CN109765322B

CN109765322B - Method for constructing characteristic spectrum of schizonepeta and quality detection method

Info

Publication number: CN109765322B
Application number: CN201811646360.3A
Authority: CN
Inventors: 迟玉明; 姚璐; 王淑英; 储兆铮; 李铮; 颜士华
Original assignee: Beijing Zhongyan Tongrentang Chinese Medicine R & D Co ltd
Current assignee: Beijing Tongrentang Pharmaceutical Co Ltd; Beijing Zhongyan Tongrentang Chinese Medicine R & D Co ltd
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2022-02-15
Anticipated expiration: 2038-12-29
Also published as: CN109765322A

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a characteristic spectrum construction method and a quality detection method of schizonepeta, wherein a characteristic spectrum of non-volatile components of schizonepeta is established by controlling liquid chromatography conditions, mobile phase compositions and an elution program; establishing a herba schizonepetae volatile component characteristic spectrum by selecting proper gas chromatography conditions; marking characteristic peaks of the characteristic map; measuring the content of hesperidin in herba Schizonepetae by adjusting the elution procedure of liquid chromatography; meanwhile, a schizonepeta quality control method is established, the schizonepeta is evaluated by utilizing the schizonepeta characteristic spectrum, the content of hesperidin is measured, the schizonepeta is further subjected to thin-layer chromatography identification, heavy metal and harmful element inspection, pesticide residue measurement and extract measurement, the schizonepeta is evaluated in terms of effectiveness and safety, and the quality detection method has the advantages of simplicity, rapidness, stability, reliability, high precision, good reproducibility, easiness in mastering and the like.

Description

Method for constructing characteristic spectrum of schizonepeta and quality detection method

Technical Field

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a construction method and a quality detection method of a characteristic spectrum of schizonepeta.

Background

Herba schizonepetae, being slightly warm in nature and pungent in taste, has the effects of relieving exterior syndrome, dispelling wind, promoting eruption and eliminating sores, is used for treating cold, headache, measles, rubella and pyocutaneous disease, and is one of the commonly used traditional Chinese medicinal materials. Today, the quality monitoring of Chinese medicinal materials is very important in the growing concern of the quality of the medicaments. With the development of the chromatographic technology, the quality standard of the schizonepeta medicinal material not only comprises the aspects of simple characters, identification, routine inspection, extract measurement and the like, but also comprises the measurement of a high performance liquid fingerprint. The traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification means, and is one of the quality control modes which are currently in line with the characteristics of traditional Chinese medicines and evaluate the authenticity, stability and consistency of the traditional Chinese medicines.

The fingerprint spectrum mainly has the function of reflecting the uniformity and stability of the internal quality of the traditional Chinese medicine containing complex components and the preparation thereof. The chromatograph expert believes that the higher the degree of separation, the better the peaks separated; the information processing expert also considers that the more data that is given the better. However, the international conference on coordination about the precision and accuracy of quantitative analysis says that "the more precise and more accurate the analysis result is, but the requirement of the analysis object is met", that is, the more peaks of the fingerprint are, the better the peak is, but the purpose of forming the fingerprint characteristic is achieved, and as a medicine, on the basis of ensuring the safety, the purpose of monitoring the effective components is achieved.

The main effective components of the schizonepeta are reported to comprise volatile oil, various flavonoid compounds, monoterpene glycoside and the like, wherein the main components of the volatile oil are pulegone and menthone which can be used as representative substances of the volatile oil; the hesperidin content in the flavonoid component is high, and the flavonoid component has a certain pharmacological action. The existing Chinese literature, "HPLC method simultaneously determines the content of 4 ingredients in herba schizonepetae formula particles" (Journal of Chinese Medicinal Materials, 2016 (10) month, volume 39, period 10 2285-. However, the method uses the formula particles, and the raw material schizonepeta medicinal material or decoction pieces are not controlled from the source, while the effective component pulegone is partially contained in the nonvolatile component and partially contained in the volatile oil, and the menthone is also the effective component of the schizonepeta, and the menthone is mainly contained in the volatile oil, so the method cannot carry out comprehensive quality control on the effective component of the schizonepeta.

The quality control of the schizonepeta medicinal material is further improved, safety indexes such as active ingredients, heavy metals, harmful elements, pesticide residue and the like have important influence in the practical application process, and the medication safety is influenced, so that the control of the effectiveness of the traditional Chinese medicine is more realistic by combining the traditional Chinese medicine quality control of safety control, and a comprehensive, effective and safe quality control method of the schizonepeta is established.

Disclosure of Invention

Therefore, the first technical problem to be solved by the invention is to overcome the defect that effective ingredients related to the schizonepeta characteristic map are not comprehensive enough in the prior art, so that the schizonepeta characteristic map construction method is provided, the schizonepeta characteristic map construction method comprises the construction of a non-volatile effective ingredient characteristic map and a volatile effective ingredient characteristic map, and the construction of characteristic chromatographic peaks of the effective ingredients of the schizonepeta is more comprehensive.

The second technical problem to be solved by the invention is to overcome the defect that the prior art can not carry out comprehensive quality control on the schizonepeta from two aspects of effectiveness and safety, and establish a schizonepeta quality detection method.

