CN113933436A - Method for measuring contents of various components in caulis et folium piperis nigri medicinal material - Google Patents
Method for measuring contents of various components in caulis et folium piperis nigri medicinal material Download PDFInfo
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Abstract
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for determining contents of various components in a periploca forrestii schltr medicinal material. The invention adopts a UPLC-PDA method, takes Waters CQUITYLCCEBECHC181.7 mu M (2.1 multiplied by 50mm, 1.7 mu M) as a chromatographic column, and acetonitrile-0.1 percent formic acid water solution for gradient elution, wherein the detection wavelength is 225nm and 330nm, the flow rate is 0.35mL/min, and the column temperature is 38 ℃. The method is simple, convenient, rapid, accurate and good in repeatability, can be used for measuring the content of 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid methyl ester and periplogenin in the medicinal material of the caulis et folium piperis nigri, and provides a reference basis for the quality control of the caulis et folium piperis nigri.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for determining contents of various components in a periploca forrestii schltr medicinal material.
Background
The caulis et folium Piperis Futokadsurae is dry root or whole plant of Periploca forrestii Schltr (Asclepiadaceae), is collected in quality Standard of Chinese medicinal materials and national medicinal materials (2003 edition) in Guizhou province, and is one of the commonly used medicinal materials of Miao nationality in Guizhou province. The caulis et folium Periplocae Forrestii has effects of dredging channels, promoting blood circulation and dispelling pathogenic wind, and has pharmacological effects of resisting inflammation, relieving pain and resisting rheumatoid arthritis, and can be used for treating rheumatic arthralgia and traumatic injury. The clinical application results show that: caulis et folium Periplocae Forrestii and its compound preparation have good therapeutic effect on rheumatism and rheumatoid diseases.
The caulis et folium fici Tikouae is collected in 2003 edition of quality standards of traditional Chinese medicinal materials and national medicinal materials in Guizhou province, but no standard exists for measuring the content of active ingredients of the caulis et folium fici Tikouae, so that the quality standard of the caulis et folium fici Tikouae is imperfect, and the inherent quality of the caulis et folium fici Tikouae cannot be comprehensively controlled.
The applicant has carried out chemical composition analysis of byttneria nigra, and found that the byttneria nigra contains components such as 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, methyl 3-O-caffeoylquinic acid, periplocin, and the like. Previous studies by the applicant have shown that these ingredients may be both active benefit ingredients.
Therefore, it is necessary to develop a method for simultaneously measuring the contents of various beneficial components in the caulis et folium piperis nigri, such as 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid methyl ester, periplocin and the like.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a method for measuring the content of various components in a periploca forrestii schltr medicinal material, which comprises the following steps:
a method for measuring contents of multiple components in caulis et folium Periplocae Forrestii comprises measuring with UPLC-PDA method, using Waters acquisition UPLC BEH C18(2.1 × 50mm, 1.7 μm) as chromatographic column, detecting wavelength of 225nm and 330nm, flow rate of 0.35mL/min, and column temperature of 38 deg.C. And eluted with a gradient of acetonitrile-0.1% aqueous formic acid.
Furthermore, the UPLC-PDA method takes the extract of the periploca forrestii schltr as a test solution after being diluted.
The periploca forrestii schltr medicinal material extracting solution is obtained by extracting periploca forrestii schltr with 50% methanol as a solvent, the extraction efficiency is higher when the periploca forrestii schltr medicinal material is subjected to reflux extraction with 50% methanol, and beneficial components in the periploca forrestii schltr cannot be damaged.
The dilution is to dilute the periploca forrestii medicinal material extract by 2.5 times by using distilled water. The UPLC-PDA has better measuring effect after being diluted according to the proportion.
The caulis et folium Periplocae Forrestii medicinal material is caulis et folium Periplocae Forrestii medicinal material powder sieved by a third sieve, and the caulis et folium Periplocae Forrestii medicinal material powder sieved by the third sieve has good extraction effect.
Further, the test solution is prepared by the following steps: taking 1g of the medicinal material powder of the periploca forrestii schltr which passes through a third sieve, precisely weighing, placing the medicinal material powder into a conical flask with a plug, precisely adding 20mL of 50% methanol, weighing, heating and refluxing for 2 hours, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, weighing 10mL of the subsequent filtrate, transferring the subsequent filtrate into a 25mL volumetric flask, diluting the subsequent filtrate to a scale with distilled water, and shaking up to obtain a test solution. At the moment, the extraction efficiency of the periploca forrestii medicinal material is higher, the concentration of the test solution is moderate, and the measured result is more accurate.
