CN114674947A - Detection method for rapidly and comprehensively controlling quality of pinellia ternate and magnolia officinalis decoction standard decoction - Google Patents

Abstract

The invention discloses a method for rapidly and comprehensively controlling the quality of a pinellia ternate-magnolia bark decoction standard decoction, which establishes a characteristic map through a liquid chromatography and takes scutellarin, rosmarinic acid, magnolol and honokiol as content index components. The method disclosed by the invention is simple to operate, good in reproducibility and stability, reliable in recovery rate, low in detection cost and high in detection efficiency, and provides a basis for standardizing substance standards, preparation quality and reasonable development of the pinellia ternate-magnolia bark decoction.

Description

Detection method for rapidly and comprehensively controlling quality of pinellia ternate and magnolia officinalis decoction standard decoction

Technical Field

The invention relates to a detection method of a traditional Chinese medicine preparation, in particular to a detection method for rapidly and comprehensively controlling the quality of a standard decoction of pinellia ternate and magnolia officinalis decoction.

Background

The pinellia ternate-magnolia officinalis decoction is listed in the first list of classic famous prescriptions in 2018 from jin Kui Yao L ü e of Zhang Zhongjing of Han Dynasty, is the first list of 17 th list of classic famous prescriptions, is a clinical classic proved prescription for treating globus hystericus, consists of pinellia ternate, magnolia officinalis, poria cocos, dried perilla and ginger, and has the effects of promoting qi circulation, resolving masses, lowering adverse qi and reducing phlegm. Modern clinical researches show that the pinellia ternate-magnolia bark decoction has obvious curative effects on treating gastroesophageal reflux disease, chronic gastritis, functional dyspepsia, bronchial asthma, cough, chronic pharyngitis, depression and the like.

The standard decoction is a material standard for measuring the quality consistency of the traditional Chinese medicine compound preparation and clinical decoction, and is a core idea of classical famous formula research, namely the quality of the standard decoction is directly related to the quality of the traditional Chinese medicine compound preparation after production and transformation. In the aspect of quality standard control, most of the medicinal flavors of the pinellia ternate-magnolia officinalis decoction characterized by the existing literature are mainly concentrated on the ministerial medicament of magnolia officinalis, magnolol and honokiol are mainly used as main quality control indexes, and the detection and control of other medicinal flavors or a plurality of index components of the prescription are lacked. In the aspect of characteristics or fingerprint, few documents exist at present about the research on the characteristics or fingerprint of the pinellia ternate-magnolia officinalis decoction.

Patent CN113341010A establishes a quality control method of pinellia ternate-magnolia officinalis decoction, which is respectively used for controlling the content and characteristic map method of a pinellia ternate-magnolia officinalis decoction preparation, and the content measurement establishes a determination method of 4 index components of magnolol, honokiol, rosmarinic acid and 6-gingerol, wherein the 6-gingerol component has no specificity, can be interfered by ginger processed products, and is difficult to effectively control the quality of the ginger processed pinellia ternate and the ginger in the compound preparation; the characteristic spectrum is detected by HPLC, the time is long (150 minutes), the detection cost is high, and the peak separation degree of individual characteristics is poor and needs to be optimized.

Disclosure of Invention

The invention aims to: the invention aims to provide a detection method for rapidly and comprehensively controlling the quality of a standard decoction of pinellia ternate-magnolia bark decoction, wherein the method is used for detecting 4 content index components and 12 characteristic peaks of the characteristic spectrum, so that a simple, convenient, rapid and effective detection method is provided for the quality reference of a classical famous formula pinellia ternate-magnolia bark decoction and the quality control of a compound preparation.

The technical scheme is as follows: the detection method for rapidly and comprehensively controlling the quality of the standard decoction of the pinellia ternate and magnolia officinalis decoction comprises the following steps of:

(1) preparation of a test solution: extracting standard decoction of BANXIAHOUPU decoction with organic solvent, shaking, filtering, and collecting filtrate to obtain the sample solution;

(2) preparation of reference solutions: dissolving scutellarin, rosmarinic acid, magnolol and honokiol reference substance with solvent to obtain reference substance solution;

(3) sample introduction detection: detecting the sample solution and the reference solution by a liquid chromatograph under the following two chromatographic conditions (a) and (b);

(4) calculating the content: calculating the content of rosmarinic acid, magnolol and honokiol by adopting the map obtained by the chromatographic condition in (a) in (3) through an external standard method, and calculating the content of scutellarin by adopting the map obtained by the chromatographic condition in (b) in (3) through the external standard method;

(5) Establishing a characteristic spectrum: detecting a plurality of batches of the test sample solution of the standard decoction of the pinellia ternate and magnolia officinalis decoction, taking the chromatogram obtained by adopting the chromatographic condition in the step (3) and selecting chromatographic peaks existing in different batches of the standard decoction of the pinellia ternate and magnolia officinalis decoction as characteristic peaks to formulate the characteristic chromatogram.

The detection method for rapidly and comprehensively controlling the quality of the pinellia ternate-magnolia bark decoction standard decoction comprises water decoction, extract, concentrated solution and freeze-dried powder of the pinellia ternate-magnolia bark decoction.

The detection method for rapidly and comprehensively controlling the quality of the pinellia ternate-magnolia officinalis decoction standard decoction specifically comprises the following two chromatographic conditions:

(a) octadecylsilane chemically bonded silica chromatographic column, using methanol as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, and performing gradient elution (0-4 min, 2-7% A, 4-13 min, 7-17% A, 13-19 min, 17% A, 19-23 min, 17-23% A, 23-38 min, 23-34% A, 38-42 min, 34% A, 42-55 min, 34-45% A, 55-68 min and 45-90% A); (b) octadecylsilane chemically bonded silica chromatographic column, and acetonitrile-0.2% formic acid as mobile phase for isocratic elution.

The detection method for rapidly and comprehensively controlling the quality of the pinellia ternate-magnolia officinalis decoction standard decoction further comprises the following steps: (6) carrying out sample injection detection in the step (3) on a to-be-detected sample solution, comparing a spectrum obtained by adopting the chromatographic condition in the step (3) with a characteristic spectrum, identifying the number of characteristic peaks, and determining the similarity; meanwhile, the quality of the standard decoction is comprehensively evaluated by taking scutellarin, rosmarinic acid, magnolol and honokiol as content index components.

The detection method for rapidly and comprehensively controlling the quality of the pinellia ternate-magnolia bark decoction standard decoction comprises the following steps that (1) the organic solvent is a methanol water solution with the volume concentration of 30-75%, the extraction time is 15-45 minutes, and the extraction mode comprises ultrasonic treatment, shaking extraction and heating reflux; the extraction volume is 15 ml-50 ml.

The detection method for rapidly and comprehensively controlling the quality of the pinellia ternate magnolia officinalis decoction standard decoction is characterized in that the liquid chromatograph in the step (3) is an ultra-high performance liquid chromatograph.

The detection method for rapidly and comprehensively controlling the quality of the standard decoction of the pinellia ternate-magnolia bark decoction is characterized in that (3) an ultraviolet detector is adopted in the step (a), the column temperature is 20-30 ℃, the flow rate is 0.25-0.4 ml per minute, and the detection wavelength is 280-330 nm; (b) an ultraviolet detector is adopted, the column temperature is 20-30 ℃, the flow rate is 0.25-0.4 ml per minute, and the detection wavelength is 280-330 nm.