The invention discloses a method for constructing a herba schizonepetae characteristic spectrum, which comprises a method for constructing a non-volatile component characteristic spectrum and a method for constructing a volatile component characteristic spectrum, wherein the method comprises the following steps:

A. the method for constructing the non-volatile component characteristic spectrum comprises the following steps:

preparation of test solution I: preparing a schizonepeta test sample into a test sample solution I;

preparation of reference solution I: preparing a herba schizonepetae reference medicinal material into a reference medicinal material solution I; preparing a reference substance solution I of the hesperidin reference substance;

chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, and using octadecyl bonded silica gel as chromatographic column filler; taking acetonitrile as a mobile phase A, and taking a phosphoric acid aqueous solution with the volume fraction of 0.2% as a mobile phase B for elution;

the determination method comprises the following steps: precisely absorbing the reference substance solution I and the test solution I respectively, injecting into a liquid chromatograph, and measuring to obtain the final product; and

B. the construction method of the volatile component characteristic map comprises the following steps:

preparation of test solution II: preparing volatile oil prepared from a schizonepeta test sample to prepare a test sample solution II;

preparation of reference solution II: preparing reference substance solution II of reference substance from pulegone and menthone respectively;

chromatographic conditions and system applicability test: measuring by gas chromatography, using N2 as carrier gas and PEG as stationary phase, and controlling injection port temperature, detector temperature, column temperature and flow rate;

the determination method comprises the following steps: precisely absorbing the reference substance solution II and the sample solution II respectively, injecting into a gas chromatograph, and measuring to obtain the final product.

Preferably, the method also comprises the construction of a comparison characteristic spectrum, and the method comprises the steps of analyzing the characteristic spectrum of at least 15 batches of the obtained test solution I by utilizing a traditional Chinese medicine chromatography fingerprint similarity evaluation system of the State pharmacopoeia Committee to generate a non-volatile component comparison characteristic spectrum; and/or

And analyzing the gas phase characteristic spectrum of at least 15 batches of the obtained test solution II by using a traditional Chinese medicine chromatogram fingerprint similarity evaluation system of the State pharmacopoeia Committee to generate a volatile component comparison characteristic spectrum.

Preferably, the non-volatile component characteristic spectrum comprises 6 characteristic peaks, and the characteristic peaks are as follows: the No. 1 peak is hesperidin, the No. 2 peak is rosmarinic acid, the No. 3 peak, the No. 4 peak and the No. 5 peak are common characteristic peaks, and the No. 6 peak is pulegone; taking the peak No. 1 as a reference peak, and the relative retention time of each peak is respectively as follows: peak No. 1: 1.00; peak No. 2: 1.07; peak No. 3: 1.09; peak No. 4: 1.20; peak No. 5: 1.25; peak No. 6: 1.64 of; and/or

The volatile component characteristic spectrum comprises 3 characteristic peaks, wherein the characteristic peaks are as follows: the No. 1 peak is menthone, the No. 2 peak is pulegone, and the No. 3 peak is a common characteristic peak; taking the peak No. 2 as a reference peak, and the relative retention time of each peak is respectively as follows: peak No. 1: 0.67; peak No. 2: 1.00; peak No. 3: 1.50.

preferably, in the method for constructing the non-volatile component feature map, the elution mode is a gradient elution mode, and the gradient elution procedure is as follows: 0-15min, mobile phase A: the volume ratio of the mobile phase B is 8-12%: 92-88% → 12-16%: 88 to 84 percent; 15-40min, mobile phase A: the volume ratio of the mobile phase B is 12-16%: 88-84% → 24-28%: 76 to 72 percent; 40-55min, mobile phase A: the volume ratio of the mobile phase B is 24-28%: 76-72% → 58-62%: 42 to 38 percent; 55-60min, mobile phase A: the volume ratio of the mobile phase B is 58-62%: 42-38% → 83-87%: 17 to 13 percent; 60-65min, mobile phase A: the volume ratio of the mobile phase B is 83-87%: 17-13% → 83-87%: 17 to 13 percent;

preferably, the gradient elution procedure is: 0-15min, mobile phase A: the volume ratio of the mobile phase B is 10%: 90% → 14%: 86 percent; 15-40min, mobile phase A: the volume ratio of the mobile phase B is 14%: 86% → 26%: 74 percent; 40-55min, mobile phase A: the volume ratio of the mobile phase B is 26%: 74% → 60%: 40 percent; 55-60min, mobile phase A: the volume ratio of the mobile phase B is 60%: 40% → 85%: 15 percent; 60-65min, mobile phase A: the volume ratio of the mobile phase B is 85%: 15% → 85%: 15 percent;

preferably, the chromatographic conditions are: the column temperature is 25-40 ℃; the flow rate is 0.8-1.2 ml/min; the detection wavelength is 278-288 nm;

preferably, the chromatographic conditions are: the column temperature was 40 ℃; the flow rate is 1 ml/min; the detection wavelength was 283 nm.

Preferably, in the method for constructing the volatile component characteristic map, the column temperature is controlled to be increased in a gradient manner, and the gradient temperature increasing program is as follows: the initial temperature is 55-65 deg.C, and is maintained for 1-3 min; the temperature is raised from 55-65 ℃ to 115-125 ℃, and then the temperature is kept for 4-6min, and the temperature raising rate is 1.5-2.5 ℃/min; the temperature is increased from 115 ℃ to 125 ℃ to 155 ℃ to 165 ℃, and the temperature increase rate is 2.5-3.5 ℃/min; the temperature is increased from 155 ℃ to 165 ℃ to 235 ℃ to 245 ℃, and the temperature increase rate is 4.5-5.5 ℃/min;

preferably, the gradient temperature-increasing program is: the initial temperature is 60 ℃, and the temperature is kept for 2 min; the temperature is increased from 60 ℃ to 120 ℃, the temperature is kept for 5min, and the temperature increasing rate is 2 ℃/min; the temperature is increased from 120 ℃ to 160 ℃, and the temperature increasing rate is 3 ℃/min; the temperature is increased from 160 ℃ to 240 ℃, and the temperature increasing rate is 5 ℃/min;

preferably, the temperature of the injection port is 245-255 ℃, the temperature of the detector is 245-255 ℃, and the total flow rate is 3.5-3.9 ml/min;

preferably, the injection port temperature is 250 deg.C, the detector temperature is 250 deg.C, and the total flow rate is 3.7 ml/min.

