CN112666281B

CN112666281B - Production method of lung clearing and toxin expelling soup established based on fingerprint model

Info

Publication number: CN112666281B
Application number: CN202011406163.1A
Authority: CN
Inventors: 方铁铮; 陆锡傍; 姚振弘; 赵留栓; 龙飞; 廖嘉媛; 杨凯
Original assignee: Guangdong Yipiantian Pharmaceutical Group Pharmaceutical Co ltd; Guangdong Zhidao Medicine Technology Co ltd
Current assignee: Guangdong Yipiantian Pharmaceutical Group Pharmaceutical Co ltd; Guangdong Zhidao Medicine Technology Co ltd
Priority date: 2020-12-03
Filing date: 2020-12-03
Publication date: 2022-10-14
Anticipated expiration: 2040-12-03
Also published as: CN112666281A

Abstract

The invention provides a production method of lung clearing and toxin expelling soup established based on a fingerprint spectrum model, which comprises the steps of searching a fingerprint spectrum system with optimal chromatographic parameters by using a standard sample in advance, testing a standard reference sample of a single medicine by using the chromatography system, searching and determining fingerprint peaks of a small amount of specific components at one time, thereby establishing a simplified qualitative and quantitative fingerprint spectrum model, and utilizing the model to produce the lung clearing and toxin expelling soup; the simplified fingerprint pattern model can preliminarily qualitatively screen unqualified lung-clearing and toxin-expelling soup products, can carry out quantitative analysis on qualified products, and then combines a more comprehensive thin-layer chromatography identification method to carry out comprehensive analysis on qualified products; the invention has the technical effects of simplifying the quality detection process, retaining the effect of accurate quantitative analysis and improving the detection efficiency.

Description

Production method of lung-clearing and toxin-expelling soup established based on fingerprint model

Technical Field

The invention relates to a quality control method of a preparation process of lung clearing and toxin expelling soup, in particular to a quality control method for identifying traditional Chinese medicinal materials, controlling process parameters and analyzing the quality of a finished product for a process and a preparation of lung clearing and toxin expelling soup by a fingerprint method, and a method for producing the lung clearing and toxin expelling soup according to the quality control method.

Background

The lung-heat clearing and toxin expelling soup is prepared from traditional Chinese medicine classic formulas, and comprises Maxingshigan soup, blackberry lily ephedra soup, xiaochaihu soup and Wuling powder, and has mild nature and taste. The main medicine components of the prescription are as follows: 9g of ephedra, 6g of honey-fried licorice root, 9g of almond, 15-30 g of gypsum (decocted first), 9g of cassia twig, 9g of rhizoma alismatis, 9g of polyporus umbellatus, 9g of bighead atractylodes rhizome, 15g of poria cocos, 16g of radix bupleuri, 6g of scutellaria baicalensis, 9g of ginger processed pinellia tuber, 9g of ginger, 9g of aster, 9g of flos farfarae, 9g of blackberry lily, 6g of asarum, 12g of Chinese yam, 6g of immature bitter orange, 6g of dried orange peel and 9g of wrinkled gianthyssop herb.

In order to effectively monitor the quality of the traditional Chinese medicine components, the identification and effective content measurement of the raw material components are required. The thin layer identification method is a common method in the establishment of a medicine quality standard, a proper stationary phase is coated on a glass plate, plastic or an aluminum sheet during identification so as to form a uniform thin layer, and the thin layer is developed and then compared with a chromatogram ratio shift value (Rf) obtained by a proper reference substance according to the same method so as to complete the identification and the content determination of the medicine.

In recent years, the method for detecting the traditional Chinese medicine components by utilizing the fingerprint spectrum technology becomes a high-efficiency and convenient quality detection or monitoring method. The fingerprint technology is to perform liquid chromatography analysis on a traditional Chinese medicine extraction solution by using High Performance Liquid Chromatography (HPLC), and form a fingerprint specific to the traditional Chinese medicine through specific peak lines and combinations of the peak lines of the chromatogram for representing specific components of the traditional Chinese medicine, thereby completing quality detection or monitoring of the traditional Chinese medicine preparation.

For example, zhengruweng (high performance liquid chromatography fingerprint analysis of xiaochupu granules, evaluation and analysis of medication in chinese hospital, vol.17, no. 8, 2017) reports that fingerprint analysis of xiaochupu granules and 7 Chinese medicines (dangshen, liquorice, jujube, ginger, radix bupleuri, radix scutellariae and pinellia ternate) composed of the xiaochupu granules is performed by using HPLC, and distribution of 26 peaks among 35 common peaks in a common mode of the fingerprint analysis of xiaochupu granules in the 7 Chinese medicines is studied; according to the common peak characteristics corresponding to each medicinal material, the using condition of the bupleurum tenue particle Chinese medicinal material can be reflected to a certain extent, and the product quality can be distinguished from the medicinal material aspect. However, this method requires a large number of tests to test the common characteristic peaks, and at the same time, the number of detected fingerprint peaks is as large as 26, which causes a cumbersome and inconvenient process for creating a fingerprint.

Weihuizhen (the study on multi-wavelength switching fingerprint spectra of Maxingshigan decoction, vol.23, no. 1 of Shizhen national medicine 2012) reports that an Agilent1100-DAD detector is utilized, the mobile phase is acetonitrile-0.1% phosphoric acid, gradient elution is carried out, and the detection wavelength is 0-32min; 32-40min; 40-50min; the sample amount is 10 mul, the column temperature is 25 ℃, the flow rate is 1ml/min, 12 common fingerprint peaks in the Maxingshigan decoction are determined, and an HPLC fingerprint pattern model is established. The method can reflect the use condition of the traditional Chinese medicine of the ephedra, apricot, gypsum and licorice decoction to a certain extent, and can distinguish the product quality from the perspective of the traditional Chinese medicine. However, this method requires testing the common characteristic peaks through a large number of experiments while detecting as many as 12 fingerprint peaks, which causes a cumbersome and inconvenient process for creating a fingerprint.

The invention discloses a method for detecting the quality of ephedra, almond and gypsum decoction by infrared fingerprint spectrum, thin-layer qualitative identification and HPLC content measurement, wherein ephedrine hydrochloride, pseudoephedrine hydrochloride and amygdalin are used as characteristic peaks to establish an HPLC fingerprint spectrum model, so as to obtain better detection effect. However, the method needs to perform preliminary screening on infrared fingerprint and thin-layer layered analysis in advance to obtain fewer 3 characteristic fingerprint peaks, and the whole process is also complicated and inconvenient for establishing the fingerprint.

The invention patent CN201711009683.7, the name of the invention, "a preparation process of a traditional Chinese medicine preparation", discloses a preparation method of a Xiaochaihu decoction preparation by combining HPLC fingerprint detection, which comprises the step of selecting characteristic fingerprint according to all HPLC mass spectrum peaks of bupleurum, scutellaria, ginseng, prepared pinellia, honey-fried licorice root, ginger and Chinese date for the prepared Xiaochaihu decoction granule extract, thereby respectively establishing respective characteristic fingerprint for 7 components and obtaining better detection effect. Based on the detection method, the invention also establishes a new preparation method of the small bupleurum decoction preparation. However, this method requires testing the fingerprint characteristic peaks of 7 drugs through a large number of tests, which causes a cumbersome and inconvenient process for establishing the fingerprint.

In addition, at present, no report on related prior art for establishing a fingerprint spectrum to detect the quality of the wuling san and the belamcanda root and rhizome ephedra decoction is available for a while.

Therefore, in the prior art, for completing quality detection or monitoring of traditional Chinese medicines by using a fingerprint spectrum and a traditional Chinese medicine preparation method established by using the method, characteristic fingerprint peaks of a plurality of medicinal flavors are required to be found and established generally. However, the lung clearing and toxin expelling soup is prepared by combining and adjusting 4 ancient prescriptions, wherein the medicinal material components can be more than 20, and the detectable active components can be more than one hundred. Considerable screening work is required if the different active ingredients in the finished granules are identified one by one and the characteristic fingerprint peaks are determined. Meanwhile, no report about the related prior art of establishing a fingerprint to detect the quality of the wuling san and the belamcanda chinensis ephedra decoction exists for a while, so more technical obstacles are brought to establishing a fingerprint detection method of the lung clearing and toxin expelling decoction.

In addition, as the lung-clearing and toxin-expelling soup belongs to a newly searched and determined prescription and has no unified production standard temporarily, the production processes of various manufacturers are different according to actual conditions, which may bring certain adverse effects on the standardized production of the traditional Chinese medicine preparation.

Therefore, a method for quality monitoring of the product quality of lung-clearing and toxin-expelling soup by a fingerprint spectrum method and a standardized production method based on the quality monitoring method are needed.

Disclosure of Invention

Therefore, the first purpose of the invention is to provide a production method of lung clearing and toxin expelling soup based on a fingerprint model, which comprises the following steps: taking the medicine: the main medicine components of the prescription are as follows: 9g of ephedra, 6g of honey-fried licorice root, 9g of almond, 15-30 g of gypsum (decocted first), 9g of cassia twig, 9g of rhizoma alismatis, 9g of polyporus umbellatus, 9g of bighead atractylodes rhizome, 15g of poria cocos, 16g of radix bupleuri, 6g of scutellaria baicalensis, 9g of ginger processed pinellia tuber, 9g of ginger, 9g of aster, 9g of flos farfarae, 9g of blackberry lily, 6g of asarum, 12g of Chinese yam, 6g of immature bitter orange, 6g of dried orange peel and 9g of wrinkled gianthyssop herb; the production method of the method comprises the following steps:

(1) Establishing a simplified qualitative fingerprint spectrum mass spectrum model, which comprises the following steps:

(1) weighing appropriate amount of single standard reference medicinal materials of Scutellariae radix, fructus Aurantii Immaturus, flos Farfarae, glycyrrhrizae radix and rhizoma Belamcandae, decocting with water for 30min, filtering, and collecting filtrate to obtain single reference medicinal solution;

(2) injecting a single medicinal material solution 20 mu l for detection to obtain a chromatogram;

(3) analyzing fingerprint parameters of 5 single medicines, and establishing simplified fingerprint pattern model, wherein the time of emergence of baical skullcap root fingerprint peak is about 27min, 29min, 33min, 78min, 86min, 88min and 104min; the peak emergence time of fingerprint peak of fructus Aurantii Immaturus is about 42min, 47min, 53min, 61min; the peak emergence time of the fingerprint peak of the coltsfoot flower is about 46min and 65min; the time to peak of the fingerprint peak of licorice was about 82min; the peak emergence time of the belamcanda chinensis fingerprint peak is about 95min;

(2) Taking 1.0g of sample particles, precisely weighing, placing in a 50ml volumetric flask, adding water for dissolving, fixing the volume, shaking up, filtering, and taking a subsequent filtrate;

(3) Injecting 20 mu l of sample solution for detection to obtain a chromatogram;

(4) Comparing whether the chromatographic peaks flowing out of the sample solution and the qualitative fingerprint model within 25-105min of peak-out time are consistent or not, if so, carrying out next detection, and if not, directly judging that the sample to be detected is unqualified;

(5) Respectively injecting 10 μ l sample of the test solution and naringin reference substance, hesperidin reference substance, neohesperidin reference substance, baicalin reference substance, and wogonoside reference substance, and detecting to obtain chromatogram;

(6) Comparing whether the chromatographic peaks of the test solution and the reference solution flowing out in 45-90min are consistent, if so, judging that the sample to be detected meets the primary quality standard, and if not, directly judging that the sample to be detected is unqualified;

(7) Comparing the peak intensity difference of the test solution and the reference solution in the step (6), and determining the content of naringin, hesperidin, neohesperidin, baicalin and wogonoside in the test solution according to the standard concentration of the reference solution;

wherein in any of the above steps, the chromatographic parameters are: column temperature: 30 ℃, sample introduction: 20 μ l, flow rate: 1ml/min, detection wavelength: 278nm, mobile phase:

in one embodiment, the liquid chromatograph is selected from the group consisting of an Agilent1100 high performance liquid chromatograph, and the column is selected from the group consisting of Agela Venuscil MP C18 (4.6X 250mm,5 μm).

In any of the above embodiments, in step (5), a suitable amount of naringin control, hesperidin control, neohesperidin control, baicalin control, and wogonoside control is precisely weighed and added with methanol to prepare solutions containing naringin 40 μ g, hesperidin 40 μ g, neohesperidin 40 μ g, wogonoside 40 μ g, and baicalin 100 μ g per 1ml, respectively, to obtain a control solution;

in any of the above embodiments, wherein step (5) and step (6) are used to establish a quantitative fingerprint chromatogram model, the peak appearance time of the fingerprint peak of the naringin to be detected is about 47min; the peak appearance time of the fingerprint peak of the hesperidin to be detected is about 52min; the peak emergence time of the fingerprint peak of the neohesperidin to be detected is about 60min; the peak emergence time of the fingerprint peak of the baicalin (S) to be detected is about 77min; the peak appearance time of the fingerprint peak of the wogonoside to be measured was about 87min.

In any of the above embodiments, wherein the process of determining consistency in step (6) comprises: taking the corresponding peak of the baicalin reference substance peak as an S peak, and calculating the relative retention time and the relative peak area of each main peak and the S peak; the similarity between the test sample fingerprint and the comparison fingerprint is not less than 0.90, and the relative retention time and the relative peak area are within +/-10% of the specified value, which indicates that the sample to be tested meets the primary quality standard, and if the standard is not met, the sample to be tested is directly judged to be unqualified.

In any of the above embodiments, before the detection method for fingerprint quality preliminary screening, a preliminary test for measuring chromatographic conditions and system applicability is further included, which includes: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid is taken as a mobile phase B, and gradient elution is carried out according to the following parameters;

wherein the detection wavelength is 278nm; the column temperature is 30 ℃; the flow rate is 1.0ml/min, and the number of theoretical plates is not less than 5000 calculated according to baicalin peak.

The second purpose of the invention is to provide a method for rapidly detecting the lung-heat clearing and toxin expelling decoction by combining fingerprint spectrum and thin-layer chromatography, which comprises the following steps:

the above-mentioned detection steps (1) to (7), and,

the thin-layer chromatography detection method for carrying out grouping developing agent on the sample to be detected which passes through the quality primary screening comprises the following steps:

(8) Taking a sample filtrate to be detected, adding an extracting agent for extraction, and combining extract liquor;

(9) Evaporating the extractive solution in water bath, and dissolving the residue with methanol to obtain sample solution; preparing positive control solution and negative control solution in parallel by the same method;

(10) Respectively sucking a test solution, a positive control solution and a negative control solution, respectively placing the test solution, the positive control solution and the negative control solution on the same silica gel G thin-layer plate, respectively developing the test solution, the positive control solution and the negative control solution on a thin-layer identification developing agent, and taking out the test solution;

(11) Adding color developing solution, heating with hot air until the color development of spots is clear, and inspecting in sunlight or ultraviolet light;

(12) Comparing the spots of the test sample, the positive control and the negative control, and if the spots of the same color appear at the corresponding positions of the test sample and the positive control and the spots of no interference appear at the negative position, judging that the test sample contains the medicine components with the same or similar quality as the positive control; wherein the content of the first and second substances,

for ephedra and immature bitter orange, the upper layer solution of n-butanol-glacial acetic acid-water (4;

for scutellaria and ginger, the ratio of toluene-ethyl acetate-methanol-formic acid (10;

for asarum, belamcanda rhizome, cassia twig, coltsfoot flower, tangerine peel and aster, after the first unfolding with ethyl acetate-methanol-water (100;

for alisma orientale, chloroform-ethyl acetate-formic acid (6;

for bupleurum and licorice, chloroform-methanol-water (13.

