CN114674947B - Detection method for rapidly and comprehensively controlling quality of pinellia tuber magnolia bark Shang Biaozhun decoction - Google Patents

Abstract

The invention discloses a detection method for rapidly and comprehensively controlling quality of decoction of pinellia ternate and magnolia officinalis Shang Biaozhun, which is characterized in that a characteristic spectrum is established by a liquid chromatography method, and scutellarin, rosmarinic acid, magnolol and honokiol are used as content index components. The method has the advantages of simple operation, good reproducibility and stability, reliable recovery rate, low detection cost and high detection efficiency, and is expected to provide basis for standardization of the standard of pinellia ternate magnolia officinalis Shang Wuzhi, preparation quality and reasonable development.

Description

Detection method for rapidly and comprehensively controlling quality of pinellia tuber magnolia bark Shang Biaozhun 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 decoction of pinellia ternate and magnolia officinalis Shang Biaozhun.

Background

The pinellia tuber and magnolia bark decoction is listed in the ' Jinkui ' province of Zhang Zhongjing ' in 2018 in the first catalog of classical formula 17, is a clinical classical proved recipe for treating ' globus hystericus ', and is composed of pinellia tuber, magnolia bark, 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 tuber magnolia bark decoction has remarkable curative effects in the aspects of treating gastroesophageal reflux disease, chronic gastritis, functional dyspepsia, bronchial asthma, cough, chronic pharyngitis, antidepressant and the like.

The standard decoction is a material standard for measuring the quality consistency of the traditional Chinese medicine compound preparation and the clinical decoction, and is the core concept of the study of the classical prescription, namely the quality of the standard decoction is directly related to the quality of the traditional Chinese medicine compound preparation after the conversion. In the quality standard control surface, most of the characteristic medicinal herbs in the prior literature of pinellia tuber magnolia bark decoction are mainly concentrated in ministerial medicine magnolia bark, magnolol and honokiol are mainly used as quality control indexes, and detection and control of other medicinal herbs or a plurality of index components of a prescription are absent. In terms of characteristics or fingerprints, there are few studies on pinellia ternate magnolia officinalis Shang Tezheng or fingerprints.

The patent CN113341010A establishes a quality control method of the pinellia tuber magnolia bark decoction, which is respectively used for controlling the content and the characteristic spectrum method of the pinellia tuber magnolia bark decoction preparation, and the content measurement establishes a measurement method of 4 index components of magnolol, honokiol, rosmarinic acid and 6-gingerol, wherein the 6-gingerol component has no specificity and can be interfered by ginger processed products (ginger processed products), so that the quality of the ginger processed products and ginger in the compound can not be effectively controlled; 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 decoction of pinellia ternate magnolia officinalis Shang Biaozhun, and the detection method is used for measuring characteristic patterns of 4 content index components and 12 characteristic peaks, so that a simple, convenient, rapid and effective detection method is provided for quality control of standard and compound preparations of classical name pinellia ternate magnolia officinalis Shang Wuzhi.

The technical scheme is as follows: the invention discloses a detection method for rapidly and comprehensively controlling quality of pinellia tuber magnolia bark Shang Biaozhun decoction, which comprises the following steps:

(1) Preparation of test solution: extracting rhizoma Pinelliae and cortex Magnolia officinalis Shang Biaozhun decoction with organic solvent, shaking, filtering, and collecting filtrate to obtain the sample solution;

(2) Preparation of reference solution: dissolving scutellarin, rosmarinic acid, magnolol and honokiol reference substances in a solvent to obtain reference substance solution;

(3) And (3) sample injection 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 using the spectrum obtained under the chromatographic condition of (a) in (3) and calculating the content of scutellarin by using the spectrum obtained under the chromatographic condition of (b) in (3) through an external standard method;

(5) Establishing a characteristic map: detecting sample solutions of multiple batches of rhizoma Pinelliae and Magnolia officinalis Shang Biaozhun decoction, taking the spectrum obtained by adopting the chromatographic conditions of (a) in (3), selecting chromatographic peaks existing in different batches of rhizoma Pinelliae and Magnolia officinalis Shang Biaozhun decoction as characteristic peaks, and making the characteristic spectrum.

The method for detecting the quality of the pinellia tuber magnolia bark Shang Biaozhun decoction comprises the steps of decocting the pinellia tuber magnolia bark decoction in water or extracting solution, extractum or concentrated solution and freeze-dried powder.

The detection method for rapidly and comprehensively controlling the quality of the pinellia tuber magnolia bark Shang Biaozhun decoction comprises the following specific chromatographic conditions:

(a) Octadecylsilane chemically bonded silica chromatographic column, methanol as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, 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, 45-90% A); (b) Octadecylsilane chemically bonded silica column, eluting with acetonitrile-0.2% formic acid as flow equality.

The detection method for rapidly and comprehensively controlling the quality of the pinellia tuber magnolia bark Shang Biaozhun decoction further comprises the following steps: (6) Sample injection detection in the step (3) is carried out on the sample solution to be detected, the spectrum obtained by the chromatographic condition in the step (3) is compared with the characteristic spectrum, the number of characteristic peaks is identified, and the similarity is determined; meanwhile, scutellarin, rosmarinic acid, magnolol and honokiol are taken as content index components to comprehensively evaluate the quality of the standard decoction.

The method for detecting the quality of the decoction of the pinellia ternate and magnolia officinalis Shang Biaozhun comprises the steps of (1) using 30-75% methanol aqueous solution as an organic solvent, extracting for 15-45 minutes, and performing ultrasonic treatment, shaking extraction and heating reflux; the extraction volume is 15 ml-50 ml.

The method for detecting the quality of the decoction of pinellia tuber magnolia officinalis Shang Biaozhun is characterized in that the liquid chromatograph in the step (3) is an ultra-high performance liquid chromatograph.

In the detection method for rapidly and comprehensively controlling the quality of the pinellia tuber magnolia bark Shang Biaozhun decoction, an ultraviolet detector is adopted in (a) in (3), 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) The 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.