Preferably, the preparation method of the test solution I or the reference solution I of the reference drug comprises the following steps: preparing herba Schizonepetae sample or herba Schizonepetae reference material into powder, precisely weighing 0.5-1g, adding solvent 15-30ml, sealing, extracting, cooling, adding the solvent to compensate the lost weight, shaking, filtering the supernatant, and collecting the filtrate to obtain sample solution I or reference material solution I;

preferably, the preparation method of the reference substance solution I of the control substance comprises the following steps: dissolving hesperidin control in the solvent to obtain reference solution I containing 55-65 μ g/ml hesperidin;

preferably, the test sample is selected from raw medicinal materials or decoction pieces of herba schizonepetae;

preferably, the solvent is methanol, ethanol or water;

preferably, the extraction treatment adopts ultrasonic treatment for 30-90min, reflux for 30-120min or dipping for 12-24 h;

preferably, the control reference solution I is a control reference solution I containing 60 mug/ml hesperidin.

Preferably, the reference substance solution II is a control solution containing 3-6mg/ml pulegone and 3-6mg/ml menthone, and the solvent is ethyl acetate;

the preparation method of the test medicinal material solution II comprises the following steps: taking a schizonepeta tenuifolia sample, preparing volatile oil by an A method for measuring 2204 volatile oil according to general rules of the four ministry of the pharmacopoeia of the people's republic of China 2015 edition, precisely absorbing 25-50ul of volatile oil, adding ethyl acetate to a constant volume of 2ml, removing water and drying to obtain the schizonepeta tenuifolia volatile oil.

The invention also provides a herba schizonepetae quality detection method, which comprises the step of performing quality detection on herba schizonepetae products by adopting the herba schizonepetae characteristic spectrum construction method.

Preferably, the schizonepeta product to be detected is detected by operating the schizonepeta product to be detected according to the schizonepeta characteristic map construction method and the same method, so that a obtained schizonepeta non-volatile component characteristic map and a obtained schizonepeta volatile component characteristic map are obtained, wherein the non-volatile characteristic map presents 6 characteristic peaks and corresponds to 6 characteristic peaks in a reference substance characteristic map of a reference medicinal material, and the retention time of peaks 1 and 6 is consistent with that of a reference substance peak of a reference substance; the volatile component characteristic spectrum presents 3 characteristic peaks, wherein the peak 1 and the peak 2 are consistent with the retention time of a reference substance peak of a reference substance.

Preferably, the method also comprises the step of measuring the content of the schizonepeta, and comprises the following steps:

test solution III: preparing a schizonepeta test sample into a test sample solution III;

control solution: dissolving hesperidin control in solvent to obtain control solution;

chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, and using octadecyl bonded silica gel as chromatographic column filler; using acetonitrile as a mobile phase A, using phosphoric acid water solution with volume fraction of 0.2% as a mobile phase B for gradient elution, and performing gradient elution according to the following procedures: 0-28min, mobile phase A: the volume ratio of the mobile phase B is 17-19%: 83-81% → 17-19%: 83 to 81 percent; 28-29min, mobile phase A: the volume ratio of the mobile phase B is 17-19%: 83-81% → 84-86%: 16 to 14 percent; 29-39min, mobile phase A: the volume ratio of the mobile phase B is 84-86%: 16-14% → 84-86%: 16 to 14 percent; the column temperature is 25-35 ℃; the flow rate is 0.8-1.2 ml/min; the detection wavelength is 278-288 nm; the theoretical plate number is not less than 3000 calculated according to hesperidin;

the determination method comprises the following steps: precisely sucking the reference solution and the test solution III, respectively, injecting into a liquid chromatograph, and measuring.

Preferably, the method further comprises a step of identifying by thin layer chromatography, a step of measuring heavy metals and harmful elements and/or a step of measuring extracts:

C. the identification step of the nonvolatile components by thin layer chromatography comprises the following steps:

taking 0.5-1.0g of coarse powder of a schizonepeta test sample and 0.5-1.0g of coarse powder of a schizonepeta control medicinal material respectively, adding 15-25ml of ethyl acetate, carrying out ultrasonic treatment for 15-25 minutes, filtering, volatilizing the filtrate to 1ml to serve as a test sample solution IV and a control medicinal material solution, sucking 1-5ul of the filtrate respectively, respectively dropping the filtrate on the same thin-layer plate, developing the laminate in a developing agent, taking out the laminate, airing the laminate, and viewing the laminate under ultraviolet light with the wavelength of 361nm-371 nm;

preferably, the thin layer plate is a silica gel G plate or an H plate;

preferably, the developing solvent is a chloroform-ethyl acetate-glacial acetic acid mixed solution, and the volume ratio of the developing solvent to the chloroform-ethyl acetate-glacial acetic acid mixed solution is (9-11) to 1 (0.3-0.8); and/or