In one embodiment, for ephedra and immature bitter orange, ammonia water is added and extraction is performed with n-butanol for 2 times in steps (8) and (9), the extracts are combined, evaporated to dryness in a water bath, and the residue is dissolved with methanol as a test solution. In another embodiment, the solution of 0.5% ninhydrin in ethanol is sprayed on step (11) and baked at 105 ℃ until the spots become clear. In a specific embodiment, in the step (8), 2g of finished product particles are taken, 20ml of water is added for dissolving, 1ml of ammonia water is added, n-butyl alcohol is used for extracting for 2 times, 10ml of n-butyl alcohol is used for each time, the extract liquor is combined, water bath is evaporated to dryness, and the residue is dissolved by 1ml of methanol to be used as a test solution; and (9) taking 0.5g of each of the positive control medicinal materials and the negative control medicinal materials, respectively adding a proper amount of water, extracting under reflux for 60 minutes, cooling, filtering, concentrating the filtrate to 20ml, respectively adding 1ml of ammonia water, extracting with n-butyl alcohol for 2 times, 15ml each time, combining the extract solutions, evaporating in a water bath, and dissolving the residue with 1ml of methanol to obtain a control medicinal material solution.

In one embodiment, for Scutellaria baicalensis Georgi and Zingiber officinale Roscoe, after adding hydrochloric acid in steps (8) and (9), extracting with ethyl acetate for 2 times, combining the extracts, evaporating to dryness in water bath, and dissolving the residue with methanol to obtain a test solution. In another embodiment, step (11) is sprayed with a 2% vanillin sulfuric acid solution and baked at 105 ℃ until the spots are clear. In a specific embodiment, in the step (8), 2g of finished product particles are taken, 20ml of water is added for dissolving, 1ml of hydrochloric acid is added for extracting for 2 times by using ethyl acetate, 10ml of hydrochloric acid is added for each time, the extracts are combined, water bath is carried out for drying by distillation, and the residue is dissolved by using 1ml of methanol to be used as a test solution; and (9) taking 0.3g of each of the positive control drug and the negative control drug, adding 20ml of ethyl acetate, carrying out ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, dissolving the residue by using 1ml of methanol, and taking the residue as a control drug solution.

In one embodiment, as to asarum, blackberry lily, cassia twig, tussilago farfarfara, tangerine peel and aster, ethyl acetate is added in steps (8) and (9) for extraction for 2 times, the extracts are combined, evaporated to dryness in a water bath, and the residue is dissolved with methanol as a test solution. In another embodiment, the step (12) is that the same fluorescent spots appear on the corresponding positions of the chromatogram of the control drug, and the control drug is sprayed with an aluminum trichloride test solution and is placed under an ultraviolet lamp for inspection. In a specific embodiment, in the step (8), 2g of finished product particles are taken, 20ml of water is added for dissolving, ethyl acetate is used for extracting for 2 times, 10ml of the solution is added for each time, the extracts are combined, water bath is carried out for drying, and the residue is dissolved by 1ml of methanol to be used as a test solution; in the step (9), 0.5g of each of the positive and negative control medicinal materials is taken, a proper amount of water is added respectively, reflux extraction is carried out for 60 minutes, cooling is carried out, filtration is carried out, filtrate is concentrated to 20ml, ethyl acetate is used for extraction for 2 times, 20ml is used for each time, extraction liquid is combined, water bath evaporation is carried out, and residues are dissolved by 1ml of methanol to be used as a control medicinal material solution.

In one embodiment, for Alismatis rhizoma, petroleum ether (60-90 deg.C) is added in steps (8) and (9) for extraction for 2 times, the extracts are combined, evaporated to dryness in water bath, and the residue is dissolved in methanol to obtain a sample solution. In another embodiment, step (11) is sprayed with 10% ethanol sulfate solution, heated at 105 deg.C until the spots are clearly developed, and inspected under UV light. In a specific embodiment, in the step (8), 2g of finished product particles are taken, 20ml of water is added for dissolving, petroleum ether (60-90 ℃) is used for extracting for 2 times, 20ml of the solution is used for extracting each time, the extract liquid is combined, the water bath is evaporated to dryness, and 1ml of methanol is used for dissolving residues to be used as a test solution; and (9) taking 0.5g of each of the positive control medicinal materials and the negative control medicinal materials, adding a proper amount of water respectively, performing reflux extraction for 60 minutes, cooling, filtering, concentrating the filtrate to 20ml, extracting for 2 times by using petroleum ether (60-90 ℃) and 20ml each time, combining the extract, evaporating in a water bath, and dissolving residues by using 1ml of methanol to serve as a control medicinal material solution.

In one embodiment, for bupleuri radix and Glycyrrhrizae radix, ethyl acetate or n-butanol is added in steps (8) and (9) for extraction for 2 times, the extracts are combined, evaporated to dryness in water bath, and the residue is dissolved with methanol as a test solution. In another embodiment, the solution of 1% p-dimethylaminobenzaldehyde in 10% ethanol is sprayed in step (11) and hot air is blown to the spots to develop a clear color, which can be seen in sunlight and ultraviolet light, respectively. In a specific embodiment, in the step (9), 0.5g of licorice control drug is taken, an appropriate amount of water is added, reflux extraction is performed for 60 minutes, cooling is performed, filtration is performed, the filtrate is concentrated to 20ml, extraction is performed for 2 times by using ethyl acetate, 20ml of each time, the extracts are combined, evaporation is performed in a water bath, and the residue is dissolved by using 1ml of methanol to serve as the control drug solution. In another specific embodiment, 1.0g of radix bupleuri control drug is taken, an appropriate amount of water is added, reflux extraction is carried out for 60 minutes, cooling is carried out, filtration is carried out, filtrate is concentrated to 20ml, n-butanol is used for extraction for 2 times, 20ml of the filtrate is used for each time, extract liquor is combined, ammonia test solution is used for washing for 2 times, n-butanol layer is discarded, water bath evaporation is carried out, and residue is dissolved by 1ml of methanol to be used as radix bupleuri control drug solution.

In any of the above embodiments, the silica gel G thin layer plate is selected from a german Merck silica gel G plate or a Qingdao marine chemical silica gel G plate. In a preferred embodiment, the silica gel G thin layer plate is selected from german Merck silica gel G plate.

The third purpose of the invention is to provide a preparation method of a decoction preparation for clearing lung and expelling toxin, which comprises the steps of adding water into twenty-one medicines for decocting twice, filtering, merging decoction, decompressing and concentrating filtrate into clear paste, adding a proper amount of maltodextrin, drying or crushing to prepare granules of the decoction preparation for clearing lung and expelling toxin, monitoring the quality of the prepared granules, and guiding the standardized production of products according to the quality monitoring result, wherein the quality monitoring or detecting step is characterized by comprising the following steps of:

the above steps (1) - (7), or (8) - (12), or (1) - (12).

In any of the above preparation methods, the method comprises weighing the following Chinese medicinal components in parts by weight:

decocting twice with water, each time for 1 hour, filtering, merging decoction, decompressing and concentrating filtrate to obtain clear paste with the relative density of 1.03-1.05 (70 ℃), adding a proper amount of maltodextrin, drying or crushing, and preparing 1000g of granules.

In a specific embodiment, wherein 10 times of water is added for the first time, soaking is carried out for 0.5 hour, and decocting is carried out for 1.0 hour; adding 10 times of water for the second time, decocting for 1.0 hr, mixing decoctions, and filtering;

in any of the above embodiments, wherein the filtrate is concentrated under reduced pressure at 75 ℃ ± 5 ℃ to a clear paste with a density of 1.03-1.05 (70 ℃), and centrifuged at 10000 rpm/min;

in any of the above embodiments, the centrifuged fluid extract is taken, added with a proper amount of maltodextrin (40 g for each prescription, and the solid content of the centrifuged fluid extract is about 10.5% of the prescription), dissolved, mixed, and spray-dried to obtain extract powder;

in any of the above embodiments, the extract powder is taken, added with a proper amount of binder (water), boiled and granulated, and granulated into 1000g;

in any of the above embodiments, the particles are packaged in 10 g/bag in composite film packages.

In any of the above technical solutions, the quality monitoring includes detecting and evaluating the quality of the product, and determining whether the quality of the product is qualified, thereby determining whether to continue or interrupt the production process of the product, and guiding the production of the product in a standardized manner. In a specific embodiment, after completion of the whole grain process, samples are randomly withdrawn and identified by thin layer chromatography using the aforementioned set of developing agents to determine whether to continue or interrupt the production process of the product.

The principle of the invention is as follows: the invention breaks through the traditional research mode that a fingerprint model enough for representing various specific medicinal materials can be established only by repeatedly searching and screening suitable fingerprint peaks for multiple components in the traditional Chinese medicinal composition, and firstly proposes: the fingerprint chromatogram system with the best chromatogram parameter is searched by using a standard sample, then the standard reference sample of a single medicine is tested by using the chromatogram system, and the fingerprint peaks of a small amount of specific components are searched and determined at one time, thereby establishing a simplified qualitative and quantitative fingerprint chromatogram model. The simplified fingerprint pattern model can preliminarily qualitatively screen the lung-clearing and toxin-expelling soup products with unqualified quality, and can carry out quantitative analysis on qualified products. And then, the qualified quality product is comprehensively analyzed by combining a more comprehensive thin-layer chromatography identification method, so that the technical effects of simplifying the quality detection process, retaining the effect of accurate quantitative analysis and improving the detection efficiency are achieved.

Compared with the prior art, the invention has the following advantages:

the prior fingerprint spectrum method needs to determine the characteristic spectrums of various components in the traditional Chinese medicine, but the invention belongs to the invention of 4 ancient formulas, the corresponding medicinal materials are as many as 21, and the fingerprint peaks of the components serving as the characteristic spectrums can be as many as hundreds of types, so that great technical difficulty exists in completing the identification of the medicinal materials by fingerprint spectrums. The invention searches the fingerprint chromatogram system with the best chromatogram parameter in advance, then selects 5 components in 5 representative medicines through the determined fingerprint chromatogram system, and searches and determines the fingerprint peak of a small amount of specific components at one time, thereby establishing a simplified fingerprint spectrum model. The method has the function of primary screening detection, and can be used for carrying out comprehensive thin-layer chromatography in accordance with the requirement and directly eliminating nonconformity. Specifically, the method comprises the following steps:

1. the method comprises the steps of establishing a fingerprint model by a two-step method, wherein in the first step, five medicinal materials which have good division and are not easy to interfere in a chromatogram are selected to establish a qualitative primary screening chromatographic model; and secondly, on the basis, selecting 5 glycosides with good graduation and difficult interference as reference to establish a preliminary screening chromatographic model for quantification, and comparing the corresponding peak intensities so as to determine the content of the corresponding glycosides in the product to be detected.

2. The invention overcomes the influence of fingerprint spectrums of wuling san and belamcanda chinensis-ephedra decoction which are not reported in the prior art, firstly searches and optimizes chromatographic parameters, and then tries to determine characteristic components which can be used as fingerprint peaks by using single medicinal taste, thereby establishing a stable and efficient chromatographic prescreening system which can carry out rapid and simple qualitative and quantitative analysis on products.

3. In order to further improve the detection stability of the chromatographic primary screening system, the invention also introduces a preliminary test for measuring chromatographic conditions and system applicability before the detection method of fingerprint quality primary screening is carried out so as to determine that the used chromatographic system is reliable and effective.

4. In order to further detect whether all the medicines are contained in the composition, on the basis, the primary screening system is further combined with a predetermined more comprehensive TLC detection method to perform qualitative and quantitative analysis on the qualified medicine composition again, so as to determine whether all the medicines meeting the quality requirements are contained. In particular, the method of manufacturing a semiconductor device,

(1) The TLC detection method included in the method of the invention refers to the standards of main medicinal materials in a prescription recorded in Chinese pharmacopoeia, relevant standards of relevant traditional Chinese medicine preparations and literature data, carries out a great exploration attempt, omits the quality monitoring of partial components which do not need or have small influence on the product quality, selects a standard method different from the recommended standard method of Chinese pharmacopoeia, carries out a combination test on various developing agents of main ephedra, bitter apricot seed, honey-fried licorice root, cassia twig, rhizoma alismatis, rhizoma atractylodis macrocephalae, radix bupleuri, radix scutellariae, ginger, aster, coltsfoot flower, blackberry lily, asarum, immature bitter orange, dried orange peel, wrinkled gianthyssop herb and other components, realizes the simultaneous detection of various traditional Chinese medicine components by a few identifying developing agent groups, and optimizes and simply monitors the quality on the basis of ensuring the identifying effect.

(2) At present, no report about the quality of TLC detection of the complex synthesis is available, so the invention is the first invention research on systematic TLC detection quality in China.

(3) The TLC method for detecting the quality of the traditional Chinese medicine is a mature method, the detection means is conventional, but in the face of a quality control method which has so many medicines to be detected and needs major research and breakthrough in a short time, a large amount of time and materials are still paid, and the grouped thin-layer chromatography method is creatively tested, so that the method has high technical prospect.

(4) The invention proves that the standard method of Chinese pharmacopoeia is not suitable for thin layer analysis for detecting the multi-component composition through a large amount of test data, so that the thin layer analysis needs to be searched and verified one by one. Therefore, the inventors conducted a large number of experimental screens using the following research approaches:

(5) The existing developing agent is still used for individual medicinal flavors, but the medicinal flavor (such as radix scutellariae) is creatively combined with other medicinal flavors, and the technical effect that the same developing agent can be used for simultaneously distinguishing a plurality of medicinal flavors is obtained through screening through a research mode;

(6) For the components of asarum, blackberry lily, cassia twig, coltsfoot flower, dried orange peel and aster, the invention unexpectedly discovers that a secondary developing solvent system can be selected to clearly detect the components through a large number of experiments. Tests show that the secondary developing solvent system can analyze and detect 6 components at one time, and the components do not interfere with each other, so that the detection time and cost are effectively saved;

(7) For some components which have similar components and are difficult to analyze in the same developing solvent, the invention respectively applies different developing solvents to perform chromatographic identification (such as dried orange peel and immature bitter orange) through experiments or under the condition of not adding extra developing solvent combinations, or the components are proved to have high similarity of isomers through the experiments, so that the identification can be completed only by selecting one with the best separation effect (such as aster and bighead atractylodes rhizome);

(8) As for the developing solvent and the chromatography method which are recommended by Chinese pharmacopoeia, such as the Alisma orientale, and the like, tests show that the better detection effect can be obtained by selecting different developing solvents and matching with an improved test sample.

(9) Through the creative screening thought and a large number of comparison tests, the invention can omit unnecessary chromatographic separation components and can realize accurate quantitative and qualitative analysis of all components. The developing solvent system has good repeatability, high stability in high-temperature and low-temperature environments, high detection quality, saved detection cost and time, and stable quality detection effect.

(10) It should be noted that the invention abandons the detection of gypsum and the components of largehead atractylodes rhizome, wrinkled gianthyssop herb, almond and the like, and proposes only 5 developing agent groups, namely, the quality monitoring and evaluation can be carried out on 13 components, and a quality standard monitoring method which is enough to evaluate 20 components of the lung-clearing and toxin-expelling soup is established, thereby effectively improving the production process.

5. In addition, in the quality detection method, a large number of specific detection data point values are involved, so that although the protection range is narrower, unqualified products with poor or insufficient effects are eliminated through various specific detection data point values, and the quality detection method is very suitable for establishing a stable quality control standard and meets the requirements of traditional Chinese medicine production.