According to the detection method for rapidly and comprehensively controlling the quality of the decoction of the pinellia ternate magnolia officinalis Shang Biaozhun, the characteristic map in (5) is provided with 12 characteristic peaks F1-F12, the relative retention time F2 is 1, the relative retention time of F1 is 0.330-0.385, the relative retention time F9 is 1, and the relative retention time of 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 tuber magnolia officinalis Shang Biaozhun decoction is characterized in that the thin layer detection is performed after the liquid chromatography detection.

The detection method for rapidly and comprehensively controlling the quality of the pinellia tuber magnolia officinalis Shang Biaozhun decoction comprises the following steps of:

(1) Preparation of a thin layer of sample solution: extracting rhizoma Pinelliae and cortex Magnolia officinalis Shang Biaozhun decoction with organic solvent, shaking, filtering, evaporating to dryness, and dissolving with methanol to obtain the sample solution;

(2) Preparation of a thin layer control solution: perilla leaf control medicinal material solution, rhizoma Zingiberis recens control medicinal material solution; preparing a negative control group with purple perilla leaves and ginger, adding methanol for dissolution, and taking the solution as a negative control solution according to a pinellia tuber and magnolia bark decoction formula;

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

The standard decoction of the pinellia tuber magnolia bark decoction comprises water decoction (extract), 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 officinal magnolia bark, 12g of poria cocos, 6g of perilla leaf and 15g of ginger, respectively placing in each decocting pot, adding 1200-1600 mL of water, soaking for 30-40min, boiling with strong fire, keeping boiling with slow fire until the decoction is 600-800 mL, stopping heating, and filtering with a 200-350 mesh standard sieve to obtain an extract. Concentrating the extractive solution under reduced pressure (55-75deg.C) to specific gravity of 1.01-1.05 to obtain concentrated solution. Freeze drying the concentrated solution to obtain the dried powder of pinellia tuber magnolia bark Shang Dong.

The beneficial effects are that: (1) According to the invention, an ultra-high liquid chromatography is adopted, chromatographic conditions are reasonably optimized, a characteristic spectrum of 12 characteristic peaks of the freeze-dried powder of the pinellia tuber magnolia bark decoction is established, the content of index components is detected, a novel analysis means is provided for quality control of a standard of the pinellia tuber magnolia bark Shang Wuzhi, and the method is an effective means for overall quality control of the pinellia tuber magnolia bark Shang Biaozhun decoction. (2) The method takes scutellarin, rosmarinic acid, magnolol and honokiol as content index components, establishes a characteristic map and performs thin-layer identification, and is suitable for the standard of the classical formula pinellia tuber magnolia Shang Wuzhi prepared by water decoction or extraction. (3) The method has the advantages of simple operation, good reproducibility and stability, reliable recovery rate, low detection cost and high detection efficiency, and is expected to provide basis for standardization of the standard of pinellia ternate magnolia officinalis Shang Wuzhi, the market of preparations and reasonable development. (4) Compared with the invention of the patent CN113341010A, the detection of the newly added scutellarin content index has important significance on the half Xia Houpiao Shang Wuzhi standard 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 patterns are met. The established characteristic spectrum measuring method can measure 12 characteristics of 4 medicinal herbs, can not only characterize the components of the monarch drug ginger processed pinellia tuber, the ministerial drug magnolia officinalis, the assistant and guide drug perilla leaf and ginger, but also can measure 3 specific components (magnolol, honokiol and rosmarinic acid) in the magnolia officinalis and the perilla leaf at the same time.

Drawings

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

FIG. 2 is a diagram showing the detection of a scutellarin content measurement control;

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

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

FIG. 5 is a chromatogram of various extraction modes for determining rosmarinic acid content, total amount of magnolol and honokiol;

FIG. 6 is a chromatogram of different extraction modes for scutellarin content determination;

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

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

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

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

FIG. 11 is a reference characteristic chromatogram of 15 batches of pinellia ternate magnolia officinalis Shang Wuzhi;

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

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

FIG. 14 is a specific investigation chromatogram of scutellarin content determination;

FIG. 15 is a chromatogram of various flow rate determinations for rosmarinic acid content, magnolol and honokiol;

FIG. 16 is a chromatogram of different flow rate surveys of scutellarin content determination;

FIG. 17 is a chromatogram of different chromatographic column temperature measurements for rosmarinic acid content, total amount of magnolol and honokiol;

FIG. 18 is a chromatogram of different chromatographic column temperature investigation for scutellarin content determination;

FIG. 19 is a chromatogram of various chromatographic column assays for rosmarinic acid content, magnolol and total honokiol;

FIG. 20 shows thin layer identification of different preparation methods of Perillae herba and rhizoma Zingiberis recens in rhizoma Pinelliae and cortex Magnolia officinalis decoction; wherein 1-3-pinellia tuber magnolia bark decoction (JDG-BXHP-20210525-F1-3), S1-S3-perilla leaf reference medicine and S4-S6-dried ginger reference medicine;

FIG. 21 shows thin layer identification of different sample application amounts of Perillae herba and rhizoma Zingiberis recens in rhizoma Pinelliae and Magnolia officinalis decoction, wherein 1-rhizoma Pinelliae and Magnolia officinalis decoction (JDG-BXHP-20210525-F1-3): 1 (1. Mu.l), 2 (3. Mu.l), 3 (5. Mu.l), 4 (8. Mu.l) 5; 4-purple perilla-lack negative control, 6-purple ginger-lack negative control, S1-purple perilla control medicinal material and S2-dried ginger control medicinal material;

FIG. 22 shows the specific thin layer identification of Perillae herba and rhizoma Zingiberis recens in rhizoma Pinelliae and Magnolia officinalis decoction, wherein 1-rhizoma Pinelliae and Magnolia officinalis decoction (F1), 2-rhizoma Pinelliae and Magnolia officinalis decoction (F2), 3-rhizoma Pinelliae and Magnolia officinalis decoction (F3), 4-Perilla herba lack negative control, 5-rhizoma Zingiberis recens lack negative control, S1-Perilla herba control, and S2-rhizoma Zingiberis control;

