CN109828064B - HPLC fingerprint image establishing method for effective part group of periploca forrestii schltr - Google Patents
HPLC fingerprint image establishing method for effective part group of periploca forrestii schltr Download PDFInfo
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Abstract
The invention discloses a method for establishing an HPLC fingerprint of an effective part group of periploca forrestii schltr. The method has the advantages of simplicity, stability, high precision, good reproducibility, etc.; the method can also comprehensively reflect the types and the quantity of chemical components contained in the effective part group of the periploca forrestii schltr, and further carry out overall description and evaluation on the quality of the periploca forrestii schltr. The effective part group fingerprint spectrum of the periploca forrestii detected by the method has relatively more chemical components, moderate height proportion of each characteristic peak, stable base line, better separation degree and peak shape and higher column efficiency.
Description
Technical Field
The invention relates to a method for establishing an HPLC fingerprint map of an effective part group of periploca forrestii schltr, belonging to the technical field of medicines.
Background
Caulis et folium Periplocae Forrestii Schltr, belonging to Periploca of Asclepiadaceae (Asplepiadaceae), is collected from 2003 edition of quality standards of Chinese medicinal materials and national medicinal materials in Guizhou province, has effects of dispelling pathogenic wind and removing dampness, promoting blood circulation, dredging collaterals, eliminating arthralgia, relieving pain, relieving rigidity of muscles, activating collaterals, dispelling pathogenic wind and removing dampness; can be used for treating rheumatic arthritis, traumatic injury, gastralgia, dyspepsia, amenorrhea, and dysentery. The caulis et folium piperis nigri is one of the commonly used Miao medicine, and is an important component medicine of caulis et folium piperis nigri wind-dispelling and collateral-activating capsules, caulis et folium piperis nigri tendon-extending and bone-penetrating liquid and caulis et folium piperis nigri tendon-extending and bone-penetrating spray.
The traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification means, is established on the basis of the systematic research of the chemical components of the traditional Chinese medicine, and is mainly used for evaluating the authenticity, the excellence and the stability of the quality of the semi-finished products of the traditional Chinese medicine and the traditional Chinese medicine preparation. The traditional Chinese medicine and the preparation thereof are all multi-component complex systems, so that the quality of the traditional Chinese medicine and the preparation thereof is evaluated by adopting a detection method which is adaptive to the traditional Chinese medicine and can provide rich identification information, and the establishment of the traditional Chinese medicine fingerprint spectrum can comprehensively reflect the types and the quantities of chemical components contained in the traditional Chinese medicine and the preparation thereof, thereby integrally describing and evaluating the quality of the medicine. On the basis, if the research on the spectrum effect is further carried out, the quality of the traditional Chinese medicine and the drug effect thereof can be really combined, which is helpful for clarifying the action mechanism of the traditional Chinese medicine. At present, no report on HPLC fingerprint spectrum research work of the effective part group of the periploca forrestii is available.
Disclosure of Invention
The invention aims to provide a method for establishing an HPLC fingerprint map of an effective part group of periploca forrestii schltr. The method has the advantages of simplicity, stability, high precision, good reproducibility, etc.; the method can also comprehensively reflect the types and the quantity of chemical components contained in the periploca forrestii schltr, and further carry out overall description and evaluation on the quality of the periploca forrestii schltr. The periploca forrestii fingerprint spectrum detected by the method has relatively more chemical components, moderate height proportion of each characteristic peak, stable base line, good separation degree and peak shape and high column efficiency.
