CN114994220B - Construction method of fingerprint spectrum of Qiqingbaidu granule, determination method of component content of Qiqingbaidu granule and application of Qiqingbaidu granule - Google Patents
Construction method of fingerprint spectrum of Qiqingbaidu granule, determination method of component content of Qiqingbaidu granule and application of Qiqingbaidu granule Download PDFInfo
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Abstract
The invention belongs to the field of component detection, and particularly relates to a construction method of a fingerprint spectrum of Qiqing Baidu granule, a component content measuring method and application thereof. In the invention, the sample solution of the Qiqingbaidu particles is subjected to high performance liquid chromatography analysis to obtain the fingerprint of the Qiqingbaidu particles; the content information of the components is obtained by an external standard method, so that qualitative and quantitative analysis of the components of the Qiqingbaidu granules is realized, and the method particularly relates to simultaneous qualitative and quantitative analysis of the following 7 components: (R, S) -epigoitrin, baicalin, wogonin, pulsatilla saponin B4, polygonin and emodin provide reliable and quantifiable standards for quality assessment of Qiqingbaidu granules. The chromatogram obtained by the method has the characteristics of stable baseline, high spectrum peak separation degree, strong specificity and good peak symmetry.
Description
Technical Field
The invention belongs to the field of component detection, and particularly relates to a construction method of a fingerprint spectrum of Qiqing Baidu granule, a component content measuring method and application thereof.
Background
The QIQINGBAIDU granule has effects of clearing heat and detoxicating, eliminating dampness and relieving diarrhea, and can be used for treating diarrhea due to damp-heat, and pullorum disease. The Qiqing Baidu granule prescription contains the traditional Chinese medicine components such as radix scutellariae, giant knotweed, chinese pulsatilla root, radix sophorae flavescentis, radix isatidis, rhizoma drynariae, folium isatidis, sucrose, dextrin and the like. The medicine effect and the safety of the product are related to the components and the content thereof, but the content of each component is not easy to control due to the complex components, so that the quality control of the finished product is relatively difficult.
The veterinary pharmacopoeia (2010 two parts) discloses a method for identifying the components of baicalin, polygonum cuspidatum, emodin, indigo and indirubin in Qiqingbaidu granules by using a thin layer chromatography, which has complicated steps and does not quantify the components. Therefore, the quality control of the Qiqing antiphlogistic granule does not reach the quantitative standard, and other components in the Qiqing antiphlogistic granule cannot be qualitatively and quantitatively determined. Therefore, a high-efficiency detection method of Qiqingbaidu particles capable of simultaneously qualitatively and quantitatively is needed at present, and technical support is provided for quality control of Qiqingbaidu particles.
Disclosure of Invention
Aiming at the problems that the detection steps of the Qiqing baijiu granules are complicated and multiple components cannot be simultaneously qualitatively and quantitatively detected in the prior art, the invention provides a construction method of the Qiqing baijiu granules, a component content measuring method and application thereof, and particularly relates to the detection of the qualitative and quantitative of 7 components in Qiqing baijiu granules, namely (R, S) -epigoitrin, baicalin, wogonin, pulsatilla saponin B4, polygonin and emodin.
The invention adopts a High Performance Liquid Chromatography (HPLC) method to manufacture the fingerprint of the Qiqing baidu granule, analyzes the content of the Qiqing baidu granule components and provides reference for quality control.
In order to achieve the above purpose, the method specifically comprises the following technical scheme:
the method for constructing the fingerprint of the Qiqingbaidu granule comprises the following steps of preparing a Qiqingbaidu granule sample solution, and performing high performance liquid chromatography on the Qiqingbaidu granule sample solution to obtain the fingerprint of the Qiqingbaidu granule; chromatographic conditions for high performance liquid chromatography include: the C-18 chromatographic column is a stationary phase, acetonitrile is taken as a mobile phase A, and 0.05-0.2% phosphoric acid aqueous solution is taken as a mobile phase B.
The chromatographic conditions for high performance liquid chromatography also include: the C-18 chromatographic column is used as a stationary phase, acetonitrile is used as a mobile phase A, a 0.05-0.2% phosphoric acid aqueous solution is used as a mobile phase B, the detection wavelengths are respectively 201nm and 245nm, and the sum of the volume fractions of the mobile phase A and the mobile phase B is 100% during gradient elution, wherein the volume fraction of the A phase changes as follows within 0-150 min: 0-25min,2% -5%A phase; 25-45min,5% -15% phase A; 45-70min,15% phase A; 70-80min,15% -25% of phase A; 80-100min,25% phase A; 100-105min,25% -30% of phase A; 105-110min,30% phase A; 110-120min,30% -40% of phase A; 120-130min,40% -45% of phase A; 130-140min,45% phase A; 140-150min,45% -80% of phase A.
The inventor of the present invention found in a great number of fingerprint studies of Qiqingbaidu particles that by eluting with acetonitrile as mobile phase a and 0.05-0.2% phosphoric acid aqueous solution as mobile phase B under the specific detection wavelength under the specific elution conditions described above, a fingerprint containing characteristic peaks of (R, S) -epigoitrin, baicalin, baicalein, wogonin, pulsatilla saponin B4, polygonin and emodin, which have high purity and good peak separation degree and do not contain other hetero peaks, can be obtained.
The fingerprint fully shows the information of the traditional Chinese medicine components and chemical components of the Qiqingbaidu granule, and provides basis for the content determination, detection and identification of the components.
Under the specific chromatographic condition, the method can identify seven medicinal materials (radix scutellariae, polygonum cuspidatum, pulsatilla chinensis, radix sophorae flavescentis, radix isatidis, rhizoma dryopteris crassirhizomae and folium isatidis) contained in the formula of the Qiqing baidu granule, and the quantitative analysis can be realized by using 7 characteristic chemical components of 4 medicinal materials, namely baicalin, baicalein and wogonin of radix scutellariae, pulsatilla chinensis saponin B4 of radix pulsatillae, polygonin of polygonum cuspidatum and emodin of radix isatidis, and (R, S) -epigoitrin of radix isatidis. The characteristic absorption peaks of the kuh-seng and the rhizoma dryopteris crassirhizomae can be used for quality control reference.