Drawings

Fig. 1 to 12: a development result graph determined by groping the existing developer system in example 3;

fig. 13 to fig. 21: in example 4, a test sample and a control verification result chart is carried out on the fumbling developing agent system;

fig. 22-fig. 28: example 5 results plot of the effect of analyzing low temperature factor on the determined developer system;

fig. 29 to fig. 35: example 6 results plot of the analysis of the effect of high humidity factor on the determined spreader system;

fig. 36 to fig. 42: example 7 results plot of the analysis of the effect of low moisture factor on the identified spreader system;

fig. 43-fig. 47: a process diagram for determining optimal fingerprint parameters in example 8;

fig. 48 to fig. 50: in the embodiment 9, a figure of a qualitative fingerprint spectrum mass spectrum model of a primary screening of the lung clearing and toxin expelling decoction is established;

fig. 51-fig. 55: in example 10, the fingerprint spectrum of qualitative and quantitative detection is performed on a sample to be detected;

FIG. 56: example 11 relates to a schematic flow diagram of preparing lung clearing and toxin expelling soup for quality detection.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

Example 1, test apparatus and feedstock

1. Instrument and reagent

1.1 instruments

Thin-layer automatic imager (CAMAG TLC VIUALIZER 2, switzerland CAMAG Co.), thin-layer PLATE heating PLATE (CAMAG TLC PLATE HEAT III, switzerland CAMAG Co.), electronic balance (METTLER ME2002E, mettler-Tollido instruments (Shanghai) Co.), electronic balance (METTLER MS 204TS, mettler-Tollido instruments (Shanghai) Co., ltd.), ultrasonic cleaner (KQ-300 DE, kunshan ultrasonic instruments Co., ltd.), medical centrifuge (Hunan instruments H1850 Hunan instruments laboratory Instrument development Co., ltd.), constant temperature water bath (HWS-26 type), plane chromatography spotter (SPDY-1A, nanjing Mackery scientific instruments Co., ltd.), silica G thin-layer PLATE (Qingdao maritime), silica G thin-layer PLATE (Merck German)

1.2 reagents and reagents

Baikal skullcap root control medicinal materials (batch number: 120955-201309), 6-gingerol control products (batch number: 111833-201806), immature bitter orange control medicinal materials (batch number: 120936-201606), ephedra herb control medicinal materials (batch number: 121051-201606), bitter apricot kernel control medicinal materials (batch number: 121554-201804), asarum herb control medicinal materials (batch number: 121204-201606), cassia twig control medicinal materials (batch number: 121191-201605), coltsfoot flower control medicinal materials (batch number: 121449-201816), blackberry lily control medicinal materials (batch number: 120994-201801), dried orange peel control medicinal materials (batch number: 120969-201510), aster tataricus control medicinal materials (batch number: 120081956-200505), bupleurum root control medicinal materials (batch number: 120992-201509), honey-fried licorice root control medicinal materials (batch number: 120904-201519) and alisma rhizome control medicinal materials (batch number: 121201803) are purchased in China pharmaceutical biological product procurement institute, and other reagents are analytical pure research.

The lung-clearing and toxin-expelling decoction granules (batch Nos. 2020033001, 2020033002 and 2020033003) are provided by Guangdong Zhidao medicine science and technology Co., ltd.

2. Preparation of solutions

2.1 preparation of test solutions

(1) Taking 2g of the granule of the decoction for clearing lung-heat and removing toxic substances, dissolving in 20ml of water, adding 1ml of ammonia water, extracting with n-butanol for 2 times, 20ml each time, mixing extractive solutions, evaporating in water bath, and dissolving the residue with 1ml of methanol to obtain a sample solution of herba Ephedrae and fructus Aurantii Immaturus.

(2) Taking 2g of the granule of the decoction for clearing lung-heat and removing toxic substances, dissolving in 20ml of water, adding 1ml of hydrochloric acid, extracting with ethyl acetate for 2 times, 20ml each time, mixing the extractive solutions, evaporating in water bath, and dissolving the residue with 1ml of methanol to obtain a sample solution of Scutellariae radix and rhizoma Zingiberis recens.

(3) Taking 2g of the granule of the lung-heat clearing and toxin expelling decoction, dissolving in 20ml of water, extracting with ethyl acetate for 2 times, 20ml each time, combining the extracts, evaporating to dryness in water bath, and dissolving the residue with 1ml of methanol to obtain a test solution of asarum, blackberry lily, cassia twig, coltsfoot flower, dried orange peel and aster.

(4) Taking 2g of the lung-clearing and toxin-expelling decoction particles, adding 20ml of water for dissolving, extracting for 2 times by 20ml each time by using petroleum ether (60-90 ℃), combining the extract liquor, evaporating to dryness in a water bath, and dissolving residues by using 1ml of methanol to be used as a sample solution of the rhizoma alismatis.

(5) Dissolving 2g of the granule of QINGFEIPAIDU decoction in 20ml of water, extracting with ethyl acetate or n-butanol for 2 times (20 ml each time), mixing extractive solutions, evaporating in water bath, and dissolving the residue with 1ml of methanol to obtain test solution of bupleuri radix and Glycyrrhrizae radix

2.2 preparation of reference medicinal materials and reference solutions

(1) Taking herba Ephedrae, fructus Aurantii Immaturus and bupleuri radix control materials 0.5g respectively, adding appropriate amount of water, reflux extracting for 60min, cooling, filtering, concentrating the filtrate to 20ml, adding 1ml ammonia water respectively, extracting with n-butanol for 2 times, 20ml each time, mixing the extractive solutions, evaporating in water bath, and dissolving the residue with 1ml methanol to obtain herba Ephedrae and fructus Aurantii Immaturus control material solution.

(2) Taking 0.5g of each of control medicinal materials such as liquorice, asarum, blackberry lily, cassia twig, common coltsfoot flower, dried orange peel, aster, baical skullcap root and the like, respectively adding a proper amount of water, refluxing and extracting for 60 minutes, cooling, filtering, concentrating the filtrate to 20ml, extracting for 2 times by using ethyl acetate, 20ml each time, combining the extract liquid, evaporating in a water bath to dryness, and dissolving the residue by using 1ml of methanol to obtain the control medicinal material solution of the asarum, the blackberry lily, the cassia twig, the common coltsfoot flower, the dried orange peel, the aster and the baical skullcap root.

(3) Taking 0.5g of rhizoma alismatis as a control medicinal material, respectively adding a proper amount of water, carrying out reflux extraction for 60 minutes, cooling, filtering, concentrating the filtrate to 20ml, extracting for 2 times by using petroleum ether (60-90 ℃) and 20ml each time, combining the extract, drying the extract by distillation in a water bath, and dissolving residues by using 1ml of methanol to obtain the rhizoma alismatis control medicinal material solution.

(4) Adding methanol into 6-gingerol control sample to obtain a solution containing 0.5mg per 1ml as control solution.

(5) Taking radix bupleuri and Glycyrrhrizae radix each 0.5g, adding appropriate amount of water, respectively, extracting with n-butanol for 2 times, 20ml each time, mixing extractive solutions, evaporating in water bath, and dissolving residue with 1ml methanol to obtain Alismatis rhizoma control solution.

2.3 preparation of negative control solution

Weighing a prescription lacking the medicinal flavors of ephedra, immature bitter orange, scutellaria baicalensis, ginger, honey-fried licorice root, cassia twig, asarum, blackberry lily, radix bupleuri, rhizoma alismatis, coltsfoot flower and the like according to the prescription proportion, decocting according to a standard preparation method, and respectively preparing a negative control solution lacking the medicinal flavors of ephedra, immature bitter orange, scutellaria baicalensis, ginger, honey-fried licorice root, cassia twig, asarum, blackberry lily, radix bupleuri, coltsfoot flower and the like, a control solution lacking pericarpium citri reticulatae, immature bitter orange double negative (according to a preparation method of a pericarpium citri reticulatae test sample) and a control solution lacking aster and bighead atractylodes rhizome double negative (according to a preparation method of an aster test sample).

Example 2 searching and screening of different extraction solvents (i.e. extractants) according to the Chinese pharmacopoeia

Referring to the identification items of liquorice, scutellaria, dried ginger, immature bitter orange, blackberry lily, asarum, dried orange peel, patchouli, bupleurum capsules and the like in the first edition of Chinese pharmacopoeia 2015, ethyl acetate and n-butanol extraction and different treatment methods are considered to treat the lung-clearing toxin-expelling decoction granules.

1. Method for preparing test solution

(1) Dissolving the granule sample (batch No. 2020033001) in 20ml of water, extracting with ethyl acetate for 2 times (20 ml each time), mixing the ethyl acetate extracts, evaporating in water bath, and dissolving the residue in 1ml of methanol to obtain sample solution 1.

(2) Extracting the residual water layer with n-butanol for 2 times (20 ml each time), mixing n-butanol extractive solutions, evaporating in water bath, and dissolving the residue with 1ml methanol to obtain sample solution 2.

(3) Dissolving the granule sample (batch No. 2020033001) in 20ml of water, extracting with n-butanol for 2 times (20 ml each time), mixing n-butanol extractive solutions, evaporating in water bath, and dissolving the residue in 1ml of methanol to obtain sample solution 3.

(4) Dissolving the granule sample (batch No. 2020033001) in 20ml of water, adding HCl 1ml, extracting with ethyl acetate for 2 times (20 ml each time), mixing ethyl acetate extractive solutions, evaporating to dryness in water bath, and dissolving the residue in 1ml of methanol to obtain sample solution 4.

(5) Dissolving the granule sample (batch No. 2020033001) in 20ml of water, extracting with n-butanol for 2 times (20 ml each time), mixing n-butanol extractive solutions, washing with ammonia solution for 2 times (20 ml each time), mixing n-butanol layers, evaporating in water bath, and dissolving the residue in 1ml of methanol to obtain sample solution 5.

2. Preparation of drug control solution

According to the prescription amount, 9g of ephedra, 6g of honey-fried licorice root, 9g of cassia twig, 9g of rhizoma alismatis, 9g of bighead atractylodes rhizome, 16g of radix bupleuri, 6g of radix scutellariae, 9g of ginger, 9g of aster, 9g of tussilago farfara, 9g of blackberry lily, 6g of asarum, 6g of immature bitter orange, 6g of dried orange peel and 9g of agastache rugosus in the prescription are respectively added with 1000ml of water, decocted for 1 hour and filtered. The filtrate was concentrated to 400ml for use.

(1) Herba ephedrae medicinal material control solution: taking 20ml of the herba Ephedrae decoction, extracting with ethyl acetate for 2 times, 20ml each time, mixing ethyl acetate extractive solutions, evaporating in water bath, and dissolving residue with 1ml of methanol to obtain herba Ephedrae control solution 1; adding 2ml of ammonia water into the residual water layer, extracting with n-butanol for 2 times (20 ml each time), mixing n-butanol solutions, evaporating in water bath, and dissolving the residue with 1ml of methanol to obtain herba Ephedrae control solution 2.

(2) Extracting the rest materials with 20ml of decoction with ethyl acetate for 2 times, each time 20ml, mixing the ethyl acetate extractive solutions, evaporating in water bath, dissolving the residue with 1ml of methanol to obtain respective medicinal material control solutions 1; extracting the residual water layer with n-butanol for 2 times (20 ml each time), mixing n-butanol solutions, evaporating to dryness in water bath, and dissolving the residue with 1ml methanol as control solution 2.

Example 3 analysis of different thin layer identification spreaders

Referring to the identification items of liquorice, scutellaria, dried ginger, immature bitter orange, bitter apricot seed, blackberry lily, asarum, dried orange peel, cablin potchouli herb, bupleurum capsule and the like in the first edition of Chinese pharmacopoeia 2015, the following existing developing agents are researched in a mode:

(1) "Licorice" identification developing agent: ethyl acetate-formic acid-glacial acetic acid-water (15;

(2) Identification developing agent for scutellaria baicalensis: toluene-ethyl acetate-methanol-carboxylic acid (10;

(3) Identifying developing agent for dried ginger: petroleum ether (60 to 90 ℃) -chloroform-ethyl acetate (2;

(4) Identification developing agent of immature bitter orange: n-butanol-glacial acetic acid-water (4;

(5) "Belamcanda" identification development agent: chloroform-butanone-methanol (3;

(6) The asarum identification developing agent: petroleum ether (ii) -ethyl acetate (3);

(7) Identifying developing agent for dried orange peel: ethyl acetate-methanol-water (100;

(8) Identification developing agent of patchouli: petroleum ether (i) -ethyl acetate-glacial acetic acid (95;

(9) The identification developing agent of the Xiaochaihu capsule comprises the following components: chloroform-methanol-water (13.

Grouping embodiment:

example 3.1 identification of developing agent by "licorice", test samples and test articles were selected, respectively, and developed with ethyl acetate-formic acid-glacial acetic acid-water (15

The development results on the Merck precast slab are shown in FIG. 1, where A is an examination at 365nm for no coloration; b is inspection under the condition of developing color by 10 percent sulfuric acid ethanol of 365 nm; c is 10% sulfuric acid ethanol color development under natural light, T:25 ℃, RH:60 percent.

Strip schematic:

1. cassia twig 2, dried orange peel 3, asarum herb 4, belamcanda rhizome 5, licorice root 6, bighead atractylodes rhizome 7, scutellaria root 8, bitter apricot seed 9, alisma rhizome 10, aster 11, coltsfoot flower 12, wrinkled gianthyssop 13, immature bitter orange 14, ephedra herb 15, ginger 16, bupleurum root 17, test solution 4, test solution 5

Analysis shows that:

(1) before developing under 365nm ultraviolet, the bright blue fluorescent spots of the bighead atractylodes rhizome, the aster tataricus and the immature bitter orange interfere with each other, and the immature bitter orange and the ephedra are respectively provided with one blue fluorescent spot without interference.

(2) Under the condition of natural light color development of ethanol sulfate, each component has a serious trailing interference band, bright blue fluorescent spots of liquorice, bighead atractylodes rhizome, aster and immature bitter orange after color development under 365nm ultraviolet interfere with each other, 1 blue fluorescent spot of immature bitter orange does not interfere, and 2 yellow fluorescent spots of liquorice do not interfere.

In summary, the conventional licorice identification developer is not suitable as a developer for combined detection because it cannot be used for TLC analysis of not only licorice components but also other drugs.

Example 3.2 identification of ginger by use of "ginger" developing agent, petroleum ether (60-90 ℃) -chloroform-ethyl acetate (2

The developed result of the Merck preformed sheet, shown in FIG. 2, is inspected at 254nm for A; b is an undeveloped view at 365 nm; c is 5% vanillin sulfuric acid color development.

Strip schematic:

Analysis shows that:

(1) the blackberrykiky rhizome has a spot under 254nm, no interference exists, the test solution 1 is selected as the test solution, and other medicinal materials cannot be identified.

(2) Ginger does not show any result at 254nm and 365nm, and the color development is clearer only in vanillin.

(3) The scutellaria baicalensis shows a more obvious single band at 254nm and 365nm, and can clearly separate a plurality of interference bands without tails under the condition of vanillin coloration.

In conclusion, although the developing agent can identify ginger under the condition of vanillin development, it does not show any result at 254nm and 365nm, which shows no indication effect on the final vanillin. Meanwhile, other herbs are not prominent either in heavy or single bands.

However, unexpectedly, the developing agent can identify ginger and scutellaria under the condition of vanillin development, and whether the same developing agent can be used for the two components is predicted.

Example 3.3 identification of developing agent with "scutellaria", toluene-ethyl acetate-methanol-formic acid (10

According to the results of example (2) and fig. 2, scutellaria identification developing agent is selected to detect scutellaria and ginger, and tangerine peel, bitter apricot kernel and wrinkled giant hyssop components are detected at the same time. The results of the development of the Merck precast slab are shown in FIG. 3, where A is an examination at 254nm for no coloration; b is an inspection under the condition of developing no color at 365 nm; c is 5% vanillin sulfuric acid color development daylight viewing, T:25 ℃, RH:60 percent.