The detection method for rapidly and comprehensively controlling the quality of the standard decoction of the pinellia ternate and magnolia officinalis decoction comprises the step (5) that the characteristic map has 12 characteristic peaks F1-F12, the relative retention time F2 is 1, the relative retention time F1 is 0.330-0.385, the relative retention time F9 is 1, and the relative retention time F3-F12 is 0.580-0.620, 0.640-0.652, 0.655-0.680, 0.700-0.730, 0.790-0.820, 0.830-0.865, 1.000, 1.370-1.420, 1.430-1.495 and 1.470-1.530 respectively.

The detection method for rapidly and comprehensively controlling the quality of the pinellia ternate-magnolia bark decoction standard decoction is characterized in that thin-layer detection is carried out after liquid chromatography detection.

The detection method for rapidly and comprehensively controlling the quality of the pinellia ternate-magnolia officinalis decoction standard decoction specifically comprises the following steps of:

(1) preparation of thin-layer test solution: extracting standard decoction of BANXIAHOUPU decoction with organic solvent, shaking, filtering, evaporating, and dissolving in methanol to obtain test solution;

(2) preparation of thin-layer control solution: folium Perillae reference medicinal material solution, and rhizoma Zingiberis recens reference medicinal material solution; preparing a perilla leaf and ginger-deficient negative control group according to a pinellia tuber and magnolia bark decoction formula, and adding methanol to dissolve the perilla leaf and ginger-deficient negative control group to obtain a negative control solution;

(3) and (3) determination: sucking the sample solution, the negative control solution and the control solution, dropping on the same silica gel G thin layer plate, developing with toluene-ethyl acetate-formic acid as developing agent, taking out, air drying, and inspecting under ultraviolet light.

The pinellia ternate and magnolia officinalis decoction standard decoction comprises water decoction (extracting solution), extract (concentrated solution) and freeze-dried powder, and the specific preparation method comprises the following steps: weighing 15g of ginger processed pinellia tuber, 9g of ginger processed magnolia bark, 12g of poria cocos, 6g of perilla leaf and 15g of ginger, respectively placing the materials in each decocting pot, adding 1200-1600 mL of water, soaking for 30-40min, boiling with strong fire, keeping the boiling with slow fire until the decoction is 600-800 mL, stopping heating, and filtering with a standard sieve of 200-350 meshes to obtain an extracting solution. Concentrating the extract under reduced pressure (55-75 ℃) until the specific gravity is 1.01-1.05 to obtain a concentrated solution. And (5) freeze-drying the concentrated solution to obtain the pinellia ternate-magnolia bark decoction freeze-dried powder.

Has the advantages that: (1) the method adopts the ultra-high liquid chromatography, reasonably optimizes the chromatographic conditions, establishes the characteristic spectrums of 12 characteristic peaks of the pinellia ternate and magnolia officinalis decoction freeze-dried powder, detects the content of index components, provides a new analysis means for the quality control of the substance reference of the pinellia ternate and magnolia officinalis decoction, and is an effective means for the overall quality control of the standard decoction of the pinellia ternate and magnolia officinalis decoction. (2) The method takes scutellarin, rosmarinic acid, magnolol and honokiol as content index components, establishes a characteristic map and identifies a thin layer, and is suitable for the material standard of the classical famous formula pinellia ternate magnolia bark decoction prepared by water decoction or extraction. (3) The method disclosed by the invention is simple to operate, good in reproducibility and stability, reliable in recovery rate, low in detection cost and high in detection efficiency, and provides a basis for standardizing substance standards of the pinellia ternate and magnolia officinalis decoction, and reasonably developing the preparation market. (4) Compared with the patent CN113341010A, the method for detecting the content index of scutellarin has important significance for the quality reference of the cortex magnoliae officinalis decoction and the quality control index of the preparation thereof. The sample processing method is simple, and a set of sample processing method is adopted, so that the detection requirements of 4 index components and characteristic maps are met. The established characteristic spectrum determination method can determine 12 characteristics of 4 medicinal materials, not only can represent the components of monarch drug rhizoma pinelliae, ministerial drug cortex magnoliae officinalis and assistant drug folium perillae and rhizoma zingiberis, but also can simultaneously determine 3 special components (magnolol, honokiol and rosmarinic acid) in cortex magnoliae officinalis and folium perillae.

Drawings

FIG. 1 is a detection diagram of a pinellia ternate and magnolia officinalis decoction freeze-dried powder characteristic spectrum reference substance;

FIG. 2 is a detection chart of scutellarin content determination control;

FIG. 3 is a chromatogram of different extraction solvents used for determination of the content of rosmarinic acid, magnolol and the total amount of honokiol;

FIG. 4 is a chromatogram of different extraction solvents for determining scutellarin content;

FIG. 5 is a chromatogram of determination of the content of rosmarinic acid, magnolol and the total amount of honokiol by different extraction methods;

FIG. 6 is a chromatogram of different extraction methods for determining scutellarin content;

FIG. 7 is a chromatogram of the total amount of rosmarinic acid, magnolol, and honokiol determined using different extraction volumes;

FIG. 8 is a chromatogram of different extraction volumes for determining scutellarin content;

FIG. 9 is a chromatogram of determination of the content of rosmarinic acid, magnolol, and the total amount of honokiol using different extraction times;

FIG. 10 is a chromatogram of content determination of scutellarin with different extraction times;

FIG. 11 is a chromatogram of 15 batches of ban Xia Hou Po Tang based on the reference feature of the material;

FIG. 12 is a linear relationship diagram of scutellarin, rosmarinic acid, magnolol, and honokiol;

FIG. 13 is a survey chromatogram of the total determination specificity of rosmarinic acid content, magnolol, and honokiol;

FIG. 14 is a chromatogram for investigating specificity of determining scutellarin content;

FIG. 15 is a chromatogram for investigation of the content of rosmarinic acid, the determination of the total amount of magnolol and honokiol at different flow rates;

FIG. 16 is a chromatogram for examining the content of scutellarin at different flow rates;

FIG. 17 is a chromatogram for investigation of the content of rosmarinic acid, the total content of magnolol and honokiol by measuring different temperatures of chromatographic columns;

FIG. 18 is a chromatogram for temperature investigation of different chromatographic columns for determining scutellarin content;

FIG. 19 is a chromatogram for investigation of different chromatographic columns for determination of the content of rosmarinic acid, the total amount of magnolol and honokiol;

FIG. 20 is a thin-layer identification of perilla and ginger in ban Xia Hou Po decoction by different preparation methods; wherein 1-3-pinellia ternate and mangnolia officinalis decoction (JDG-BXHP-20210525-F1-3), S1-S3-perilla leaf contrast medicine, S4-S6-dried ginger contrast medicine;

FIG. 21 is a thin-layer identification of different sample amounts of Perillae herba and rhizoma Zingiberis recens in BANXIAHOUPU decoction, wherein 1-BANXIAHOUPU decoction (JDG-BXHP-20210525-F1-3): 1 (1. mu.l), 2 (3. mu.l), 3 (5. mu.l), 4 (8. mu.l) 5; 4-perilla lacking negative control, 6-ginger lacking negative control, S1-perilla control medicinal material, and S2-rhizoma zingiberis control medicinal material;