FIG. 23 shows different temperature thin layer discrimination of Perillae herba and rhizoma Zingiberis recens in rhizoma Pinelliae and Magnolia officinalis decoction, wherein 1-rhizoma Pinelliae and Magnolia officinalis decoction (F1) 2-rhizoma Pinelliae and Magnolia officinalis decoction (F2), 3-rhizoma Pinelliae and Magnolia officinalis decoction (F3), 4-Perilla herba lack negative control, 5-rhizoma Zingiberis recens lack negative control, S1-Perilla herba control, and S2-Zingiberis rhizoma control;

FIG. 24 shows the thin-layer discrimination of different humidity levels of Perillae herba and rhizoma Zingiberis recens in rhizoma Pinelliae and Magnolia officinalis decoction, wherein 1-rhizoma Pinelliae and Magnolia officinalis decoction (F1) 2-rhizoma Pinelliae and Magnolia officinalis decoction (F2), 3-rhizoma Pinelliae and Magnolia officinalis decoction (F3), 4-Perilla herba lack negative control, 5-rhizoma Zingiberis recens lack negative control, S1-Perilla herba control, and S2-rhizoma Zingiberis control;

FIG. 25 shows identification of thin-layer plates of different manufacturers of Perillae herba and rhizoma Zingiberis recens in rhizoma Pinelliae and Magnolia officinalis decoction, wherein 1-rhizoma Pinelliae and Magnolia officinalis decoction (F1), 2-rhizoma Pinelliae and Magnolia officinalis decoction (F2), 3-rhizoma Pinelliae and Magnolia officinalis decoction (F3), 4-Perilla herba lack negative control, 5-Perilla herba lack negative control, S1-Perilla herba control, and S2-Zingiberis rhizoma control.

Detailed Description

Example 1

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

1. Reagents and samples

Scutellarin (lot number 110842-201609) is purchased from Chinese food and drug institute for content measurement, and the content is 91.7%, and no treatment is needed before use.

Rosmarinic acid (lot 111871-201505) was purchased from the institute of food and drug testing in China for content determination, and the content was 98.5% based on the total content, and no treatment was required before use.

Magnolol (lot 110729-201714) is purchased from China food and drug institute for content measurement, and the content is 100.0% and no treatment is needed before use.

Honokiol (lot number 110730-201614) is purchased from Chinese food and drug institute for content measurement, and the content is 99.3%, and no treatment is needed before use.

Caffeic acid (lot number 110885-200102) is purchased from Chinese food and drug institute for content measurement, and the content is 99.7%, and no treatment is needed before use.

6-gingerol (lot No. 111833-202007) is purchased from China food and drug institute for content measurement, the content is 99.3%, and the treatment is not needed before use.

The medicinal materials, decoction pieces and pinellia tuber magnolia bark decoction freeze-dried powder are provided by Tianjiang pharmaceutical industry limited company in Jiangyin city.

2. Preparation of control solution

And (3) taking a proper amount of scutellarin reference substance, precisely weighing, adding 50% methanol to prepare a solution containing 10 mug of scutellarin per 1ml, and obtaining a scutellarin reference substance solution. Taking appropriate amounts of rosmarinic acid reference substance, magnolol reference substance and honokiol reference substance, precisely weighing, and adding 50% methanol to obtain mixed reference substance solution containing 22 μg rosmarinic acid, 10 μg magnolol and 10 μg scutellarin per 1 ml. The scutellarin reference substance is taken, precisely weighed, and 50% methanol is added to prepare a reference substance solution with 9 mug per 1 ml. In addition, the caffeic acid reference substance and 6-gingerol reference substance are weighed in proper amounts, and 50% methanol is added to prepare 10 mug caffeic acid and 10 mug 6-gingerol reference substance solution.

3. Preparation of test solutions

About 0.2g of freeze-dried powder of the product is taken, precisely weighed, placed in a conical bottle with a plug, added with 25ml of 50% methanol, sealed, weighed, subjected to ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, cooled, weighed again, subjected to shaking uniformly, filtered, and taken to obtain the subsequent filtrate.

4. Determination of analytical methods

4.1 characterization and content determination chromatographic conditions

Octadecylsilane chemically bonded silica is used as a filler (column length is 100mm, inner diameter is 2.1mm, 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), flow rate is 0.3 ml.min-1, column temperature is 25 ℃, sample injection amount is 2 mu l, and detection wavelength is 280nm.

Mixing control solution containing scutellarin, rosmarinic acid, magnolol, and magnolol, caffeic acid, and 6-gingerol, and detecting with the graph shown in figure 1. The result shows that the detection baseline is stable, and the separation degree of the target components is good.

4.2 chromatographic conditions for determining the content of scutellarin

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

And (2) carrying out sample injection detection on the scutellarin reference substance solution, wherein the spectrum is shown in figure 2. The result shows that the detection baseline is stable, the separation degree of the target component is good, and the detection time can be completed within 15 minutes.

4.3 preparation of sample solutions

(1) Investigation of the extraction solvent

Conclusion: when methanol, 75% methanol, 50% methanol and 10% methanol are used as extraction solvents, the contents of scutellarin and rosmarinic acid in the methanol are low, the contents of magnolol and honokiol in the 10% methanol are low, and the difference between the contents of 50% methanol and 75% methanol is not large; considering that rosmarinic acid is a water-soluble component, magnolol and honokiol are fat-soluble components, and considering the solubility of each component, 50% methanol is selected as an extraction solvent for measuring the content of baicalin, rosmarinic acid, magnolol and honokiol in pinellia ternate magnolia Shang Ye.

The investigation method comprises the following steps: taking about 0.2g of the powder, 4 groups of the powder in parallel, precisely weighing 2 parts of each group, placing the powder into a conical bottle with a plug, precisely adding 10% methanol, 50% methanol, 75% methanol and 25ml of each of the methanol, sealing, weighing the powder, performing ultrasonic treatment (power is 250W and frequency is 40 kHz) for 30 minutes, cooling, weighing the powder, supplementing the weight loss with corresponding solvent, shaking the powder uniformly, filtering the mixture, and taking subsequent filtrate. 2 μl of each test solution is precisely sucked and injected into a liquid chromatograph, the sample is injected according to the chromatographic condition under the item "4.1", the rosmarinic acid content, the magnolol and the total honokiol content are calculated, the results are shown in Table 1 and FIG. 3, the sample is injected according to the chromatographic condition under the item "4.2", and the scutellarin content is calculated, and the results are shown in Table 2 and FIG. 4.