Strip schematic:

1. scutellariae radix control 2, rhizoma Zingiberis recens control 3, pericarpium Citri Tangerinae control 4, and test solution 1

5. Test solution 2, semen Armeniacae amarum control 7, herba Agastaches control

The analysis shows that:

(1) the scutellaria baicalensis and the ginger can be identified and can be basically separated, wherein the ginger shows clear single bands in 5 percent vanillin sulfuric acid color development sunlight.

(2) The background interference of the test sample is large, and the treatment of the test sample needs to be further optimized.

(3) Pericarpium Citri Reticulatae, semen Armeniacae amarum, and herba Pogostemonis have a number of streaking bands that cannot be used with this deployment system.

Therefore, considering that the developing agents of the examples (2) to (3) have better developing effects on the ginger and the scutellaria baicalensis, and more in line with the inventive concept of reducing the combination of the developing agents, it was determined that the developing agent of the ginger does not use the developing agent recommended by pharmacopeia, but uses the scutellaria baicalensis.

Example 3.4 identification of developing agents with "immature bitter orange": n-butanol-glacial acetic acid-water (4

The chromatographic results of the 16 kinds of medicinal materials and the two kinds of test solutions are shown in FIG. 4-1, wherein A is an examination under 254nm of no color development; b is an examination under the condition of developing no color at 365nm, T:25 ℃, RH:60 percent.

Strip schematic:

Analysis shows that:

(1) under 254nm, zhishi and Huang-Cen are clear with less interference, but other herbs are blurred and difficult to separate

(2) The observation at 365nm shows that the Chinese ephedra stripe is very clear and has no interference band, but the immature bitter orange has more interference bands, and the radix scutellariae has serious tailing, which indicates that the developing agent is not suitable.

Therefore, the immature bitter orange needs to be subjected to ninhydrin ethanol color development and recheck, and meanwhile, the recheck effect of the ephedra herb component is analyzed.

The results are shown in FIG. 4-2, where A is an examination at 254nm for no coloration; b is 0.5% ninhydrin ethanol coloration natural light, T:25 ℃, RH:60 percent.

Each strip shows schematically:

1. immature bitter orange 1 (added with ammonia water and n-butanol for extraction) 2. Immature bitter orange 2 (added with ammonia water and ethyl acetate for extraction);

3. ephedra 1 (adding ammonia water and n-butanol for extraction) 4, ephedra 2 (adding ammonia water and ethyl acetate for extraction);

5. a test sample (chloroform extraction with ammonia water) 6. A test sample (ethyl acetate extraction with ammonia water);

7. a test sample 2, a test sample (extracted by adding ammonia water and n-butanol);

analysis shows that:

(1) the ninhydrin ethanol is developed, compared with the extraction with the ammonia water and the ethyl acetate, the chromatography results of the immature bitter orange and the ephedra herb extracted by the ammonia water and the n-butyl alcohol are clearer, and the results are both better than the comparison results of the test products extracted by the ammonia water and the ethyl acetate or the ammonia water and the n-butyl alcohol

(2) The ninhydrin ethanol color development result of the ephedra herb is unexpectedly superior to the ninhydrin ethanol color development result of the immature bitter orange.

Therefore, immature bitter orange developing agent can be selected, meanwhile, the Chinese ephedra is developed and analyzed, and ninhydrin ethanol color development is recommended to be used as a final chromatographic analysis means.

Example 3.5 "belamcanda" identification development agent: chloroform-butanone-methanol (3

The results are shown in FIG. 5, in which A was not developed at 254nm and B was not developed at 365nm (T: 25 ℃ RH: 60%).

Each strip shows schematically:

1. cassia twig 2, dried orange peel 3, asarum herb 4, blackberry lily 5, licorice root 6, bighead atractylodes rhizome 7, scutellaria baicalensis 8, bitter apricot seed 9, rhizoma alismatis 10, aster 11, common coltsfoot flower 12, wrinkled gianthyssop 13, immature bitter orange 14, ephedra herb 15, ginger 16, bupleurum 17, test solution 118 and test solution 2

Analysis shows that:

(1) at 254nm only belamcanda rhizome and immature bitter orange could be detected, the other components were either interfering severely or could not be displayed.

(2) At 365nm, only Atractylodis rhizoma and Aster tataricus could be detected, but they lacked the evidence at 254 nm.

In view of the above, the tectonic agent of the shoot does not meet the inventive concept of reducing the combination of the tectonic agents, and therefore it was determined that the tectonic agent of the shoot does not use the pharmacopoeia recommended development agent.

Example 3.6 "asarum" identification development reagent: petroleum ether (ii) -ethyl acetate (3

The results are shown in FIG. 6, where A is an examination at 254nm for no coloration; b is detection under 365 nm; c is vanillin development, T:25 ℃, RH:60 percent.

Each strip shows schematically:

1. test solution 1, cassia twig 3, dried orange peel 4, asarum 5, blackberry lily 6, bighead atractylodes rhizome 7, test solution 1

8. Alisma orientale 9, aster 10, coltsfoot flower 11, wrinkled gianthyssop 12, immature bitter orange 13 and test solution 1

The analysis shows that:

(1) at 254nm, only asarum was detectable, and the other components either interfered severely or aggregated together without forward separation to form separate bands.

(2) At 365nm, separate bands appeared in Asarum, cassia twig, test solution 1 and Aster tataricus, but other components were either severely disturbed or aggregated together without forward separation to form separate bands.

(3) Under the condition of vanillin display, the isolated bands of asarum are unclear, but the other components have almost no visible isolated bands.

In summary, the spreading agent of asarum is not in accordance with the inventive concept of reducing the combination of spreading agents, and therefore, it was determined that the spreading agent of asarum does not use the spreading agent recommended by pharmacopoeia.

Example 3.7 for the remaining ingredients of undetermined developing solvent, "orange peel" was chosen to identify the developing solvent: ethyl acetate-methanol-water (100.

The results are shown in FIG. 7-1, in which A is an examination at 254nm for no coloration; b365nm, T:25 ℃, RH:60 percent.

Each strip shows schematically:

1. test solution 1, cassia twig 3, dried orange peel 4, asarum 5, blackberry lily 6, bighead atractylodes rhizome 7 and test solution 1

The analysis shows that:

(1) asarum, blackberrylily rhizome and coltsfoot flower at 254nm show clearly, and have no interference basically.

(2) The asarum, the coltsfoot flower and the cassia twig under 365nm are clearly displayed, basically have no interference and can be used as an identification method.

In conclusion, the first development with ethyl acetate-methanol-water (10.

Therefore, the second expansion is considered.

The results are shown in FIG. 7-2, in which A is an examination at 254nm for no coloration; b is inspection under the condition of developing no color at 365 nm; c is the color development of the aluminum trichloride test solution under 365nm, T:25 ℃, RH:60 percent.

Strip schematic:

8. Alisma orientale 9, aster tataricus 10, common coltsfoot flower 11, wrinkled Gianthyssop 12, immature bitter orange 13 and test solution 1

Analysis shows that:

(1) FIGS. 7-2A and 7-2B, which are comparative experiments, show the same results as FIGS. 7-1A and 7-1B under the conditions of the secondary developing agent, and illustrate the developing effects of the secondary developing agent at 254nm and 365nm as the primary developing agent.

(2) In FIG. 7-2C, the Aster tataricus band was observed to have a definite tailing under 365nm color development in the aluminum trichloride test solution, but the main band was very prominent and isolated and no tailing, which is very suitable for the secondary developing agent.

(3) In fig. 7-2C, cassia twig, dried orange peel, asarum and blackberry lily have no trailing interference zone and good separation effect. Although the coltsfoot flower has a certain trailing, the main belt is very protruding and separated and has no trailing, so that the secondary spreading agent is very suitable for the secondary spreading agent.

(4) In FIGS. 7-2C, although the white atractylodes rhizome has clear separation bands, it is similar to the results of Aster tataricus and is likely to cause interference, and it is necessary to analyze the thin layer chromatography behavior of the interference components of both.

(5) In FIGS. 7-2B and 7-2C, the same components of hesperidin and the like are used in tangerine peel and immature bitter orange, so that the results of the tangerine peel and the immature bitter orange are similar, and the same developing agent is not easy to use. However, in the developing agent, compared with immature bitter orange, the dried orange peel has clear band separation and no trailing interference band, so that the developing agent can be selected for analyzing the dried orange peel.

Example 3.8 chromatographic behavior study of Aster tataricus and Atractylodes macrocephala interfering components

According to the results of example (7), the chromatographic behavior of interfering components of Aster tataricus and Atractylodes macrocephala was analyzed, and an appropriate developing agent and a component to be tested were determined.

The results are shown in FIG. 8, where 8-A is petroleum ether (II) -ethyl acetate (3; 8-B is ethyl acetate-methanol-water (100; 8-C is toluene-ethyl acetate-formic acid-water (20; 8-D is chloroform-butanone-methanol (3.

The analysis shows that:

selecting 4 kinds of thin layer development conditions, wherein the main spots of the aster and the white atractylodes rhizome are coincided in three ranges of Rf 0.1-0.3, rf 0.4-0.6 and Rf 0.7-0.9, but the fluorescence intensities of the aster and the white atractylodes rhizome are different, considering that the aster and the white atractylodes rhizome may be isomers,

therefore, both Aster and Atractylodes cannot be detected simultaneously by the same system of developing agent.

Combining the results of example (7) and fig. 7, it was determined that aster was selected to be suitable for the secondary opener system because the band of aster was more prominent and separable than that of atractylodis macrocephalae.

In summary, it was determined that, for asarum, belamcanda rhizome, cassia twig, coltsfoot flower, tangerine peel and aster, after the first development with ethyl acetate-methanol-water (100: 17

Example 3.9 identification of "patchouli" developing agents: petroleum ether (i) -ethyl acetate-glacial acetic acid (95;

the results are shown in FIG. 9, in which A is an examination at 365 nm; b is the observation under the developing sunlight of ferric trichloride, T:25 ℃, RH:60 percent.

Strip schematic:

8. Alisma orientale 9, aster 10, coltsfoot flower 11, patchouli 12, immature bitter orange 13 and test solution 1

Analysis shows that:

(1) at 365nm, only cassia twig and test solution 1 present clear separation bands, and the other components are agglomerated together and cannot be separated, so that only cassia twig can be detected, and other medicinal material components cannot be detected;

(2) after ferric trichloride develops, the components of the agastache rugosus medicinal material control and the test sample are agglomerated together and cannot be separated, so that any spot presenting separation cannot be detected.

In conclusion, the production process of the product is water decoction, and volatile oil is not additionally extracted, so that agastache rugosus cannot be detected and cannot be identified. Also, the developer system cannot be used for chromatographic analysis of other components.

Example 3.10 identification of bupleurum tenue capsules developing agent: chloroform-methanol-water (13

The results are shown in FIG. 10, where A is an examination at 254nm for no coloration; b is an inspection under the condition of developing no color at 365 nm; c is inspection of dimethylaminobenzaldehyde sulfuric acid ethanol under 365nm of color development; d is the content of T:25 ℃, RH:60 percent.

Strip schematic:

1. bupleurum root 2, test solution 5, licorice root 4, test solution 3, blackberry lily 6, test solution 1

Analysis shows that:

(1) the bupleurum and the liquorice present clear separation bands under four display conditions, and the main bands are not interfered with each other;

(2) the blackberry lily can be clearly separated under the condition of the color development display of dimethylaminobenzaldehyde sulfuric acid ethanol, but the color development effect is not as good as that of bupleurum and liquorice.

And (4) conclusion: the developing agent can separate bupleuri radix, glycyrrhrizae radix, and rhizoma Belamcandae. However, as the separable components of the bupleurum, the liquorice and the blackberry lily are more, in order to avoid the interference of the components of the blackberry lily with the detection result and also to consider that the blackberry lily can be already included in the detection group of the secondary developing solvent, the developing solvent is recommended to be used for detecting the bupleurum and the liquorice.

Example 3.11 using a developing agent different from alisma identified in chinese pharmacopoeia: chloroform-ethyl acetate-formic acid (6.3.5) chinese pharmacopoeia 2015 for alisma thin layer analysis: cyclohexane-ethyl acetate (1)

The results are shown in FIG. 11, where A is an examination at 254nm for no coloration; b is an inspection under the condition of developing no color at 365 nm; c is a test result under the condition that 10% sulfuric acid ethanol develops color at 365nm, T:25 ℃, RH:60 percent.

Strip schematic:

1. test solution 5, cassia twig 3, dried orange peel 4, asarum 5, blackberry lily 6, liquorice 7, bighead atractylodes rhizome 8, scutellaria 9, bitter apricot kernel 10 and rhizoma alismatis

11. Aster 12, coltsfoot flower 13, wrinkled gianthyssop 14, immature bitter orange 15, ephedra 16, ginger 17, radix bupleuri 18 and test solution 3;

the analysis shows that:

(1) belamcanda chinensis, radix Scutellariae, and flos Farfarae can show separated spots under 254nm and 365nm, zelesia chinensis can not show separated spots, and only the control shows multiple spots;

(2) under the condition of 365nm color development of 10% sulfuric acid ethanol, a plurality of separated spots appear on the alisma medicinal material, although a plurality of spots also appear on the control, the trailing interference band is serious, and the test solution 1 needs to be changed for further verification.

And (4) conclusion: the developing agent can be used for separating rhizoma Belamcandae, scutellariae radix, and flos Farfarae. However, due to the fact that the belamcanda chinensis, radix scutellariae and flos farfarae have more separable components, in order to avoid the components from interfering with the detection result, the rhizoma alismatis is recommended to be further verified by using the developing agent considering that the components can be already included in the detection group with the developing agent determined previously.

Example 3.12 preparation verification of Alisma orientale test sample

The results are shown in FIG. 12, where A is the test using sample 1; and B is the ratio of T:25 ℃, RH:60 percent.

Strip schematic:

1. test sample 12, drug control 3, negative control

Analysis shows that:

(1) through the test results, the test article 1 is adopted for verification, and the result shows that the identification cannot be carried out.

(2) According to the method reported by Chuaiyurong (research on quality standard of Huoluotong oral liquid, china medical science 2015,5 (7): 50), 20ml of water is added to a particle sample (batch No. 2020033001) for dissolution, petroleum ether (60-90 ℃) is added for extraction for 2 times, 20ml of water is added for each time, extraction liquid is combined, evaporation is carried out, and 1ml of methanol is added to residues for dissolution, so that a sample solution Ym is obtained. The control medicinal materials and the negative control are prepared by the same method, and the result shows that the identification can be realized.

In conclusion, for Alisma orientale, it is recommended to use an improved developing agent, and an improved test solution Ym is required to obtain a good detection effect.

Example 4 repeated validation of the optimal developer group for the Lung clearing and toxin expelling decoction

In conclusion, the invention tests five groups of developing agents of ephedra, honey-fried licorice root, cassia twig, rhizoma alismatis, radix bupleuri, radix scutellariae, ginger, radix asteris, flos farfarae, blackberry lily, asarum, immature bitter orange and dried orange peel.

As for the medicinal material components of which the developing agent is not determined, or which are isomeric with other components, they are not easily distinguished, and thus are not conveniently analyzed (e.g., atractylodes macrocephala koidz and aster);

or effective medicinal components (such as herba Agastaches) are not extracted due to preparation process;

or an inorganic substance belonging to an unidentifiable analysis (e.g. gypsum);

or belongs to adjuvant drug component, and is not main drug (such as rhizoma Dioscoreae) for focus detection;

or have similar compositions and effects, and are difficult to analyze in the same developer system (e.g., poria and Polyporus).