D. The volatile component identification step by thin layer chromatography comprises the following steps:

taking a proper amount of coarse powder of a schizonepeta test sample, measuring according to a volatile oil measuring method 2204 of the four ministry of communications on pharmacopoeia 2015 edition of the people's republic of China, dividing into 0.1-0.3 ml of volatile oil, adding ethyl acetate to dilute the volatile oil to 1.5-4.5 ml, and taking the volatile oil as a test sample solution V. And (3) adding ethyl acetate into (-) -menthone and pulegone reference substances to prepare a solution containing 5-30 mg of the (-) -menthone and pulegone per 1ml, and taking the solution as a reference substance solution. Sucking 1-5ul of each, respectively dropping on the same thin-layer plate, developing in a developing agent, taking out, air drying, spraying 2-8% vanillin sulfuric acid ethanol solution, and heating at 105 deg.C until the color of spots is clear;

preferably, the thin layer plate is a silica gel G plate or an H plate;

preferably, the developing solvent is petroleum ether (60-90 ℃) ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is (11-13) to 1; and/or;

E. the determination of the heavy metals and the harmful elements comprises the determination of the contents of heavy metals of lead, cadmium, arsenic, mercury and copper, and comprises the following steps:

the pesticide residue determination comprises the content determination of total hexachloro cyclohexane, total dichlorodiphenyl trichloroethane and pentachloronitrobenzene; and/or

F. The step of extract determination comprises:

the extract determination operation steps are as follows: 2-5g of coarse powder of a schizonepeta test medicinal material is taken, precisely weighed, placed in a 250-300 ml conical flask, precisely added with 100ml of a solvent, sealed, cold soaked, shaken all the time within the first 6 hours, then kept stand for 18 hours, rapidly filtered by a drying filter, precisely taken with 20ml of a subsequent filtrate, placed in an evaporating dish dried to constant weight, dried on a water bath, dried at 105 ℃ for 3 hours, placed in a dryer for cooling for 30 minutes, rapidly and precisely weighed, and the content (%) of extract in the test sample is calculated by using the dried product; wherein the solvent is ethanol, and the volume ratio of the solvent is 50-90%

The technical scheme of the invention has the following advantages:

1. the method for constructing the herba schizonepetae characteristic spectrum comprises the construction of a non-volatile effective component characteristic spectrum and a volatile effective component characteristic spectrum, on one hand, the non-volatile effective component characteristic spectrum comprises characteristic chromatographic peaks of hesperidin, rosmarinic acid and pulegone, and the volatile effective component characteristic spectrum comprises characteristic chromatographic peaks of pulegone and pulegone, wherein the pulegone can coexist with non-volatile effective components and volatile effective components, and through liquid chromatography analysis and gas chromatography analysis, the pulegone in a medicinal material can be effectively evaluated and detected, and other effective components in the herba schizonepetae can be pertinently treated: the comprehensive detection of the rosmarinic acid, the hesperidin and the menthone is carried out, the rosmarinic acid, the hesperidin and the menthone comprise non-volatile effective components and volatile effective components, the chromatographic peak is completely separated, and the peak shape is good and free of interference.

On the other hand, the characteristic spectrum of the non-volatile effective components of the schizonepeta is constructed by adopting a high performance liquid chromatography, the separation efficiency is high, the sensitivity is good, the speed is high, and after a plurality of tests, the acetonitrile (A) -0.2% phosphoric acid water (B) is taken as a mobile phase, and target substances of rosmarinic acid, hesperidin and pulegone can be completely separated; the method is characterized in that the gas chromatography and the high performance liquid chromatography are mutually supplemented to analyze volatile effective components in the schizonepeta, chromatographic conditions adopted by a plurality of tests are matched with a temperature raising program, so that menthone and pulegone are well separated, the gas chromatography and the liquid chromatography are combined, the non-volatile effective components and the volatile effective components of the schizonepeta can be comprehensively separated, the effective components of the schizonepeta can be comprehensively separated and detected, chromatographic peaks of the effective components are fully separated, the peak shape is good and is free of interference, and the characteristic spectrum detection method has the advantages of simplicity, rapidness, stability, reliability, high precision, good reproducibility, easiness in mastering and the like.

2. According to the construction method of the herba schizonepetae characteristic spectrum, the herba schizonepetae raw medicinal materials and the herba schizonepetae decoction pieces are used as test samples, so that the quality of the medicinal materials can be effectively monitored from the source of the medicinal materials, and the actual application value of the characteristic spectrum is improved; the pretreatment of a sample to be tested by a liquid chromatography test method adjusts a solvent and extraction operation, and the preferable scheme uses methanol as the solvent and reflux as the extraction operation, so that the dissolving effect of the effective components is good, the dissolving amount is obviously increased, the preparation process is easy to filter and treat, and the sensitivity of chromatographic analysis is increased; the invention adopts a volatile component preparation method in pharmacopoeia, the method is mature, the prepared volatile component has high purity, ethyl acetate is dissolved and diluted to obtain proper concentration suitable for analysis, the ethyl acetate is used as a background solvent, the test result is not interfered, the gas phase detection result is accurate, the sensitivity is high, and the speed is high.

3. According to the method for constructing the characteristic spectrum of the schizonepeta, in the liquid chromatographic analysis process, through multiple tests, acetonitrile (A) -0.2% phosphoric acid water (B) is used as a mobile phase, the adopted detection wavelength is 278nm-288nm, the separation degree of nonvolatile effective components of the schizonepeta is good in the wavelength range, the characteristic spectrum peaks of all the effective components of the schizonepeta can be well separated by matching with a gradient elution program, the obtained characteristic spectrum base line is stable, the peak shape is good, in the gas chromatographic analysis process, the adopted heating program is obtained through multiple test evaluations, and under the heating program, the separation degree of the gas chromatographic peaks is good, and the method is simple and rapid.