TABLE 1 comparison of the content of rosmarinic acid, the total amount of magnolol and honokiol with different extraction solvents

Table 2 comparison of different extraction solvents for scutellarin content determination

(2) Investigation of the extraction method

Conclusion: when the ultrasonic treatment, the heating reflux and the shaking extraction are adopted, the contents of scutellarin, rosmarinic acid, magnolol and honokiol are not obviously different, which indicates that the extraction efficiencies are similar in different times, and the ultrasonic treatment is selected in consideration of simple and convenient operation.

The investigation method comprises the following steps: taking about 0.2g of the powder, 3 groups of powder, each group is 2 parts in parallel, precisely weighing, placing the powder into a conical flask with a plug, precisely adding 25ml of 50% methanol, sealing, weighing, respectively performing ultrasonic treatment (power 250W and frequency 40 kHz), heating, refluxing, shaking and extracting for 30 minutes, cooling, weighing again, supplementing the weight loss with 50% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product. 2 μl of each test solution is precisely sucked up and injected into a liquid chromatograph, the sample is injected according to the chromatographic condition under the item "4.1", the rosmarinic acid content, the magnolol and the total honokiol content are calculated, the results are shown in Table 3 and FIG. 5, the sample is injected according to the chromatographic condition under the item "4.2", and the scutellarin content is calculated, and the results are shown in Table 4 and FIG. 6.

TABLE 3 comparison of the way in which the total amount of rosmarinic acid, magnolol and honokiol were measured and extracted

Table 4 comparison of extraction modes for scutellarin content determination

(3) Investigation of the extraction solvent volume

Conclusion: when the extraction volume is 15ml, 25ml and 50ml, the contents of scutellarin, rosmarinic acid, magnolol and honokiol are not quite different, which indicates that the extraction is sufficient when the extraction solvent is 15 ml. In view of the differences between the samples of different batches, 25ml of solvent was chosen for the addition to ensure adequate extraction.

The investigation method comprises the following steps: taking about 0.2g of the powder, 3 groups of powder, each group is 2 parts in parallel, precisely weighing, placing the powder into a conical flask with a plug, precisely adding 15ml, 25ml and 50ml of 50% methanol, sealing, weighing, performing ultrasonic treatment (power is 250W and frequency is 40 kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with 50% methanol, shaking uniformly, filtering, and taking the subsequent filtrate. 2 μl of each test solution is precisely sucked up and injected into a liquid chromatograph, the sample is injected according to the chromatographic condition under the item "4.1", the rosmarinic acid content, the magnolol and the total honokiol content are calculated, the results are shown in Table 5 and FIG. 7, the sample is injected according to the chromatographic condition under the item "4.2", and the scutellarin content is calculated, the results are shown in Table 6 and FIG. 8.

TABLE 5 comparison of the total measured extraction volumes of rosmarinic acid content, magnolol and honokiol

Table 6 comparison of extraction volumes for scutellarin content determination

(4) Investigation of extraction time

Conclusion: different ultrasonic treatment times show that the scutellarin, the rosmarinic acid content, the magnolol and the total honokiol content are not quite different, and the extraction time is 15 minutes, so that the extraction is more complete. Taking into account the differences between different batches of samples, the extraction time took 30 minutes to ensure adequate extraction.

The investigation method comprises the following steps: taking about 0.2g of the powder, 3 groups of powder, each group is 2 parts in parallel, precisely weighing, placing the powder into a conical flask with a plug, precisely adding 25ml of 50% methanol, sealing the mixture, weighing, respectively carrying out ultrasonic treatment (with the power of 250W and the frequency of 40 kHz) for 15 minutes, 30 minutes and 45 minutes, cooling, weighing again, supplementing the weight loss with 50% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product. 2 μl of each test solution is precisely sucked up and injected into a liquid chromatograph, the sample is injected according to the chromatographic condition under the item "4.1", the rosmarinic acid content, the magnolol and the total honokiol content are calculated, the results are shown in Table 7 and FIG. 9, the sample is injected according to the chromatographic condition under the item "4.2", and the scutellarin content is calculated, the results are shown in Table 8 and FIG. 10.

TABLE 7 comparison of the rosemaric acid content, the total amount of magnolol and honokiol determination extraction time

Table 8 comparison of extraction time for scutellarin content determination

The preparation method of the final determined test sample solution comprises the following steps:

taking about 0.2g of the powder, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of 50% methanol, sealing, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking, filtering, and collecting the subsequent filtrate.

(5) Detection of different batches

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

table 9 results of total amount content of different batches of pinellia ternate magnolia Shang Wuzhi reference scutellarin, rosmarinic acid, magnolol and honokiol

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TABLE 10 different batches of pinellia ternate and Magnolia officinalis Shang Wuzhi reference characteristic peak area data

TABLE 11 relative peak area data for different batches of pinellia ternate and Magnolia officinalis Shang Wuzhi reference characteristics

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Table 12 different batches of pinellia ternate magnolia officinalis Shang Wuzhi reference characteristic retention time data

TABLE 13 relative retention time data for different batches of pinellia ternate and Magnolia officinalis Shang Wuzhi reference characteristics

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Example 2

Methodological verification of content measurement construction method

1. Linear relationship

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

Precisely sucking rosmarinic acid reference solution (with the concentration of 21.6109 mug/ml), magnolol reference solution (with the concentration of 9.78 mug/ml), and honokiol reference solution (with the concentration of 9.8704 mug/ml) 0.2, 0.5, 1.0, 1.5, 2, 3, 4, 5 and 6 mul, injecting into a liquid chromatograph, injecting sample according to chromatographic conditions under the item "4.1", taking the peak area as the ordinate, taking the injection amount as the abscissa, drawing a standard curve, and obtaining a regression equation: rosmarinic acid y= -5669.0013x+61960.758, r=0.99999, magnolol y= -1615.9369x+4197770.926, r= 0.99998, and magnolol y= -1504.4128x+4429822.829, r= 0.99998, the results are shown in table 14, fig. 12. Accurately sucking scutellarin reference solution (concentration of 18.5784 μg/ml) 0.1, 0.2, 0.5, 1, 2, and 3 μl, injecting into liquid chromatograph, sampling under chromatographic condition under item "4.2", taking peak area as ordinate, and sampling amount as abscissa, and drawing standard curve. And (3) solving a regression equation: scutellarin y=143.32x+0.0246, r= 0.99998, the results are shown in table 15, fig. 12.