And carrying out a repeatability verification test of a test product and a negative-positive control on the lung-clearing and toxin-expelling decoction according to the determined developing agent combination.

Example 4.1 verification of the developing agent of ephedra and immature bitter orange: n-butanol-glacial acetic acid-water (4

Taking 2g of the product granule, dissolving in 20ml of water, adding 1ml of ammonia water, extracting with n-butanol for 2 times, 10ml each time, mixing extractive solutions, evaporating in water bath, and dissolving residue with 1ml of methanol to obtain test solution. Taking herba Ephedrae and fructus Aurantii Immaturus control materials 0.5g each, adding appropriate amount of water respectively, reflux extracting for 60min, cooling, filtering, concentrating the filtrate to 20ml, adding 1ml ammonia water respectively, extracting with n-butanol for 2 times, 15ml each time, mixing the extractive solutions, evaporating in water bath, and dissolving the residue with 1ml methanol to obtain herba Ephedrae and fructus Aurantii Immaturus control material solution. According to a thin-layer chromatography test (0502 of the four ministry of the ministry of health in the 'Chinese pharmacopoeia' 2015 edition), sucking 2 microlitres of each of the three solutions, respectively dropping the solutions on the same silica gel G thin-layer plate, pre-saturating for 30 minutes by taking an n-butyl alcohol-glacial acetic acid-water (4).

The results are shown in FIG. 13, T:25 ℃, RH: and 64 percent.

Strip shows that:

1. herba Ephedrae negative control 2, herba Ephedrae medicinal material control 3, test sample (batch No. 2020033001)

4. Sample (batch No. 2020033002) 5, sample (batch No. 2020033003) 6, and fructus Aurantii Immaturus as control

7. Immature bitter orange negative control

The analysis shows that:

(1) under this developer assay, the positive control main band is prominent without any interference bands. Negative control is not interfered, which indicates that the system has no detection error;

(2) although the herba ephedrae and fructus aurantii immaturus samples have a certain tailing, the negative control has no interference, and the main band has the same prominent main band compared with the positive control.

And (4) conclusion: the developing agent can be used for simultaneously detecting the ingredients of the ephedra herb and the immature bitter orange in three samples, and the detection result is accurate, so that the interference bands of negative control can be clearly distinguished.

Example 4.2 validation of developing agents for scutellaria baicalensis and ginger: toluene-ethyl acetate-methanol-carboxylic acid (10

Dissolving 2g of the granule in 20ml of water, adding 1ml of hydrochloric acid, extracting with ethyl acetate for 2 times (10 ml each time), mixing the extractive solutions, evaporating in water bath, and dissolving the residue in 1ml of methanol to obtain a sample solution. Taking 0.3g Scutellariae radix reference medicinal material powder, adding ethyl acetate 20ml, ultrasonic treating for 30min, filtering, evaporating filtrate to dryness, and dissolving residue with 1ml methanol to obtain reference medicinal material solution. Adding methanol into 6-gingerol control to obtain a solution containing 0.5mg per 1ml as control solution. According to a thin-layer chromatography (0502 of the four ministry of the book of Chinese pharmacopoeia 2015), 2 ul of each of the test solution and the control solution, 4 ul of 6-gingerol control solution are respectively spotted on the same silica gel GF254 thin-layer plate, a toluene-ethyl acetate-methanol-formic acid (10.

The results are shown in FIG. 14, T:25 ℃, RH: and 64 percent.

Strip shows that:

1. scutellaria baicalensis negative control 2. Scutellaria baicalensis medicinal material control 3. Test sample (batch No. 2020033001)

4. Sample (batch No. 2020033002) 5, sample (batch No. 2020033003) 6, and rhizoma Zingiberis recens negative control

7.6-gingerol

Analysis shows that:

(1) when the positive control is detected under an ultraviolet lamp (254 nm), the main band of the positive control is prominent without any interference band. Negative control is not interfered, which indicates that the system has no detection error;

(2) when the test is carried out under an ultraviolet lamp (254 nm), the three test samples have the same band type, but a clear and prominent main band is added compared with the positive control of radix scutellariae;

(3) after heating at 105 ℃ in 2% vanillin-sulfuric acid solution, the three samples were equally banded and the aforementioned extra clearly prominent main band was identical to the 6-gingerol positive control.

And (4) conclusion: the developing agent can be used for detecting the components of radix scutellariae and ginger in three samples at the same time, and the detection result is accurate, especially the component of ginger can be accurately identified.

Example 4.3 verification of developing Agents for Asarum sieboldii, belamcanda chinensis, cassia twig, farfaran flower, citrus reticulata and Aster tataricus

Dissolving 2g of the granule in 20ml of water, extracting with ethyl acetate for 2 times (20 ml each time), mixing extractive solutions, evaporating in water bath, and dissolving the residue in 1ml of methanol to obtain sample solution. Taking 0.5g of each of herba asari, rhizoma Belamcandae, ramulus Cinnamomi, flos Farfarae, pericarpium Citri Tangerinae and radix Asteris as reference medicinal materials, adding appropriate amount of water, reflux-extracting for 60min, cooling, filtering, concentrating the filtrate to 20ml, extracting with ethyl acetate for 2 times, 20ml each time, mixing the extractive solutions, evaporating in water bath, and dissolving the residue with 1ml of methanol to obtain the reference medicinal solution. According to a thin-layer chromatography (0502 of the four parts of the book of pharmacopoeia of China 2015), 2-5 mul of each solution is absorbed and respectively spotted on the same silica gel GF254 thin-layer plate, the solution is spread to about 3cm by taking ethyl acetate-methanol-water (100). The results are shown in FIGS. 15 to 19.

The identification result of pericarpium Citri Tangerinae is shown in FIG. 15, wherein A is 365nm before color development; b is 365nm inspection after the color development of the aluminum trichloride.

Strip shows that:

1. pericarpium Citri Tangerinae double yin contrast 2. Pericarpium Citri Tangerinae medicinal material contrast 3. Test sample (batch No. 2020033001)

4. Sample (batch No. 2020033002) 5 sample (batch No. 2020033003)

The analysis shows that:

(1) the negative and positive control bands were typed more, but did not interfere with each other, indicating that they could be used for comparative analysis of the test samples.

(2) Compared with the positive control, the test sample also shows a strip with the same color and size at the same position below, which indicates that the test sample contains the expected medicinal ingredients.

(3) In the same position above the negative control, multiple bands of the same brightness and size appear in the sample, indicating that the sample contains some of the same components as the negative control.

And (4) conclusion: the combined developing agent can detect the medicinal components of the dried orange peel, but the tested product contains up to 21 medicinal components, the bands are more in types, and the dried orange peel can be identified by carefully comparing positive and negative controls.

The identification result of Aster is shown in FIG. 16, wherein A is 365nm before color development; b is 365nm inspection after aluminum trichloride color development, T:25 ℃, RH: and 64 percent.

Strip schematic:

1. radix Asteris double-negative control 2 radix Asteris medicinal material control 3 sample (batch No. 2020033001)

4. Sample (batch No. 2020033002) 5 sample (batch No. 2020033003)

Analysis shows that:

(2) In the same position above the positive control, the sample also shows a band of the same size, indicating that the sample contains the expected drug component.

(3) In the same position below, the test article appears as a plurality of bands of the same color and size as compared to the negative control, indicating that the test article contains some of the same components as the negative control.

And (4) conclusion: the combined developing agent can detect the components of the dried orange peel medicinal materials, but needs to compare positive and negative controls carefully.

The identification results of rhizoma Belamcandae and herba asari are shown in FIG. 17, wherein A is thin-layer chromatogram of rhizoma Belamcandae identification at 254nm; b is 365nm inspection asarum identification thin-layer spectrum, T:25 ℃, RH: and 64 percent.

Strip schematic:

1. belamcanda chinensis negative control 2. Belamcanda chinensis medicinal material control 3. Test sample (batch No. 2020033001)

4. Test sample (batch No. 2020033002) 5, test sample (batch No. 2020033003) 6, asarum negative control

7. Herba asari control

Analysis shows that:

(1) the positive control band of the blackberry lily is far smaller than that of the negative control, and the upper part and the lower part of the blackberry lily are respectively provided with a prominent main band which is not interfered with the negative control, so that the positive control band and the lower part of the blackberry lily can be used for comparing and analyzing a test sample; the asarum positive control band is far smaller than the asarum negative control band, and a prominent main band is arranged below each asarum positive control band, so that the asarum positive control band and the asarum negative control band do not interfere with each other, and the asarum positive control band can be used for comparing and analyzing a test sample;

(2) compared with the positive control of the blackberry lily, the test sample also presents a strip with the same size at the upper part and the lower part, which indicates that the test sample contains the expected medicinal ingredients; compared with the asarum positive control, the test sample also presents a strip with the same size at the same position below, which indicates that the test sample contains the expected medicinal material components;

(3) compared with the blackberry lily negative control, the sample has a plurality of strips with the same brightness and size at the same position at the bottom, which indicates that the sample contains some components which are the same as those of the negative control; as compared with asarum negative control, at the same position on the top, the test sample has several bands of the same brightness and size, indicating that the test sample contains some components identical to those of the negative control

And (4) conclusion: the combined developing agent can detect the ingredients of belamcanda chinensis and asarum, but needs to compare positive and negative controls carefully.

The results of the cassia twig identification are shown in fig. 18. Strip schematic:

1. ramulus Cinnamomi negative control 2, ramulus Cinnamomi control 3, and test sample (batch No. 2020033001)

4. Sample (batch No. 2020033002) 5 sample (batch No. 2020033003)

Analysis shows that:

(1) the negative control bands were typed more, but the positive control had only 1 major band at the top, without interfering with each other, indicating that it could be used for comparative analysis of the test article.

(2) Compared with the positive control, at the same position at the top, the test sample also shows a band with the same color and size, indicating that the test sample contains the expected medicinal ingredients.

(3) In the same position except the uppermost position, the test article showed multiple bands of the same brightness and size as compared with the negative control, indicating that the test article contained a large amount of the same components as the negative control.

And (4) conclusion: the combined developing agent can detect the ingredients of the cassia twig medicinal materials, and can quasi-group identify the ingredients of the cassia twig by combining with positive control.

The result of identifying flos Farfarae is shown in FIG. 19, in which A is 254nm before aluminum trichloride development; b is 365nm inspection after aluminum trichloride color development, T:25 ℃, RH: and 64 percent.

Strip schematic:

1. tussilago farfara negative control 2. Tulago farfara medicinal material control 3. Test article (batch number: 2020033001)

4. Sample (batch No. 2020033002) 5 sample (batch No. 2020033003)

The analysis shows that:

(1) the negative control bands were typed more, but the positive control had only 1 major band in the middle and below, without interfering with each other, indicating that it could be used for comparative analysis of the test article.

(2) Compared with the positive control, the test sample also presents a strip with the same color and size at the same position in the middle and lower part, which indicates that the test sample contains the expected medicinal ingredients.

(3) In comparison with the negative control, at the same positions except the above, the test article showed multiple bands of the same brightness and size, indicating that the test article contained a large amount of the same components as the negative control.

And (4) conclusion: the combined developing agent can detect the ingredients of cassia twig medicinal materials, and can identify the embodiment 4.4 of the ingredients of cassia twig and verify the developing agent of rhizoma alismatis by standard groups by combining positive control: chloroform-ethyl acetate-carboxylic acid (6

Taking 2g of the product granules, adding 20ml of water for dissolving, extracting for 2 times by 20ml each time by using petroleum ether (60-90 ℃), combining the extract liquor, evaporating in a water bath to dryness, and dissolving residues by using 1ml of methanol to be used as a test solution. Taking 0.5g of alisma orientale as a reference medicinal material, respectively adding a proper amount of water, refluxing and extracting for 60 minutes, cooling, filtering, concentrating the filtrate to 20ml, extracting for 2 times by using petroleum ether (60-90 ℃) and 20ml each time, combining the extract, evaporating in a water bath to dryness, and dissolving residues by using 1ml of methanol to obtain a reference medicinal material solution. According to the thin-layer chromatography (0502 of the four ministry of the national pharmacopoeia 2015), 2 mul of the test solution and the control solution are respectively absorbed and respectively spotted on the same silica gel G thin-layer plate, chloroform-ethyl acetate-formic acid (6: 3.5.

The identification results of alisma orientale are shown in figure 20.

Strip schematic:

1. alisma orientale negative control 2 Alisma orientale medicinal material control 3. Test sample (batch No. 2020033001)

4. Sample (batch No. 2020033002) 5 sample (batch No. 2020033003)

The analysis shows that:

(2) Compared with the positive control, at the same position below, the test sample also presents a strip with the same color and size, which indicates that the test sample contains the expected medicinal ingredients.

(3) In the same position except the above, the test article showed multiple bands of the same brightness and size as compared with the negative control, indicating that the test article contained a large amount of the same components as the negative control.

And (4) conclusion: the combined developing agent can detect the components of the alisma orientale medicinal materials, but the test sample contains up to 21 medicinal materials, the occurrence of banding is more, and the positive control and the negative control need to be carefully compared for identification.

Example 4.5, validation of spreading agents for bupleurum and licorice: chloroform-methanol-water (13

Taking 0.5g of licorice control medicinal material, adding a proper amount of water, carrying out reflux extraction for 60 minutes, cooling, filtering, concentrating the filtrate to 20ml, extracting with ethyl acetate for 2 times, 20ml each time, combining the extracts, evaporating in a water bath, and dissolving the residue with 1ml of methanol to obtain the licorice control medicinal material solution. Taking another bupleurum root reference medicinal material 1.0g, adding a proper amount of water, refluxing and extracting for 60 minutes, cooling, filtering, concentrating the filtrate to 20ml, extracting with n-butanol for 2 times, 20ml each time, combining the extract, washing with ammonia test solution for 2 times, discarding, collecting the n-butanol layer, evaporating in water bath, and dissolving the residue with 1ml of methanol to obtain the bupleurum root reference medicinal material solution. According to a thin-layer chromatography (0502 of the four ministry of the national pharmacopoeia 2015), respectively absorbing and identifying 2 mul of the test solution, the radix bupleuri control drug and the liquorice control drug solution under the items (1) and (2), respectively dropping the solutions on the same silica gel G thin-layer plate, taking out and airing the solutions, spraying a 10% sulfuric acid ethanol solution of 1% p-dimethylaminobenzaldehyde on the solution, blowing hot air until the spots are clear in color, respectively inspecting the solutions under sunlight and ultraviolet light (365 nm), wherein the spots of the same color appear in the chromatogram of the test sample (1) at the positions corresponding to the chromatogram of the radix bupleuri control drug; in the chromatogram of the test sample (2), spots with the same color appear at the positions corresponding to the chromatogram of the licorice control drug, and the negative is not interfered.

The results of the identification of licorice and bupleurum are shown in fig. 21, T:25 ℃, RH: and 64 percent.

Strip shows that:

1. licorice root negative control 2. Licorice root drug control 3. Test sample (batch No. 2020033001)

4. Test sample (batch No. 2020033002) 5 test sample (batch No. 2020033003) 6 Bupleurum negative control

7. Radix bupleuri reference 8, sample (batch No. 2020033001) 9, sample (batch No. 2020033002)

10. Sample (batch No. 2020033003)

The analysis shows that:

(1) the liquorice positive control strip is far smaller than the negative control strip, and the upper part and the middle part of the liquorice positive control strip are respectively provided with a prominent main strip which is not interfered with the negative control strip, so that the liquorice positive control strip can be used for comparing and analyzing a test sample; the positive and negative control bands of bupleurum are more types, but do not interfere with each other, which indicates that the positive and negative control bands can be used for comparing and analyzing the test sample. (ii) a

(2) Compared with the licorice positive control, the sample also presents 2 strips with the same size at the same position in the middle part, which indicates that the sample contains the expected medicinal material components; compared with the positive control of radix bupleuri, the test sample also presents a plurality of strips with the same size at the same position in the middle part, which indicates that the test sample contains the expected medicinal material components;

(3) compared with the liquorice negative control, the test sample basically has no strips at the same position, which indicates that the components of the test sample and the liquorice negative control are greatly different; compared with the Bupleurum negative control, 1 strip with the same brightness and size appears on each sample at the same position above and below, which indicates that the sample contains some components same as those in the negative control.