Further, the UPLC-PDA method uses a mixed solution of 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, methyl 3-O-caffeoylquinic acid, and periplogenin dissolved in 50% methanol as a control solution.
Further, the control solution contains 1, 3-O-dicaffeoylquinic acid at a concentration of 50.0. mu.g/mL, 3, 4-O-dicaffeoylquinic acid at a concentration of 16.0. mu.g/mL, 3, 5-O-dicaffeoylquinic acid at a concentration of 20.0. mu.g/mL, 4, 5-O-dicaffeoylquinic acid at a concentration of 40.0. mu.g/mL, 5-O-caffeoylquinic acid at a concentration of 100.0. mu.g/mL, 3-O-caffeoylquinic acid at a concentration of 80.0. mu.g/mL, 4-O-caffeoylquinic acid at a concentration of 120.0. mu.g/mL, 3-O-caffeoylquinic acid methyl ester at a concentration of 64.0. mu.0. mu.g/mL, and periplogenin at a concentration of 80.0. mu.g/mL.
Further, the reference substance solution is prepared by the following method:
(1) respectively precisely weighing 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid methyl ester and periplogenin, placing in a 10mL measuring flask, adding 50% methanol for dissolving and fixing volume to scale to obtain 1, 3-O-dicaffeoylquinic acid with concentration of 1.25mg/mL, 3, 4-O-dicaffeoylquinic acid with concentration of 0.400mg/mL and 3, 5-O-dicaffeoylquinic acid with concentration of 0.500mg/mL, The concentration of 4, 5-O-dicaffeoylquinic acid is 1.00mg/mL, the concentration of 5-O-caffeoylquinic acid is 2.50mg/mL, the concentration of 3-O-caffeoylquinic acid is 2.00mg/mL, the concentration of 4-O-caffeoylquinic acid is 3.00mg/mL, the concentration of 3-O-caffeoylquinic acid methyl ester is 1.60mg/mL, and the concentration of periplogenin is 2.00 mg/mL;
(2) respectively and precisely measuring the 9 reference substance stock solutions, diluting the reference substance stock solutions, putting the diluted reference substance stock solutions into a 25mL volumetric flask, adding 50% methanol to dilute the diluted reference substance stock solutions to a scale, and shaking the mixed reference substance solutions uniformly to obtain a mixed reference substance solution, wherein the concentration of 1, 3-O-dicaffeoylquinic acid is 50.0 mu g/mL, the concentration of 3, 4-O-dicaffeoylquinic acid is 16.0 mu g/mL, the concentration of 3, 5-O-dicaffeoylquinic acid is 20.0 mu g/mL, the concentration of 4, 5-O-dicaffeoylquinic acid is 40.0 mu g/mL, the concentration of 5-O-caffeoylquinic acid is 100.0 mu g/mL, the concentration of 3-O-caffeoylquinic acid is 80.0 mu g/mL, the concentration of 4-O-caffeoylquinic acid is 120.0 mu g/mL, and, The concentration of 3-O-caffeoylquinic acid methyl ester is 64.0 μ g/mL, and the concentration of periplogenin is 80.0 μ g/mL.
Further, the UPLC-PDA method, using Waters ACQUITY UPLC BEH C18 (2.1X 50mm, 1.7 μm) as chromatographic column, detecting wavelength is 225nm and 330nm, flow rate is 0.35mL/min, column temperature is 38 deg.C, sample amount is 10 μ L, mobile phase is acetonitrile (A) -0.1% formic acid water solution (B) to carry out gradient elution, elution procedure is as follows:
the method comprises the following specific operation steps:
(1) preparing test solution and reference solution
Preparation of a test solution:
taking 1g of the medicinal material powder of the periploca forrestii schltr which passes through a third sieve, precisely weighing, placing the medicinal material powder into a conical flask with a plug, precisely adding 20mL of 50% methanol, weighing, heating and refluxing for 2 hours, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, weighing 10mL of the subsequent filtrate, transferring the subsequent filtrate into a 25mL volumetric flask, diluting the subsequent filtrate to a scale with distilled water, and shaking up to obtain a test solution.
