CN102183604A - Method and application for building fingerprint of cortex fraxini or extract thereof - Google Patents

Abstract

The invention discloses a method for using high-performance capillary electrophoresis (HPCE) to establish the fingerprint of the medicinal material Qinpi or its extract, which comprises the following steps: (a) preparation of the test solution: take the medicinal material Qinpi or its extract, add organic Solvent extraction, constant volume, is the test solution; (b) preparation of reference solution: prepare daidzein solution as internal standard; (c) electrophoresis analysis conditions: use dihydrogen phosphate-borax as buffer stock solution, Its pH value is between 7.8-9.2, preferably 8.6; acetonitrile is used as an organic modifier; the detection wavelength is between 200-400nm, preferably 210nm; the working voltage is between 5-30kV, preferably 20kV (d) Assay method: get an appropriate amount of sample solution to be analyzed, pack into sample bottle successively and measure by HPCE, record the electrophoresis spectrum; (e) Data analysis: take the retention time of internal standard as 1, calculate the relative peak of each spectrum Migration time, taking the peak area of the internal standard as 1, calculate the relative peak area of each peak. The invention provides a new method for more quickly and effectively identifying counterfeit and inferior products of bark bark or its extracts on the market and distinguishing commercial medicinal materials from different production areas.

Description

The establishment method and application of the fingerprint of Qinpi medical material or its extract

technical field

The invention relates to a method for establishing a fingerprint of a traditional Chinese medicine, in particular to a method for establishing and an application of a HPCE fingerprint of a medicinal material or an extract thereof.

Background technique

Chrysanthemum chinensis varies from place to place, and the sources of varieties are different, but they all belong to the genus Fraxinus of the Oleaceae family (Oleaceae), and their morphological characteristics and odor are similar to those recorded in Materia Medica. "Pharmacopoeia of the People's Republic of China" 2010 edition stipulates that Bark Bark is the dry branch bark of Fraxinus rhynchophylla Hance, Fraxinus chinensis Roxb., Fraxinus szabaona Lingelsh. or Fraxinus stylosa Lingelsh. or dry skin. Among them, the bitter ash tree is distributed in Jilin, Liaoning, Hebei, Henan and other places. Ash trees are distributed in North China, Northwest China, East China, Central South, Southwest China and Liaoning. The pointed-leaf ash tree is distributed in Hebei, Shaanxi, Shanxi, Gansu, and Hunan. Suzhu ash tree is distributed in Shaanxi. Commodity medicinal materials mainly include Northeast Qinpi or Liaoning Qinpi (Ash Tree), Sichuan Qinpi (Ash Tree), Shaanxi Baidian Qinpi (Suzhu Ash Tree), Henan Qinpi, etc.

Qinpi has the effects of clearing heat and detoxification, clearing liver and improving eyesight, and is used for heat dysentery, diarrhea, red and white discharge, red and swollen eyes, and nebula. Modern pharmacological studies have shown that Qinpi has various effects such as anti-pathogenic microorganisms, anti-inflammation, diuresis, anti-coagulant and anti-allergy, cough, phlegm, asthma, central inhibition, and anti-tumor. It can be used clinically to treat bacillary dysentery, chronic bronchitis, Inflammation and so on.

Fructus Fructus mainly contains coumarin compounds such as Fructus A, Fructus B, Fructus Glycoside, Frucetine and other coumarin compounds. The Chinese Pharmacopoeia stipulates that the base source of Fructus Fructus medicinal materials includes 4 kinds of plants of the genus Oleaceae. Due to the fact that Cortex chinensis is a multi-source variety, and the geographical environment of the original plant distribution is different, the quality of commercial medicinal materials from different origins and sources is quite different. At the same time, the types of Pittia chinensis and mixed counterfeit medicinal materials are also complicated, which urgently needs to be clarified. Therefore, it is extremely important to establish an objective, comprehensive and rapid method to evaluate its intrinsic quality. So far, most of the internal quality control of chinensis adopts the content determination method of fennecine A and quincetrin B, and only one document involves the research on the fingerprint spectrum of quincetrin that can reveal the inner quality, but this study uses the HPLC method, and the analysis time is long ( 75min), the consumption of organic solvents is large, and there are many shortcomings such as large environmental pollution. For this reason, the present invention provides a method for constructing fingerprints of Pittia chinensis medicinal materials or their extracts using HPCE as an analysis method, and provides a new analysis method for the overall control of the internal quality of Pittonia chinensis, so as to achieve a more rapid and effective identification of Pittip chinensis or its extracts on the market. The purpose of counterfeiting and shoddy products of pharmaceuticals and distinguishing commercial medicinal materials from different production areas.