Matrine and oxymatrine, which are characteristic components of radix Sophorae Flavescentis, are not shown in the chromatogram, and may form salt under the acidic mobile phase condition, and the retention capacity of C-18 chromatographic column is reduced. The indirubin which is a characteristic component of the dyers woad leaf has smaller polarity, the indirubin cannot be effectively enriched under the treatment condition of the sample, and the dyers woad leaf reference medicinal material solution has no obvious absorption peak under the chromatographic condition.
The inventor finds that the selection of specific mobile phases and gradient elution conditions during chromatographic detection can obviously influence the number of characteristic peaks, the peak purity and the peak separation degree of the fingerprint, namely, the adoption of different mobile phases or different gradient elution conditions can cause the absence of the related peaks of (R, S) -epigoitrin, baicalin, baicalein, wogonin, pulsatilla saponin B4, polydatin or emodin, so that seven components of (R, S) -epigoitrin, baicalin, baicalein, wogonin, pulsatilla saponin B4, polygonin and emodin cannot be simultaneously measured.
Preferably, the chromatographic conditions of the high performance liquid chromatography further include: the column temperature is 25-35 ℃, the sample injection amount is 5-15 mu L, and the flow rate of the mobile phase is 0.5-1.5mL/min.
Further preferably, the 0.1% phosphoric acid aqueous solution is mobile phase B phase, the column temperature is 30 ℃, the sample injection amount is 10 mu L, the flow rate of the mobile phase is 1mL/min, and the obtained chromatogram has better baseline stability, separation degree and peak symmetry.
Preferably, the preparation method of the test solution of the Qiqingbaidu granule comprises the following steps: mixing the Qiqingbaidu particles with a solvent, and performing ultrasonic extraction or reflux extraction to obtain a sample solution; the solvent is alcohol or alcohol-water mixed solution.
Further preferably, the alcohol is methanol.
Preferably, the concentration of the Qiqingbaidu particles is 100-300g/L.
Further preferably, the concentration of the Qiqingbaidu particles is 200g/L.
A method for detecting component content of QINGBAIDU granule comprises preparing QINGBAIDU granule test solution, and performing high performance liquid chromatography to obtain fingerprint of QINGBAIDU granule; calculating the content of the component to be detected by an external standard method through the peak area of the fingerprint; chromatographic conditions for high performance liquid chromatography include: acetonitrile is used as a mobile phase A phase, and 0.05-0.2% phosphoric acid aqueous solution is used as a mobile phase B phase.
Preferably, the component to be detected is at least one of (R, S) -epigoitrin, baicalin, baicalein, wogonin, pulsatilla saponin B4, polydatin and emodin.
The chromatographic conditions for high performance liquid chromatography also include: the C-18 chromatographic column is a stationary phase, and the detection wavelength is 201nm and 245nm respectively; when the gradient elution is carried out, the sum of the volume fractions of the mobile phase A and the mobile phase B is 100%, wherein the volume fraction of the mobile phase A changes as follows within 0-150 min: 0-25min,2% -5%A phase; 25- > 45min,5% → 15% phase a; 45-70min,15% phase A; 70-80min,15% -25% of phase A; 80-100min,25% phase A; 100-105min,25% -30% of phase A; 105-110min,30% phase A; 110-120min,30% -40% of phase A; 120-130min,40% -45% of phase A; 130-140min,45% phase A; 140-150min,45% -80% of phase A.
The chromatographic conditions of the high performance liquid chromatography analysis also comprise: the column temperature is 25-35 ℃, the sample injection amount is 5-15 mu L, and the flow rate of the mobile phase is 0.5-1.5mL/min.
Further preferably, the 0.1% phosphoric acid aqueous solution is mobile phase B, the column temperature is 30 ℃, the sample injection amount is 10 mu L, the flow rate is 1mL/min, and the obtained chromatogram has better baseline stability, separation degree and peak symmetry.
Preferably, the external standard method establishes a linear regression equation of each component by calculating the area of the spectrum peak of the high performance liquid chromatography and taking the area as an ordinate (y) and the concentration of the component as an abscissa (x).
The R value of the linear regression equation of each component obtained by the method is very close to or equal to 1, the linearity is good, and a reliable basis is provided for the quantification of the component.
The external standard method comprises a reference substance solution, wherein the reference substance solution is any one of the following solutions A-E:
a: controlling the medicinal material solution;
b: a negative sample solution;
c: mixing the reference substance solution;
d: mixing the control solution and the control medicinal material solution;
e: the control solution and the negative sample solution were mixed.
The HPLC spectrograms of the sample solution and the reference medicinal material solution are analyzed to obtain the traditional Chinese medicine component information of the Qiqingbaidu granule.
By analyzing HPLC spectrograms of the sample solution and the negative sample solution, the Chinese medicinal component information of the Qiqingbaidu granule can be obtained.
The chemical composition information of the Qiqing toxin-vanquishing particles can be obtained by analyzing the HPLC spectrograms of the sample solution and the mixed reference solution.
By analyzing HPLC spectrograms of the sample solution, the mixed reference substance solution and the reference medicinal material solution, the traditional Chinese medicine component information, chemical component and content information of the Qiqingbaidu granule can be obtained.
By analyzing HPLC spectrograms of the sample solution, the mixed reference solution and the negative sample solution, the traditional Chinese medicine component information, the chemical component and the content information of the Qiqingbaidu granule can be obtained.
The traditional Chinese medicine comprises at least one of the following components: radix Scutellariae, rhizoma Polygoni Cuspidati, radix Pulsatillae, radix Sophorae Flavescentis, radix Isatidis, rhizoma Dryopteris Crassirhizomatis and folium Isatidis.
The chemical components comprise at least one of the following components: (R, S) -epigoitrin, polydatin, baicalin, pulsatilla saponin B4, baicalin, wogonin and emodin.