In order to solve the technical problems, the invention adopts the following technical scheme: an HPLC fingerprint map establishing method of an effective part group of periploca forrestii comprises the following steps:
(1) preparation of mixed control solution: taking chlorogenic acid, cryptochlorogenic acid and isochlorogenic acid C standard, diluting with methanol to constant volume to obtain chlorogenic acid, cryptochlorogenic acid and isochlorogenic acid C with concentration of 0.005-0.015 g.mL-1Mixing the reference solution;
(2) preparation of a test solution: accurately weighing 100.0g of periploca forrestii schltr coarse powder, performing reflux extraction with ethanol, performing suction filtration, concentrating, and performing constant volume of 1000mL with distilled water to obtain a sample solution; precisely weighing D101 macroporous adsorption resin, filling the D101 macroporous adsorption resin into a chromatographic column, washing with distilled water until the liquid flowing out from the lower end of the chromatographic column is clear and colorless, loading a sample solution into the chromatographic column for adsorption, eluting with distilled water after complete adsorption, then eluting with ethanol, collecting the ethanol part flowing out from the chromatographic column, concentrating, drying and grinding to obtain periploca forrestii effective part group powder; precisely weighing the effective fraction group powder of caulis et folium Periplocae Forrestii, diluting to constant volume with methanol, filtering with 0.45 μm microporous membrane, and collecting the filtrate to obtain sample solution;
(3) making a fingerprint spectrum: chromatographic conditions are as follows: a chromatographic column: xtimate C184.6mm × 250mm, 5 μm, acetonitrile (A) -0.1% phosphoric acid water solution (B) as mobile phase, gradient elution with detection wavelength of 360nm and flow rate of 1 mL/min-1The column temperature is 38 ℃, and the sample injection amount is 10 mu L; recording the spectrogram to obtain an HPLC fingerprint of the effective fraction group of the caulis et folium Periplocae Forrestii;
(4) and (3) standard fingerprint spectrum confirmation: according to the method provided by the above, HPLC fingerprint spectra are established for a plurality of batches of effective fractions of the periploca forrestii schltr, 24 common peaks are determined by analysis and comparison, and the common peaks form fingerprint characteristics of the effective fractions of the periploca forrestii schltr and are used as standard fingerprint spectra of the effective fractions of the periploca forrestii schltr.
In the aforementioned method for establishing an HPLC fingerprint of an effective fraction group of periploca forrestii, the mixed control solution is prepared by: taking chlorogenic acid, cryptochlorogenic acid and isochlorogenic acid C standard, and diluting with methanol to constant volume to obtain chlorogenic acid, cryptochlorogenic acid and isochlorogenic acid C with concentration of 0.01 mg/mL-1The control solutions were mixed.
In the aforementioned method for establishing an HPLC fingerprint of an effective fraction group of periploca forrestii, the sample solution is prepared by: accurately weighing 100.0g of periploca forrestii schltr coarse powder, performing reflux extraction for three times by using 70% ethanol, wherein the added ethanol is 8 times, 8 times and 6 times respectively, the extraction time is 1.5h, 1.5h and 1.0h respectively, combining the three filtrates, performing suction filtration, concentrating to 180-volume 220mL, and performing constant volume to 1000mL by using distilled water to obtain a sample solution; accurately weighing 100g of D101 macroporous adsorption resin, filling the resin into a chromatographic column, washing with distilled water until the liquid flowing out from the lower end of the chromatographic column is clear and colorless, loading 1000mL of sample solution into the chromatographic column for adsorption, eluting with 1200mL of distilled water after complete adsorption, then eluting with 2500mL of 70% ethanol, collecting 70% ethanol part flowing out from the chromatographic column, concentrating, drying and grinding to obtain periploca forrestii effective part group powder; precisely weighing 50.0mg of periploca forrestii schltr effective fraction group powder, diluting to 10mL with 70% methanol, filtering with 0.45 μm microporous membrane, and collecting filtrate to obtain test solution.
In the aforementioned method for establishing an HPLC fingerprint of an effective fraction group of periploca forrestii, the gradient elution is as follows: the mobile phase is acetonitrile (A) -0.1 percent phosphoric acid water solution (B); the gradient elution procedure was: 0-15 min, 11% -11% A; 15-30 min, 11% -18% A; 30-40 min, 18% -18% A; 40-50 min, 18% -23% A; 50-60 min, 23% -25% A; 60-85 min, 25% -32% A.