Preparation of control solutions:
respectively precisely weighing 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid methyl ester and periplogenin, placing in a 10mL measuring flask, adding 50% methanol for dissolving and fixing volume to scale to obtain 1, 3-O-dicaffeoylquinic acid with concentration of 1.25mg/mL, 3, 4-O-dicaffeoylquinic acid with concentration of 0.400mg/mL and 3, 5-O-dicaffeoylquinic acid with concentration of 0.500mg/mL, The concentration of 4, 5-O-dicaffeoylquinic acid is 1.00mg/mL, the concentration of 5-O-caffeoylquinic acid is 2.50mg/mL, the concentration of 3-O-caffeoylquinic acid is 2.00mg/mL, the concentration of 4-O-caffeoylquinic acid is 3.00mg/mL, the concentration of 3-O-caffeoylquinic acid methyl ester is 1.60mg/mL, and the concentration of periplogenin is 2.00 mg/mL. Precisely measuring the 9 reference substance stock solutions respectively, diluting, placing into the same 25mL volumetric flask, adding 50% methanol to dilute to scale, and shaking to obtain mixed reference substance solution. The concentration of 1, 3-O-dicaffeoylquinic acid is 50.0 μ g/mL, the concentration of 3, 4-O-dicaffeoylquinic acid is 16.0 μ g/mL, the concentration of 3, 5-O-dicaffeoylquinic acid is 20.0 μ g/mL, the concentration of 4, 5-O-dicaffeoylquinic acid is 40.0 μ g/mL, the concentration of 5-O-caffeoylquinic acid is 100.0 μ g/mL, the concentration of 3-O-caffeoylquinic acid is 80.0 μ g/mL, the concentration of 4-O-caffeoylquinic acid is 120.0 μ g/mL, the concentration of 3-O-caffeoylquinic acid methyl ester is 64.0 μ g/mL, and the concentration of periploca is 80.0 μ g/mL
(2) The determination process comprises the following steps: respectively taking the mixed reference substance solution and the test solution, and determining by using UPLC-PDA to obtain chromatograms, wherein the detection wavelengths are 225nm and 330nm respectively, and the chromatogram conditions are as follows: the column was Waters ACQUITYUPLC BEH C18 (2.1X 50mm, 1.7 μm). The column temperature was 38 ℃, the flow rate was 0.35mL/min, and the mobile phase was acetonitrile (a) -0.1% aqueous formic acid (B) for gradient elution, the elution procedure was as follows:
according to the technical scheme, linear relation investigation, precision experiment, repeatability experiment, stability experiment and sample adding recovery rate experiment are respectively carried out.
Screening of sample preparation method:
in order to select a stable sample preparation method, the extraction mode of the periploca forrestii is subjected to a screening experiment by taking a solvent, an extraction mode and extraction time as variables, and the results are as follows:
from the above data, it can be seen that: when 50% methanol is used as a solvent, the extraction efficiency is highest; compared with the water bath reflux extraction, the ultrasonic extraction has higher reflux extraction efficiency; the extraction effect of refluxing for 1h is poor, and the extraction effect of refluxing for 2h is not greatly different from that of refluxing for 3 h and 4 h.
And (3) screening the measurement wavelength:
because the components to be detected in the caulis et folium Periplocae Forrestii all have ultraviolet absorption, an ultraviolet detector is selected to detect the components to be detected.
Full wavelength scanning was performed by PDA detector, and the maximum absorption wavelength of each component was as follows:
| composition (I) | Wavelength of maximum absorption (nm) | |
| 1, 3-O- | 328nm | |
| 3, 4-O- | 330nm | |
| 3, 5-O- | 327nm | |
| 4, 5-O-dicaffeoylquinic acid | 336nm | |
| 5-O-caffeoylquinic acid | 331nm | |
| 3-O-caffeoylquinic acid | 332nm | |
| 4-O-caffeoylquinic acid | 329nm | |
| 3-O-Caffeoylquinic acid methyl ester | 326nm | |
| Periploca sepium aglycone | 225nm |
From the data, the maximum absorption wavelength of periplogenin is greatly different from the maximum absorption wavelength of other components, detection at the same wavelength is considered, the detection sensitivity is obviously influenced, the content of the 9 components is determined by selecting double wavelengths (225nm and 330nm) comprehensively, periplogenin is determined under the condition of 225nm, the rest 8 components are determined under the condition of 330nm, and the purpose of simultaneously determining various chemical components of different types under the same chromatographic condition is achieved.