Preferably, the preparation method of the sample solution comprises the following steps: mixing the Qiqingbaidu particles with a solvent, and performing ultrasonic extraction or reflux extraction to obtain a sample solution; the solvent is alcohol or alcohol-water mixed solution.
Preferably, the preparation method of the control medicinal material solution comprises the following steps: mixing the medicinal materials with a solvent, and performing ultrasonic extraction or reflux extraction to obtain a control medicinal material solution; the solvent is alcohol or alcohol-water mixed solution.
Preferably, the concentration of the medicinal materials is 2-15g/L.
The medicinal materials are one of baikal skullcap root, giant knotweed, pulsatilla root, kuh-seng, radix isatidis, rhizoma dryopteris crassirhizomae and dyers woad leaf.
The control medicinal material solution is one of the following solutions: radix Scutellariae control medicinal solution, radix Pulsatillae control medicinal solution, rhizoma Polygoni Cuspidati control medicinal solution, radix Sophorae Flavescentis control medicinal solution, radix Isatidis control medicinal solution, folium Isatidis control medicinal solution, and rhizoma Dryopteris Crassirhizomatis control medicinal solution.
Preferably, the preparation method of the negative sample solution comprises the following steps: mixing a negative sample with a solvent, and performing ultrasonic extraction or reflux extraction to obtain a negative sample solution; the negative sample is the sample remaining in the absence of any one of the following components: radix Scutellariae, rhizoma Polygoni Cuspidati, radix Pulsatillae, radix Sophorae Flavescentis, radix Isatidis, rhizoma Dryopteris Crassirhizomatis and folium Isatidis; the solvent is alcohol or alcohol-water mixed solution.
The negative sample solutions include the following: radix Scutellariae negative sample solution, radix Begoniae Laciniatae Weng Yinxing sample solution, rhizoma Polygoni Cuspidati negative sample solution, radix Sophorae Flavescentis negative sample solution, radix Isatidis She Yinxing sample solution, and rhizoma Dryopteris Crassirhizomatis negative sample solution.
The mass ratio of the baikal skullcap root, giant knotweed, pulsatilla root, kuh-seng, radix isatidis, rhizoma dryopteris crassirhizomae and dyers woad leaf in the negative sample is (1-4): 1-2): 0.5-1): 1-2: 1-3: 0.5-1): 1-2, wherein the mass of any one of the components which is missing is regarded as 0.
Further preferably, the mass ratio of the baikal skullcap root, giant knotweed, pulsatilla root, kuh-seng, isatis root, rhizoma dryopteris crassirhizomae and dyers woad leaf is 4:1.3:0.875:1.75:2.66:0.87:1.75, wherein the mass of any one of the components which is absent is regarded as 0.
Preferably, the preparation method of the mixed reference substance solution comprises the following steps: dissolving (R, S) -epigoitrin, polydatin, baicalin, pulsatilla saponin B4, baicalin, wogonin and emodin in solvent to obtain mixed reference solution; the solvent is alcohol or alcohol-water mixed solution.
Further preferably, the components and the concentrations thereof in the mixed reference solution are as follows: 0.0003-0.015mg/mL (R, S) -epigoitrin, 0.008-0.4mg/mL polydatin, 0.08-4mg/mL baicalin, 0.02-1mg/mL pulsatilla saponin B4,0.01-0.5mg/mL baicalein, 0.004-0.2mg/mL wogonin, 0.0015-0.075mg/mL emodin.
Still more preferably, the components and their concentrations in the mixed reference solution are as follows: 0.003mg/mL (R, S) -epigoitrin, 0.08mg/mL polydatin, 0.8mg/mL baicalin, 0.2mg/mL pulsatilla saponin B4,0.1mg/mL baicalein, 0.04mg/mL wogonin, 0.015mg/mL emodin.
Preferably, the ultrasonic extraction time is 20-90min.
Further preferably, the ultrasonic extraction time is 30min.
Preferably, the ultrasonic extraction time is room temperature.
The reflux extraction time is 20-90min.
Further preferably, the reflux extraction time is 30min.
Further preferably, the alcohol is methanol.
The construction method of the fingerprint of the Qiqingbaidu granule or the detection method of the component content of the Qiqingbaidu granule is applied to the quality control of the Qiqingbaidu granule.
The construction method or the detection method of the fingerprint of the Qiqingbaidu granule can determine the traditional Chinese medicine component information and the chemical component information, in particular to qualitative and quantitative application of 7 chemical components of (R, S) -epigoitrin, polygonin, baicalin, pulsatilla saponin B4, baicalein, wogonin and emodin, and has better prospect in the aspect of quality control of the Qiqingbaidu granule.
Compared with the prior art, the invention has the following advantages:
(1) The invention establishes the fingerprint spectrum of the Qiqing antiphlogistic granule, and provides reference for quality control.
(2) The method can efficiently and accurately perform qualitative and quantitative analysis on (R, S) -epigoitrin, polygonin, baicalin, pulsatilla saponin B4, baicalein, wogonin and emodin in the Qiqing Baidu granules.
(3) The method can efficiently determine the information of the traditional Chinese medicine components in the Qiqingbaidu granules, in particular to 7 traditional Chinese medicine components of radix scutellariae, giant knotweed, chinese pulsatilla root, radix sophorae flavescentis, radix isatidis, rhizoma dryopterygii and folium isatidis.
(4) The chromatogram obtained by the method has stable baseline, good spectrum peak separation degree, symmetrical peaks and strong specificity.
(5) The method can conveniently and rapidly detect and identify the quality of the Qiqingbaidu particles, and provide reliable and quantitative standards for quality control.
Drawings
FIG. 1 shows HPLC patterns of a sample solution, each control medicinal material solution and a mixed control solution at 245nm wavelength, wherein S1-S9 respectively represent the HPLC patterns of the sample solution, the Baical skullcap root control medicinal material solution, the Chinese pulsatilla root control medicinal material solution, the giant knotweed control medicinal material solution, the kuh-seng control medicinal material solution, the radix isatidis control medicinal material solution, the dyers woad leaf control medicinal material solution, the rhizoma drynariae control medicinal material solution and the mixed control solution.