FIG. 22 is a thin layer identification of the specificity of perilla and ginger in ban Xia Hou Po decoction, wherein 1-ban Xia Hou Po decoction (F1), 2-ban Xia Hou Po decoction (F2), 3-ban Xia Hou Po decoction (F3), 4-lack of perilla negative control, 5-lack of ginger negative control, S1-perilla control, S2-Zingiberis rhizoma control;

FIG. 23 is a thin-layer identification of perilla and ginger at different temperatures in Banxia Houpo Tang, wherein 1-Banxia Houpo Tang (F1), 2-Banxia Houpo Tang (F2), 3-Banxia Houpo Tang (F3), 4-lacking perilla negative control, 5-lacking ginger negative control, S1-perilla control, S2-lacking ginger control;

FIG. 24 is a thin-layer identification of purple perilla and ginger at different humidity in ban Xia Hou Po decoction, wherein 1-ban Xia Hou decoction (F1), 2-ban Xia Hou Po decoction (F2), 3-ban Xia Hou decoction (F3), 4-lack purple perilla negative control, 5-lack ginger negative control, S1-purple perilla control, S2-Zingiberis rhizoma control;

FIG. 25 shows identification of thin-layer plates of perilla and ginger of different manufacturers in ban Xia Hou Po decoction, wherein 1-ban Xia Hou decoction (F1), 2-ban Xia Hou Po decoction (F2), 3-ban Xia Hou decoction (F3), 4-lack of perilla negative control, 5-lack of ginger negative control, S1-perilla control, S2-dry ginger control.

Detailed Description

Example 1

Method for measuring content of standard decoction of pinellia ternate and magnolia officinalis decoction and construction of fingerprint

1. Reagents and samples

Scutellarin (batch No. 110842-201709) was purchased from China institute for testing and testing food and drug, and the content of scutellarin is 91.7%, and no treatment is needed before use.

Rosmarinic acid (batch No. 111871-.

Magnolol (batch No. 110729-201714) was purchased from the institute for testing food and drug, and was used for content determination, and the content was calculated as 100.0%, and no treatment was required before use.

Honokiol (batch No. 110730-.

Caffeic acid (batch No. 110885-200102) was purchased from China institute for food and drug assay for content determination, the content was 99.7%, and no treatment was required before use.

6-gingerol (batch No. 111833-202007) was purchased from China institute for testing and determining food and drug, and its content was 99.3%, and no treatment was required before use.

The medicinal materials, decoction pieces, and lyophilized powder of BANXIAHOUPU decoction are provided by TIANJIANG pharmaceutical industry GmbH of Jiangyin city.

2. Preparation of control solutions

Taking a proper amount of scutellarin reference substance, precisely weighing, and adding 50% methanol to obtain solution containing 10 μ g of scutellarin per 1ml to obtain scutellarin reference substance solution. Accurately weighing appropriate amount of rosmarinic acid reference substance, magnolol reference substance, and honokiol reference substance, and adding 50% methanol to obtain mixed reference substance solution containing rosmarinic acid 22 μ g, magnolol 10 μ g, and scutellarin 10 μ g per 1 ml. Taking scutellarin reference substance, precisely weighing, and adding 50% methanol to make into reference substance solution containing 9 μ g per 1 ml. Additionally, a proper amount of caffeic acid reference substance and 6-gingerol reference substance are weighed, and 50% methanol is added to prepare 10 mug of caffeic acid and 10 mug of 6-gingerol reference substance solution.

3. Preparation of test solution

Weighing about 0.2g of the lyophilized powder, accurately weighing, placing in a conical flask with a plug, adding 25ml of 50% methanol, sealing, weighing, ultrasonically treating (power 250W, frequency 40kHz) for 30 min, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking, filtering, and collecting the filtrate.

4. Determination of analytical methods

4.1 characterization and content determination chromatographic conditions

Octadecylsilane chemically bonded silica is used as a filling agent (the column length is 100mm, the inner diameter is 2.1mm, the particle size is 1.8 mu m), methanol is used as a mobile phase A, 0.1% phosphoric acid aqueous solution is used as a mobile phase B, gradient elution is carried out (0-4 min, 2-7% A, 4-13 min, 7-17% A, 13-19 min, 17% A, 19-23 min, 17-23% A, 23-38 min, 23-34% A, 38-42 min, 34% A, 42-55 min, 34-45% A, 55-68 min, 45-90% A), the flow rate is 0.3 ml.min < -1 >, the column temperature is 25 ℃, the sample injection amount is 2 mu l, and the detection wavelength is 280 nm.

Detecting mixed reference solution containing scutellarin, rosmarinic acid, magnolol, and magnolol, caffeic acid, and 6-gingerol by sample injection, and detecting the chromatogram with figure 1. The results show that the detection baseline is stable, and the separation degree of the target components is good.

4.2 Scutellarein glycoside content determination chromatographic conditions

Octadecylsilane chemically bonded silica gel as filler (column length 100mm, inner diameter 2.1mm, particle diameter 2.2 μm), acetonitrile-0.2% formic acid (18: 82) as eluent, and flow rate of 0.35 ml/min^-1The column temperature was 25 ℃, the amount of sample was 2. mu.l, and the detection wavelength was 330 nm.

Detecting the scutellarin control solution by sample injection, and obtaining a map shown in figure 2. The results show that the detection baseline is stable, the separation degree of the target components is good, and the detection can be completed within 15 minutes.

4.3 preparation of test solutions

(1) Examination of extraction solvent

And (4) conclusion: when methanol, 75% methanol, 50% methanol and 10% methanol are used as extraction solvents, the content of scutellarin and rosmarinic acid in the methanol is low, the content of magnolol and honokiol in the 10% methanol is low, and the content difference between the 50% methanol and the 75% methanol is not large; considering that rosmarinic acid is a water-soluble component, magnolol and honokiol are fat-soluble components, and the solubility of each component is considered, 50% methanol is selected as an extraction solvent for measuring the content of scutellarin, rosmarinic acid, magnolol and honokiol in the pinellia ternate and magnolia officinalis decoction.

The investigation method comprises the following steps: taking about 0.2g of the powder, paralleling 4 groups, paralleling 2 parts of each group, precisely weighing, placing in a conical flask with a plug, precisely adding 10% methanol, 50% methanol, 75% methanol and 25ml of methanol respectively, sealing the plug, weighing, ultrasonically treating (power 250W, frequency 40kHz) for 30 minutes, cooling, weighing again, complementing the weight loss with corresponding solvent, shaking uniformly, filtering, and taking the subsequent filtrate. Precisely sucking 2 μ l of each test solution, injecting into liquid chromatograph, injecting sample under chromatography condition of "4.1", calculating rosmarinic acid content, magnolol and honokiol total content, and calculating scutellarin content under chromatography condition of "4.2", and table 2 and figure 4.

TABLE 1 comparison of rosmarinic acid content, total magnolol and honokiol determination for different extraction solvents

TABLE 2 comparison of different extraction solvents for determining scutellarin content

(2) Examination of extraction methods

And (4) conclusion: when ultrasonic treatment, heating reflux and shaking extraction are adopted, the contents of scutellarin, rosmarinic acid, magnolol and honokiol are not obviously different, the extraction efficiency at different time is similar, and the ultrasonic treatment is selected in consideration of the simple and convenient ultrasonic treatment operation.