TABLE 14 peak area and sample injection amount relationship of rosmarinic acid, magnolol and honokiol control

Table 15 relation between peak area and sample injection amount of scutellarin reference substance

2. Precision test

2.1 Instrument precision test

Results: the peak area RSD is less than 2.0%, and the instrument precision test is good.

2 mu l of a standard sample solution of the accurate pinellia ternate magnolia officinalis Shang Wuzhi is injected into a liquid chromatograph, sample injection is continuously performed for 6 times according to the chromatographic condition under the item "4.1", measured values of peak areas of rosmarinic acid, magnolol and honokiol are recorded, sample injection is continuously performed for 6 times according to the chromatographic condition under the item "4.2", measured values of peak areas of scutellarin are recorded, and relative standard deviation is calculated, and the result is shown in Table 16.

Table 16 instrument precision test

2.2 repeatability test

Results: the scutellarin, the rosmarinic acid content, the magnolol and the total honokiol content RSD are less than 2.0 percent, and the repeatability test is good.

Taking about 0.2g of samples of the same batch, precisely weighing, parallel weighing 6 parts, preparing samples according to the preparation method of the sample solution under the item "4.3", carrying out sample injection analysis according to the chromatographic condition under the item "4.1", measuring the peak areas of rosmarinic acid, magnolol and honokiol, calculating the total content of rosmarinic acid, magnolol and honokiol and RSD values, carrying out sample injection analysis according to the chromatographic condition under the item "4.2", measuring the peak areas of scutellarin, and calculating the peak areas of scutellarin and RSD values, wherein the results are shown in Table 18.

TABLE 17 repeatability test of determination of rosmarinic acid content, magnolol and total amount of honokiol

Repeatability test of Table 18 scutellarin content determination

2.3 intermediate precision

Results: the scutellarin, the content of the superimposed aromatic acid, the magnolol and the total content RSD of the honokiol are less than 2.0 percent, and the intermediate precision test is good.

Taking 3 parts of samples of the same batch, preparing samples by different experimenters according to the preparation method of the sample solution under the item "4.3", respectively injecting 2 μl of the sample on the same instrument at different times according to the chromatographic conditions under the item "4.1", measuring the area values of rosmarinic acid, magnolol and honokiol, calculating the content of rosmarinic acid, the total content of magnolol and honokiol and RSD, and the results are shown in Table 19. 2 μl of each sample was introduced into each instrument at different times under the chromatographic conditions under item "4.2", the scutellarin area value was measured, and the scutellarin content and RSD were calculated, and the results are shown in table 20.

TABLE 19 intermediate precision test for determination of rosmarinic acid content, magnolol and total amount of honokiol

Intermediate precision test for measuring content of scutellarin in surface 20

3. Accuracy test

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

0.1g of samples with known contents (0.29% of rosmarinic acid, 0.10% of magnolol and 0.12% of honokiol) are taken, 9 parts are parallel, the samples are precisely weighed, mixed reference substance solutions corresponding to the rosmarinic acid, the magnolol and the honokiol contained in 0.1g of samples are respectively added, the sample-adding recovery sample solution is prepared according to the preparation method of the sample solution under the item of 4.3, the sample-adding recovery sample solution is sampled according to the chromatographic condition under the item of 4.1, 2 μl of sample is respectively sampled, and the recovery rates and RSD of rosmarinic acid, magnolol and honokiol are calculated according to the following formulas, and the results are shown in tables 21-23. 0.1g of a sample with a known content (the scutellarin content is 0.11%) is taken, 9 parts are weighed in parallel, the scutellarin reference substance solution contained in 0.1g of the sample is respectively added, the sample is prepared according to the preparation method of the sample solution under the item of 4.3, the sample is recycled, the sample is injected according to the chromatographic condition under the item of 4.2, 2 μl of the sample is respectively injected, and the scutellarin recovery rate and RSD are calculated according to the following formula, and the result is shown in Table 24.

Table 21 rosmarinic acid accuracy test

TABLE 22 magnolol accuracy test

TABLE 23 honokiol accuracy test

Accuracy test of Table 24 scutellarin

4. Specificity test

Conclusion: from the results, the blank solvent has no interference to the measurement of scutellarin, rosmarinic acid, magnolol and honokiol, and the method has strong specificity.

Taking blank solvent, reference substance solution, rhizoma Pinelliae Magnolia officinalis Shang Wuzhi reference sample solution, and performing sample injection under chromatographic conditions under item "4.1", and the result is shown in figure 13. Sample injection was performed under the chromatographic conditions under item "4.2", and the results are shown in FIG. 14.

5. Durability test

5.1 stability test

Results: the test solution has good stability (RSD% < 2.0%) within 48 hours.

Taking 1 part of sample, preparing a test solution according to the preparation method of the test solution under the item "4.3", sampling the sample under the chromatographic conditions under the item "4.1" for 0, 2, 4, 6, 8, 12, 18, 24 and 48 hours, measuring the peak area value, and calculating the RSD value. The results are shown in Table 25. Sample is injected under the chromatographic condition of '4.2' item in 0, 2, 4, 6, 8, 12, 18, 24 and 48 hours, 2 μl is injected, peak area value is measured, and RSD value is calculated. The results are shown in Table 26.

TABLE 25 determination of the content of rosmarinic acid, the total amount of magnolol and honokiol test sample stability test determination results

Table 26 determination of scutellarin content test sample stability test

5.2 different flow Rate investigation

Results: the flow rate is 0.25 to 0.35 ml.min ^-1 The measurement results were not different, and the durability was good.