4. The schizonepeta quality detection method provided by the invention comprises the safety quality detection and the effectiveness quality detection of schizonepeta, wherein the safety quality detection comprises the detection of heavy metals and harmful elements and the measurement of pesticide residue amount, and the detection is carried out by a method in pharmacopeia, and the detection method is standard and has good detection sensitivity and accuracy; the effective quality detection comprises the steps of detecting a characteristic map of a sample by using the non-volatile effective component detection method and the volatile effective component detection method, establishing the characteristic map as a contrast, wherein the characteristic map has a characteristic chromatographic peak and is used as a contrast of the characteristic map of the schizonepeta medicinal material to be controlled, and simply evaluating the effective component condition of the medicinal material to be detected through similar performance; the effectiveness quality detection also comprises the content determination of hesperidin, which is supplemented with the detection items of the characteristic map to perform qualitative and quantitative quality evaluation on the schizonepeta tenuifolia sample, and also comprises the identification of thin-layer chromatography and the determination of extract.

5. The schizonepeta quality detection method provided by the invention is characterized in that the hesperidin content determination method adopts the preparation process of the test solution I to prepare a test solution III for content determination, and adjusts the elution procedure to best present the liquid chromatographic peak of hesperidin, so that the hesperidin content detection result is accurate and the sensitivity is high.

6. The thin-layer chromatography identification in the schizonepeta quality detection method optimizes the existing method in pharmacopeia, ethyl acetate is used as a solvent, trichloromethane-ethyl acetate-glacial acetic acid is used as a developing agent, a color developing agent is not used, fluorescence inspection is used instead, fluorescent spots obtained under fluorescence are clear, the separation degree is good, the identification method is stable and reliable, the repeatability is good, and the treatment method has the advantages of simplicity in operation, time saving and the like.

7. The volatile oil thin-layer chromatography identification method in the schizonepeta quality detection method provided by the invention has the advantages that schizonepeta volatile oil is taken as a test sample, petroleum ether (60-90 ℃) and ethyl acetate are taken as developing agents, spots obtained by color development are clear, the separation degree is good, effective components such as menthone and pulegone can be clearly identified, and the identification method is stable and reliable and has good repeatability.

8. According to the method for detecting the quality of the schizonepeta, the extract is detected by adopting the existing method in pharmacopoeia, 50-90% of ethanol is used as a solvent, active ingredients in the solvent can be well dissolved out, and the accuracy and the sensitivity of the extract detection are improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a characteristic spectrum of a reference solution I of a reference drug in example 1;

FIG. 2 is a characteristic spectrum of a reference substance solution I of the hesperidin control in example 1;

FIG. 3 is a characteristic map of the test solution I in example 1;

FIG. 4 is a characteristic map of the test solution II in example 1;

FIG. 5 is a comparison spectrum of the nonvolatile components of Schizonepeta tenuifolia of example 4;

FIG. 6 is a comparison spectrum of volatile components of Schizonepeta tenuifolia according to example 4;

FIG. 7 is a liquid chromatogram of the hesperidin control solution of example 5;

FIG. 8 is a liquid chromatogram of the test solution III in example 5;

FIG. 9 is a regression curve of the hesperidin content determination in example 5;

FIG. 10 is a thin layer chromatogram of example 8;

FIG. 11 is a thin layer chromatogram of example 11;

FIG. 12 is a thin layer chromatogram obtained by a pharmacopoeia method in an experimental example;

FIG. 13 is a low temperature investigation thin layer chromatogram of the thin layer chromatography detection method in the experimental example;

FIG. 14 is a thin-layer chromatogram for 18% humidity examination of the thin-layer chromatography detection method in the experimental example;

FIG. 15 is a thin layer chromatogram for humidity examination at 72% of the thin layer chromatography detection method in the experimental example;

FIG. 16 is a thin layer chromatogram for a specific investigation of a thin layer chromatography detection method in an experimental example;

FIG. 17 is a thin-layer chromatogram of a silica gel G thin-layer plate for durability test of thin-layer chromatography in the laboratory;

FIG. 18 is a thin layer chromatogram of silica gel G thin layer plate of the research institute of chemical industry of the cigarette end market for durability examination of the thin layer chromatography detection method in the experimental example;

FIG. 19 is a thin layer chromatogram obtained by examining the reproducibility of the thin layer chromatography detection method in the experimental example by an experimenter;

FIG. 20 is a thin layer chromatogram obtained by another experimenter through the reproducibility inspection of the thin layer chromatography detection method in the experimental example;

FIG. 21 is a low temperature investigation thin layer chromatogram of the volatile oil thin layer chromatography detection method in the experimental example;

FIG. 22 is a thin-layer chromatogram for humidity investigation at 18% of the volatile oil thin-layer chromatography detection method in the experimental example;

FIG. 23 is a thin-layer chromatogram for humidity investigation at 72% of the volatile oil thin-layer chromatography detection method in the experimental example;

FIG. 24 is a thin-layer chromatogram for specificity test of the volatile oil thin-layer chromatography in the experimental example;

FIG. 25 is a thin-layer chromatogram of a silica gel G thin-layer plate in Qingdao ocean chemical branch plant for durability examination of a volatile oil thin-layer chromatography detection method in an experimental example;