The investigation method comprises the following steps: taking about 0.2g of the powder of the product, weighing 3 groups in parallel, weighing 2 parts of each group precisely, placing the mixture in a conical flask with a plug, adding 25ml of 50% methanol precisely, sealing the plug, weighing the weight, performing ultrasonic treatment (power 250W and frequency 40kHz), heating, refluxing, shaking and extracting for 30 minutes, cooling, weighing the weight again, complementing the weight loss with 50% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the product. Precisely sucking 2 μ l of each test solution, injecting into liquid chromatograph, injecting sample under chromatography condition of "4.1", calculating rosmarinic acid content, magnolol and honokiol total content, and calculating scutellarin content under chromatography condition of "4.2", and table 4 and fig. 6.

TABLE 3 comparison of rosmarinic acid content, magnolol and honokiol Total amount determination and extraction methods

TABLE 4 comparison of scutellarin content determination and extraction modes

(3) Examination of volume of extraction solvent

And (4) conclusion: when the extraction volume is 15ml, 25ml and 50ml, the contents of scutellarin, rosmarinic acid, magnolol and honokiol are not very different, which indicates that the extraction is sufficient when the extraction solvent is 15 ml. To ensure adequate extraction, 25ml of solvent was chosen to account for sample variation from batch to batch.

The investigation method comprises the following steps: taking about 0.2g of the powder of the product, weighing 3 groups in parallel, weighing 2 parts of each group precisely, placing the mixture in a conical flask with a plug, adding 15ml, 25ml and 50ml of 50% methanol precisely, sealing the plug, weighing the mixture, carrying out ultrasonic treatment (power of 250W and frequency of 40kHz) for 30 minutes, cooling the mixture, weighing the mixture again, complementing the weight loss with 50% methanol, shaking the mixture evenly, filtering the mixture, and taking the subsequent filtrate to obtain the product. Precisely sucking 2 μ l of each test solution, injecting into liquid chromatograph, injecting sample under chromatography condition of "4.1", calculating rosmarinic acid content, magnolol and honokiol total content, and calculating scutellarin content under chromatography condition of "4.2", and table 6 and figure 8.

TABLE 5 comparison of rosmarinic acid content, total magnolol and honokiol content for determination of extraction volume

TABLE 6 comparison of scutellarin content determination and extraction volume

(4) Investigation of extraction time

And (4) conclusion: different ultrasonic treatment times have small differences of the content of scutellarin and rosmarinic acid, the total content of magnolol and honokiol, which indicates that the extraction is sufficient when the extraction time is 15 minutes. To ensure adequate extraction, taking into account the differences between batches of samples, extraction times of 30 minutes were used.

The investigation method comprises the following steps: taking about 0.2g of the powder of the product, weighing 3 groups in parallel, weighing 2 parts of each group precisely, placing the mixture in a conical flask with a plug, precisely adding 25ml of 50% methanol, sealing the plug, weighing the weight, performing ultrasonic treatment (power 250W and frequency 40kHz) for 15 minutes, 30 minutes and 45 minutes respectively, cooling the mixture, weighing the weight again, complementing the weight loss with 50% methanol, shaking the mixture evenly, filtering the mixture, and taking the subsequent filtrate to obtain the product. Precisely sucking 2 μ l of each test solution, injecting into liquid chromatograph, injecting sample under chromatography condition of "4.1", calculating rosmarinic acid content, magnolol and honokiol total content, and calculating scutellarin content under chromatography condition of "4.2", and table 8 and figure 10.

TABLE 7 comparison of rosmarinic acid content, magnolol and honokiol Total amount determination extraction time

TABLE 8 comparison of scutellarin content determination and extraction time

Finally, the preparation method of the sample solution is determined as follows:

taking about 0.2g of the powder, accurately weighing, placing in a conical flask with a plug, accurately adding 25ml of 50% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (with the power of 250W and the frequency of 40kHz) for 30 minutes, cooling, weighing again, complementing the loss weight with 50% methanol, shaking up, filtering, and taking the subsequent filtrate.

(5) Detection of different batches

Taking 15 samples of different batches, preparing samples according to a preparation method of a test solution under the item '4.3', carrying out sample injection analysis under the chromatographic condition under the item '4.1', measuring peak areas and characteristic data of common peaks of rosmarinic acid, magnolol and honokiol, calculating the content, the relative peak area and the relative retention time, and obtaining results shown in tables 9-13 and 11, carrying out sample injection analysis under the chromatographic condition under the item '4.2', recording the peak area of scutellarin, calculating the content of the scutellarin, and obtaining the scutellarin content shown in the table 9. The specific results are as follows:

TABLE 9 Total content results of scutellarin, rosmarinic acid, magnolol and honokiol in different batches of BANXIAHOUPU decoction

TABLE 10 Standard characteristic peak area data of different batches of rhizoma Pinelliae and cortex Magnolia officinalis decoction

TABLE 11 relative peak area data of standard characteristics of different batches of BANXIA HOUPU decoction materials

TABLE 12 data of reference characteristic retention time of different batches of rhizoma Pinelliae and cortex Magnolia officinalis decoction

TABLE 13 data of relative retention time of reference characteristics of different batches of rhizoma Pinelliae and cortex Magnolia officinalis decoction

Example 2

Methodology validation of assay construction method

1. Linear relation

The results show that: the sampling amount of scutellarin is in the range of 0.00186-0.05574 mu g, the sampling amount of rosmarinic acid is in the range of 0.00432-0.12967 mu g, the sampling amount of magnolol is in the range of 0.00196-0.05868 mu g, and the sampling amount of honokiol is in the range of 0.00197-0.05922 mu g, and the sampling amount and the peak area are in a good linear relation.

Precisely sucking rosmarinic acid reference substance solution (with the concentration of 21.6109 mu g/ml), magnolol reference substance solution (with the concentration of 9.78 mu g/ml) and honokiol reference substance solution (with the concentration of 9.8704 mu g/ml)0.2, 0.5, 1.0, 1.5, 2, 3, 4, 5 and 6 mu l, injecting into a liquid chromatograph, injecting sample according to the chromatographic condition under the item of '4.1', drawing a standard curve by taking the peak area as the ordinate and the sample injection amount as the abscissa, and solving a regression equation: rosmarinic acid Y ═ 5669.0013x +6161960.758, R ═ 0.99999, magnolol Y ═ 1615.9369x +4197770.926, R ═ 0.99998, honokiol Y ═ 1504.4128x +4429822.829, R ═ 0.99998, the results are shown in table 14 and fig. 12. Precisely sucking scutellarin control solution (with concentration of 18.5784 μ g/ml)0.1, 0.2, 0.5, 1, 2, and 3 μ l, injecting into liquid chromatograph, injecting sample under chromatographic condition of "4.2", and drawing standard curve with peak area as ordinate and sample amount as abscissa. Solving a regression equation: scutellarin Y is 143.32x +0.0246, and R is 0.99998, the results are shown in table 15 and fig. 12.