Contents of the invention

On the one hand, the present invention relates to a method for using HPCE to establish the fingerprint of the medicinal material Qinpi or its extract, which is characterized in that it comprises the following steps:

(a) Preparation of the test solution: get the medicinal material of Cortex chinensis or its extract, add organic solvent to extract, and constant volume is the test solution;

(b) Preparation of reference solution: prepare daidzein solution as internal standard;

(c) Electrophoresis analysis conditions: with dihydrogen phosphate-borax as buffer stock solution, its pH value is between 7.8-9.2, preferably 8.6; with acetonitrile as organic modifier; detection wavelength between 200-400nm Between, preferably 210nm; working voltage between 5-30kV, preferably 20kV;

(d) Determination method: take an appropriate amount of the sample solution to be analyzed, put it into a sample bottle successively and measure it by HPCE, and record the electrophoretic spectrum;

(e) Data analysis: take the retention time of the internal standard as 1, calculate the relative migration time of each spectral peak, and take the peak area of the internal standard as 1, calculate the relative peak area of each spectral peak.

In one embodiment of the present invention, the organic solvent in step (a) is C1-C6 lower alcohol, preferably methanol, more preferably reflux extraction or ultrasonic extraction.

In one embodiment of the present invention, wherein the concentration of dihydrogen phosphate used when preparing the buffer stock solution is 80-120mmol/L, preferably 100mmol/L, and the concentration of borax solution is 30-70mmol/L, preferably 50mmol/L L, the volume ratio of the two solutions is 2:1-1:4, preferably 1.02:1.98.

In one embodiment of the present invention, the volume ratio of buffer stock solution:water:organic modifier during chromatographic operation is 1.5:1:0.2-4:1:1, preferably 3:1:0.8.

On the other hand, the present invention also relates to the application of Qinpi fingerprint spectrum in the quality detection of Qinpi medicinal material or its extract, which is characterized in that daidzein internal standard is used as a reference peak, which includes 4 characteristic peaks, respectively:

Peak No. 7, relative migration time 0.905, RSD 0.52%;

Peak No. 8, relative migration time 0.926, RSD 0.41%;

Peak No. 11, relative migration time 1.077, RSD 1.21%;

Peak No. 15, relative migration time 1.168, RSD 1.51%;

In one embodiment of the present invention, described Qin Pi fingerprint spectrum also comprises one or more characteristic peaks, and described characteristic peak is selected from:

Peak No. 1, relative migration time 0.600, RSD 3.24%;

Peak No. 2, relative migration time 0.764, RSD 1.41%;

Peak No. 3, relative migration time 0.773, RSD 1.38%;

Peak No. 4, relative migration time 0.800, RSD 1.14%;

Peak No. 5, relative migration time 0.809, RSD 1.04%;

Peak No. 6, relative migration time 0.879, RSD 0.73%;

Peak No. 10, relative migration time 1.024, RSD 0.85%;

Peak No. 12, relative migration time 1.096, RSD 1.08%;

Peak No. 13, relative migration time 1.121, RSD 1.51%;

Peak No. 14, relative migration time 1.145, RSD 1.66%;

Peak No. 16, relative migration time 1.187, RSD 1.61%.

The present invention also relates to the application of the fingerprint of Cortex chinensis in the quality detection of Cortex chinensis medicinal material or its extract.

In one embodiment of the present invention, the quality detection means that the HPCE fingerprint of the Chinese medicinal material or its extract to be detected obtained by the method of the present invention has a similarity of 90% with the control HPCE fingerprint of the reference medicinal material or its extract. Above, preferably above 95%. The similarity means that their respective electrophoretic spectra are imported into the National Pharmacopoeia Commission 2004A version Chinese medicine chromatographic fingerprint similarity evaluation system in the form of AIA files and calculated.

Description of drawings

Fig. 1 fingerprint spectrum of Pittrichum officinalis medicinal material, wherein No. 9 peak is the internal standard peak;

Figure 220 The control fingerprints derived from Qinpi, wherein peak No. 9 is the internal standard peak;

Fig. 3 Qinpi 3D chromatogram.

Detailed ways

The samples were collected between March 2005 and March 2007. Except for the Sichuan Qianwei sample collected from the place of origin, the rest of the samples were purchased from different pharmacies and different operating points in the medicinal materials market in various cities. The same large retail pharmacy purchases every six months, and the medicinal material wholesale market purchases once a quarter, and each purchase is made at 5 different merchants, and the place of origin is provided by the merchant. See the table below for the detailed information of Qinpi medicinal materials.

Table 1 Sample source

experimental method

1 Instruments, reagents and samples

Agilent 3DCE capillary electrophoresis instrument (model: G1600A, including diode array detector and HP chromatography workstation); the effective length of fused silica capillary is 45cm (53.5cm×75μm ID, Hebei Yongnian Ruifeng Chromatography Device Co., Ltd.).