FIG. 26 is a thin layer chromatogram of silica gel G thin layer plate of the research institute of chemical industry in cigarette end for durability examination of the volatile oil thin layer chromatography detection method in the experimental example;

FIG. 27 is a thin layer chromatogram obtained by a human experimenter in the reproducibility inspection of the thin layer chromatography detection method of volatile oil in the experimental example;

FIG. 28 is a thin layer chromatogram obtained by another experimenter through the reproducibility inspection of the thin layer chromatography detection method of volatile oil in the experimental example;

FIG. 29 is a liquid chromatogram obtained by elution method 1 in the investigation of the conditions of content measurement liquid chromatography in the experimental example;

FIG. 30 is a liquid chromatogram obtained by elution method 2 in the investigation of the conditions of content measurement liquid chromatography in the experimental example;

FIG. 31 is a liquid chromatogram obtained by elution method 3 in the investigation of the conditions of content measurement liquid chromatography in the experimental example;

FIG. 32 is a liquid chromatogram obtained by elution method 4 in the investigation of the conditions of content measurement liquid chromatography in the experimental example;

FIG. 33 is a full wavelength scanning spectrum of the DAD of Schizonepeta tenuifolia in the experimental example;

FIG. 34 is a liquid chromatogram obtained by elution method 1 in the investigation of the conditions of HPLC liquid chromatography for non-volatile components in the experimental examples;

FIG. 35 is a liquid chromatogram obtained by elution method 2 in the investigation of the conditions of HPLC liquid chromatography for non-volatile components in the experimental examples;

FIG. 36 is a liquid chromatogram obtained by elution method 3 in the investigation of the conditions of HPLC liquid chromatography for non-volatile components in the experimental examples;

FIG. 37 is a control profile of non-volatile components of the experimental examples;

FIG. 38 is a gas chromatogram obtained by temperature rising program 1 of volatile component gas chromatography in the test example;

FIG. 39 is a gas chromatogram obtained by measuring temperature rising program 2 by volatile component gas chromatography in the test example;

FIG. 40 is a gas chromatogram obtained by temperature rising program 3 for volatile component gas chromatography detection in the test example;

FIG. 41 is a gas chromatogram obtained by temperature increasing program 4 for volatile component gas chromatography in the test example;

FIG. 42 is a gas chromatogram obtained by temperature rising program 5 for volatile component gas chromatography in the test example;

FIG. 43 is a control profile of volatile components in the examples.

Detailed Description

The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.

The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

The method for detecting the characteristic spectrum of the schizonepeta of the embodiment of the invention is specifically explained below.

1. The experimental instruments and reagents used in the examples of the present invention are shown in table 1.

TABLE 1 Experimental instruments and reagent statistics table

2. The invention is further illustrated by the following examples:

example 1

The embodiment provides a method for constructing a schizonepeta characteristic spectrum, which comprises a method for constructing a non-volatile component characteristic spectrum and a method for constructing a volatile component characteristic spectrum, and specifically comprises the following steps:

preparation of test solution I: taking 0.5g of tested schizonepeta medicinal material powder (screened by a No. two sieve) produced in Anhui, putting the powder into a triangular flask with a plug, adding 25ml of methanol, sealing the plug, weighing, refluxing for 30 minutes, cooling, complementing the lost weight with the methanol, shaking up, taking supernatant, filtering, and taking subsequent filtrate to obtain the schizonepeta medicinal material powder;

preparation of reference solution I: taking 0.5g of herba schizonepetae reference medicinal material powder (screened by a second sieve), placing the herba schizonepetae reference medicinal material powder in a triangular flask with a plug, adding 25ml of methanol, sealing the plug, weighing, refluxing for 30 minutes, cooling, complementing the weight loss by methanol, shaking up, taking supernate, filtering, taking subsequent filtrate to obtain reference medicinal material solution I; weighing appropriate amount of hesperidin control, adding methanol to dissolve, and making into reference solution I containing hesperidin 60 μ g per 1 ml;

chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, using octadecyl bonded silica gel as column filler, and selecting Waters column (250mm × 4.6mm, 5 μm); using acetonitrile as a mobile phase A and phosphoric acid aqueous solution with the volume fraction of 0.2% as a mobile phase B, and carrying out gradient elution according to the following procedures: 0-15min, mobile phase A: the volume ratio of the mobile phase B is 10%: 90% → 14%: 86 percent; 15-40min, mobile phase A: the volume ratio of the mobile phase B is 14%: 86% → 26%: 74 percent; 40-55min, mobile phase A: the volume ratio of the mobile phase B is 26%: 74% → 60%: 40 percent; 55-60min, mobile phase A: the volume ratio of the mobile phase B is 60%: 40% → 85%: 15 percent; 60-65min, mobile phase A: the volume ratio of the mobile phase B is 85%: 15% → 85%: 15 percent; the column temperature was 40 ℃; the flow rate is 1 ml/min; the detection wavelength is 283nm, and the number of theoretical plates is not less than 3000 calculated according to hesperidin;

the determination method comprises the following steps: precisely sucking 10 μ l of reference solution I and sample solution I respectively, injecting into liquid chromatograph, and measuring; the reference solution I of the reference medicinal material has a characteristic spectrum shown in figure 1, the reference solution I of the hesperidin reference is shown in figure 2, and the test solution I is shown in figure 3.