In the aforementioned method for establishing an HPLC fingerprint of an effective fraction of periploca forrestii, the 24 common peaks, taking chlorogenic acid as peak 3 as a reference peak, have relative retention times of 0.570, 0.595, 1.000, 1.110, 1.587, 1.851, 1.981, 2.117, 2.177, 2.436, 2.561, 2.720, 3.317, 4.092, 4.277, 4.360, 4.443, 4.580, 4.844, 4.886, 5.730, 5.879, 6.169 and 6.424, respectively.
The inventors have conducted a number of experiments and the following is a study of the detection method of the present invention
Experimental example: HPLC fingerprint spectrum research of effective part group of periploca forrestii schltr
1 HPLC fingerprint spectrum determination condition of effective part of caulis et folium Periplocae Forrestii
1.1 instruments and reagents
TABLE 1 Experimental instruments
The reagents methanol and acetonitrile are chromatographic pure, the ethanol is medical ethanol, the phosphoric acid is analytical pure, the water is double-distilled water and Wahaha purified water, and D-101 macroporous resin and the like are added.
1.2 preparation of test and control solutions
1.2.1 preparation of control solutions
Precisely weighing appropriate amount of chlorogenic acid, cryptochlorogenic acid, and isochlorogenic acid C standard, and diluting with methanol to constant volume of 0.01 mg/mL-1And (4) standard solution.
1.2.2 preparation of test solutions
Precisely weighing 100.0g of periploca forrestii coarse powder (Guizhou dragon palace), performing reflux extraction with 70% ethanol for three times, wherein the added ethanol is 8 times, 8 times and 6 times respectively, the extraction time is 1.5h, 1.5h and 1.0h respectively, combining the three filtrates, performing suction filtration, concentrating to about 200mL, and performing constant volume to 1000mL with distilled water to obtain a sample solution. Accurately weighing 100g of D101 macroporous adsorption resin, filling the resin into a chromatographic column, washing with distilled water until the liquid flowing out from the lower end of the chromatographic column is clear and colorless, loading 1000mL of sample solution into a sample for adsorption, eluting with 1200mL of distilled water after complete adsorption, then eluting with 2500mL of 70% ethanol, collecting 70% ethanol part flowing out from the chromatographic column, concentrating, drying and grinding to obtain the periploca forrestii effective part group powder. Precisely weighing 50.0mg of periploca forrestii schltr effective fraction group powder, diluting to 10mL with 70% methanol, filtering with 0.45 μm microporous membrane, and collecting filtrate to obtain test solution.
1.3 chromatographic conditions
The chromatographic column is Xtimate C18(4.6mm × 250mm, 5 μm), acetonitrile-0.1% phosphoric acid water solution as mobile phase, gradient elution (see Table 2), detection wavelength of 360nm, flow rate of 1 mL/min-1The column temperature was 38 ℃ and the amount of sample was 10. mu.L.
TABLE 2 conditions of mobile phase elution
The reference substance is subjected to sample injection inspection according to the same chromatographic conditions, and the retention time and chromatographic behavior of each chromatographic peak are compared to identify 4 chromatographic peaks which are respectively a No. 2 peak (neochlorogenic acid), a No. 3 peak (chlorogenic acid), a No. 4 peak (cryptochlorogenic acid) and a No. 15 peak (isochlorogenic acid C). See fig. 1 and 2:
2 methodology examination
In order to investigate and prove that the fingerprint measurement and evaluation method to be adopted is reliable and repeatable and accords with the traditional Chinese medicine fingerprint determination conditions, the experimental method is verified methodically. The fingerprint analysis process is complex, and the influence factors are more, so that a methodology verification experiment needs to be carried out to verify the whole process of the measurement result, reliable data information and process analysis control nodes are provided for quality control of the medicinal materials, and the aspect needs to be fully considered in research. In view of this, this section has performed methodological investigations on the precision, reproducibility and stability of the experimental methods.