TABLE 14 correlation of peak area and sample size of rosmarinic acid, magnolol, and honokiol reference substances

TABLE 15 correlation between peak area and sample amount of scutellarin control

2. Precision test

2.1 instrumental precision test

As a result: the peak area RSD is less than 2.0%, and the precision test of the instrument is good.

Injecting 2 mul of the precise pinellia ternate and magnolia officinalis decoction substance reference sample solution into a liquid chromatograph, injecting sample according to the chromatographic condition under the item of 4.1, continuously injecting sample for 6 times, recording the measured values of the peak areas of rosmarinic acid, magnolol and honokiol, injecting sample according to the chromatographic condition under the item of 4.2, continuously injecting sample for 6 times, recording the measured value of the peak area of scutellarin, and calculating the relative standard deviation, wherein the result is shown in a table 16.

TABLE 16 Instrument precision test

2.2 repeatability test

As a result: the contents of scutellarin and rosmarinic acid, the total content of magnolol and honokiol RSD are less than 2.0 percent, and the repeatability test is good.

Taking about 0.2g of the same batch of samples, precisely weighing, paralleling 6 parts, preparing the samples according to the preparation method of the test solution under the item '4.3', carrying out sample injection analysis under the chromatographic condition under the item '4.1', measuring the peak areas of the rosmarinic acid, the magnolol and the honokiol, calculating the content of the rosmarinic acid, the total content of the magnolol and the honokiol and the RSD value, and obtaining the result shown in the table 17, carrying out sample injection analysis under the chromatographic condition under the item '4.2', measuring the peak area of the scutellarin, calculating the content of the scutellarin and the RSD value, and obtaining the result shown in the table 18.

TABLE 17 repeatability tests for determination of rosmarinic acid content, magnolol and honokiol

TABLE 18 repeatability test of scutellarin content determination

2.3 intermediate precision

As a result: scutellarin, contents of the same, magnolol and total content of the same RSD (RSD less than 2.0%), and good intermediate precision test.

Taking 3 parts of the same batch of samples, preparing the samples by different experimenters according to the preparation method of the test solution under the item 4.3, respectively injecting 2 mu l of sample on the same instrument at different times according to the chromatographic condition under the item 4.1, respectively, measuring the area values of the rosmarinic acid, the magnolol and the honokiol, and calculating the content of the rosmarinic acid, the total content of the magnolol and the honokiol and the RSD, wherein the results are shown in a table 19. Separately injecting 2 μ l sample into different instruments at different time according to the chromatographic conditions of "4.2", measuring the area value of scutellarin, and calculating the content and RSD of scutellarin, the results are shown in Table 20.

TABLE 19 determination of Rosmarinic acid content, magnolol and Honokiol Total amount intermediate precision test

TABLE 20 determination of scutellarin content intermediate precision test

3. Accuracy test

As a result: the average recovery rate of scutellarin, rosmarinic acid, magnolol and honokiol is 92-105%, the RSD is less than 3.0%, and the accuracy test is good.

Taking 0.1g of a sample with known content (0.29% of rosmarinic acid, 0.10% of magnolol and 0.12% of honokiol), weighing 9 parts in parallel, respectively adding a mixed reference substance solution corresponding to rosmarinic acid, magnolol and honokiol contained in 0.1g of the sample, preparing a sample-adding and recycling sample solution according to the preparation method of the sample solution under the item of '4.3', injecting samples according to the chromatographic condition under the item of '4.1', respectively injecting 2 mu l of samples, and calculating the recovery rates and the RSD of rosmarinic acid, magnolol and honokiol according to the following formulas, wherein the results are shown in tables 21-23. Taking 0.1g of a sample with a known content (scutellarin content 0.11%), weighing 9 parts in parallel, accurately weighing, adding a control solution of scutellarin contained in the 0.1g of sample, preparing a sample recovery sample solution according to the preparation method of the sample solution under the item '4.3', injecting 2 mul of sample solution under the chromatographic condition under the item '4.2', and calculating the scutellarin recovery rate and RSD according to the following formulas, wherein the results are shown in Table 24.

TABLE 21 Rosmarinic acid accuracy test

TABLE 22 magnolol accuracy test

TABLE 23 accuracy test of honokiol

TABLE 24 accuracy test of scutellarin

4. Specificity test

And (4) conclusion: from the results, the blank solvent has no interference on the determination of scutellarin, rosmarinic acid, magnolol and honokiol, and the method has strong specificity.

Sampling blank solvent, reference solution, and reference sample solution of rhizoma Pinelliae and cortex Magnolia officinalis decoction under chromatography condition of "4.1", and finding the result shown in FIG. 13. The sample was injected under the chromatographic conditions of "4.2" and the results are shown in FIG. 14.

5. Durability test

5.1 stability test

As a result: the test solutions were stable well within 48 hours (RSD% < 2.0%).

Taking 1 part of sample, preparing a test solution according to the preparation method of the test solution under the item '4.3', injecting samples under the chromatographic condition under the item '4.1' within 0, 2, 4, 6, 8, 12, 18, 24 and 48 hours, injecting 2 mu l of sample, measuring the peak area value, and calculating the RSD value. The results are shown in Table 25. Injecting sample under chromatography condition of "4.2" at 0, 2, 4, 6, 8, 12, 18, 24, 48h, injecting 2 μ l, measuring peak area value, and calculating RSD value. The results are shown in Table 26.

TABLE 25 determination of stability of test samples for rosmarinic acid content, magnolol and honokiol

TABLE 26 determination of stability of scutellarin content in test sample

5.2 investigation of different flow rates

As a result: the flow rate is 0.25-0.35 ml/min^-1The measurement results are not different from each other, and the durability is good.

Taking 1 part of sample, preparing sample solution according to the preparation method of sample solution under item "4.3", and inspecting 0.25 ml/min ^-1、0.30ml·min^-1、0.35ml·min^-1Influence on chromatographic peak separation and content results at three flow rates. The results are shown in tables 27 and 28, fig. 15, and fig. 16.

TABLE 27 determination of Rosmarinic acid content, magnolol and Honokiol Total at different flow rates

TABLE 28 different flow rate investigation of scutellarin content determination

5.3 column temperature investigation

As a result: the column temperature is between 20 ℃ and 30 ℃, the measurement result has no obvious difference, and the durability is good.

Sample 1 part, prepare the sample solution according to the preparation method of the sample solution under item "4.3", examine three temperatures of 20 ℃, 25 ℃, 30 ℃, and see table 29, table 30, fig. 17, fig. 18.

TABLE 29 examination of the content of rosmarinic acid, the total amount of magnolol and honokiol

TABLE 30 examination of different column temperatures for determination of scutellarin content

5.4 column inspection

As a result: the chromatographic columns of three different models have good separation effect and moderate retention time, which indicates that the chromatographic columns have little influence on the measurement result of the sample.

Taking 1 part of sample, preparing a test solution according to the preparation method of the test solution under the item of '4.3', and respectively adopting a column 1: HST 3 (2.1X 100mm, 1.8 μm), column 2: eclipse Plus RRHD C18 (2.1X 100mm, 1.8 μm), column 3: the content of rosmarinic acid, magnolol and honokiol is analyzed by injecting samples into Poroshell C18(2.1 × 100mm, 1.9 μm) three chromatographic columns under the chromatographic condition of '4.1', and the analysis results are shown in Table 31 and FIG. 19. Another Acclaim ^TMRSLCPolaraDvantage II (PA2) was injected under the chromatography condition of item "4.2" to analyze the content of scutellarin, and the analysis results are shown in Table 32.