preparation of test solution II: preparing volatile oil from tested herba Schizonepetae medicinal material from Anhui by A method of 2204 volatile oil determination according to pharmacopoeia of the people's republic of China 2015 edition, precisely sucking 50 μ l, adding ethyl acetate to desired volume of 2ml, drying with anhydrous sodium sulfate, shaking, and collecting filtrate;

preparation of reference solution II: accurately weighing 0.5g of pulegone and menthone respectively, dissolving in 30ml of ethyl acetate solution, and diluting to 100ml to obtain a mixed reference substance solution containing 5mg/ml of pulegone and 5mg/ml of menthone;

chromatographic conditions and system applicability test: measured by gas chromatography, as N₂As carrier gas, PEG (DB-WAX, 30m × 0.530mm) as stationary phase, and injection port temperatureThe temperature of the detector is 250 ℃, and the temperature of the column is controlled to be increased in a gradient way, wherein the gradient temperature increasing procedure comprises the following steps: the initial temperature is 60 ℃, and the temperature is kept for 2 min; the temperature is increased from 60 ℃ to 120 ℃, and then the temperature is kept for 5min, wherein the temperature increasing rate is 2 ℃/min; the temperature is increased from 120 ℃ to 160 ℃, and the temperature increasing rate is 3 ℃/min; the temperature is increased from 160 ℃ to 240 ℃, and the temperature increasing rate is 5 ℃/min; the total flow is 3.7ml/min, and the number of theoretical plates is 3200 according to the peak of the pulegone;

the determination method comprises the following steps: precisely sucking 5 μ l of reference solution II and sample solution II respectively, injecting into gas chromatograph, and measuring; the characteristic map of the test solution II is shown in figure 4.

Example 2

The embodiment provides a method for constructing a schizonepeta characteristic spectrum, which comprises the steps of a method for constructing a non-volatile component characteristic spectrum and the steps of a method for constructing a volatile component characteristic spectrum, and specifically comprises the following steps:

preparation of test solution I: taking 1g of tested schizonepeta medicinal material powder (screened by a No. two sieve) produced in Anhui province, putting the powder into a triangular flask with a plug, adding 15ml of methanol, sealing the plug, weighing, refluxing for 60 minutes, cooling, complementing the lost weight with the methanol, shaking uniformly, taking supernatant, filtering, and taking subsequent filtrate;

preparation of reference solution I: taking 1g of herba schizonepetae reference medicinal material powder (screened by a second sieve), placing the herba schizonepetae reference medicinal material powder in a triangular flask with a plug, adding 15ml of methanol, sealing the plug, weighing, refluxing for 60 minutes, cooling, complementing the lost weight with methanol, shaking up, taking supernate, filtering, taking subsequent filtrate to obtain reference medicinal material solution I; weighing appropriate amount of hesperidin control, adding methanol to dissolve, and making into reference solution I containing 55 μ g hesperidin per 1ml control;

chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, using octadecyl bonded silica gel as column filler, and selecting Alchromobond-AQ column (250mm × 4.6mm, 5 μm); acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution with the volume concentration of 0.2% is used as a mobile phase B, and gradient elution is carried out according to the following procedures: 0-15min, mobile phase A: the volume ratio of the mobile phase B is 8%: 92% → 12%: 88 percent; 15-40min, mobile phase A: the volume ratio of the mobile phase B is 12%: 88% → 24%: 76%; 40-55min, mobile phase A: the volume ratio of the mobile phase B is 24%: 76% → 58%: 42%; 55-60min, mobile phase A: the volume ratio of the mobile phase B is 58%: 42% → 83%: 17 percent; 60-65min, mobile phase A: volume ratio of mobile phase B83%: 17% → 83%: 17 percent; the column temperature was 35 ℃; the flow rate is 0.8 ml/min; the detection wavelength is 278nm, and the number of theoretical plates is 12035 according to the hesperidin;

the determination method comprises the following steps: precisely sucking 10 μ l of reference solution I and sample solution I respectively, injecting into liquid chromatograph, and measuring.

preparation of test solution II: preparing volatile oil from tested herba Schizonepetae medicinal material from Anhui by A method of 2204 volatile oil determination in 2015 pharmacopoeia of the people's republic of China, precisely sucking 25 μ l, adding ethyl acetate to desired volume of 2ml, drying with anhydrous sodium sulfate, shaking, and collecting filtrate;

preparation of reference solution II: accurately weighing 0.3g of pulegone and menthone respectively, dissolving in 30ml of ethyl acetate solution, and fixing the volume to 100ml to obtain a mixed reference substance solution containing 3mg/ml of pulegone and 3mg/ml of menthone;

chromatographic conditions and system applicability test: measuring by gas chromatography, using N2 as carrier gas, PEG (DB-WAX, 30m × 0.530mm) as stationary phase, sample inlet temperature 245 ℃, detector temperature 245 ℃, and column temperature controlled in gradient temperature rise, wherein the gradient temperature rise procedure is as follows: keeping the initial column temperature at 55 deg.C for 1 min; the temperature is increased from 55 ℃ to 115 ℃, and then the temperature is kept for 4min, wherein the temperature increasing rate is 1.5 ℃/min; the temperature is increased from 115 ℃ to 155 ℃, and the temperature increase rate is 2.5 ℃/min; the temperature is increased from 155 ℃ to 235 ℃, and the temperature increasing rate is 4.5 ℃/min; the total flow rate is 3.5ml/min, and the number of theoretical plates is 4000 calculated according to the peak of the pulegone;

the determination method comprises the following steps: precisely sucking 5 μ l of reference solution II and sample solution II respectively, injecting into gas chromatograph, and measuring.