2.1 precision test
Accurately weighing 50.0mg of effective component group powder (Guizhou dragon palace) of the periploca forrestii, fixing the volume to 10mL by using 70% methanol, filtering the solution by using a 0.45-micrometer microporous filter membrane, taking subsequent filtrate to obtain a sample solution, continuously sampling for 6 times under the chromatographic condition of '1.3', calculating the relative retention time of each common peak and the RSD of the relative peak area to be less than 3.0%, and indicating that the instrument has good precision. See tables 3, 4, 5 and 6.
TABLE 3 retention time for precision test
TABLE 4 area of precision test Peak
TABLE 5 relative retention times for precision tests
TABLE 6 relative peak area for precision test
2.2 stability test
Accurately weighing 50.0mg of periploca forrestii schltr effective part group powder (Guizhou Long Gong), metering the volume to 10mL by using 70% methanol, filtering the solution by using a 0.45-micrometer microporous filter membrane, taking subsequent filtrate to obtain a sample solution, respectively injecting samples for 0, 2, 4, 8, 24 and 36h under the chromatographic condition of '1.3', calculating the relative retention time of each common peak and the RSD of the relative peak area to be less than 3.0%, and experiments show that the sample solution is stable within 36h at normal temperature. See tables 7, 8, 9 and 10.
TABLE 7 stability test Retention time
TABLE 8 stability test Peak area
TABLE 9 stability test relative Retention time
TABLE 10 relative peak areas for stability tests
2.3 repeatability test
Accurately weighing 50.0mg and 6 parts of periploca forrestii schltr effective part group powder (Guizhou dragon palace), fixing the volume to 10mL by using 70% methanol, filtering by using a 0.45 mu m microporous membrane, taking subsequent filtrate to obtain a sample solution, analyzing according to the chromatographic conditions under the item of 1.3 respectively, and calculating the relative retention time of each common chromatographic peak and the RSD of the relative peak area to be less than 3.0 percent, thereby indicating that the used method has good repeatability. See tables 11, 12, 13 and 14.
TABLE 11 retention time for repeatability tests
TABLE 12 peak area for repeatability tests
TABLE 13 repeatability test relative retention time
TABLE 14 relative peak area for repeatability tests
3 establishment of HPLC fingerprint of effective part group of caulis et folium Periplocae Forrestii
In order to establish the effective part group fingerprint spectrum of the Periploca forrestii, 11 batches of Periploca forrestii Schltr are collected and identified as Periploca forrestii Schltr, and the numbers of the Periploca forrestii Schltr are S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 respectively. The detailed sources of the samples are shown in Table 15. Accurately weighing 100.0g of periploca forrestii schltr medicinal material powder of 11 batches of different sources, preparing a sample solution according to the method under the item '1.2.2', injecting samples according to the chromatographic condition under the item '1.3', and recording a chromatogram. Through the chromatogram analysis of 11 batches of samples, 24 common peaks are marked, the common peaks of the effective part groups of 11 batches of the caulis et folium piperis shown in figures 3-5, and the comparison fingerprint spectrum of the common mode is shown in figure 2. The retention time and peak area of the effective part group fingerprints of 11 batches of the periploca forrestii samples are shown in tables 16 and 18, and the relative retention time and relative peak area of the effective part group fingerprints of 11 batches of the periploca forrestii samples are shown in tables 17 and 19 by taking the No. 3 peak (chlorogenic acid as a reference peak).
TABLE 1511 batch of information on caulis et folium Periplocae Forrestii samples from different sources
TABLE 1611 retention time of each common peak in fingerprint of effective fractions of caulis et folium Periplocae Forrestii
TABLE 1711 fingerprint chromatogram of effective fractions of caulis et folium Periplocae Forrestii
TABLE 1811 fingerprint spectrum of effective fractions of caulis et folium Periplocae Forrestii sample
TABLE 1911 effective part group finger print of caulis et folium Periplocae Forrestii sample
3.1 finger print similarity of effective fractions of caulis et folium Periplocae Forrestii
By using the similarity evaluation software of the national pharmacopoeia committee, the chromatogram of the sample No. S1 is used as a reference chromatogram, the average number generation method is adopted, the time window width is 0.1min, automatic matching is carried out after multi-point correction, a reference chromatogram is generated, and the HPLC fingerprint similarity of the effective position group of the caulis et folium piperis nigri in each production place is obtained, which is shown in Table 20. The similarity of effective part groups of 11 batches of the periploca forrestii schltr is within the range of 0.837-0.989, wherein the similarity of the effective part groups of 3 batches of the periploca forrestii schltr is below 0.9, and the similarity of the effective part groups of the rest 8 batches of the periploca forrestii schltr is above 0.9.