And (4) conclusion: the combined developing agent can detect the components of the medicinal materials of the liquorice and the radix bupleuri, has fewer components as the same as those of negative control, and is beneficial to the identification of the liquorice and the radix bupleuri.

Example 5 measurement of stability of temperature factor for the above monitoring method

5.1 identification test of Ephedra and immature bitter orange at low temperature

The effect of the temperature factor on the above-mentioned identification effect of the ephedra herb and immature bitter orange exfoliant combination was tested in the same manner and humidity as in example 4 at 4 c.

The results are shown in FIG. 22.

Strip shows that:

1. herba Ephedrae reference material 2, sample (batch No. 2020033001) 3, sample (batch No. 2020033002)

4. Sample (batch No. 2020033003) 5, fructus Aurantii Immaturus reference drug

Analysis shows that:

the positive, negative control and test strips remained identical with no significant difference compared to example 4.1.

5.2 differentiation test of Scutellariae radix and rhizoma Zingiberis recens at low temperature

The effect of the temperature factor on the above-described identification effect of the scutellaria baicalensis and ginger developer combination at 4 ℃ was tested according to the same method and humidity as in example 4.

The results are shown in FIG. 23, in which A is a 365nm inspection before development; b is detected at 365nm after color development.

Strip shows that:

1. scutellariae radix reference material 2, sample (batch No. 2020033001) 3, sample (batch No. 2020033002)

4. Test sample (batch No. 2020033003) 5.6-gingerol control

The analysis shows that:

compared with example 4.2, gingerol showed no visible spots at 365nm before development. The scutellaria baicalensis is detected to have visible spots at 365nm after color development, and the bands of the rest positive and negative controls and the test sample are kept consistent and have no obvious difference.

5.3 discrimination test of Cassia twig and Aster tataricus at Low temperature

The effect of the temperature factor on the above-described discrimination effect of the combination of the cassia twig and aster tataricus developing agent at 4 ℃ was tested according to the same method and humidity as in example 4.

The results are shown in FIG. 24, in which A is a 365nm inspection before development; b is detected under 365nm after the color development of the aluminum trichloride test solution.

Strip schematic:

1. ramulus Cinnamomi reference material 2, sample (batch No. 2020033001) 3, sample (batch No. 2020033002)

4. Sample (batch No. 2020033003) 5 Aster control drug

Analysis shows that:

compared with example 4.3, the bands of the positive control, the negative control and the test article are consistent and have no obvious difference.

5.4 authentication test of Belamcanda chinensis and flos Farfarae at low temperature

The effect of the temperature factor on the above-described identifying effect of the combination of belamcanda chinensis and coltsfoot flower developing agent was tested in the same manner and humidity as in example 4 under an environment of 4 ℃.

The results are shown in FIG. 25, in which A is a 254nm inspection before development; b is detection at 365nm before color development; c is detected under 365nm after the aluminum trichloride test solution develops color.

Strip schematic:

1. blackberry lily reference medicinal material 2, sample (batch No. 2020033001) 3, sample (batch No. 2020033002)

4. Test sample (batch No. 2020033003) 5 reference medicinal material of flos Farfarae

Analysis shows that:

5.5 identification test of pericarpium Citri Tangerinae and herba asari at low temperature

The effect of temperature factor on the identification effect of the combination of pericarpium Citri Reticulatae Chachiensis and herba asari Forbesii at 4 ℃ was tested according to the same method and humidity as in example 4.

The results are shown in FIG. 26.

Strip shows that:

1. pericarpium Citri Tangerinae reference material 2, sample (batch No. 2020033001) 3, sample (batch No. 2020033002)

4. Test sample (batch No. 2020033003) 5 herba asari as reference material

The analysis shows that:

5.6 Alisma orientale discrimination test at Low temperature

The effect of temperature factor on the identifying effect of alisma orientale spreading agent combination at 4 ℃ environment was tested according to the same method and humidity of example 4.

The results are shown in FIG. 27.

Strip schematic:

1. alisma orientale reference material 2. Sample (batch No. 2020033001)

3. Sample (batch No. 2020033002) 4 sample (batch No. 2020033003)

The analysis shows that:

in comparison with example 4.4, the test article also exhibited a band of the same color and size at the same position below as compared to the positive control, indicating that the band remained identical to the positive control with no significant difference.

5.7 discrimination test of Licorice root and Bupleurum root at low temperature

The effect of temperature factor on the discrimination effect of the combinations of licorice and bupleurum root developing agents at 4 c was tested according to the same method and humidity as in example 4.

The results are shown in FIG. 28, where A is the daylight after color development; b is detected at 365nm after color development.

Strip schematic:

1. glycyrrhrizae radix reference material 2, sample (batch No. 2020033001) 3, sample (batch No. 2020033002)

4. Test sample (batch No. 2020033003) 5 radix bupleuri reference material 6 test sample (batch No. 2020033001)

7. Sample (batch No. 2020033002) 8 sample (batch No. 2020033003)

The analysis shows that:

the positive, negative control and test strips remained identical with no significant difference compared to example 4.5.

Example 6 stability determination of high humidity factor for the above monitoring method

6.1 identification test of Ephedra and immature bitter orange under high humidity factor

Following the same procedure and humidity as in example 4, the humidity was measured at RH: the influence of the humidity factor in the environment of 88% on the identification effect of the composition of the ephedra and immature bitter orange developing agents.

The results are shown in FIG. 29.

Strip shows that:

4. Sample (batch No. 2020033003) 5 fructus Aurantii Immaturus control

Analysis shows that:

6.2 Scutellaria baicalensis and ginger identification test under high humidity factor

Following the same procedure and humidity as in example 4, the humidity was measured at RH: influence of humidity factor under 88% environment on identification effect of the radix Scutellariae and rhizoma Zingiberis recens developer composition.

The results are shown in FIG. 30, in which A is a 365nm inspection before development; b is detected at 365nm after color development.

Strip schematic:

4. Test sample (batch No. 2020033003) 5.6-gingerol control

The analysis shows that:

the positive, negative control and test strips remained identical with no significant difference compared to example 4.

6.3 discrimination test of Cassia twig and Aster tataricus under high-humidity factor

Following the same procedure and humidity as in example 4, the humidity was measured at RH: influence of humidity factor in 88% environment on the identification effect of the composition of ramulus Cinnamomi and radix Asteris exfoliant.

The results are shown in FIG. 31, in which A is a 365nm inspection before development; b is detected under 365nm after the color development of the aluminum trichloride test solution.

Strip schematic:

1. ramulus Cinnamomi reference material 2, test sample (batch No. 2020033001) 3, test sample (batch No. 2020033002)

4. Sample (batch No. 2020033003) 5 Aster control drug

Analysis shows that:

compared with example 4, the bands of the positive control, the negative control and the test article are consistent and have no obvious difference.

6.4 identification test of Belamcanda chinensis and flos Farfarae under high humidity factor

Following the same procedure and humidity as in example 4, the humidity was measured at RH: the influence of the humidity factor in the environment of 88% on the identification effect of the combination of the blackberrykiky rhizome and the common coltsfoot flower developing agent.

The results are shown in FIG. 32, where A is 254nm inspection before development; b is detection at 365nm before color development; c is detected under 365nm after the aluminum trichloride test solution develops color.

Strip schematic:

Analysis shows that:

6.5 identification test of pericarpium Citri Tangerinae and herba asari under high humidity factor

Following the same procedure and humidity as in example 4, the humidity was measured at RH: influence of humidity factor under 88% environment on identification effect of pericarpium Citri Tangerinae and herba asari developer composition.

The results are shown in FIG. 33.

Strip schematic:

4. Test sample (batch No. 2020033003) 5 herba asari as reference material

Analysis shows that:

6.6 Alisma orientale discrimination test under high humidity factor

Following the same procedure and humidity as in example 4, the humidity was measured at RH: the influence of humidity factor under 88% environment on the identification effect of the alisma orientale developing agent composition.

The results are shown in FIG. 34.

Strip schematic:

1. alisma orientale reference material 2. Sample (batch No. 2020033001)

3. Test article (batch No. 2020033002) 4 test article (batch No. 2020033003)

Analysis shows that:

6.7 differentiation test of liquorice and radix bupleuri under high humidity factor:

following the same procedure and humidity as in example 4, the humidity was measured at RH: the influence of the humidity factor under the environment of 88 percent on the identification effect of the liquorice and radix bupleuri developing agent combination.

The results are shown in FIG. 35, where A is a day-light examination after color development; b is detected at 365nm after color development.

Strip schematic:

4. Test sample (batch No. 2020033003) 5 radix bupleuri reference medicinal material 6 test sample (batch No. 2020033001)

7. Sample (batch No. 2020033002) 8 sample (batch No. 2020033003)

Analysis shows that:

Example 7 stability determination of Low moisture factor for the above monitoring method

7.1 identification test of Ephedra sinica and immature bitter orange under low-humidity factor

Following the same procedure and temperature as in example 4, the measured RH: the influence of the humidity factor in the environment of 32% on the identification effect of the ephedra herb and immature bitter orange developing agent combination.

The results are shown in fig. 36, where the bands are schematic:

4. Sample (batch No. 2020033003) 5 fructus Aurantii Immaturus control

Analysis shows that:

7.2 differentiation test of Scutellariae radix and rhizoma Zingiberis recens under low humidity factor

Following the same procedure and temperature of example 4, the test was carried out at RH: influence of humidity factor in 32% environment on identification effect of radix Scutellariae and rhizoma Zingiberis recens developer composition

The results are shown in FIG. 37, in which A is a 365nm inspection before development; b is detected at 365nm after color development.

Strip schematic:

4. Test sample (batch No. 2020033003) 5.6-gingerol control

Analysis shows that:

7.3 differentiation test of ramulus Cinnamomi and radix Asteris under Low-humidity factor

Following the same procedure and temperature of example 4, the test was carried out at RH: influence of humidity factor in 32% environment on the identification effect of the composition of ramulus Cinnamomi and radix Asteris exfoliants.

The results are shown in FIG. 38, in which A is a 365nm inspection before development; b is the detection at 365nm after the color development of the aluminum trichloride test solution.

Strip schematic:

4. Sample (batch No. 2020033003) 5 Aster control drug

The analysis shows that:

7.4 Belamcanda chinensis and flos Farfarae identification test under low humidity factor

Following the same procedure and temperature as in example 4, the measured RH: the influence of the humidity factor in the environment of 32% on the identification effect of the combination of the blackberrykiky rhizome and the coltsfoot flower developing agent.

The results are shown in FIG. 39, in which A is a 254nm inspection before development; b is detection at 365nm before color development; c is detected under 365nm after the aluminum trichloride test solution develops color.

Strip schematic:

1. blackberry lily reference medicinal material 2, sample (batch number: 2020033001) 3, sample (batch number: 2020033002)

4. Sample (batch No. 2020033003) 5 reference flos Farfarae

Analysis shows that:

7.5 identification test of dried orange peel and asarum under low-humidity factor

Following the same procedure and temperature of example 4, the test was carried out at RH: influence of humidity factor in 32% environment on identification effect of pericarpium Citri Tangerinae and herba asari developer composition.

The results are shown in FIG. 40.

Strip schematic:

1. reference material 2, sample (batch No. 2020033001) 3, sample (batch No. 2020033002)

4. Test sample (batch No. 2020033003) 5 herba asari as control

Analysis shows that:

7.6 Alisma orientale discrimination test under low humidity factor

Following the same procedure and temperature as in example 4, the measured RH: influence of humidity factor in 32% environment on identification effect of the alisma orientale developing agent composition.

The results are shown in FIG. 41.

Strip shows that:

1. alisma orientale reference material 2. Test sample (batch No. 2020033001)

3. Sample (batch No. 2020033002) 4 sample (batch No. 2020033003)

Analysis shows that:

7.7 identification test of radix Glycyrrhizae and radix bupleuri under low humidity factor

Following the same procedure and temperature of example 4, the test was carried out at RH: influence of humidity factor in 32% environment on identification effect of the radix Glycyrrhizae and radix bupleuri developer composition.

The results are shown in FIG. 42, where A is a day-light examination after color development; b is detected at 365nm after color development.

Strip schematic:

7. Sample (batch No. 2020033002) 8 sample (batch No. 2020033003)

Analysis shows that:

Combining the results of example 6 and example 7 shows that: the method can effectively separate the medicinal materials under the conditions of normal temperature and low humidity and normal temperature and high humidity, does not influence the identification of the corresponding medicinal materials, and has no influence on the test due to the temperature.

Example 8 determination of optimization conditions for finger-print chromatography

Grouping experiment 8.1 chromatographic condition and system applicability pre-test

Octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 278nm; the column temperature is 30 ℃; the flow rate was 1.0ml/min. The number of theoretical plates should not be less than 5000 calculated according to baicalin peak.

Grouping experiment 8.2 selection of mobile phase System

8.2.1. Acetonitrile-0.1% phosphoric acid system

8.2.1.1 Instrument conditions

Liquid chromatograph: agilent1100 high performance liquid chromatograph

A chromatographic column: agela Venusil MP C18 (4.6X 250mm, 5m)

Column temperature: 30 deg.C

Sample injection amount: 20 μ l

Flow rate: 1ml/min

Detection wavelength: 278nm

Mobile phase:

8.2.1.2 standard sample preparation method

Taking 1.0g of a standard sample, precisely weighing, placing in a 50ml volumetric flask, adding water for dissolving, fixing the volume, shaking up, filtering, and taking a subsequent filtrate to obtain the product.

8.2.1.3 results

The chromatographic peak of the test sample is basically finished at 45min, and the mobile phase can be continuously adjusted, as shown in fig. 43: chromatogram of acetonitrile-0.1% phosphoric acid system

8.3.2 methanol-0.1% phosphoric acid system

8.3.2.1 apparatus conditions

A liquid chromatograph: agilent1100 high performance liquid chromatograph

A chromatographic column: agela Venusil MP C18 (4.6X 250mm, 5m)

Column temperature: 30 deg.C

Sample introduction amount: 20 μ l

Flow rate: 1ml/min

Detection wavelength: 278nm

Mobile phase:

8.3.2.2 Standard test article preparation method

Precisely weighing 1.0g of sample, placing in a 50ml volumetric flask, adding water for dissolving, fixing the volume, shaking up, filtering, and taking the subsequent filtrate.

8.3.2.3 results

The chromatographic peak of the sample is basically discharged in 70min, the column pressure is large, and the flowing phase proportion is inconvenient to adjust, as shown in figure 44: chromatogram of methanol-0.1% phosphoric acid system

8.3.3 conclusion

From the above experimental results, the acetonitrile-0.1% phosphoric acid system was selected as the composition of the mobile phase in consideration of the necessity of further adjustment of the chromatographic peak.

Grouping experiment 8.4 investigation of mobile phase gradients:

after the composition of the mobile phase is determined to be acetonitrile-0.1 percent phosphoric acid, different mobile phase gradients are inspected, and the separation effects of chromatographic peaks are compared.