FIG. 2 is an HPLC chart of a sample solution, each control medicinal material solution and a mixed control solution under the wavelength of 201nm, wherein S1-S9 respectively represent the HPLC charts of the sample solution, the baical skullcap root control medicinal material solution, the Chinese pulsatilla root control medicinal material solution, the giant knotweed control medicinal material solution, the lightyellow sophora root control medicinal material solution, the indigowoad root control medicinal material solution, the dyers woad leaf control medicinal material solution, the rhizoma drynariae control medicinal material solution and the mixed control solution.
FIG. 3 shows HPLC patterns of a sample solution, each negative sample solution and a mixed reference solution at 245nm wavelength, wherein S1-S9 respectively represent the HPLC patterns of the sample solution, the Baikal skullcap root negative sample solution, the Baikal skullcap root Weng Yinxing sample solution, the giant knotweed negative sample solution, the kuh-seng negative sample solution, the isatis root negative sample solution, the dyers woad She Yinxing sample solution, the rhizoma drynariae negative sample solution and the mixed reference solution.
FIG. 4 shows HPLC patterns of a sample solution, each negative sample solution and a mixed reference solution at a wavelength of 201nm, wherein S1-S9 respectively represent the HPLC patterns of the sample solution, the Baikal skullcap root negative sample solution, the Baikal skullcap root Weng Yinxing sample solution, the giant knotweed negative sample solution, the kuh-seng negative sample solution, the isatis root negative sample solution, the dyers woad She Yinxing sample solution, the rhizoma drynariae negative sample solution and the mixed reference solution.
FIG. 5 is an HPLC plot of a self-made QiPod particle sample solution at 245nm wavelength and 5 batches of QiPod particle sample solutions, wherein S1 represents the self-made QiPod particle sample solution and S2-S6 represent the HPLC plots of batches 2102001, 2103001, 2104001, 2105001, 2106001, respectively.
FIG. 6 is an HPLC plot of a self-made Qiqing toxin-stripping particle sample solution at a wavelength of 201nm and 5 batches of Qiqing toxin-stripping particle sample solutions, wherein S1 represents the self-made Qiqing toxin-stripping particle sample solution and S2-S6 represent the HPLC plots of batches 2102001, 2103001, 2104001, 2105001, 2106001, respectively.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described by means of specific examples.
Material, instrument and test method
Seven clear toxin-vanquishing particles (Guangdong big organism Co., ltd., lot No. 2006007), radix Scutellariae traditional Chinese medicine formulation particles (Jiangyin Tianjiang pharmaceutical Co., ltd., lot No. 20081721), rhizoma Polygoni Cuspidati traditional Chinese medicine formulation particles (Jiangyin Tianjiang pharmaceutical Co., ltd., lot No. 20120501), radix Pulsatillae traditional Chinese medicine formulation particles (Jiangyin Tianjiang pharmaceutical Co., ltd., lot No. 19091631), radix Sophorae Flavescentis traditional Chinese medicine formulation particles (Jiangyin Tianjiang pharmaceutical Co., ltd., lot No. 20071721), radix Isatidis traditional Chinese medicine formulation particles (Jiangyin Tianjiang pharmaceutical Co., ltd., lot No. 20040641), rhizoma Dryopteris traditional Chinese medicine formulation particles (Jiangyin Tianjiang pharmaceutical Co., ltd., lot No. 20026934), folium Isatidis traditional Chinese medicine formulation particles (Jiangyin Tianjiang pharmaceutical Co., ltd.); (R, S) -Legioyichun (Chengman Biotechnology Co., ltd.), polydatin (China medicine Biotechnology institute), baicalin (China food and medicine institute), pulsatilla saponin B4 (Chengman Biotechnology Co., ltd.), baicalin (China medicine Biotechnology institute), wogonin (Chengman Biotechnology Co., ltd.), emodin (China food and medicine institute); high performance liquid chromatograph (Agilent 1260 Infinicity II), detector (VWD, agilent G7114A), chromatographic column (phenylomenex C-18).
Taking a C-18 chromatographic column as a stationary phase, acetonitrile as a mobile phase A, 0.05-0.2% phosphoric acid aqueous solution as a mobile phase B, carrying out gradient elution at a column temperature of 25-35 ℃, a sample injection amount of 5-15 mu L, a mobile phase flow rate of 0.5-1.5mL/min and detection wavelengths of 201nm and 245nm respectively, wherein the mobile phase gradient program is shown in the following table 1:
TABLE 1 gradient of mobile phases
Example 1
1. Preparation of mixed control solution
Accurately weighing (R, S) -epigoitrin, polydatin, baicalin, pulsatilla saponin B4, baicalein, wogonin and emodin, diluting with methanol to obtain 6 mixed reference solutions (each mixed reference solution contains 7 components), and numbering 1-6. The specific components and contents in the numbers 1-6 are as follows: the concentrations of (R, S) -epigoitrin are respectively 0.0003, 0.0006, 0.0015, 0.003, 0.006 and 0.015mg/mL, the concentrations of polydatin are respectively 0.008, 0.016, 0.04, 0.08, 0.16 and 0.4mg/mL, the concentrations of baicalin are respectively 0.08, 0.16, 0.4, 0.8, 1.6 and 4mg/mL, the concentrations of pulsatilla saponin B4 are respectively 0.02, 0.04, 0.1, 0.2 and 0.4mg/mL, the concentrations of baicalein are respectively 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5mg/mL, the concentrations of wogonin are respectively 0.004, 0.008, 0.02, 0.04, 0.08 and 0.2mg/mL, and the concentrations of emodin are respectively 0.0015, 0.003, 0.0075, 0.03 and 0.015mg/mL.