In order to select proper chromatographic conditions, different chromatographic columns, different mobile phase systems, different mobile phase pH values and different mobile phase elution gradients are considered, repeated experiments are carried out for a plurality of times, and finally acetonitrile and 0.1% formic acid aqueous solution are selected as mobile phases for gradient elution. Under the conditions, 9 chemical components in the periploca forrestii schltr are better separated.
In the experiment, the content of 9 chemical components, namely 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid methyl ester and periplogenin, in the caulis et folium Periplocae Forrestii is determined by adopting a UPLC-PDA technology. The method is simple and convenient to process, and can achieve good separation of 9 components under the same chromatographic condition within only 12 minutes. Proved by methodology, the method has good linearity, good repeatability, precision, stability and recovery rate, and can be used for quality control of the periploca forrestii schltr medicinal material. The analysis of the measurement results of a plurality of batches of the periploca forrestii dunn proves that the content fluctuation of 9 components is large, so that the quality of the periploca forrestii dunn needs to be controlled by a plurality of indexes.
Compared with the prior art, the invention has the technical effects that:
the invention adopts a UPLC-PDA method, uses Waters ACQUITY UPLC BEH C18 (2.1X 50mm, 1.7 μm) as a chromatographic column, and acetonitrile-0.1% formic acid water solution for gradient elution, wherein the detection wavelengths are 225nm and 330nm, the flow rate is 0.35mL/min, and the column temperature is 38 ℃. According to the method, 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid methyl ester and periplocin in the medicinal material of the caulis et folium piperis nigri can be separated simultaneously in 12 minutes, and the separation effect is good. The sample recovery rate of the method is 99.4-100.6%, and the RSD is less than or equal to 3.0%. The method is simple, convenient, rapid, accurate and good in repeatability, can be used for content determination of 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid methyl ester and periplogenin in the medicinal material of caulis et folium Periplocae Forrestii, and provides reference basis for quality control of caulis et folium Periplocae Forrestii.
Drawings
Fig. 1 is a diagram of a system suitability experiment UPLC.
Wherein, the corresponding relation of each absorption peak is as follows: peak 1 corresponds to periplogenin; peak 2 corresponds to 5-O-caffeoylquinic acid; peak 3 corresponds to 3-O-caffeoylquinic acid; peak 4 corresponds to 4-O-caffeoylquinic acid; peak 5 corresponds to 3, 4-O-dicaffeoylquinic acid; peak 6 corresponds to 3, 5-O-dicaffeoylquinic acid; peak 7 corresponds to 4, 5-O-dicaffeoylquinic acid; peak 8 corresponds to 1, 3-O-dicaffeoylquinic acid; peak 9 corresponds to methyl 3-O-caffeoylquiniate.
Detailed Description
The technical solution of the present invention is further defined below with reference to the specific embodiments, but the scope of the claims is not limited to the description.
Example (b):
instruments and materials:
an Acquity UPLC-PDA system (Waters, containing a binary ultra high pressure gradient pump, autosampler, chromatographic column incubator, array diode detector, Empower workstation); EL204 ten thousandth electronic balance (mettler-toledo instruments shanghai ltd); ultra pure water machines (Sichuan Volter science and technology development Co., Ltd.); DK-98-II type electric heating constant temperature water bath (Tester instruments, Inc. of Tianjin).
Methanol (analytically pure), acetonitrile (chromatographically pure), formic acid (chromatographically pure); the experimental caulis et folium periplocae medicinal material is identified as the dry root and stem of caulis et folium periplocae for restii schltr by crude drugs from the institute of pharmacy of medical university of Guizhou medical university and professor Liuchun flower of the university of medicinal plant.
Preparation of a test solution:
taking 1g of the medicinal material powder of the periploca forrestii schltr which passes through a third sieve, precisely weighing, placing the medicinal material powder into a conical flask with a plug, precisely adding 20mL of 50% methanol, weighing, heating and refluxing for 2 hours, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, weighing 10mL of the subsequent filtrate, transferring the subsequent filtrate into a 25mL volumetric flask, diluting the subsequent filtrate to a scale with distilled water, and shaking up to obtain a test solution.