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

Table 27 determination of rosmarinic acid content, total amount of magnolol and honokiol different flow rates were examined

Table 28 determination of scutellarin content different flow rates for investigation

5.3 column temperature investigation

Results: the column temperature is between 20 ℃ and 30 ℃, the measurement results have no obvious difference, and the durability is good.

1 part of sample is taken, and the sample solution is prepared according to the preparation method of the sample solution under the item "4.3", and three temperatures of 20 ℃, 25 ℃ and 30 ℃ are examined, and the results are shown in Table 29, table 30, FIG. 17 and FIG. 18.

Table 29 investigation of the total amount of rosmarinic acid, magnolol and honokiol for measuring different column temperatures

Investigation of Table 30 scutellarin content determination different column temperatures

5.4 column chromatography investigation

Results: the separation effect of the chromatographic columns of three different types is good, the retention time is moderate, and the influence of the chromatographic columns on the measurement result of the sample is small.

Taking 1 part of sample, preparing a test solution according to the preparation method of the test solution under the item "4.3", and respectively adopting a column 1: HSS T3 (2.1X100 mm,1.8 μm), column 2: eclipse Plus RRHD C18 (2.1X100 mm,1.8 μm), column 3: three columns of Poroshell C18 (2.1X100 mm,1.9 μm) were subjected to sample injection under the chromatographic conditions under the term "4.1" to analyze the contents of rosmarinic acid, magnolol, and honokiol, and the analysis results are shown in Table 31 and FIG. 19. Another Acclaim is taken ^TM RSLCPolarAdvantage II (PA 2) the scutellarin content was analyzed by chromatography under the conditions of "4.2", and the analysis results are shown in Table 32.

TABLE 31 comparison of the content of rosmarinic acid, the total amount of magnolol and honokiol with different chromatographic columns

Comparison of different chromatographic columns for measuring the content of scutellarin in Table 32

Example 3

Methodological verification of feature construction methods

1. Precision test

1.1 Instrument precision test

Results: the relative peak area of the common peak and the relative retention time RSD are less than 5.0%, and the precision test of the instrument is good.

2 mu l of a standard sample solution of the accurate pinellia ternate magnolia officinalis Shang Wuzhi is injected into a liquid chromatograph, sample injection is carried out according to the chromatographic condition under the item "4.1", continuous sample injection is carried out for 6 times, measurement values of 12 common peak-peak areas and retention time are recorded, the relative standard deviation is calculated, and the results are shown in tables 33-36.

Table 33 results of Peak area for precision test

Table 34 results of precision test relative peak area

Table 35 results of precision test retention time

Table 36 results of precision test relative retention time

1.2 repeatability test

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

Taking about 0.2g of samples of the same batch, precisely weighing, preparing 6 parts in parallel, preparing the samples according to the preparation method of the sample solution under the item "4.3", analyzing by sample injection according to the chromatographic condition under the item "4.1", measuring the peak areas and retention times of 12 common peaks, calculating the RSD values of the relative peak areas and the relative retention times, and the results are shown in tables 37-40.

Table 37 results of the repeatability test peak areas

Table 38 relative peak area results of repeatability test

Table 39 repeatability test retention time results

Table 40 relative retention time results of repeatability test

1.3 intermediate precision

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

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

Table 41 results of peak area for intermediate precision test

Table 42 results of intermediate precision test relative peak area

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Table 43 intermediate precision test retention time results

Table 44 results of intermediate precision test relative retention time

2. Specificity test

Conclusion: from the results, the blank solvent has no interference to the measurement of the characteristics, and the method has strong specificity.

Taking blank solvent, negative control solution, rhizoma Pinelliae Magnolia officinalis Shang Wuzhi reference sample solution, and performing sample injection under chromatographic conditions under item "4.1", and the result is shown in figure 13.

3. Stability test

Results: the test solution has good stability (RSD% < 5.0%) within 48 hours.

Taking 1 part of a sample, preparing a test solution according to the preparation method of the test solution under the item "4.3", sampling 2 μl under the item "4.1" at 0, 2, 4, 6, 8, 12, 18, 24 and 48 hours, measuring the peak areas and retention times of 12 common peaks, calculating RSD values of the relative peak areas and the relative retention times, and obtaining the results 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 measurement results of Standard decoction

1. Preparation of Standard decoction

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

2. Preparation of test solutions

Preparing an extracting solution sample solution: precisely sucking rhizoma Pinelliae and cortex Magnolia officinalis Shang Diqu liquid 15ml to 25ml volumetric flask, adding methanol, performing ultrasonic treatment, fixing volume to scale mark, shaking, and collecting filtrate.

Preparing a concentrated solution sample solution: accurately weighing rhizoma Pinelliae and cortex Magnolia officinalis Shang Nongsu liquid 3 g-20 ml in a volumetric flask, adding 50% methanol, performing ultrasonic treatment, fixing volume to scale mark with 50% methanol, shaking, and collecting filtrate.

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

3. Preparation of reference solutions

Adding methanol into appropriate amount of scutellarin, rosmarinic acid, magnolol, and honokiol reference substance to obtain mixed reference substance solution containing scutellarin 40 μg, rosmarinic acid 40 μg, magnolol 60 μg, and magnolol 20 μg per 1 ml.

4. Content determination

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

TABLE 49 results of extract, concentrate and lyophilized powder contents

Example 5

Construction of thin-layer identification and measurement method for pinellia tuber magnolia bark Shang Biaozhun decoction

1. Instrument, reagent and sample

A thin layer automated imager (CAMAG TLC VISUALIZER); ultrasonic cleaners (KQ-250E, kunshan ultrasonic instruments Co., ltd.); a thousandth balance (AR 223CN, ohus); silica gel G thin layer plate (national pharmaceutical group chemical reagent limited); GKC Water bath (Nantong Huatai laboratory instruments Co., ltd.); HY-4 speed regulating multipurpose oscillator (Jintan Kexing instruments and factories).

Toluene (Shanghai Lingfeng chemical Co., ltd.), sulfuric acid (Shanghai Lingfeng chemical Co., ltd.), ethyl acetate (Guogong chemical Co., ltd.), formic acid (Guogong chemical Co., ltd.), methanol (Guogong chemical Co., ltd.) are all analytically pure, water.