TABLE 2011 batch caulis et folium Periplocae Forrestii sample HPLC fingerprint similarity
3.2 calibration of common peaks
1. The reference source and lot number are shown in Table 21.
TABLE 21 reference Source and batch number
2. The above reference substances are analyzed by injecting sample under the same chromatographic conditions, and compared with retention time and chromatographic behavior of each chromatographic peak, 4 chromatographic peaks are identified, which are respectively No. 2 peak (neochlorogenic acid), No. 3 peak (chlorogenic acid), No. 4 peak (cryptochlorogenic acid) and No. 15 peak (isochlorogenic acid C). See fig. 5 and 6.
4 results and conclusions
1 optimization of chromatographic conditions
1.1 determination of the mobile phase
The chromatogram map is prepared by reflecting the chemical component quantity and component ratio in the medicinal materials as much as possible under the chromatogram conditions of the fingerprint map, and comparing and selecting the following four mobile phases on the basis of the previous research for obtaining the characteristic fingerprint map of the effective part group of the periploca forrestii:
a first group: acetonitrile-0.1% phosphoric acid aqueous solution was measured according to different gradients.
Second group: tetrahydrofuran-0.1% phosphoric acid in water was measured according to different gradients.
Third group: methanol-0.1% phosphoric acid aqueous solution was measured according to different gradients.
And a fourth group: methanol-0.2% phosphoric acid aqueous solution was measured according to different gradients.
As can be known from the chromatogram, the acetonitrile-0.1% phosphoric acid aqueous solution is used as the mobile phase, the gradient elution effect is best, the chromatogram has more chromatographic peaks, the resolution is better, and the retention time is moderate, so the acetonitrile-0.1% phosphoric acid aqueous solution is selected as the mobile phase.
1.2 investigation of effective fraction group HPLC fingerprint column temperature of caulis et folium Periplocae Forrestii
In order to obtain a better chromatogram, the column temperature was also examined in this experiment, and the column temperatures were respectively 30 ℃, 35 ℃, 38 ℃ and 40 ℃, and by comparing the chromatograms, it was found that the chromatographic peak separation was the best at a column temperature of 38 ℃, so the column temperature was finally selected to be 38 ℃ in this study.
1.3 selection of detection wavelength
In the experiment, four wavelengths of 220 nm, 254 nm, 274 nm and 360nm are selected for measurement, more chromatographic peaks appear under 360nm, and most chromatographic peaks have stronger absorption, so 360nm is selected as the detection wavelength in the experiment.
1.4 examination of sample solvent
The test sample solvents of 60% methanol, 70% methanol and 80% methanol are respectively examined, and the obtained chromatogram shows that when the solvent is 70% methanol, the peak area of each main chromatographic peak does not influence with the increase of the solvent dosage, so the experiment finally determines that the concentration of the test sample solvent is 70% methanol.
The tests show that the determination method is stable and reliable, the fingerprint spectrum is relatively stable, and the method can be used for controlling the quality of the effective part group of the periploca forrestii schltr.
Compared with the prior art, the method has the advantages of simplicity, convenience, stability, high precision, good reproducibility and the like; the method can also comprehensively reflect the types and the quantity of chemical components contained in the effective part group of the periploca forrestii schltr, and further carry out overall description and evaluation on the quality of the periploca forrestii schltr. The effective part group fingerprint spectrum of the periploca forrestii detected by the method has relatively more chemical components, moderate height proportion of each characteristic peak, stable base line and good separation degree, peak shape and column efficiency.