TABLE 31 comparison of rosmarinic acid content, total magnolol and honokiol determination for different chromatographic columns

TABLE 32 comparison of different chromatographic columns for determining scutellarin content

Example 3

Methodological validation of feature construction methods

1. Precision test

1.1 instrumental precision test

As a result: the relative peak area and the relative retention time RSD of the common peak are less than 5.0 percent, and the precision test of the instrument is good.

Injecting 2 mu l of a precise pinellia ternate and magnolia officinalis decoction substance reference sample solution into a liquid chromatograph, injecting samples according to the chromatographic condition under the item of '4.1', continuously injecting the samples for 6 times, recording the measured values of 12 common peak-to-peak areas and retention time, and calculating the relative standard deviation, wherein the result is shown in tables 33-36.

TABLE 33 precision test Peak area results

TABLE 34 relative peak area results for precision tests

TABLE 35 precision test retention time results

TABLE 36 precision test relative retention time results

1.2 repeatability test

As a result: the relative peak area and the relative retention time RSD of the common peak are less than 5.0 percent, and the repeatability test is good.

About 0.2g of the same batch of samples are precisely weighed and are parallelly distributed by 6 parts, the samples are prepared according to the preparation method of the test solution under the item '4.3', the sample injection analysis is carried out according to the chromatographic condition under the item '4.1', the peak areas and the retention times of 12 common peaks are measured, and the RSD values of the relative peak areas and the relative retention times are calculated, and the results are shown in tables 37-40.

TABLE 37 peak area results of repeatability tests

TABLE 38 relative peak area results of repeatability tests

TABLE 39 repeatability test retention time results

TABLE 40 repeatability test relative retention time results

1.3 intermediate precision

As a result: the relative peak area and relative retention time RSD of the common peak are less than 5.0 percent, and the intermediate precision test is good.

Taking 3 parts of the same batch of samples, preparing the samples by different experimenters according to a preparation method of a test solution under the item '4.3', respectively injecting 2 mu l of sample on the same instrument at different times according to chromatographic conditions under the item '4.1', measuring peak areas and retention times of 12 common peaks, and calculating RSD values of the relative peak areas and the relative retention times, wherein the results are shown in tables 41-44.

TABLE 41 area results of intermediate precision test peaks

TABLE 42 relative peak area results of intermediate precision tests

TABLE 43 results of intermediate precision test retention time

TABLE 44 relative retention time results for intermediate precision tests

2. Specificity test

And (4) conclusion: as can be seen from the results, the blank solvent did not interfere with the determination of the characteristics, and the method was highly specific.

Sampling blank solvent, negative control solution, and rhizoma Pinelliae and cortex Magnolia officinalis decoction reference sample solution under chromatography condition of "4.1", and finding the result shown in FIG. 13.

3. Stability test

As a result: the test solutions were stable well within 48 hours (RSD% < 5.0%).

Taking 1 part of sample, preparing a test solution according to the preparation method of the test solution under the item '4.3', injecting 2 mu l of sample under the item '4.1' in 0, 2, 4, 6, 8, 12, 18, 24 and 48h, measuring the peak areas and retention times of 12 common peaks, and calculating the RSD values of the relative peak areas and the relative retention times, wherein the results are shown in tables 45-48.

TABLE 45 stability test Peak area results

TABLE 46 stability test relative peak area results

TABLE 47 stability test Retention time results

TABLE 48 stability test relative Retention time results

Example 4: content determination result of standard decoction

1. Preparation of standard decoction

Weighing 15g of ginger processed pinellia tuber, 9g of ginger processed magnolia bark, 12g of poria cocos, 6g of perilla leaf and 15g of ginger, respectively placing the materials in each decocting pot, adding 1200ml of water, soaking for 30min, boiling with strong fire, boiling with slow fire until the volume of decoction is 600ml, stopping heating, and filtering with a standard sieve of 200 meshes to obtain an extracting solution. Concentrating the extracting solution under reduced pressure (60 ℃) until the specific gravity is 1.01-1.05 to obtain concentrated solution. And (5) freeze-drying the concentrated solution to obtain the pinellia ternate-magnolia bark decoction freeze-dried powder.

2. Preparation of test solution

Preparing an extracting solution test solution: precisely sucking 15ml to 25ml of pinellia ternate and magnolia officinalis decoction extracting solution in a volumetric flask, adding methanol, carrying out ultrasonic treatment, fixing the volume to a scale mark, shaking up, and taking a subsequent filtrate to obtain the traditional Chinese medicine.

Preparing a concentrated solution test solution: precisely weighing the pinellia ternate and magnolia officinalis decoction concentrated solution 3g to 20ml in a volumetric flask, adding 50% methanol, performing ultrasonic treatment, fixing the volume to a scale mark with 50% methanol, shaking up, and taking a subsequent filtrate to obtain the traditional Chinese medicine.

Preparing a freeze-dried powder test solution: weighing about 0.2g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 50% methanol, sealing the plug, weighing, ultrasonically treating (power 250W, frequency 40kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, and taking the subsequent filtrate.

3. Preparation of reference solutions

Taking appropriate amount of scutellarin, rosmarinic acid, magnolol and honokiol as reference substances, and adding methanol to obtain mixed reference substance solution containing 40 μ g of scutellarin, 40 μ g of rosmarinic acid, 60 μ g of magnolol and 20 μ g of magnolol per 1 ml.

4. Determination of content

Detecting the test solution and the reference solution by adopting characteristic chromatographic conditions, recording peak areas, and calculating the contents and transfer rates of the compound extracting solution, the concentrated solution, the freeze-dried powder scutellarin, rosmarinic acid, magnolol and honokiol.

TABLE 49 results of extract, concentrate, and lyophilized powder contents

Example 5

Construction of identification and determination method for standard decoction thin layer of pinellia ternate and magnolia officinalis decoction

1. Instruments, reagents and samples

Thin layer automated imager (CAMAG TLC VIUALIZER); an ultrasonic cleaner (KQ-250E, ultrasonic instruments Co., Ltd., Kunshan, Ltd.); one thousandth of a balance (AR223CN, aohaus); silica gel G thin layer plates (national drug group chemical agents limited); GKC temperature-controlled water bath (Nantong Huatai laboratory instruments Co., Ltd.); HY-4 speed-adjustable multi-purpose oscillator (Kangxing apparatus, jin Tan).

Toluene (shanghai Linfeng Chemicals Co., Ltd.), sulfuric acid (shanghai Linfeng Chemicals Co., Ltd.), ethyl acetate (national drug group Chemicals Co., Ltd.), formic acid (national drug group Chemicals Co., Ltd.), and methanol (national drug group Chemicals Co., Ltd.) were all analytically pure and water.

The perilla leaf control drug (120914-201712) is purchased from China food and drug testing institute.

The dried ginger reference drug (120942-201911) was purchased from the Chinese institute for food and drug testing.

The lyophilized powder of BANXIAHOUPU decoction and the lyophilized powder of negative control are provided by TIANJIANG pharmaceutical industry GmbH of Jiangyin city.