The folium Perillae control medicinal material (120914-201712) is purchased from Chinese food and drug verification institute.

The rhizoma Zingiberis control drug (120942-201911) was purchased from China food and drug inspection institute.

The pinellia tuber magnolia bark decoction freeze-dried powder and the negative control freeze-dried powder are provided by Tianjiang pharmaceutical industry Co.

2. Preparation of control solution

Taking 1g of perilla leaf control medicinal material, adding 20ml of water for dissolution, filtering, extracting the filtrate with ethyl acetate twice, each time 20ml of the filtrate, combining ethyl acetate solutions, evaporating to dryness, and adding 0.5ml of methanol for dissolution to obtain a perilla control medicinal material solution.

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

Taking 1g of each of the negative control of the deficiency of perilla leaf and the deficiency of ginger, adding 20ml of methanol, carrying out ultrasonic treatment (power 250W,40 kHz) for 30 minutes, filtering, evaporating to dryness, and adding 1ml of methanol for dissolution to obtain a negative control solution.

3. Preparation of test solutions

Taking 1g of dried powder of pinellia ternate and magnolia officinalis Shang Dong, adding 20ml of methanol, performing ultrasonic treatment (power 250W,40 kHz) for 30 minutes, filtering, evaporating to dryness, and adding 1ml of methanol for dissolution to obtain a sample solution.

4. Determination of analytical methods

4.1 determination of thin layer authentication

Sucking the sample solution, negative control solution and control medicinal solution, spotting 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 leaves and the ginger has specificity.

4.2 preparation of sample solutions

(1) Investigation of the preparation method

Conclusion: in the chromatograms of different preparation methods of the test sample of the pinellia tuber magnolia bark decoction and the dried ginger reference medicinal material, each spot is clear and has little difference. The ultrasonic and heating reflux chromatographic spots in the perilla leaf control medicinal material are consistent, the difference between the ultrasonic and heating reflux chromatographic spots and the solution of the test sample is larger, and the extracted control solution is consistent with the chromatographic spots of the test sample. Therefore, ultrasonic treatment is selected as a test sample of the pinellia tuber magnolia bark decoction, a dried ginger control medicinal material and a negative control preparation method, and ethyl acetate extraction is selected as a perilla control medicinal material preparation method.

The investigation method comprises the following steps: 1g of freeze-dried powder of pinellia tuber and magnolia bark decoction is taken, 2 parts of freeze-dried powder are added in parallel, 20ml of methanol is added, ultrasonic treatment (power 250W,40 kHz) is carried out, heating reflux is carried out for 30 minutes, filtration and evaporation are carried out, and 1ml of methanol is added for dissolution to be used as a test solution. Dissolving 1g of dried powder of pinellia ternate and magnolia officinalis Shang Dong in 20ml of water, extracting twice with 20ml of ethyl acetate each time, combining ethyl acetate solutions, evaporating to dryness, and dissolving with 1ml of methanol to obtain a sample solution. Preparing control medicinal material solutions of folium Perillae control medicinal material and rhizoma Zingiberis recens control medicinal material 1g each in parallel with 3 parts. The sample solution, the negative control solution and the control medicinal solution are sucked and spotted on the same silica gel G thin layer plate, and are spread by using toluene-ethyl acetate-formic acid (6:2:1) as a spreading agent, taken out, dried and inspected under ultraviolet light (365 nm), as shown in figure 20.

(2) Investigation of different spotting volumes

Conclusion: when the sample application amount of the pinellia tuber magnolia bark decoction is 3-5 mu l, the chromatographic spots are clear and no other interference exists, so the sample application amount of the pinellia tuber magnolia bark decoction is 5 mu l.

The investigation method comprises the following steps: taking 1g of dried powder of pinellia ternate and magnolia officinalis Shang Dong, adding 20ml of methanol, carrying out ultrasonic treatment (power 250W,40 kHz), heating and refluxing for 30 minutes, filtering, evaporating to dryness, and adding 1ml of methanol for dissolution to obtain a sample solution. Taking 1g of each of the purple perilla leaf-lacking, ginger-lacking and dried ginger reference medicinal materials, and preparing a negative reference and dried ginger reference medicinal material solution by the same method. Dissolving folium Perillae control 1g in water 20ml, extracting with ethyl acetate twice, each time 20ml, mixing ethyl acetate solutions, evaporating to dryness, and dissolving with methanol 1ml to obtain folium Perillae control solution. The sample solution, the negative control solution and the control medicinal solution are sucked and spotted on the same silica gel G thin layer plate, and are spread by using toluene-ethyl acetate-formic acid (6:2:1) as a spreading agent, taken out, dried and inspected under ultraviolet light (365 nm), as shown in figure 21.

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

collecting rhizoma Pinelliae Magnolia officinalis decoction lyophilized powder, perilla frutescens negative control, rhizoma Zingiberis recens negative control 1g, adding methanol 20ml, ultrasonic treating (power 250W,40 kHz) for 30 min, filtering, evaporating to dryness, and adding methanol 1ml for dissolving to obtain test solution. Taking 1g of dried ginger reference medicinal material, and preparing 1ml of dried ginger reference medicinal material solution by the same method. Taking 1g of perilla leaf control medicinal material, adding 20ml of water for dissolution, filtering, extracting the filtrate with ethyl acetate twice, each time 20ml of the filtrate, combining ethyl acetate solutions, evaporating to dryness, and adding 0.5ml of methanol for dissolution to obtain a perilla control medicinal material solution. According to thin layer chromatography (rule 0502 of Chinese pharmacopoeia 2020 edition), sucking 5 μl of each solution, respectively spotting on the same silica gel G thin layer plate, spreading 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 sample at positions corresponding to those of the chromatogram of the control drug.

Example 6

Methodological verification of thin layer build methods

1. Specificity test of samples

Results: the positions corresponding to the chromatogram of the pinellia tuber magnolia bark Shang Sepu, the purple perilla-lacking negative, the ginger-lacking negative and the control medicinal material show spots with the same color, and the negative control is free from interference. The thin layer method has good specificity

5 μl of pinellia tuber magnolia bark decoction, perilla herb lack negative control, ginger lack negative control, perilla herb control medicinal material and dried ginger control medicinal material solution are respectively spotted on the same silica gel G thin layer plate, and are spread by using toluene-ethyl acetate-formic acid (6:2:1) as a developing agent, taken out, dried and inspected under ultraviolet light (365 nm), as shown in figure 22.