Drawings
FIG. 1 is a chromatogram of a high performance liquid chromatography test of a control;
FIG. 2 is a high performance liquid chromatography chromatogram of a caulis et folium Periplocae Forrestii sample;
FIG. 3 is HPLC fingerprint of 11 batches of effective fractions of caulis et folium piperis nigri;
FIG. 4 is an HPLC control spectrum of effective fraction group of 11 batches of caulis et folium piperis nigri;
FIG. 5 is an HPLC profile of a mixed control;
figure 6 is a control fingerprint of consensus pattern.
The present invention will be further described with reference to the following examples.
Detailed Description
Example 1:
an establishment method of an HPLC fingerprint map of an effective part group of periploca forrestii comprises the following steps:
an HPLC fingerprint map establishing method of an effective part group of periploca forrestii comprises the following steps:
(1) preparation of mixed control solution: taking chlorogenic acid, cryptochlorogenic acid and isochlorogenic acid C standard, and diluting with methanol to constant volume to obtain chlorogenic acid, cryptochlorogenic acid and isochlorogenic acid C with concentration of 0.01 mg/mL-1Mixing the reference solution;
(2) preparation of a test solution: accurately weighing 100.0g of periploca forrestii schltr coarse powder, performing reflux extraction for three times by using 70% ethanol, wherein the added ethanol is 8 times, 8 times and 6 times respectively, the extraction time is 1.5h, 1.5h and 1.0h respectively, combining the three filtrates, performing suction filtration, concentrating to 180-volume 220mL, and performing constant volume to 1000mL by using distilled water to obtain a sample solution; accurately weighing 100g of D101 macroporous adsorption resin, filling the resin into a chromatographic column, washing with distilled water until the liquid flowing out from the lower end of the chromatographic column is clear and colorless, loading 1000mL of sample solution into the chromatographic column for adsorption, eluting with 1200mL of distilled water after complete adsorption, then eluting with 2500mL of 70% ethanol, collecting 70% ethanol part flowing out from the chromatographic column, concentrating, drying and grinding to obtain periploca forrestii effective part group powder; precisely weighing 50.0mg of periploca forrestii schltr effective fraction group powder, diluting to 10mL with 70% methanol, filtering with 0.45 μm microporous membrane, and collecting filtrate to obtain sample solution;
(3) making a fingerprint spectrum: chromatographic conditions are as follows: a chromatographic column: xtimate C18× 250mm of 4.6mm and 5 mu m, acetonitrile (A) -0.1 percent of phosphoric acid aqueous solution (B) as a mobile phase, and gradient elution, wherein the gradient elution procedure comprises the steps of 0-15 min, 11-11 percent of A, 15-30 min, 11-18 percent of A, 30-40 min, 18-18 percent of A, 40-50 min, 18-23 percent of A, 50-60 min, 23-25 percent of A, 60-85 min, 25-32 percent of A, detection wavelength of 360nm, flow rate of 1 mL/min-1The column temperature is 38 ℃, and the sample injection amount is 10 mu L; recording the spectrogram to obtain an HPLC fingerprint of the effective fraction group of the caulis et folium Periplocae Forrestii;
(4) and (3) standard fingerprint spectrum confirmation: according to the method provided by the above, HPLC fingerprint spectra are established for a plurality of batches of effective fractions of the periploca forrestii schltr, 24 common peaks are determined by analysis and comparison, and the common peaks form fingerprint characteristics of the effective fractions of the periploca forrestii schltr and are used as standard fingerprint spectra of the effective fractions of the periploca forrestii schltr. The 24 common peaks, with chlorogenic acid as reference peak No. 3, have relative retention times of 0.570, 0.595, 1.000, 1.110, 1.587, 1.851, 1.981, 2.117, 2.177, 2.436, 2.561, 2.720, 3.317, 4.092, 4.277, 4.360, 4.443, 4.580, 4.844, 4.886, 5.730, 5.879, 6.169 and 6.424, respectively.