2. Preparation of control medicinal solution
Radix scutellariae control medicinal material solution: taking 0.4g of baical skullcap root traditional Chinese medicine formula particles (each 1g corresponds to 5g of decoction pieces), adding 40mL of methanol, heating and reflux-extracting for 30min, filtering, taking filtrate, evaporating solvent of the filtrate to dryness, adding 80% methanol (mixed solution of 20% water and 80% methanol) for dissolution, and fixing the volume to 25mL. As a control medicinal solution of radix Scutellariae.
Polygonum cuspidatum control medicinal material solution: taking 0.15g of polygonum cuspidatum traditional Chinese medicine formula particles (each 1g is equivalent to 15g of decoction pieces), and treating by referring to a method of a scutellaria baicalensis control medicinal material solution to serve as a polygonum cuspidatum control medicinal material solution.
Pulsatilla chinensis control medicinal material solution: taking 0.085g of pulsatilla root traditional Chinese medicine formula particles (each 1g corresponds to 20g of decoction pieces) and treating by referring to a radix scutellariae control medicinal material solution method to obtain pulsatilla root control medicinal material solution.
Kuh-seng control medicinal material solution: taking 0.12g of kuh-seng traditional Chinese medicine formula particles (each 1g is equivalent to 10g of decoction pieces), and treating by referring to a method of a radix scutellariae control medicinal material solution to obtain a kuh-seng control medicinal material solution.
Radix isatidis control medicinal material solution: taking 0.53g of isatis root traditional Chinese medicine formula particles (each 1g is equivalent to 7.5g of decoction pieces), and treating by referring to a radix scutellariae control medicinal material solution method to obtain isatis root control medicinal material solution.
Rhizoma dryopteris crassirhizomae control medicinal material solution: taking 0.0875g of rhizoma dryopteris crassirhizomae traditional Chinese medicine formula particles (each 1g is equivalent to 15g of decoction pieces), and treating by referring to a radix scutellariae control medicinal material solution method to obtain a rhizoma dryopteris crassirhizomae control medicinal material solution.
Folium Isatidis control medicinal material solution: taking 0.53g of dyers woad leaf traditional Chinese medicine formula particles (each 1g is equivalent to 5g of decoction pieces), and treating by referring to a radix scutellariae control medicinal material solution method to obtain dyers woad leaf control medicinal material solution.
3. Preparation of test solutions
Precisely weighing 10g of Qiqing Baidu granule powder, adding 50mL of methanol, weighing, ultrasonically extracting for 30min, standing to room temperature, weighing, adding methanol to complement lost weight, filtering, taking 30mL of subsequent filtrate, evaporating the filtrate to dryness, adding 80% methanol into residues for dissolution, and using 80% methanol to fix volume to 25mL to obtain a sample solution.
4. Preparation of negative sample solution
4.1 baikal skullcap root negative sample solution: taking 0.175g of dyers woad leaf traditional Chinese medicine formula particles, 0.087g of rhizoma dryopteris crassirhizomae traditional Chinese medicine formula particles, 0.133g of polygonum cuspidatum traditional Chinese medicine formula particles, 0.0875g of Chinese pulsatilla root traditional Chinese medicine formula particles, 0.175g of radix sophorae flavescentis traditional Chinese medicine formula particles and 0.266g of radix isatidis traditional Chinese medicine formula particles, adding 40mL of methanol, heating, refluxing and extracting for 30min, filtering, evaporating filtrate, dissolving residues in 80% methanol, and using 80% methanol to reach a constant volume of 25mL to serve as a radix scutellariae negative sample solution.
4.2 Polygonum cuspidatum negative sample solution: taking 0.175g of dyers woad leaf traditional Chinese medicine formula granule, 0.087g of rhizoma dryopteris crassirhizomae traditional Chinese medicine formula granule, 0.4g of radix scutellariae traditional Chinese medicine formula granule, 0.0875g of radix pulsatillae traditional Chinese medicine formula granule, 0.175g of radix sophorae flavescentis traditional Chinese medicine formula granule and 0.266g of radix isatidis traditional Chinese medicine formula granule, and treating the same by referring to the preparation method of radix scutellariae negative sample solution to be used as the polygonum cuspidatum negative sample solution.
4.3 white head Weng Yinxing sample solution: taking 0.175g of dyers woad leaf traditional Chinese medicine formula granule, 0.087g of rhizoma dryopteris crassirhizomae traditional Chinese medicine formula granule, 0.133g of polygonum cuspidatum traditional Chinese medicine formula granule, 0.4g of radix scutellariae traditional Chinese medicine formula granule, 0.175g of radix sophorae flavescentis traditional Chinese medicine formula granule and 0.266g of radix isatidis traditional Chinese medicine formula granule, and treating the same by referring to a radix scutellariae negative sample solution preparation method to obtain a white head Weng Yinxing sample solution.
4.4 Sophora flavescens negative sample solution: taking 0.175g of dyers woad leaf traditional Chinese medicine formula granule, 0.087g of rhizoma dryopteris crassirhizomae traditional Chinese medicine formula granule, 0.133g of polygonum cuspidatum traditional Chinese medicine formula granule, 0.0875g of Chinese pulsatilla root traditional Chinese medicine formula granule, 0.4g of radix scutellariae traditional Chinese medicine formula granule and 0.266g of radix isatidis traditional Chinese medicine formula granule, and treating the dyers woad leaf traditional Chinese medicine formula granule by referring to the preparation method of radix scutellariae negative sample solution to be used as a radix sophorae flavescentis negative sample solution.
4.5 Isatis root negative sample solution: taking 0.175g of dyers woad leaf traditional Chinese medicine formula granule, 0.087g of rhizoma dryopteris crassirhizomae traditional Chinese medicine formula granule, 0.133g of polygonum cuspidatum traditional Chinese medicine formula granule, 0.0875g of Chinese pulsatilla root traditional Chinese medicine formula granule, 0.4g of radix scutellariae traditional Chinese medicine formula granule and 0.175g of radix sophorae flavescentis traditional Chinese medicine formula granule, and treating the dyers woad leaf traditional Chinese medicine formula granule by referring to the preparation method of radix scutellariae negative sample solution to be used as the radix isatidis negative sample solution.