2. Preparation of control solutions

Dissolving folium Perillae control material 1g in water 20ml, filtering, extracting the filtrate with ethyl acetate twice (20 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving with methanol 0.5ml to obtain Perillae herba control material solution.

Taking 0.5g of a dried ginger control medicinal material, adding 20ml of methanol, carrying out ultrasonic treatment (power of 250W and 40kHz) for 30 minutes, filtering, evaporating to dryness, and adding 0.5ml of methanol to dissolve to obtain a dried ginger control medicinal material solution.

Taking 1g of each of the negative controls of folium Perillae and rhizoma Zingiberis recens, adding 20ml of methanol, performing ultrasonic treatment (power 250W, 40kHz) for 30 minutes, filtering, evaporating to dryness, and dissolving with 1ml of methanol to obtain negative control solution.

3. Preparation of test solution

Taking 1g of pinellia ternate-magnolia bark decoction freeze-dried powder, adding 20ml of methanol, carrying out ultrasonic treatment (power of 250W and 40kHz) for 30 minutes, filtering, evaporating to dryness, and adding 1ml of methanol for dissolving to obtain a sample solution.

4. Determination of analytical methods

4.1 determination of thin layer identification

Sucking the sample solution, negative control solution and control solution, dropping on the same silica gel G thin layer plate, developing with toluene-ethyl acetate-formic acid (6: 2: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet light (365 nm).

The results show that the identification of the perilla leaf and the ginger has specificity.

4.2 preparation of test solutions

(1) Examination of production method

And (4) conclusion: in the chromatograms of the different preparation methods of the pinellia ternate-magnolia bark decoction test sample and the dried ginger control medicinal material, each spot is clear and has little difference. The chromatographic spots of the folium Perillae reference medicinal material by ultrasonic and heating reflux are consistent, and the difference between the chromatographic spots of the folium Perillae reference medicinal material and the chromatographic spots of the sample solution is larger, and the chromatographic spots of the reference solution obtained by extraction are consistent with the chromatographic spots of the sample solution. Therefore, ultrasonic treatment is selected as a pinellia ternate-magnolia bark decoction sample, a rhizoma zingiberis reference medicinal material and a negative reference preparation method, and ethyl acetate extraction is selected as a perilla frutescens reference medicinal material preparation method.

The investigation method comprises the following steps: taking 1g of each pinellia ternate-magnolia bark decoction freeze-dried powder, adding 20ml of methanol into each 2 parts of the pinellia ternate-magnolia bark decoction freeze-dried powder, carrying out ultrasonic treatment (power is 250W and 40kHz), heating and refluxing for 30 minutes, filtering, evaporating to dryness, and adding 1ml of methanol to dissolve the mixture to obtain a sample solution. Dissolving pinellia ternate and magnolia officinalis decoction freeze-dried powder 1g in water 20ml, extracting with ethyl acetate twice, 20ml each time, combining ethyl acetate solutions, evaporating to dryness, and dissolving with methanol 1ml to obtain a test solution. Taking 1g of each of the perilla leaf control medicinal material and the ginger control medicinal material, and paralleling 3 parts to obtain a control medicinal material solution by the same method. Sucking the above test solution, negative control solution, and control solution, dropping on the same silica gel G thin layer plate, developing with toluene-ethyl acetate-formic acid (6: 2: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet light (365nm), as shown in FIG. 20.

(2) Investigation of different dot patterns

And (4) conclusion: when the spot size of the pinellia ternate and magnolia officinalis decoction is 3-5 mul, the chromatographic spot is clear, and no other interference exists, so that the spot size of the pinellia ternate and magnolia officinalis decoction is selected to be 5 mul.

The investigation method comprises the following steps: taking 1g of lyophilized powder of rhizoma Pinelliae and cortex Magnolia officinalis decoction, adding 20ml of methanol, performing ultrasonic treatment (power 250W, 40kHz), heating and refluxing for 30 minutes, filtering, evaporating to dryness, and dissolving with 1ml of methanol to obtain sample solution. Taking 1g of the comparison medicinal materials of the perilla leaf, the ginger and the dried ginger respectively, and preparing a negative comparison and dried ginger comparison medicinal material solution by the same method. Dissolving folium Perillae control material 1g in water 20ml, extracting with ethyl acetate twice (20 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving with methanol 1ml to obtain folium Perillae control material solution. Sucking the above test solution, negative control solution, and control solution, dropping on the same silica gel G thin layer plate, developing with toluene-ethyl acetate-formic acid (6: 2: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet light (365nm), as shown in FIG. 21.

The thin-layer identification method for determining the perilla frutescens and the ginger in the pinellia ternate-magnolia bark decoction comprises the following steps:

taking pinellia ternate-magnolia bark decoction freeze-dried powder, purple perilla deficiency negative control and ginger deficiency negative control 1g, adding 20ml of methanol, carrying out ultrasonic treatment (power 250W, 40kHz) for 30 minutes, filtering, evaporating to dryness, and adding 1ml of methanol for dissolving to obtain a sample solution. 1ml of rhizoma Zingiberis control solution is prepared by the same method with 1g of rhizoma Zingiberis control. Dissolving folium Perillae control material 1g in water 20ml, filtering, extracting the filtrate with ethyl acetate twice (20 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving with methanol 0.5ml to obtain Perillae herba control material solution. Performing thin layer chromatography (general 0502 of 2020 th edition in Chinese pharmacopoeia), sucking 5 μ l of the above solutions, respectively dropping on the same silica gel G thin layer plate, developing with toluene-ethyl acetate-formic acid (6: 2: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet light (365 nm). Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.

Example 6

Methodological validation of thin layer construction methods

1. Specificity test of samples

As a result: the spots with the same color appear at the corresponding positions of the chromatogram of the pinellia ternate magnolia officinalis decoction, the chromatogram of the perilla frutescens, the chromatogram of the ginger and the control medicinal material, and the negative control has no interference. The thin layer method has good specificity

Sucking 5 μ l solutions of rhizoma Pinelliae and cortex Magnolia officinalis decoction, herba Perillae deficient negative control, rhizoma Zingiberis recens deficient negative control, Perillae herba control and Zingiberis rhizoma control, respectively dropping on the same silica gel G thin layer plate, developing with toluene-ethyl acetate-formic acid (6: 2: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet light (365nm) as shown in figure 22.

2. Durability test

2.1 investigation of different temperatures

As a result: the temperature has no obvious influence on the identification of the purple perilla and ginger thin layers in the semi-summer magnolia officinalis decoction, which shows that the thin layer identification method has good durability to different temperatures.

Sucking 5 μ l solutions of rhizoma Pinelliae and cortex Magnolia officinalis decoction, fructus Perillae deficiency negative control, rhizoma Zingiberis recens deficiency negative control, fructus Perillae control medicinal material, and Zingiberis rhizoma control medicinal material, respectively dropping on the same silica gel G thin layer plate, respectively spreading with toluene-ethyl acetate-formic acid (6: 2: 1) as developing agent at low temperature and normal temperature, taking out, air drying, and inspecting under ultraviolet light (365nm), as shown in FIG. 23.