Claims (3)
1. An HPLC fingerprint map establishment method of an effective part group of periploca forrestii is characterized by comprising the following steps: the method comprises the following steps:
(1) preparation of mixed control solution: taking chlorogenic acid, cryptochlorogenic acid and isochlorogenic acid C standard, diluting with methanol to constant volume to obtain chlorogenic acid, cryptochlorogenic acid and isochlorogenic acid C with concentration of 0.005-0.015 mg.mL-1Mixing the reference solution;
(2) the test solution was prepared by: accurately weighing 100.0g of periploca forrestii schltr coarse powder, performing reflux extraction for three times by using 70% ethanol, wherein the added ethanol is 8 times, 8 times and 6 times respectively, the extraction time is 1.5h, 1.5h and 1.0h respectively, combining the three filtrates, performing suction filtration, concentrating to 180-volume 220mL, and performing constant volume to 1000mL by using distilled water to obtain a sample solution; accurately weighing 100g of D101 macroporous adsorption resin, filling the resin into a chromatographic column, washing with distilled water until the liquid flowing out from the lower end of the chromatographic column is clear and colorless, loading 1000mL of sample solution into the chromatographic column for adsorption, eluting with 1200mL of distilled water after complete adsorption, then eluting with 2500mL of 70% ethanol, collecting 70% ethanol part flowing out from the chromatographic column, concentrating, drying and grinding to obtain periploca forrestii effective part group powder; precisely weighing 50.0mg of periploca forrestii schltr effective fraction group powder, diluting to 10mL with 70% methanol, filtering with 0.45 μm microporous membrane, and collecting filtrate to obtain sample solution;
(3) the fingerprint is prepared by subjecting a chromatographic column to XTmatec 18, 4.6 × 250mm, 5 μm, acetonitrile as phase A and 0.1% phosphoric acid solution as phase B to gradient elution at a detection wavelength of 360nm and a flow rate of 1 mL/min-1The column temperature is 38 ℃, and the sample injection amount is 10 mu L; recording the chromatogram to obtain blackHPLC fingerprint of the effective part group of the caulis Marsdeniae Tenacissimae; the gradient elution procedure is: 0-15 min, 11% → 11% A; 15-30 min, 11% → 18% A; 30-40 min, 18% → 18% A; 40-50 min, 18% → 23% A; 50-60 min, 23% → 25% A; 60-85 min, 25% → 32% A;
(4) and (3) standard fingerprint spectrum confirmation: according to the method provided by the above, HPLC fingerprint spectra are established for a plurality of batches of effective fractions of the periploca forrestii schltr, 24 common peaks are determined by analysis and comparison, and the common peaks form fingerprint characteristics of the effective fractions of the periploca forrestii schltr and are used as standard fingerprint spectra of the effective fractions of the periploca forrestii schltr.
2. The method for establishing an HPLC fingerprint of the effective fraction group of periploca forrestii schltr of claim 1, wherein the HPLC fingerprint comprises: the mixed control solution was prepared by: taking chlorogenic acid, cryptochlorogenic acid and isochlorogenic acid C standard, and diluting with methanol to constant volume to obtain chlorogenic acid, cryptochlorogenic acid and isochlorogenic acid C with concentration of 0.01 mg/mL-1The control solutions were mixed.
3. The method for establishing an HPLC fingerprint of the effective fraction group of periploca forrestii schltr of claim 1, wherein the HPLC fingerprint comprises: the 24 common peaks, with chlorogenic acid as reference peak No. 3, have relative retention times of 0.570, 0.595, 1.000, 1.110, 1.587, 1.851, 1.981, 2.117, 2.177, 2.436, 2.561, 2.720, 3.317, 4.092, 4.277, 4.360, 4.443, 4.580, 4.844, 4.886, 5.730, 5.879, 6.169 and 6.424, respectively.
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| 黑骨藤超高效液相色谱特征图谱的研究;赵喜玲 等;《药物分析杂志》;20161231;第36卷(第11期);摘要，第1964-1965页 * |
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