Preparation of control solutions:
accurately weighing 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid methyl ester and periplogenin respectively, placing in a 10mL measuring flask, adding 50% methanol for dissolving, and fixing the volume to the scale. Respectively obtaining 1, 3-O-dicaffeoylquinic acid with the concentration of 1.25mg/mL, 3, 4-O-dicaffeoylquinic acid with the concentration of 0.400mg/mL, 3, 5-O-dicaffeoylquinic acid with the concentration of 0.500mg/mL, 4, 5-O-dicaffeoylquinic acid with the concentration of 1.00mg/mL, 5-O-caffeoylquinic acid with the concentration of 2.50mg/mL, 3-O-caffeoylquinic acid with the concentration of 2.00mg/mL, 4-O-caffeoylquinic acid with the concentration of 3.00mg/mL, 3-O-caffeoylquinic acid methyl ester with the concentration of 1.60mg/mL and periplocin with the concentration of 2.00 mg/mL. Precisely measuring the 9 reference substance stock solutions respectively, diluting, placing into the same 25mL volumetric flask, adding 50% methanol to dilute to scale, and shaking to obtain mixed reference substance solution. The concentration of 1, 3-O-dicaffeoylquinic acid is 50.0. mu.g/mL, the concentration of 3, 4-O-dicaffeoylquinic acid is 16.0. mu.g/mL, the concentration of 3, 5-O-dicaffeoylquinic acid is 20.0. mu.g/mL, the concentration of 4, 5-O-dicaffeoylquinic acid is 40.0. mu.g/mL, the concentration of 5-O-caffeoylquinic acid is 100.0. mu.g/mL, the concentration of 3-O-caffeoylquinic acid is 80.0. mu.g/mL, the concentration of 4-O-caffeoylquinic acid is 120.0. mu.g/mL, the concentration of 3-O-caffeoylquinic acid methyl ester is 64.0. mu.g/mL, and the concentration of periploca is 80.0. mu.g/mL.
Chromatographic conditions are as follows:
the column was a Waters ACQUITY UPLC BEH C18 (2.1X 50mm, 1.7 μm). The column temperature was 38 ℃, the flow rate was 0.35mL/min, the mobile phase was acetonitrile (A) -0.1% formic acid aqueous solution (B) for gradient elution, wherein the detection wavelengths were 225nm and 330nm, respectively, and the sample size was 10. mu.L. The elution procedure is shown in the table below.
TABLE 1 mobile phase gradient elution Table
And (3) system adaptability examination:
the mixed control solution and the test solution are 10 μ L each, and the active ingredients are separated well by UPLC-PDA determination under the above chromatographic conditions, as shown in figure 1.
And (3) linear relation investigation:
the mixed control solutions 1, 2, 4, 6, 8 and 10. mu.L were precisely pipetted, measured under the above chromatographic conditions, and the sample volumes were plotted as abscissa and peak areas as ordinate to obtain working curves, the results of which are shown in Table 2.
TABLE 2 regression equation and Linear Range
And (3) precision experiment:
taking the same sample solution, carrying out sample injection for 6 times continuously, calculating peak areas of 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, methyl 3-O-caffeoylquinic acid and periplogenin, and determining that RSD is 1.1%, 1.3%, 1.5%, 1.4%, 1.3%, 1.2% and 1.4% respectively. The results show good precision of the instrument. Taking the same sample, analyzing by sample injection for 3 days continuously, the peak areas RSD of 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, methyl 3-O-caffeoylquinic acid and periplogenin were calculated to be 1.9%, 1.5%, 1.8%, 2.1%, 1.8%, 1.6%, 1.9%, 2.0% and 1.8%.
And (3) repeatability experiment:
taking 6 parts of the same batch of medicinal materials, precisely weighing, preparing the test solution according to the preparation method of the test solution, and respectively measuring. The average contents of 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, methyl 3-O-caffeoylquinic acid, and periplocin in 6 parts of the test solution were 1.1354, 0.8375, 0.6875, 1.1821, 2.315, 1.634, 2.5841, 0.9347, and 1.8952mg/g, respectively, and the contents resulted in RSD of 1.2%, 2.4%, 2.5%, 1.7%, 1.3%, 1.9%, 1.1%, 1.8%, and 1.7%, respectively, which indicated good reproducibility.