4.6 sample solution of dyers woad She Yinxing: taking 0.175g of radix sophorae flavescentis traditional Chinese medicine formula particles, 0.087g of rhizoma dryopteris crassirhizomae traditional Chinese medicine formula particles, 0.133g of polygonum cuspidatum traditional Chinese medicine formula particles, 0.0875g of Chinese pulsatilla root traditional Chinese medicine formula particles, 0.4g of radix scutellariae traditional Chinese medicine formula particles and 0.266g of radix isatidis traditional Chinese medicine formula particles, and treating the mixture by referring to a radix scutellariae negative sample solution preparation method to obtain a dyers woad She Yinxing sample solution.
4.7 rhizoma Dryopteris Crassirhizomatis negative sample solution: taking 0.175g of dyers woad leaf traditional Chinese medicine formula granule, 0.175g of lightyellow sophora root traditional Chinese medicine formula granule, 0.133g of giant knotweed rhizome traditional Chinese medicine formula granule, 0.0875g of Chinese pulsatilla root traditional Chinese medicine formula granule, 0.4g of baical skullcap root traditional Chinese medicine formula granule and 0.266g of indigowoad root traditional Chinese medicine formula granule, and treating the dyers woad leaf traditional Chinese medicine formula granule by referring to a preparation method of a baical skullcap root negative sample solution to be used as a dryopteris crassirhizomes negative sample solution.
5. Optimization of chromatographic conditions
5.1 optimization of mobile phase B concentration: the test sample of '3' is subjected to high performance liquid chromatography detection analysis, a C-18 chromatographic column is used as a stationary phase, acetonitrile is used as a mobile phase A phase, detection wavelengths are respectively 201nm and 245nm, the column temperature is 30 ℃, the flow rate is 1mL/min, the sample injection amount is 10 mu L, the gradient elution program is shown in the table 1, the mobile phase B is a phosphoric acid aqueous solution, only the concentration of phosphoric acid (the concentration range is 0.05-0.2%) is changed, and the influence of different concentrations of phosphoric acid on a chromatogram is examined. The results show that when the concentrations of phosphoric acid are respectively 0.05%, 0.1% and 0.2%, the spectrogram base line, the peak symmetry, the separation degree and the specificity of the sample solution are all good.
5.2 optimization of mobile phase flow rate: the test solution of '3' is subjected to high performance liquid chromatography detection analysis, a C-18 chromatographic column is used as a stationary phase, acetonitrile is used as a mobile phase A phase, a mobile phase B is 0.1% phosphoric acid aqueous solution, the detection wavelength is 201nm and 245nm respectively, the column temperature is 30 ℃, the sample injection amount is 10 mu L, the gradient elution program is shown in a table 1, and the influence of different liquid flow rates on the chromatogram is examined by only changing the mobile phase flow rate (0.5-1.5 mL/min) of the chromatogram. The results show that the spectrogram base line, the peak symmetry, the separation degree and the specificity of the sample solution are all good when the flow rates are 0.5mL/min, 1.0mL/min and 1.5mL/min.
5.3 optimization of column temperature: the sample solution of '3' is subjected to high performance liquid chromatography detection analysis, a C-18 chromatographic column is used as a stationary phase, acetonitrile is used as a mobile phase A, a mobile phase B is 0.1% phosphoric acid aqueous solution, the detection wavelength is 201nm and 245nm respectively, the sample injection amount is 10 mu L, the gradient elution program is shown in Table 1, the flow rate is 1mL/min, the column temperature is only changed, and the influence of different column temperatures (20-35 ℃) on the chromatogram is examined. The results show that the spectrum base line, the peak symmetry, the separation degree and the specificity of the sample solution are all better when the column temperature is 25 ℃,30 ℃ and 35 ℃.
5.4 optimizing the sample injection amount: the sample solution to be tested of '3' is subjected to high performance liquid chromatography detection analysis, a C-18 chromatographic column is used as a stationary phase, acetonitrile is used as a mobile phase A phase, detection wavelengths are respectively 201nm and 245nm, a gradient elution program is shown in table 1, a mobile phase B is 0.1% phosphoric acid aqueous solution, the flow rate is 1mL/min, the column temperature is 30 ℃, the sample injection amount (5-15 mu L) is only changed, and the influence of different sample injection amounts on the chromatogram is examined. The results show that when the sample injection amount is 5 mu L, 10 mu L and 15 mu L, the spectrogram base line, the peak symmetry, the separation degree and the specificity of the sample solution are all good.
5.5 optimization of detection wavelength: the sample solution of "3" was subjected to high performance liquid chromatography detection analysis, using a C-18 column as stationary phase, acetonitrile as mobile phase a phase, mobile phase B phase as 0.1% phosphoric acid aqueous solution, gradient elution procedure as shown in table 1, flow rate of 1mL/min, column temperature of 30 ℃, sample injection amount of 10 μl, and only the detection wavelength (190 nm-260 nm) at the time of chromatography detection was changed, and the influence of different detection wavelengths on the chromatogram was examined. The results show that when the detection wavelengths are 201nm and 245nm respectively, the number of spectral peaks of components simultaneously displayed in the spectrogram of the sample solution is the largest, the specificity is strong, the peak type separation degree is good, and if other wavelengths are adopted as the detection wavelengths, the phenomena of incomplete spectral peak display, poor separation degree or asymmetric peak type of certain components exist.
6. Fingerprint establishment and component attribution
The liquid chromatography detection parameters were as follows: the C-18 chromatographic column is a stationary phase, acetonitrile is a mobile phase A, a 0.1% phosphoric acid aqueous solution is a mobile phase B, the detection wavelengths are 201nm and 245nm respectively, the column temperature is 30 ℃, the flow rate is 1mL/min, and the gradient of the mobile phase is shown in Table 1. The analysis was performed using the mixed control solution No. 4 in example 1 as the mixed control solution in this example, namely: the concentration of (R, S) -epigoitrin in the mixed control solution is 0.003mg/mL, the concentration of polydatin is 0.08mg/mL, the concentration of baicalin is 0.8mg/mL, the concentration of pulsatilla root saponin is 0.2mg/mL, the concentration of baicalein is 0.1mg/mL, the concentration of wogonin is 0.04mg/mL, and the concentration of emodin is 0.015mg/mL.