8.4.1 apparatus conditions

A liquid chromatograph: agilent1100 high performance liquid chromatograph

A chromatographic column: agela Venusil MP C18 (4.6X 250mm, 5m)

Column temperature: 30 deg.C

Sample injection amount: 20 μ l

Flow rate: 1ml/min

Detection wavelength: 278nm

Mobile phase:

8.4.2 standard sample preparation method

8.4.3 results:

according to the chromatogram under each mobile phase gradient condition (as shown in figure 45: fingerprint under different mobile phase gradients), the best condition is method five, so that the gradient condition is used as the detection condition of the fingerprint of the lung-clearing and toxin-expelling decoction.

Grouping experiment 8.5 determination of detection wavelength

And comparing the detection wavelengths of the test sample solutions according to the determined mobile phase gradient conditions, and respectively comparing the maximum absorption wavelengths of 210nm, 250nm, 278nm and 325nm in the main chromatographic peaks of the test samples.

8.5.1 apparatus conditions

A liquid chromatograph: agilent1100 high performance liquid chromatograph

A chromatographic column: agela Venusil MP C18 (4.6X 250mm, 5m)

Column temperature: 30 deg.C

Sample introduction amount: 20 μ l

Flow rate: 1ml/min

Detection wavelength: 278nm

Mobile phase:

8.5.2 standard sample preparation method

8.5.3 results

According to the chromatograms under different detection wavelength conditions (such as fingerprints under different detection wavelengths in fig. 46), the best detection wavelength is 278nm, so that 278nm is used as the detection wavelength of the fingerprint of the lung-clearing and toxin-expelling decoction.

8.6 Final optimization parameters of fingerprint method

The best fingerprint spectrum conditions obtained according to the experiments are as follows, and the standard fingerprint spectrum is shown in figure 47 (the standard fingerprint spectrum of lung-clearing and toxin-expelling soup).

Liquid chromatograph: agilent1100 high performance liquid chromatograph

And (3) chromatographic column: agela Venusil MP C18 (4.6X 250mm, 5m)

Column temperature: 30 deg.C

Sample introduction amount: 20 μ l

Flow rate: 1ml/min

Detection wavelength: 278nm

Mobile phase:

example 9 determination of Standard fingerprint Pattern model

9.1 medicinal material attribution of chromatogram fingerprint peak

9.1.1 chromatographic conditions

A liquid chromatograph: agilent1100 high performance liquid chromatograph

A chromatographic column: agela Venusil MP C18 (4.6X 250mm, 5m)

Column temperature: 30 deg.C

Sample introduction amount: 20 μ l

Flow rate: 1ml/min

Detection wavelength: 278nm

Mobile phase:

9.1.2 preparation of test solutions

9.1.3 preparation of Single medicinal material solution

Respectively taking appropriate amount of medicinal materials according to the prescription proportion, decocting with water for 30min, filtering, and collecting the filtrate to obtain single medicinal material solution.

According to the most suitable TLC drug taste determined in examples 1-7, the single drug material is selected from the group consisting of Scutellariae radix control drug (lot number: 120955-201309), 6-gingerol control drug (lot number: 111833-201806), citrus aurantium fruit control drug (lot number: 120936-201606), ephedra sinica Stapf control drug (lot number: 121051-201606), asarum sieboldii Miq control drug (lot number: 121204-201606), cinnamomum cassia twig control drug (lot number: 121191-201605), farfarana control drug (lot number: 121449-201816), blackberry lily control drug (lot number: 120994-201801), citrus reticulata Tangerine peel control drug (lot number: 12096969-201510), aster tataricus control drug (lot number: 120956-200505), radix bupleuri control drug (lot number: 120992-201509), radix Glycyrrhizae Preparata control drug (lot number: 120904-201519) and Alisma orientale control drug (lot number: 120121-201803) are all identified in biological products research institute, and other biological reagents are analyzed.

9.1.4 chromatographic fingerprint peaks

And respectively injecting 20 mul of sample solution and single medicinal material solution for detection to obtain chromatograms, and attributing chromatograms with similar retention time and similar spectrogram on the chromatograms of the sample and the single medicinal material, wherein the result is shown in figure 48 (a qualitative fingerprint spectrum mass spectrum model of a primary screen of lung-clearing and toxin-expelling soup). The result shows that the qualitative fingerprint spectrum mass spectrum model for performing quality preliminary screening on the lung-clearing and toxin-expelling decoction can be selected to be established, wherein the qualitative fingerprint spectrum mass spectrum model is from scutellaria baicalensis, immature bitter orange, common coltsfoot flower, liquorice and blackberry lily and has the advantages of good discrimination, prominent fingerprint peaks and small mutual interference.

9.2 component determination in chromatogram fingerprint Peak Picture model

9.2.1 chromatographic conditions

A liquid chromatograph: agilent1100 high performance liquid chromatograph

A chromatographic column: agela Venusil MP C18 (4.6X 250mm, 5m)

Column temperature: 30 deg.C

Sample introduction amount: 20 μ l

Flow rate: 1ml/min

Detection wavelength: 278nm

Mobile phase:

9.2.2 preparation of test solutions

9.2.3 preparation of control solutions

Taking appropriate amount of naringin reference substance, hesperidin reference substance, neohesperidin reference substance, baicalin reference substance, and wogonoside reference substance, precisely weighing, and adding methanol to obtain solutions containing naringin 40 μ g, hesperidin 40 μ g, neohesperidin 40 μ g, wogonoside 40 μ g, and baicalin 100 μ g per 1 ml. .

9.2.4 identification of the composition of the chromatographic Peak

Respectively injecting sample solution to be tested and reference solution 10 μ l for detection to obtain chromatogram, and comparing the chromatogram of the test sample and the chromatogram of the reference to obtain the results shown in figure 49 and figure 50. As a result, the peaks of the five control samples can be found in the chromatogram of the test sample. Shown in the figure: 6: naringin; 7: hesperidin; 8: neohesperidin; 9: baicalin (S); 12: wogonoside.

Example 10 detection of a product to be tested Using the determined Standard fingerprint model

10.1 qualitative preliminary screening detection of fingerprint pattern model for different Chinese medicinal granules

The test article 1, the test article 2, the test article 3, the test article 4 and the test article 5 are respectively selected from the lung-heat clearing and toxin expelling decoction particles, the small bupleurum decoction particles, the kudzuvine root decoction particles, the liquorice heart-fire purging decoction particles and the angelica sinensis Sini decoction particles in a blind mode.

Weighing 1g of the above 5 unknown test products, respectively, precisely weighing, placing in a 50ml volumetric flask, adding water to dissolve and fix the volume, shaking up, filtering, and taking the subsequent filtrate to obtain the final product.

10 μ l of sample was injected for chromatogram detection according to the following parameters:

a liquid chromatograph: agilent1100 high performance liquid chromatograph

A chromatographic column: agela Venusil MP C18 (4.6X 250mm, 5m)

Column temperature: 30 deg.C

Sample introduction amount: 20 μ l

Flow rate: 1ml/min

Detection wavelength: 278nm

Mobile phase:

fingerprints of 5 unknown samples to be tested were obtained according to the methods of examples 8 to 9 (FIGS. 51 to 55).

The detection is performed according to the confirmed fingerprint method, the spectrum is derived, the similarity with the qualitative fingerprint model established in the embodiment 9 is calculated according to the traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2004 version A), and the result is shown as follows.

Name(s)	Sample to be tested 1	Sample 2 to be tested	Sample to be tested 3	Sample to be tested 4	Sample to be tested 5
						Similarity of the two	0.563	0.991	0.291	0.581	0.201

Wherein, the similarity of the fingerprint of the sample 2 to be detected is more than 0.90, and the similarity of the rest samples is far lower than 0.90, so that the sample 2 to be detected is the lung-clearing and toxin-expelling soup.

By checking the predicted sample names, it is determined that the samples 1-5 (fig. 51-55) to be tested are XIAOCHAIHU decoction granule, QINGFEIXIETING decoction granule, GEGEN decoction granule, GANCAOXINGZHI decoction granule, and DANGGUISINISHI decoction granule, respectively.

The result shows that the qualitative mass spectrum model established in the embodiment 9 can identify the lung clearing and toxin expelling soup.

10.2 qualitative preliminary screening detection of fingerprint pattern model for different batches of lung-heat clearing and toxin expelling decoction granules

According to the method of the embodiment 10.1, 10 batches of lung clearing and toxin expelling decoction particles are respectively measured according to a proposed fingerprint method, the spectrum is derived, the similarity is calculated according to a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2004A version), and the result is shown as follows.

The result shows that the similarity of the fingerprint spectra of different batches of lung clearing and toxin expelling decoction granules is more than 0.93, so the similarity is set to be not less than 0.90.

10.3 quantitative preliminary screening detection of fingerprint pattern model for certain batch of lung-heat clearing and toxin expelling decoction granules

Taking appropriate amount of naringin reference substance, hesperidin reference substance, neohesperidin reference substance, baicalin reference substance, and wogonoside reference substance, precisely weighing, and adding methanol to obtain solutions containing naringin 40 μ g, hesperidin 40 μ g, neohesperidin 40 μ g, wogonoside 40 μ g, and baicalin 100 μ g per 1 ml.

According to the method of example 9, a sample solution (lot No. 20200330) to be tested and a naringin control, a hesperidin control, a neohesperidin control, a baicalin control, and a wogonoside control were injected in an amount of 10. Mu.l and detected to obtain a chromatogram.

Comparing the peak intensity difference between the test solution and the reference solution, and determining the contents of naringin, hesperidin, neohesperidin, baicalin and wogonoside in the test solution according to the standard concentration of the reference solution, the results are as follows:

example 11 preparation method of lung clearing and toxin expelling soup relating to quality monitoring process

Example 11.1 optimization of extraction Process

Orthogonal experiments are adopted, the baicalin transfer rate and the paste yield are taken as investigation indexes, and the optimal process extraction conditions are screened.

Selecting decoction time, decoction times and water addition amount which influence the extraction effect as main investigation factors, and performing a 4-factor 3 horizontal orthogonal optimization test, wherein the test scheme is shown in table 1.

Weighing 100g of decoction pieces according to the proportion of the prescription, weighing 9 parts of the decoction pieces in parallel, extracting according to the table 2, filtering the extracting solution, measuring the volume of the filtrate, and recording. Calculating the extraction rate and index component content of the extractive solution, and showing the results in the table

Table 1: orthogonal experiment factor level gauge

Table 2: orthogonal experiment table

Table 3: results of orthogonal experiments

The results in Table 3 were analyzed for variance, and are shown in Table 4.

Table 4: analysis of variance table

Note: f0.05 (2, 2) =19.00, F0.01 (2, 2) =99.00; * : p < 0.05, x: p is less than 0.01;

according to range analysis and variance analysis, the three factors have significant influence on the cream yield, and the preferable scheme is A2B2C3; three factors have significant influence on the transfer rate of baicalin, the preferred scheme is A2B2C3, and in order to save cost and reduce energy consumption, the A2B2C2 is selected as the optimal process, namely decocting for 2 times, adding 10 times of water each time, and decocting for 1 hour each time.

EXAMPLE 11.2 screening study of concentration temperature

The stability under different concentration temperature conditions is evaluated by concentrating the extract at 60 deg.C, 75 deg.C and 90 deg.C and detecting its index components.

(1) Sample preparation

Weighing 100g of decoction pieces according to the proportion of the prescription, preparing a sample according to the determined extraction method of the decoction for clearing lung and expelling toxin, dividing the prepared extracting solution into 3 parts, each part is 250ml, respectively carrying out reduced pressure concentration at 60 ℃, 75 ℃ and 90 ℃ to 50ml, and then diluting the sample to 250ml, thus obtaining three investigation samples.

(2) Detection assay

The 3 samples re-diluted after concentration were subjected to baicalin content measurement, and the difference before and after concentration was compared.

(3) Results of the experiment

Table 5: index components of sample under different concentration temperature conditions

	Baicalin content (mg/g)
		Unconcentrated sample	1.21
Concentrating at 60 deg.C	1.20
		Concentrating at 75 deg.C	1.19
Concentrating at 90 deg.C	1.21

The results show that the concentration is carried out at 60 ℃, 75 ℃ and 90 ℃, the different concentration temperatures have no influence on the content of the baicalin which is an index component in the liquorice diarrhea decoction, and the results show that the sample is concentrated at 60-90 ℃, and the sample is kept stable.

Example 11.3 screening Studies for impurity removal

The conventional impurity removal modes of the extracting solution comprise 2 modes, namely screen filtration and centrifugal impurity removal, and the 2 impurity removal modes are compared.

(1) Mode of removing impurities

Mode of removing impurities	Parameter(s)
		Screen filtration	300 mesh
High speed centrifugation	10000rpm/min

(2) Sample(s)

Weighing 100g of decoction pieces according to the proportion of the prescription, preparing a sample according to the determined extraction method of the decoction for clearing lung and expelling toxin, taking 2 parts of extracting solution, respectively adopting a 300-mesh screen to filter and adopting a 10000rpm/min centrifugation mode for 5 minutes, and detecting the sample after impurity removal.

(3) Detection assay

Detecting 2 samples according to a method for measuring the content of baicalin in the lung-clearing and toxin-expelling decoction.

(4) Results of the experiment

Table 6: appearance character of 2 impurity removal modes

Mode of removing impurities	Appearance character	Standing for precipitation
			Centrifugation	Clarification	No precipitate is generated after standing
Filtration	Turbidity	Standing to generate precipitate

Table 7:2 index component comparison of impurity removal modes

The results show that the baicalin content in the liquid medicine is not obviously different in the 2 impurity removal modes by adopting a centrifugal mode and a 300-mesh screen filtration mode, but the sample obtained by the filtration method is turbid in appearance, precipitates are generated in the process of placement, and the centrifugal sample does not have the problems of turbidity and precipitation, so that the centrifugal impurity removal mode is superior to the 300-mesh screen filtration mode, and centrifugation is selected as the impurity removal mode.

Example 11.4 screening study of drying regimes

And (3) performing two drying modes of quasi-spray drying and reduced pressure drying to select a proper drying mode.

(1) Sample preparation

Weighing 100g of decoction pieces according to the proportion of the prescription, paralleling 2 parts, preparing an extracting solution according to an extracting process, concentrating under reduced pressure (65 ℃), adding maltodextrin, and drying. And (4) investigating the influence of vacuum drying and spray drying on the index content of the extracting solution.

(2) Detection assay

Detecting 2 samples according to a method for measuring the content of baicalin in the lung-heat clearing and toxin expelling decoction.

(3) Results of the experiment

Table 7: results of different drying modes

Drying mode	Total amount of baicalin (mg/g)
		Spray drying (165 ℃ -175 ℃ C.)	1.53
Drying under reduced pressure (75 ℃ C.)	1.48

From the above results, it can be seen that the baicalin content in the sample in the spray drying mode is slightly higher than that in the sample in the reduced pressure mode because the spray drying is instantaneous high temperature drying, the sample is heated for a short time, and the component is less affected by heat, so the spray drying is selected as the drying mode.

Example 11.5 adjuvant screening study

The common auxiliary materials for the traditional Chinese medicine granules comprise soluble starch, maltodextrin, lactose and the like, the type of the auxiliary materials can influence the formability, the stability and the total preparation amount of finished products, and the type of the auxiliary materials is necessary to be considered. 10g of granules are prepared according to 50g of crude drugs, and the influence of the auxiliary materials on the granulation process is examined under the condition of adopting a boiling granulation mode.

