n-butyl alcohol respectively and sequentially for 3 times, combining 3 times of ethyl acetate extracting solutions, concentrating under reduced pressure, evaporating to dryness in a water bath, vacuum drying, grinding to powder to obtain an ethyl acetate part of periploca forrestii, accurately weighing 23-27mg of the ethyl acetate part of periploca forrestii to a 10m L volumetric flask, adding 2m L of methanol, carrying out ultrasonic treatment for 1-3min, cooling, adding methanol to a constant volume, shaking uniformly, and filtering through a 0.20-0.24 mu m filter membrane to obtain a continuous filtrate to obtain a test solution.
3. The method for content determination and cluster analysis of caffeoyl quinic acid components in caulis et folium Periplocae Forrestii as claimed in claim 2, is characterized in that the test solution is prepared by precisely weighing 16.0g of medicinal material sample powder, adding 70% ethanol solution 400m L according to the material-to-liquid ratio of 1:25, heating and refluxing for 3 times, 1h each time, combining the extract solutions, evaporating to dryness in 70 ℃ water bath, adding 400m L water to obtain suspension, extracting with equal amount of petroleum ether, chloroform, ethyl acetate and n-butanol respectively for 3 times, combining the 3 times of ethyl acetate extract, concentrating under reduced pressure, evaporating to dryness in water bath, vacuum drying, grinding to powder to obtain HGT-C part, precisely weighing HGT-C part powder 25mg to 10m L volumetric flask, adding methanol 2m L, performing ultrasonic treatment for 2min, cooling, adding methanol to scale, shaking up, performing microfiltration with 0.22 μm to obtain a subsequent filtrate, and obtaining the test solution.
4. The method for content determination and cluster analysis of caffeoyl quinic acid components in caulis et folium Periplocae Forrestii as claimed in claim 1, wherein: the caffeoyl quinic acids include neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C.
5. The method for content determination and cluster analysis of caffeoyl quinic acid components in caulis et folium Periplocae Forrestii as claimed in claim 1, wherein: the cluster analysis is to perform cluster analysis on the periploca forrestii medicinal material sample by using the content of caffeoyl quinic acid components as variables, applying SPSS24.0 statistical software, adopting a group mean number coupling method and using the squared Euclidean distance as a distance formula of sample similarity.
6. The method of claim 1, wherein the principal component analysis and the partial least squares discriminant analysis are performed by using SIMCA14.1 to obtain P L S-DA score chart, 3D chart and VIP chart of caulis et folium Periplocae.
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