Detecting under the same chromatographic conditions by adopting a method of HPLC (high Performance liquid chromatography) spectrum comparison of a sample solution, a negative sample solution, each control medicinal material solution and a mixed control substance solution, identifying chromatographic peaks by taking retention time as an identification standard, and carrying out preliminary attribution analysis on common peaks, wherein the results are shown in figures 1-4. As can be seen from fig. 1 to 4, (1) according to HPLC spectra of the sample solution and the control medicinal material solution (or the sample solution and the negative sample solution), the qiqingbaidu granule contains the following 7 traditional Chinese medicine components: radix Scutellariae, rhizoma Polygoni Cuspidati, radix Pulsatillae, radix Sophorae Flavescentis, radix Isatidis, rhizoma Dryopteris Crassirhizomatis and folium Isatidis. (2) According to HPLC spectrograms of the sample solution and the mixed reference solution, the Qiqingbaidu granule contains 7 chemical components of (R, S) -epigoitrin, polygonin, baicalin, pulsatilla saponin B4, baicalin, wogonin and emodin. (3) The Qiqingbaidu granule comprises the following information by analyzing HPLC spectrograms of the sample solution, the mixed reference solution and the reference medicinal material solution (or the sample solution, the mixed reference solution and the negative sample solution): (a) 7 ingredients were identified: in fig. 1, peak No. 1 (R, S) -epigoitrin is derived from isatis root, peak No. 2 polydatin and peak No. 15 emodin are derived from polygonum cuspidatum, peak No. 5 baicalin, peak No. 12 baicalin and peak No. 13 wogonin are derived from scutellaria baicalensis; in fig. 2, the 20 # peak pulsatilla saponin B4 is derived from pulsatilla. (b) Unidentified consensus peaks 3, 4, 6, 7, 9, 11, 14 and 19 were derived from baikal skullcap root, peaks 8 and 10 were derived from giant knotweed, peak 16 was derived from kuh-seng, peak 17 was derived from isatis root, peak 18 was derived from rhizoma dryopteris crassirhizomatis. (4) According to HPLC spectrograms of the sample solution and the mixed reference substance solution, a fingerprint of the Qiqing antiphlogistic granule is initially established, and a reference can be provided for quality control.
Example 2
Establishing a linear regression equation of each component
In this example, the detection parameters of HPLC are as follows: the C-18 chromatographic column is a stationary phase, acetonitrile is a mobile phase A, a 0.1% phosphoric acid aqueous solution is a mobile phase B, the detection wavelengths are 201nm and 245nm respectively, the column temperature is 30 ℃, the flow rate is 1mL/min, the sample injection volume is 10 mu L, and the gradient of the mobile phase is shown in Table 1.
Taking the mixed reference substance solutions with different concentrations of No. 1-6 in the embodiment 1, and carrying out liquid chromatography analysis by using the liquid chromatography detection parameters. The collected liquid chromatography was subjected to determination of retention time of peaks of (R, S) -epigoitrin, baicalin, wogonin, pulsatilla saponin B4, polygonin and large Huang Supu according to the method in example 1, and the spectral peak areas of the above components were recorded, and a linear regression equation was established by taking the spectral peak area of each component as an ordinate (y) and the concentration of each component in the mixed control as an abscissa (x), to examine the linear range of the content of 7 components. The results are shown in Table 2 below:
table 2 7 Linear index of the components and their contents
Example 3
Determination of Qiqingbaidu granule components and content thereof in different types and different batches
The laboratory self-made Qiqing baidu granule sample: taking 0.4g of baikal skullcap root traditional Chinese medicine formula particles, 0.175g of dyers woad leaf traditional Chinese medicine formula particles, 0.087g of rhizoma dryopterygii traditional Chinese medicine formula particles, 0.133g of giant knotweed rhizome traditional Chinese medicine formula particles, 0.0875g of Chinese pulsatilla root traditional Chinese medicine formula particles, 0.175g of lightyellow sophora root traditional Chinese medicine formula particles, 0.266g of isatis root traditional Chinese medicine formula particles, adding 40mL of methanol, heating and reflux extracting for 30min, filtering, evaporating filtrate, adding 80% of methanol into residues for dissolving, and using 80% of methanol for constant volume to 25mL to serve as a sample solution of the self-prepared Qiqing toxin-vanquishing particles in a laboratory.
The content determination was performed on 7 components of 5 batches (from Guangdong university Biotechnology Co., ltd., batch No. 2006007) and laboratory-self-made Qiqing toxin-vanquishing particle samples, wherein the preparation method of the Qiqing toxin-vanquishing particle HPLC test sample solution of different batches was referred to the preparation method of the test sample solution in example 1. The liquid chromatography detection parameters are as follows: gradient elution was carried out with a C-18 chromatographic column as stationary phase, acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, the detection wavelength was 201nm and 245nm, the column temperature was 30℃and the flow rate was 1mL/min, and the mobile phase gradient was as shown in Table 1 below. The components and the content of the Qiqingbaidu granules in different batches are analyzed according to the HPLC detection result, and the results are shown in Table 3:
table 35 seven-component Qiqingbaidu granule and 7-component Qiqingbaidu granule prepared in laboratory and content thereof
On the one hand, the spectrogram of the detection result shows that the liquid chromatograph has stable baseline, short analysis time, symmetrical peak type and strong specificity. On the other hand, according to the detection result, the seven medicinal materials (baicalin, polygonum cuspidatum, pulsatilla chinensis, radix sophorae flavescentis, radix isatidis, rhizoma dryopterygii and folium isatidis) contained in the formula of the Qiqing baidu granule can be quantitatively analyzed by using the method of the invention, and 7 characteristic components of the 4 medicinal materials can be quantitatively analyzed, namely baicalin, baicalein and wogonin of the baical skullcap root, pulsatilla chinensis saponin B4 of the pulsatilla chinensis, polydatin and emodin of the polygonum cuspidatum and (R, S) -epigoitrin of the radix isatidis. The kuh-seng (No. 16 peak) and the rhizoma dryopteris crassirhizomae (No. 18 peak) also have characteristic absorption peaks for quality control reference under the method.