Microfuge 18 desktop centrifuge (Beckman Coulter Co., Ltd., USA); Boda 1004 ultrasonic cleaner (Shenzhen Boda Ultrasonic Engineering Equipment Co., Ltd.); TGL.16G desktop high-speed centrifuge (Shanghai Medical Analytical Instrument Factory); BP210D One-hundred-thousandth electronic analytical balance (Sartorius, Germany); Milli-Q ultrapure water device (Millipore, USA); filter device and microporous membrane (0.45 μm).

2 solution preparation

2.1 Preparation of buffer solution

Accurately weigh 1.3609g KH ₂ PO ₄ in a 100mL small beaker, dissolve it with an appropriate amount of water and set the volume in a 100mL measuring bottle to prepare a potassium dihydrogen phosphate buffer solution with a concentration of 100mmol/L.

Accurately weigh 1.9068g of sodium tetraborate (Na ₂ B ₄ O ₇ 10H ₂ O) in a 100mL small beaker, dissolve it with an appropriate amount of water and set the volume in a 100mL measuring bottle to prepare a borax buffer solution with a concentration of 50mmol/L .

Precisely draw 100mmol/L potassium dihydrogen phosphate and 50mmol/L borax solution, and prepare potassium dihydrogen phosphate-borax buffer solution (pH=8.6) stock solution at a ratio of 1.02:1.98. Before use, the running buffer solution was prepared in the following ratio: buffer stock solution-water-acetonitrile (3:1:0.8).

2.2 Preparation of reference solution

Preparation of internal standard solution: Accurately weigh 100mg of daidzein into a 100mL small beaker, dissolve it with an appropriate amount of methanol and set the volume in a 100mL measuring bottle, and prepare a solution containing 1mg of daidzein per 1mL.

Preparation of stock solution of reference substance: Accurately weigh 4.8 mg of aquinetin, 5.6 mg of fencetin, 1.96 mg of fencetrin, and 3.76 mg of fencetrin B respectively, put them in a 25mL measuring bottle, add an appropriate amount of methanol to ultrasonically dissolve and dilute to the mark , Shake well, and mix the stock solutions (1 to 4 are 192.0, 224.0, 78.4, 150.4 mg/L respectively) as the reference substance.

Preparation of reference substance solution: Accurately draw 5mL of reference substance stock solution, put it in a 10mL measuring bottle, add methanol to dilute to the mark, mix well, then draw 5mL, put it in a 10mL measuring bottle, add 1mL of internal standard solution, add water to dilute to the mark, mix Evenly, pass through a 0.45 μm microporous membrane as a reference solution (48.0, 56.0, 19.6, 37.6 mg/L for 1 to 4, respectively).

2.3 Preparation of reference medicinal materials and test solution

Take Qinpi reference medicinal material and 0.25 g of the powder of the test product to be tested (passed through the No. 3 sieve of the Pharmacopoeia), accurately weighed, put into a stoppered Erlenmeyer flask, accurately add 25 mL of methanol, ultrasonic for 60 min, let cool, and filter. Precisely measure 5 mL of the continued filtrate, put it in a 10 mL measuring bottle, add 1 mL of internal standard solution, dilute to the mark with water, mix well, and pass through a 0.45 μm microporous membrane for later use.

3 detection method

Before using the capillary, wash it with 0.1mol/L NaOH solution, water, and operating buffer solution [100mmol/L potassium dihydrogen phosphate-50mmol/L borax-water-acetonitrile (1.02:1.98:1:0.8)] for 10-20 minutes each ( 55°C). Rinse for 1, 2, and 3 minutes between each injection to ensure good reproducibility. Take the prepared reference substance, reference drug solution and test solution and place them in sample bottles, and put them into the injector in turn. The electrophoresis spectrum was determined under the conditions of detection wavelength 210nm, pressure injection 20kPa·s, operating voltage 20kV(+)-(-), and column temperature 25°C.

4 Methodology Validation

In order to investigate the reliability of the analytical method, taking the medicinal material (Ash) purchased from Chengdu Tongrentang as the analytical sample, the precision of the instrumental analytical method, the stability of the sample, and the repeatability of the experimental method were studied. The results show that the method meets the requirements of Technical requirements for fingerprints (see Table 2).

Table 2 Excel software calculation results of precision, stability and repeatability similarity

5 Experimental results

This fingerprint spectrum is as shown in accompanying drawing 1, accompanying drawing 2, determines altogether 15 common peaks. Another No. 9 peak is the added internal standard peak. Through the relative migration time and the standard addition method, it is determined that No. 7 peak is azeperidin peak; The peak is the feudeletin peak; the peak No. 15 is the feudeletin peak, and the fingerprint data are shown in Table 3 and Table 4.