Table 8: effect of adjuvants on the granulation Process

The results show that the three auxiliary materials can prepare the product meeting the requirements, and the yield of the finished product has no obvious difference. Because the dissolubility of the soluble starch is slightly poor, the clarity of the finished product solution is poorer than that of the rest two solutions; lactose is easy to absorb moisture and has higher cost price, so maltodextrin is selected as an auxiliary material.

In summary, as shown in fig. 56, the preparation process of the lung-clearing and toxin-expelling soup is determined as follows:

in any of the above embodiments, the centrifuged fluid extract is taken, added with a proper amount of maltodextrin (the total amount of each prescription is 40g, and the solid content of the centrifuged fluid extract is about 10.5% of the prescription), dissolved, mixed uniformly, and spray-dried to obtain extract powder;

Wherein, after finishing the whole grain process, samples are randomly extracted, and the thin layer chromatography identification is carried out on ephedra herb, bitter apricot seed, honey-fried licorice root, cassia twig, oriental waterplantain rhizome, largehead atractylodes rhizome, chinese thorowax root, baical skullcap root, ginger, tatarian aster root, common coltsfoot flower, blackberry lily rhizome, manchurian wildginger, immature bitter orange, tangerine peel and wrinkled gianthyssop herb according to the 5 determined developing agent combinations in the embodiment 4.

And determining whether the product meets the quality standard of Chinese pharmacopoeia according to the identification result. And if the quality is judged to be qualified according to the thin-layer chromatography result, performing subsequent procedures such as total mixing and the like. And if the thin layer chromatography result is judged to be unqualified, stopping production. All raw materials and production procedures were checked.

Claims

1. A production method of lung clearing and toxin expelling soup established based on a fingerprint model comprises the following steps: the main medicine components of the prescription are as follows: 9g of ephedra, 6g of honey-fried licorice root, 9g of almond, 15-30 g of gypsum, 9g of cassia twig, 9g of rhizoma alismatis, 9g of grifola, 9g of bighead atractylodes rhizome, 15g of poria cocos, 16g of radix bupleuri, 6g of scutellaria baicalensis, 9g of ginger processed pinellia tuber, 9g of ginger, 9g of aster, 9g of tussilago farfara, 9g of blackberry lily, 6g of asarum, 12g of Chinese yam, 6g of immature bitter orange, 6g of dried orange peel and 9g of wrinkled gianthyssop herb; decocting the twenty-one medicines with water twice, filtering, combining the decoctions, concentrating the filtrate under reduced pressure to obtain clear paste, adding a proper amount of maltodextrin, drying or crushing to prepare lung-clearing and toxin-expelling decoction granules, carrying out quality detection on the prepared granules, and guiding the product to be produced in a standardized way according to the quality detection result, and is characterized in that: the quality detection method comprises the following steps:

(1) Establishing a simplified qualitative fingerprint chromatogram model, comprising:

(3) analyzing fingerprint parameters of 5 single medicines, and establishing simplified fingerprint pattern model, wherein the time of emergence of baical skullcap root fingerprint peak is about 27min, 29min, 33min, 78min, 86min, 88min and 104min; the peak emergence time of fingerprint peak of fructus Aurantii Immaturus is about 42min, 47min, 53min, 61min; the peak appearance time of the fingerprint peak of the coltsfoot flower is about 46min and 65min; the time to peak of the fingerprint peak of licorice was about 82min; the peak emergence time of the belamcanda chinensis fingerprint peak is about 95min;

(2) Taking 1.0g of sample particles, precisely weighing, placing in a 50ml volumetric flask, adding water for dissolving, fixing the volume, shaking up, filtering, and taking subsequent filtrate;

(4) Comparing whether the chromatogram of the test solution is consistent with the chromatographic peak of the qualitative fingerprint chromatogram model flowing out at the peak-off time of 25-105min, if so, carrying out the next detection, and if not, directly judging that the sample to be detected is unqualified;

(5) Respectively injecting 10 μ l sample of the test solution and naringin reference substance, hesperidin reference substance, neohesperidin reference substance, baicalin reference substance, and wogonoside reference substance solution, and detecting to obtain chromatogram;

(6) Comparing whether the chromatographic peaks and mAU values flowing out in 45-90min of the test solution and the reference solution are consistent, if so, judging that the sample to be detected meets the primary quality standard, and if not, directly judging that the sample to be detected is unqualified;

(7) Comparing the peak intensity difference of the test solution and the reference solution in the step (6), and determining the contents of naringin, hesperidin, neohesperidin, baicalin and wogonoside in the test solution according to the standard concentration of the reference solution;

wherein, the sample introduction chromatographic parameters in the steps (1), (3) and (5) are as follows:

a chromatographic column: agela Venusil MP C18, 4.6X 250mm,5 μm;

column temperature: 30 ℃, sample size: 20 μ l, flow rate: 1ml/min, detection wavelength: 278nm, mobile phase:

time (min) acetonitrile (%) 0.1% phosphoric acid (%)

0 15 85

70 20 80

110 60 40。

2. The production method of lung clearing and toxin expelling soup based on the fingerprint spectrum model as set forth in claim 1, wherein:

wherein the step (5) and the step (6) are used for establishing a quantitative fingerprint chromatogram model, in the step (5), a proper amount of naringin reference substance, hesperidin reference substance, neohesperidin reference substance, baicalin reference substance and wogonoside reference substance is precisely weighed, and methanol is added to prepare solutions containing 40 mu g of naringin, 40 mu g of hesperidin, 40 mu g of neohesperidin, 40 mu g of wogonoside and 100 mu g of baicalin per 1ml respectively so as to obtain a reference substance solution; the peak appearance time of the fingerprint peak of naringin to be detected in the test sample is about 47min, the peak appearance time of the fingerprint peak of hesperidin to be detected in the test sample is about 52min, the peak appearance time of the fingerprint peak of neohesperidin to be detected in the test sample is about 60min, the peak appearance time of the fingerprint peak of baicalin to be detected in the test sample is about 77min, and the peak appearance time of the fingerprint peak of wogonoside to be detected in the test sample is about 87min.

3. The production method of lung clearing and toxin expelling soup established based on the fingerprint spectrum model according to claim 1 or 2, characterized in that:

wherein the process of judging consistency in step (6) comprises: taking the corresponding peak of the baicalin reference substance peak as an S peak, and calculating the relative retention time and the relative peak area of each main peak and the S peak; wherein the similarity between the test sample fingerprint and the comparison fingerprint is not less than 0.90, and the relative retention time and the relative peak area are within +/-10% of the specified value, which indicates that the test sample to be tested accords with the primary quality standard, and if the standard is not met, the test sample to be tested is directly judged to be unqualified.

4. The production method of lung clearing and toxin expelling soup established based on the fingerprint spectrum model according to claim 3, is characterized in that:

wherein twenty-one medicine is decocted with water twice, wherein 10 times of water is added for the first time, and the mixture is soaked for 0.5 hour and decocted for 1.0 hour; adding 10 times of water for the second time, decocting for 1.0 hour, merging decoction, filtering, concentrating the filtrate at 70 ℃ under reduced pressure to obtain clear paste with the relative density of 1.03-1.05, adding a proper amount of maltodextrin, drying or crushing, and preparing into granules.

5. The production method of lung clearing and toxin expelling soup established based on the fingerprint spectrum model according to claim 4, is characterized in that:

wherein the total amount of each prescription is 40g, the solid content of the centrifuged clear paste is about 10.5% of the prescription amount, and then a proper amount of maltodextrin is added, dissolved, mixed evenly and spray-dried to obtain extract powder; then adding a proper amount of adhesive, boiling and granulating, finishing granules, preparing 1000g of granules, subpackaging the granules into 10 g/bag, and packaging with a composite film.

6. A method for carrying out quality control on the production of a lung-clearing and toxin-expelling decoction preparation by combining a fingerprint model and a thin-layer chromatography comprises the steps of adding water into twenty-one medicines for decocting twice, filtering, combining decoction, concentrating filtrate under reduced pressure to obtain clear paste, adding a proper amount of maltodextrin, drying or crushing to prepare lung-clearing and toxin-expelling decoction particles, carrying out quality detection on the prepared particles, and guiding the product to be produced in a standardized way according to the quality detection result, and is characterized in that: the quality detection method comprises the following steps:

the detection steps (1) to (7) of the above claim 1, and,

(8) Taking a sample aqueous solution to be detected, adding an extracting agent for extraction, and combining extract liquor;

(10) Respectively sucking a test sample solution, a positive control solution and a negative control solution, respectively placing the test sample solution, the positive control solution and the negative control solution on the same silica gel G thin-layer plate, respectively developing the test sample solution, the positive control solution and the negative control solution on a thin-layer identification developing agent, and taking out the test sample solution and the negative control solution;

(12) Comparing the spots of the test sample, the positive control and the negative control, and if the spots of the same color appear at the corresponding positions of the test sample and the positive control and the spots of no interference appear in the negative control, judging that the test sample contains the medicine components with the same or similar quality as the positive control; wherein, the first and the second end of the pipe are connected with each other,

and (3) adding ammonia water into the ephedra and the immature bitter orange in the step (8), extracting for 2 times by using n-butyl alcohol, combining extract liquor, and mixing the extract liquor by using the ratio of n-butyl alcohol: glacial acetic acid: the volume ratio of water is 4:1:5 as developing agent;

for scutellaria baicalensis and ginger, after adding hydrochloric acid in step (8), extracting with ethyl acetate for 2 times, combining the extracts, and adding toluene: ethyl acetate: methanol: the volume ratio of formic acid is 10:3:1:2 is a developing solvent;

and (3) adding ethyl acetate into asarum, blackberry lily, cassia twig, common coltsfoot flower, dried orange peel and aster in the step (8) for extraction for 2 times, combining the extracts, and adding ethyl acetate: methanol: the volume ratio of water is 100:17:13 as developing agent, then using toluene: ethyl acetate: formic acid: the volume ratio of water is 20:10:1:1, taking the upper solution of the solution as a developing agent to carry out secondary development;

and (3) adding petroleum ether with a boiling range of 60-90 ℃ into the rhizoma alismatis in the step (8), extracting for 2 times, combining the extracts, and adding chloroform: ethyl acetate: the volume ratio of formic acid is 6:3.5:0.5 solution is used as a developing agent;

for liquorice, ethyl acetate is added in the step (8) for extraction for 2 times, the extracts are combined, and the mixture is added with chloroform: methanol: the volume ratio of water is 13:6:1 as developing agent;

and (3) adding n-butanol in the step (8) for extracting for 2 times, combining the extracts, and adding chloroform: methanol: the volume ratio of water is 13:6: the solution of 1 is a developing agent.

7. The method for controlling the quality of the lung-heat clearing and toxin expelling decoction preparation by combining the fingerprint model and the thin-layer chromatography according to claim 6, wherein the method comprises the following steps:

wherein for ephedra and immature bitter orange, in the step (9), 0.5g of each of positive and negative control medicinal materials is additionally taken, a proper amount of water is respectively added, reflux extraction is carried out for 60 minutes, cooling is carried out, filtration is carried out, filtrate is concentrated to 20ml, 1ml of ammonia water is respectively added, extraction is carried out for 2 times by using n-butanol, 15ml of each time is carried out, extract liquor is combined, water bath evaporation is carried out, and residues are dissolved by using 1ml of methanol to serve as a control medicinal material solution; taking 2g of sample particles to be detected, adding 20ml of water for dissolving, adding 1ml of ammonia water, extracting for 2 times by using n-butanol, 10ml each time, combining extract liquor, evaporating by using a water bath, and dissolving residues by using 1ml of methanol to be used as a test solution; spraying 0.5% ninhydrin ethanol solution in the step (11), and drying at 105 ℃ until spots are clear; and/or the presence of a gas in the gas,

for scutellaria baicalensis and ginger, in the step (8), 2g of sample particles to be detected are taken, 20ml of water is added for dissolving, 1ml of hydrochloric acid is added for extracting for 2 times by ethyl acetate, 10ml of hydrochloric acid is added for each time, the extract liquid is combined, the extract liquid is evaporated in a water bath, and the residue is dissolved by 1ml of methanol to be used as a test solution; taking 0.3g of each of the positive and negative control medicinal materials, adding 20ml of ethyl acetate, carrying out ultrasonic treatment for 30min, filtering, evaporating filtrate to dryness, dissolving residues by using 1ml of methanol to obtain a control medicinal material solution; spraying a 2% vanillin sulfuric acid solution in the step (11), and drying at 105 ℃ until spots are clear.

8. The method for controlling the quality of lung-heat clearing and toxin expelling decoction preparation production by combining the fingerprint model and the thin layer chromatography according to claim 6 or 7, wherein for asarum, blackberrykiky rhizome, cassia twig, tussilago farfara, dried orange peel and aster, the step (8) is to take 2g of sample particles to be tested, add 20ml of water for dissolution, extract 10ml each time for 2 times, combine the extracts, evaporate the extracts in water bath, and dissolve the residues with 1ml of methanol as a test solution; taking 0.5g of each positive control medicinal material and 0.5g of each negative control medicinal material, respectively adding a proper amount of water, carrying out reflux extraction for 60 minutes, cooling, filtering, concentrating the filtrate to 20ml, respectively extracting with ethyl acetate for 2 times, 20ml each time, combining the extract, drying by evaporation in a water bath, and dissolving the residue with 1ml of methanol to obtain a control medicinal material solution; and (11) displaying the same fluorescent spots on the corresponding positions of the chromatogram of the reference medicinal material, spraying an aluminum trichloride test solution, and placing under an ultraviolet lamp for inspection.

9. The method for controlling the quality of the lung-heat clearing and toxin expelling decoction preparation by combining the fingerprint model and the thin-layer chromatography as claimed in claim 8, is characterized in that:

wherein for the rhizoma alismatis, the step (8) is to take 2g of finished product particles, add 20ml of water for dissolving, extract for 2 times by 20ml of petroleum ether with the boiling range of 60-90 ℃, combine the extract liquor, evaporate the extract liquor by distillation in a water bath, and dissolve the residue by 1ml of methanol to be used as a test solution; taking 0.5g of each of the positive and negative control medicinal materials, respectively adding a proper amount of water, refluxing and extracting for 60 minutes, cooling, filtering, concentrating the filtrate to 20ml, extracting for 2 times with petroleum ether at 60-90 ℃ and 20ml each time, combining the extract, evaporating in a water bath, and dissolving the residue with 1ml of methanol to obtain a control medicinal material solution; spraying 10% sulfuric acid ethanol solution in step (11), heating at 105 deg.C until the spots are clearly developed, and inspecting under ultraviolet light.

10. The method for controlling the quality of the lung-heat clearing and toxin expelling decoction preparation by combining the fingerprint model and the thin-layer chromatography according to claim 9, wherein the method comprises the following steps:

wherein for radix bupleuri and licorice, 0.5g of licorice control material is taken in the step (9), a proper amount of water is added, reflux extraction is carried out for 60 minutes, cooling is carried out, filtration is carried out, filtrate is concentrated to 20ml, ethyl acetate is used for extraction for 2 times, 20ml is carried out each time, extract liquor is combined, water bath evaporation is carried out, and residue is dissolved by 1ml of methanol to be used as a control material solution; taking 1.0g of radix bupleuri reference medicinal material, adding an appropriate amount of water, extracting under reflux for 60 minutes, cooling, filtering, concentrating the filtrate to 20ml, extracting with n-butanol for 2 times, 20ml each time, mixing the extracts, washing with ammonia test solution for 2 times, discarding, collecting n-butanol layer, evaporating in water bath, and dissolving the residue with 1ml of methanol to obtain radix bupleuri reference medicinal material solution;

spraying 10% sulfuric acid ethanol solution of 1% p-dimethylaminobenzaldehyde in the step (11), blowing hot air to the spots to develop clear color, and respectively inspecting under sunlight and ultraviolet light.