Matrine and oxymatrine, which are characteristic components of radix Sophorae Flavescentis, are not shown in the chromatogram, and may form salt under the acidic mobile phase condition, and the retention capacity of C-18 chromatographic column is reduced. The indirubin which is a characteristic component of the dyers woad leaf has smaller polarity, the indirubin cannot be effectively enriched under the treatment condition of the sample, and the dyers woad leaf reference medicinal material solution has no obvious absorption peak under the chromatographic condition.
Therefore, according to the method disclosed by the invention, the components and the contents of the Qiqingbaidu particles in the same type, different batches or different types can be conveniently and quickly detected, and a reliable and quantifiable standard is provided for quality evaluation of the Qiqingbaidu particles.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (5)
1. The method for constructing the fingerprint of the Qiqingbaidu granule is characterized by comprising the following steps of preparing a Qiqingbaidu granule sample solution, and performing high performance liquid chromatography on the Qiqingbaidu granule sample solution to obtain the fingerprint of the Qiqingbaidu granule; the fingerprint of the Qiqing antiphlogistic granule has 20 common peaks, wherein the 1 st common peak (R, S) -epigoitrin is derived from radix isatidis, the 2 nd common peak polygonin and the 15 th common peak chrysophain are derived from rhizoma polygoni cuspidati, the 5 th common peak baicalin, the 12 th common peak baicalin and the 13 th common peak wogonin are derived from radix scutellariae, the 20 th common peak pulsatilla saponin B4 is derived from radix pulsatillae, the 3 rd common peak, the 4 th common peak, the 6 th common peak, the 7 th common peak, the 9 th common peak, the 11 th common peak, the 14 th common peak and the 19 th common peak are derived from radix scutellariae, the 8 th common peak and the 10 th common peak are derived from rhizoma polygoni cuspidati, the 16 th common peak is derived from radix sophorae, the 17 th common peak is derived from radix isatidis and the 18 th common peak is derived from rhizoma dryopteris;
chromatographic conditions for high performance liquid chromatography include: acetonitrile is taken as a mobile phase A phase, and 0.05-0.2% phosphoric acid aqueous solution is taken as a mobile phase B phase; the C-18 chromatographic column is a stationary phase, and the detection wavelength is 201nm and 245nm respectively; when the gradient elution is carried out, the sum of the volume fractions of the mobile phase A and the mobile phase B is 100%, wherein the volume fraction of the mobile phase A changes as follows within 0-150 min: 0-25min,2% -5%A phase; 25-45min,5% -15% phase A; 45-70min,15% phase A; 70-80min,15% -25% of phase A; 80-100min,25% phase A; 100-105min,25% -30% of phase A; 105-110min,30% phase A; 110-120min,30% -40% of phase A; 120-130min,40% -45% of phase A; 130-140min,45% phase A; 140-150min,45% -80% of phase A;
the column temperature is 25-35 ℃, the sample injection amount is 5-15 mu L, and the flow rate of the mobile phase is 0.5-1.5mL/min.
2. The method for constructing the fingerprint spectrum of the Qiqingbaidu granule as claimed in claim 1, wherein the method for preparing the sample solution of the Qiqingbaidu granule comprises the following steps: mixing the Qiqingbaidu particles with a solvent, and performing ultrasonic extraction or reflux extraction to obtain a sample solution; the solvent is alcohol or alcohol-water mixed solution.
3. A method for detecting the component content of Qiqingbaidu granules, which is characterized in that the fingerprint of Qiqingbaidu granules is obtained according to the construction method of the fingerprint of Qiqingbaidu granules as defined in claim 1 or 2; calculating the content of the component to be detected by an external standard method through the peak area of the fingerprint; chromatographic conditions for high performance liquid chromatography include: acetonitrile is used as a mobile phase A phase, and 0.05-0.2% phosphoric acid aqueous solution is used as a mobile phase B phase.
4. The method for detecting the component content of the Qiqingbaidu granule as defined in claim 3, wherein the component to be detected is at least one of (R, S) -epigoitrin, baicalin, baicalein, wogonin, pulsatilla saponin B4, polydatin and emodin.
5. The use of the method for constructing a fingerprint of a Qiqingbai granule as defined in any one of claims 1-2 or the method for detecting the component content of Qiqing granule as defined in any one of claims 3-4 in quality control of Qiqing granule.
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| CN103389344A (en) * | 2012-05-09 | 2013-11-13 | 滇虹药业集团股份有限公司 | Establishment method for HPLC fingerprint spectrum of Chinese pulsatilla root medicinal material and standard fingerprint spectrum thereof |
| CN109061002A (en) * | 2018-09-18 | 2018-12-21 | 山东省兽药质量检验所（山东省畜产品质量检测中心） | Method that is a kind of while measuring scutelloside, rheum emodin, Physcion and Chrysophanol in seven clear Toxin-Vanquishing particles |
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| CN102507832A (en) * | 2011-10-26 | 2012-06-20 | 上海中医药大学 | Method for measuring high performance liquid chromatography characteristic fingerprint of isatis root and isatis root preparation |
| CN103389344A (en) * | 2012-05-09 | 2013-11-13 | 滇虹药业集团股份有限公司 | Establishment method for HPLC fingerprint spectrum of Chinese pulsatilla root medicinal material and standard fingerprint spectrum thereof |
| CN109061002A (en) * | 2018-09-18 | 2018-12-21 | 山东省兽药质量检验所（山东省畜产品质量检测中心） | Method that is a kind of while measuring scutelloside, rheum emodin, Physcion and Chrysophanol in seven clear Toxin-Vanquishing particles |
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