Collect the three-dimensional spectrum of Fructus auricola online (attached drawing 3). It can be seen intuitively from the 3D map and the contour map that the number of peaks in the electrophoresis spectrum of Fructus Fructus near 210nm is the largest, and in Fructus Fructus, Frutiflorin, Fructus A, Frucetin and Fructus B The detection results of ultraviolet absorption wavelengths of the four components of the element showed that the maximum absorption wavelengths of each component were mainly concentrated around 210nm and 365nm. In line with the determination of the wavelength of the fingerprint spectrum must follow the principle of maximizing the amount of information, further analysis and comparison of the chromatograms scanned at the wavelength of 190-400nm, the results show that the number of peaks at 210nm is more and the separation is good, which can not only fully reflect the information of active ingredients, It can also reflect the information of other ingredients in the medicinal material, and can evaluate the quality of Qinpi more comprehensively and comprehensively. Therefore, the fingerprint is determined with 210nm as the detection wavelength.

Table 3 Relative migration time of 20 batches of Qinpi common peaks

Remarks: "-" means not detected

Continued Table 3

Table 4 The relative peak area and similarity of 20 batches of Qinpi common peaks

Remarks: "-" means not detected; the total means the relative peak area sum of the characteristic peaks of Qinpi except the internal standard; r _ir is the correlation coefficient; C _ir is the cosine of the included angle.

Continued Table 4

Claims (8)

1. A method using HPCE to set up the fingerprints of the Cortex chinensis medicinal material or its extract, is characterized in that comprising the following steps: (a) Preparation of the test solution: get the medicinal material of Cortex chinensis or its extract, add organic solvent to extract, and constant volume is the test solution; (b) Preparation of reference solution: prepare daidzein solution as internal standard; (c) Electrophoresis analysis conditions: with dihydrogen phosphate-borax as buffer stock solution, its pH value is between 7.8-9.2, preferably 8.6; with acetonitrile as organic modifier; detection wavelength between 200-400nm Between, preferably 210nm; working voltage between 5-30kV, preferably 20kV; (d) Determination method: take an appropriate amount of the sample solution to be analyzed, put it into a sample bottle successively and measure it by HPCE, and record the electrophoretic spectrum; (e) Data analysis: take the retention time of the internal standard as 1, calculate the relative migration time of each spectral peak, and take the peak area of the internal standard as 1, calculate the relative peak area of each spectral peak. 2. The method for establishing fingerprints according to claim 1, wherein the organic solvent in the step (a) is a C1-C6 lower alcohol, preferably methanol, more preferably reflux extraction or ultrasonic extraction. 3. the method for establishing fingerprint according to claim 1, wherein the concentration of the dihydrogen phosphate used when preparing buffer stock solution is 80-120mmol/L, is preferably 100mmol/L, and borax solution concentration is 30-70mmol/L L is preferably 50 mmol/L, and the volume ratio of the two solutions is 2:1-1:4, preferably 1.02:1.98. 4. the method for establishing fingerprint according to claim 3, when HPCE runs, buffer stock solution: water: the volume ratio of organic modifier is 1.5: 1: 0.2-4: 1: 1, preferably 3: 1 : 0.8. 5. The application of Qinpi fingerprint spectrum in the quality detection of Qinpi medicinal material or its extract, which is characterized in that the internal standard of daidzein is used as a reference peak, which includes 4 characteristic peaks, which are: Peak No. 7, relative migration time 0.905, RSD 0.52%; Peak No. 8, relative migration time 0.926, RSD 0.41%; Peak No. 11, relative migration time 1.077, RSD 1.21%; Peak No. 15, relative migration time 1.168, RSD 1.51%; 6. application according to claim 5, described Qinpi fingerprint collection also includes one or more characteristic peaks, and described characteristic peaks are selected from: Peak No. 1, relative migration time 0.600, RSD 3.24%; Peak No. 2, relative migration time 0.764, RSD 1.41%; Peak No. 3, relative migration time 0.773, RSD 1.38%; Peak No. 4, relative migration time 0.800, RSD 1.14%; Peak No. 5, relative migration time 0.809, RSD 1.04%; Peak No. 6, relative migration time 0.879, RSD 0.73%; Peak No. 10, relative migration time 1.024, RSD 0.85%; Peak No. 12, relative migration time 1.096, RSD 1.08%; Peak No. 13, relative migration time 1.121, RSD 1.51%; Peak No. 14, relative migration time 1.145, RSD 1.66%; Peak No. 16, relative migration time 1.187, RSD 1.61%. 7. The application of the Qinpi fingerprint spectrum established by any one of claims 1-4 in the quality detection of the Qinpi medicinal material or its extract. 8. The application according to claim 7, wherein said quality detection refers to the analysis of the medicinal material to be detected or its extract by the detection method described in claim 1 steps a to d, and the obtained HPCE spectrum is consistent with that of claim 1. -4 The similarity of the Qinpi fingerprints established by any item is compared, and the similarity is more than 80%, preferably more than 90%, and more preferably more than 95%.

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