Stability test:
respectively preparing the test solution according to the preparation method of the test solution, respectively injecting samples for 0, 2, 4, 8, 12 and 24 hours, and measuring the peak areas of 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid methyl ester and periplogenin. The peak areas RSD were calculated to be 1.7%, 1.6%, 1.2%, 1.5%, 1.8%, 1.3%, 1.4% and 1.1%, respectively, indicating that the sample solutions were stable for 0 to 24 hours.
Sample application recovery rate test
According to the preparation method of the test solution, 9 parts of the test solution with 50% sampling amount are prepared, the content of each component is measured as a background value according to the repeatability of the caulis et folium Periplocae Forrestii, 50%, 100% and 150% of reference solution are respectively added, 3 parts of the test solution are prepared according to the preparation method of the test solution for each level, the recovery rate of 9 components is measured and calculated according to the chromatographic conditions, and the result is shown in table 3.
The average recovery rate of 1, 3-O-dicaffeoylquinic acid is 99.8%, and the RSD (%) is 1.8%; the average recovery rate of 3, 4-O-dicaffeoylquinic acid is 99.4%, and the RSD (%) is 1.2%; the average recovery rate of 3,5-O dicaffeoylquinic acid is 99.9 percent, and the RSD (%) is 1.4 percent; the average recovery rate of 4, 5-O-dicaffeoylquinic acid is 99.9 percent, and the RSD (%) is 1.0 percent; the average recovery rate of 5-O-caffeoylquinic acid is 100.6 percent, and the RSD (%) is 1.5 percent; the average recovery rate of 3-O-caffeoylquinic acid is 99.9 percent, and the RSD (%) is 1.6 percent; the average recovery rate of 4-O-caffeoylquinic acid is 100.2 percent, and the RSD (%) is 2.0 percent; the average recovery rate of the 3-O-caffeoylquinic acid methyl ester is 99.8 percent, and the RSD (%) is 2.3 percent; the average recovery rate of periplogenin is 99.7%, and RSD (%) is 2.1%.
TABLE 3 sample recovery test results
Sample assay
The contents of different origins of chaetomium fortunei in Guizhou were collected and measured for 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid methyl ester, periplocin and the like by the established method, and the results are shown in Table 4.
Table 4 content of 9 ingredients in different batches of periploca forrestii (mg/g, n ═ 3)
From the above, the method of the invention can well separate 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid methyl ester and periplogenin in the medicinal material of caulis et folium piperis nigri within 12 minutes. The method is simple, convenient, rapid, accurate and good in repeatability, can be used for content determination of 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid methyl ester and periplogenin in the medicinal material of the caulis et folium Periplocae Forrestii, and provides reference basis for quality control of the caulis et folium Periplocae Forrestii medicinal material.
Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above-described embodiments, and many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (10)
1. A method for measuring the contents of various components in a periploca forrestii schltr medicinal material is characterized in that the UPLC-PDA method is adopted for measuring, a Waters ACQUITY UPLC BEH C181.7 MuM (2.1 multiplied by 50mm, 1.7 Mum) is taken as a chromatographic column, the detection wavelength positions are 225nm and 330nm, the flow rate is 0.35mL/min, the column temperature is 38 ℃, and acetonitrile-0.1% formic acid aqueous solution is used for gradient elution.
2. The method for measuring the contents of various components in the periploca forrestii schltr medicinal material according to claim 1, wherein the UPLC-PDA method uses the periploca forrestii schltr medicinal material extract as a test solution after dilution.
3. The method for measuring the contents of various components of the periploca forrestii schltr medicinal material according to claim 2, wherein the periploca forrestii schltr medicinal material extracting solution is obtained by performing reflux extraction on the periploca forrestii schltr by taking 50% methanol as a solvent, and the reflux extraction time is 2 hours.
4. The method for measuring the contents of various components of the Miao national herb Periploca forrestii Schneid as claimed in claim 2, wherein the dilution is performed by diluting the periploca forrestii Schneid extract with distilled water by 2.5 times.