2. Durability test

2.1 investigation of different temperatures

Results: the temperature has no obvious influence on the thin layer identification of the purple perilla and the ginger in the pinellia tuber and magnolia bark decoction, which proves that the thin layer identification method has good durability on different temperatures.

5 mu l of pinellia tuber magnolia bark decoction, perilla-less negative control, ginger-less negative control, perilla control medicinal material and dried ginger control medicinal material solution are respectively spotted on the same silica gel G thin layer plate, and are respectively spread under the conditions of low temperature and normal temperature by using toluene-ethyl acetate-formic acid (6:2:1) as a spreading agent, taken out, dried and inspected under ultraviolet light (365 nm), as shown in figure 23.

2.2 investigation of different humidity

Results: the humidity has no influence on thin-layer identification of purple perilla and ginger in the pinellia tuber and magnolia bark decoction, and the thin-layer identification method has good durability on different humidity.

5 mu l of pinellia tuber magnolia bark decoction, perilla-less negative control, ginger-less negative control, perilla control medicinal material and dried ginger control medicinal material solution are respectively spotted on the same silica gel G thin layer plate, and are respectively spread under different humidity conditions by using toluene-ethyl acetate-formic acid (6:2:1) as a spreading agent, taken out, dried and inspected under ultraviolet light (365 nm), as shown in figure 24.

2.3 inspection of thin layer plates from different manufacturers

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

5 mu l of pinellia tuber and magnolia bark soup test sample, purple perilla negative control, ginger negative control, purple perilla control medicinal material and dried ginger control medicinal material solution are respectively absorbed and respectively spotted on the same silica gel G thin layer plate, toluene-ethyl acetate-formic acid (6:2:1) is used as a developing agent, and the mixture is respectively developed under the conditions of the thin layer plates of different manufacturers, taken out, dried and subjected to ultraviolet light (365 nm) for detection, as shown in figure 25.

Claims (6)

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

(1) Preparation of test solution: collecting decoction of rhizoma Pinelliae and cortex Magnolia officinalis Shang Biaozhun, adding extraction solvent, shaking, filtering, and collecting filtrate to obtain the sample solution;

(2) Preparation of reference solution: dissolving scutellarin, rosmarinic acid, magnolol and honokiol reference substances in a solvent to obtain reference substance solution;

(3) And (3) sample injection detection: detecting the sample solution and the reference solution by using an ultra-high performance liquid chromatograph under the following two chromatographic conditions (a) and (b); the two chromatographic conditions are specifically: (a) Octadecylsilane chemically bonded silica gel chromatographic column, methanol is used as mobile phase A,0.1% phosphoric acid aqueous solution is used as mobile phase B, gradient elution is carried out for 0-4 min, and 2% -7% of A; 4-13 min, 7-17% of A; 13-19 min,17% A; 19-23 min, 17-23% of A; 23-38 min, 23-34% of A; 38-42 min,34% A; 42-55 min, 34-45% of A; 55-68 min, 45-90% of A; (b) Octadecylsilane chemically bonded silica chromatographic column eluting with acetonitrile-0.2% formic acid as flow equality; (a) 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 nm-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 nm-330 nm;

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

(5) Establishing a characteristic map: detecting a plurality of batches of sample solutions of the pinellia ternate and magnolia officinalis Shang Biaozhun decoction, taking a spectrum obtained by adopting the chromatographic conditions of (a) in the step (3), selecting chromatographic peaks existing in different batches of the pinellia ternate and magnolia officinalis Shang Biaozhun decoction as characteristic peaks, and making a characteristic spectrum;

(6) Sample injection detection in the step (3) is carried out on the sample solution to be detected, the spectrum obtained by the chromatographic condition in the step (3) is compared with the characteristic spectrum, the number of characteristic peaks is identified, and the similarity is determined; meanwhile, scutellarin, rosmarinic acid, magnolol and honokiol are taken as content index components to comprehensively evaluate the quality of the standard decoction.

2. The method for rapidly and comprehensively controlling quality of decoction of pinellia ternate and magnolia officinalis Shang Biaozhun according to claim 1, wherein the standard decoction of the pinellia ternate and magnolia officinalis comprises water decoction, extract and freeze-dried powder of the pinellia ternate and magnolia officinalis decoction.

3. The method for detecting the quality of the decoction of the pinellia ternate and magnolia officinalis Shang Biaozhun, which is rapidly and comprehensively controlled, according to claim 1, wherein the extraction solvent in the step (1) is 30-75% methanol water solution with volume concentration, the extraction time is 15-45 minutes, and the extraction mode is ultrasonic treatment, shaking extraction or heating reflux; the extraction volume is 15 ml-50 ml.

4. The method for rapidly and comprehensively controlling the quality of decoction of magnolia officinalis Shang Biaozhun as set forth in claim 1, wherein the characteristic pattern in (5) has 12 characteristic peaks F1-F12, the relative retention time of F2 is 1, the relative retention time of F1 is 0.330-0.385, the relative retention time of F9 is 1, and the relative retention time of 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.

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

6. The method for rapidly and comprehensively controlling quality of decoction of magnolia officinalis Shang Biaozhun in pinellia ternate according to claim 5, wherein the thin layer detection comprises the following steps:

(1) Preparation of a thin layer of sample solution: extracting rhizoma Pinelliae and cortex Magnolia officinalis Shang Biaozhun decoction with organic solvent, shaking, filtering, evaporating to dryness, and dissolving with methanol to obtain the sample solution;

(2) Preparation of a thin layer control solution: perilla leaf control medicinal material solution, rhizoma Zingiberis recens control medicinal material solution; preparing a negative control group with purple perilla leaves and ginger, adding methanol for dissolution, and taking the negative control group as a negative control solution according to a pinellia tuber and magnolia bark decoction formula;

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