2.2 investigation of different humidities

As a result: the humidity has no influence on the thin layer identification of the purple perilla and the ginger in the semi-summer magnolia officinalis decoction, and the thin layer identification method has good durability to different humidities.

Sucking 5 μ l solutions of rhizoma Pinelliae and cortex Magnolia officinalis decoction, fructus Perillae deficiency negative control, rhizoma Zingiberis recens deficiency negative control, fructus Perillae control medicinal material, and Zingiberis rhizoma control medicinal material, respectively dropping on the same silica gel G thin layer plate, respectively spreading with toluene-ethyl acetate-formic acid (6: 2: 1) as developing agent under different humidity conditions, taking out, air drying, and inspecting under ultraviolet light (365nm), as shown in FIG. 24.

2.3 investigation of thin-layer plates from different manufacturers

As a result: the thin-layer plates of different manufacturers have no influence on the thin-layer identification of the purple perilla and the ginger in the pinellia and magnolia officinalis decoction, which shows that the thin-layer identification method has good durability on the thin-layer plates of different manufacturers

Sucking 5 μ l of rhizoma Pinelliae and cortex Magnolia officinalis decoction sample, lacking Perillae herba negative control, lacking rhizoma Zingiberis recens negative control, Perillae herba control medicinal material, and Zingiberis rhizoma control medicinal material solution, respectively dropping on the same silica gel G thin layer plate, respectively spreading with toluene-ethyl acetate-formic acid (6: 2: 1) as developing agent under different manufacturer thin layer plate conditions, taking out, air drying, and inspecting under ultraviolet light (365nm) as shown in FIG. 25.

Claims (10)

1. A detection method for rapidly and comprehensively controlling the quality of a pinellia ternate and magnolia officinalis decoction standard decoction is characterized by comprising the following steps:

(1) preparing a test solution: extracting standard decoction of BANXIAHOUPU decoction with organic solvent, shaking, filtering, and collecting filtrate to obtain the sample solution;

(2) preparation of reference solutions: dissolving scutellarin, rosmarinic acid, magnolol and honokiol reference substance with solvent to obtain reference substance solution;

(3) sample introduction detection: detecting the sample solution and the reference solution by a liquid chromatograph under the following two chromatographic conditions (a) and (b);

(4) Calculating the content: calculating the content of rosmarinic acid, magnolol and honokiol by adopting the chromatogram obtained under the chromatographic condition in (a) in (3) through an external standard method, and calculating the content of scutellarin by adopting the chromatogram obtained under the chromatographic condition in (b) in (3) through the external standard method;

(5) establishing a characteristic spectrum: detecting a plurality of batches of the test sample solution of the standard decoction of the pinellia ternate and magnolia officinalis decoction, taking the chromatogram obtained by adopting the chromatographic condition in the step (3) and selecting chromatographic peaks existing in different batches of the standard decoction of the pinellia ternate and magnolia officinalis decoction as characteristic peaks to formulate the characteristic chromatogram.

2. The detection method for rapidly and comprehensively controlling the quality of the pinellia ternate and magnolia bark decoction standard decoction as claimed in claim 1, wherein the pinellia ternate and magnolia bark decoction standard decoction comprises water decoction or extract, extract or concentrate and freeze-dried powder of the pinellia ternate and magnolia bark decoction.

3. The detection method for rapidly and comprehensively controlling the quality of the standard decoction of pinellia ternate and magnolia officinalis decoction according to claim 1, wherein the two chromatographic conditions are specifically as follows:

(a) octadecylsilane chemically bonded silica chromatographic column, using methanol as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, and performing gradient elution (0-4 min, 2-7% A, 4-13 min, 7-17% A, 13-19 min, 17% A, 19-23 min, 17-23% A, 23-38 min, 23-34% A, 38-42 min, 34% A, 42-55 min, 34-45% A, 55-68 min and 45-90% A); (b) octadecylsilane chemically bonded silica chromatographic column, and acetonitrile-0.2% formic acid as mobile phase for isocratic elution.

4. The detection method for rapidly and comprehensively controlling the quality of the pinellia ternate magnolia officinalis decoction standard decoction according to claim 1, which is characterized by further comprising the following steps: (6) carrying out sample introduction detection in the step (3) on a to-be-detected sample solution, comparing a spectrum obtained by adopting the chromatographic condition in the step (3) with a characteristic spectrum, identifying the number of characteristic peaks and determining the similarity; meanwhile, scutellarin, rosmarinic acid, magnolol and honokiol are used as content index components to comprehensively evaluate the quality of standard decoction.

5. The detection method for rapidly and comprehensively controlling the quality of the standard decoction of pinellia ternate and magnolia officinalis decoction according to claim 1, wherein the organic solvent in the step (1) is a methanol aqueous solution with the volume concentration of 30-75%, the extraction time is 15-45 minutes, and the extraction mode comprises ultrasonic treatment, shaking extraction and heating reflux; the extraction volume is 15ml to 50 ml.

6. The method for rapidly and comprehensively controlling the quality of the standard decoction of pinellia ternate and magnolia officinalis decoction according to claim 1, wherein the liquid chromatograph in the step (3) is an ultra-high performance liquid chromatograph.

7. The detection method for rapidly and comprehensively controlling the quality of the standard decoction of pinellia ternate and magnolia officinalis decoction according to claim 1, wherein in the step (3), an ultraviolet detector is adopted in the step (a), the column temperature is 20-30 ℃, the flow rate is 0.25-0.4 ml per minute, and the detection wavelength is 280-330 nm; (b) an ultraviolet detector is adopted, the column temperature is 20-30 ℃, the flow rate is 0.25-0.4 ml per minute, and the detection wavelength is 280nm-330 nm.

8. The method as claimed in claim 1, wherein the characteristic spectrum in (5) has 12 characteristic peaks F1-F12, the relative retention time F2 is 1, the relative retention time F1 is 0.330-0.385, the relative retention time F9 is 1, and the relative retention time F3-F12 is 0.580-0.620, 0.640-0.652, 0.655-0.680, 0.700-0.730, 0.790-0.820, 0.830-0.865, 1.000, 1.370-1.420, 1.430-1.495, 1.470-1.530, respectively.

9. The detection method for rapidly and comprehensively controlling the quality of the pinellia ternate magnolia bark decoction standard decoction according to claim 1, wherein thin layer detection is performed after liquid chromatography detection.

10. The detection method for rapidly and comprehensively controlling the quality of the standard decoction of pinellia ternate and magnolia officinalis decoction according to claim 1, wherein the thin layer detection specifically comprises the following steps:

(1) preparation of thin-layer test solution: extracting standard decoction of BANXIAHOUPU decoction with organic solvent, shaking, filtering, evaporating, and dissolving in methanol to obtain test solution;

(2) preparation of thin-layer control solution: folium Perillae reference medicinal material solution, and rhizoma Zingiberis recens reference medicinal material solution; preparing a perilla leaf and ginger-deficient negative control group according to a pinellia tuber and magnolia bark decoction formula, and adding methanol to dissolve the perilla leaf and ginger-deficient negative control group to obtain a negative control solution;

(3) And (3) determination: sucking the sample solution, the negative control solution and the control solution, dropping on the same silica gel G thin layer plate, developing with toluene-ethyl acetate-formic acid as developing agent, taking out, air drying, and inspecting under ultraviolet light.

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