5. The method for measuring the contents of various components in the periploca forrestii schltr medicinal material according to claim 2, wherein the periploca forrestii schltr medicinal material is periploca forrestii schltr medicinal material powder screened by a third sieve.
6. The method for measuring the contents of various components in the periploca forrestii schltr medicinal material according to claim 2, characterized in that the test solution is prepared by the following steps: precisely weighing 1g of periploca forrestii schltr medicinal material powder which passes through a third sieve, placing the powder into a conical flask with a plug, precisely adding 20mL of 50% methanol, weighing, heating and refluxing for 2 hours, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, weighing 10mL of continuous filtrate, transferring to a 25mL volumetric flask, diluting to a scale with distilled water, and shaking up to obtain a test solution.
7. The method for determining the contents of various components in the caulis et folium piperis nigri medicinal material of claim 1, wherein the UPLC-PDA method uses a mixed solution of 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, methyl 3-O-caffeoylquinic acid and periplogenin dissolved in 50% methanol as a control solution.
8. The method for determining the content of various components in the Heigutenus nigra Linne material of claim 7, wherein the control solution comprises 1, 3-O-dicaffeoylquinic acid at 50.0 μ g/mL, 3, 4-O-dicaffeoylquinic acid at 16.0 μ g/mL, 3, 5-O-dicaffeoylquinic acid at 20.0 μ g/mL, 4, 5-O-dicaffeoylquinic acid at 40.0 μ g/mL, 5-O-caffeoylquinic acid at 100.0 μ g/mL, 3-O-caffeoylquinic acid at 80.0 μ g/mL, 4-O-caffeoylquinic acid at 120.0 μ g/mL, 3-O-caffeoylquinic acid at 64.0 μ g/mL, and, The periplogenin concentration is 80.0 μ g/mL.
9. The method for measuring the contents of various components in the periploca forrestii schltr medicinal material according to claim 7, wherein the reference solution is prepared by the following method:
(1) respectively precisely weighing 1, 3-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid methyl ester and periplogenin, placing in a 10mL measuring flask, adding 50% methanol for dissolving and fixing volume to scale to obtain 1, 3-O-dicaffeoylquinic acid with concentration of 1.25mg/mL, 3, 4-O-dicaffeoylquinic acid with concentration of 0.400mg/mL and 3, 5-O-dicaffeoylquinic acid with concentration of 0.500mg/mL, The concentration of 4, 5-O-dicaffeoylquinic acid is 1.00mg/mL, the concentration of 5-O-caffeoylquinic acid is 2.50mg/mL, the concentration of 3-O-caffeoylquinic acid is 2.00mg/mL, the concentration of 4-O-caffeoylquinic acid is 3.00mg/mL, the concentration of 3-O-caffeoylquinic acid methyl ester is 1.60mg/mL, and the concentration of periplogenin is 2.00 mg/mL;
(2) respectively and precisely measuring the 9 reference substance stock solutions, diluting the reference substance stock solutions, putting the diluted reference substance stock solutions into a 25mL volumetric flask, adding 50% methanol to dilute the diluted reference substance stock solutions to a scale, and shaking the mixed reference substance solutions uniformly to obtain a mixed reference substance solution, wherein the concentration of 1, 3-O-dicaffeoylquinic acid is 50.0 mu g/mL, the concentration of 3, 4-O-dicaffeoylquinic acid is 16.0 mu g/mL, the concentration of 3, 5-O-dicaffeoylquinic acid is 20.0 mu g/mL, the concentration of 4, 5-O-dicaffeoylquinic acid is 40.0 mu g/mL, the concentration of 5-O-caffeoylquinic acid is 100.0 mu g/mL, the concentration of 3-O-caffeoylquinic acid is 80.0 mu g/mL, the concentration of 4-O-caffeoylquinic acid is 120.0 mu g/mL, the concentration of 3-O-caffeoylquinic acid methyl ester is 64.0 mu g/mL, The periplogenin concentration is 80.0 μ g/mL.
10. The method for measuring the contents of various components in the periploca forrestii schltr medicinal material according to claim 1, wherein the UPLC-PDA method is characterized in that the mobile phase is acetonitrile (A) -0.1% formic acid aqueous solution (B), the sample size is 10 μ L, and the elution procedure is as follows:
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