Example 3

The embodiment provides a method for constructing a schizonepeta characteristic spectrum, which comprises the steps of a method for constructing a non-volatile component characteristic spectrum and a method for constructing a volatile component characteristic spectrum, and specifically comprises the following steps:

preparation of test solution I: taking 0.8g of tested schizonepeta decoction piece powder (sieved by a No. two sieve) produced in Hebei, putting the powder into a triangular flask with a plug, adding 30ml of ethanol, sealing the plug, weighing, refluxing for 120 minutes, cooling, complementing the loss weight with ethanol, shaking uniformly, taking supernatant, filtering, and taking subsequent filtrate;

preparation of reference solution I: taking 0.8g of herba schizonepetae reference medicinal material powder (screened by a second sieve), placing the herba schizonepetae reference medicinal material powder in a triangular flask with a plug, adding 30ml of ethanol, sealing the plug, weighing, refluxing for 120 minutes, cooling, complementing the weight loss by ethanol, shaking up, taking supernate, filtering, taking subsequent filtrate to obtain reference medicinal material solution I; weighing appropriate amount of hesperidin control, adding methanol to dissolve, and making into reference solution I containing hesperidin 65 μ g per 1 ml;

chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, using octadecyl bonded silica gel as column filler, and selecting Phenomex-Luna column (250mm × 4.6mm, 5 μm); gradient elution was performed with acetonitrile as mobile phase a and 0.2% phosphoric acid in water as mobile phase B according to the following procedure: 0-15min, mobile phase A: the volume ratio of the mobile phase B is 12%: 88% → 16%: 84%; 15-40min, mobile phase A: the volume ratio of the mobile phase B is 16%: 84% → 28%: 72 percent; 40-55min, mobile phase A: the volume ratio of the mobile phase B is 28%: 72% → 62%: 38 percent; 55-60min, mobile phase A: the volume ratio of the mobile phase B is 62%: 38% → 87%: 13 percent; 60-65min, mobile phase A: the volume ratio of the mobile phase B is 87%: 13% → 87%: 13 percent; the column temperature was 25 ℃; the flow rate is 1.2 ml/min; the detection wavelength is 288nm, and the theoretical plate number is 10253 in terms of hesperidin;

preparation of test solution II: preparing volatile oil from tested herba Schizonepetae medicinal material from Anhui by A method of 2204 volatile oil determination according to pharmacopoeia of the people's republic of China 2015 edition, precisely absorbing 80 μ l, adding ethyl acetate to desired volume of 2ml, drying with anhydrous sodium sulfate, shaking, and collecting filtrate;

preparation of reference solution II: accurately weighing 0.6g of pulegone and menthone respectively, dissolving in 30ml of ethyl acetate solution, and fixing the volume to 100ml to obtain a mixed reference substance solution containing 6mg/ml of pulegone and 6mg/ml of menthone;

chromatographic conditions and system applicability test: measuring by gas chromatography, using N2 as carrier gas, PEG (DB-WAX, 30m × 0.530mm) as stationary phase, sample inlet temperature 245 ℃, detector temperature 245 ℃, and column temperature controlled in gradient temperature rise, wherein the gradient temperature rise procedure is as follows: keeping the initial column temperature at 65 deg.C for 3 min; the temperature is increased from 65 ℃ to 125 ℃, and then the temperature is kept for 6min, wherein the temperature increasing rate is 2.5 ℃/min; the temperature is increased from 125 ℃ to 165 ℃, and the heating rate is 3.5 ℃/min; the temperature is increased from 165 ℃ to 245 ℃, and the temperature increasing rate is 5.5 ℃/min; the total flow rate is 3.9ml/min, and the number of theoretical plates is 5000 according to the peak of the pulegone;

Example 4

The embodiment provides a method for constructing a herba schizonepetae characteristic spectrum, which comprises the following steps of constructing a herba schizonepetae non-volatile component comparison characteristic spectrum and a volatile component comparison characteristic spectrum:

A. the construction of the herba schizonepetae non-volatile component comparison characteristic map comprises the following steps:

randomly selecting 20 samples of different batches, measuring the characteristic chromatogram of the nonvolatile effective components of the 20 schizonepeta samples according to the characteristic chromatogram construction method for the nonvolatile effective components of the schizonepeta as described in example 1, and obtaining the comparison characteristic chromatogram of the nonvolatile effective components of the schizonepeta by utilizing a traditional Chinese medicine chromatogram fingerprint similarity evaluation system of the national pharmacopoeia committee, wherein the comparison characteristic chromatogram is shown in figure 5;

as can be seen from fig. 6, the nonvolatile component control characteristic spectrum includes 6 characteristic peaks, and the common characteristic peaks are: the No. 1 peak is hesperidin, the No. 2 peak is rosmarinic acid, the No. 3 peak, the No. 4 peak and the No. 5 peak are common characteristic peaks, and the No. 6 peak is pulegone; taking the peak No. 1 as a reference peak, and the relative retention time of each peak is respectively as follows: peak No. 1: 1.00; peak No. 2: 1.07; peak No. 3: 1.09; peak No. 4: 1.20; peak No. 5: 1.25; peak No. 6: 1.64 of; the 6 characteristic peaks correspond to 6 characteristic peaks in the characteristic chromatogram of the reference solution I of the reference medicinal material, wherein the relative retention time of the No. 1 peak and the No. 6 peak in the chromatogram of the reference solution I of the reference medicinal material is consistent;

B. the construction of the characteristic map of the volatile components of the schizonepeta comprises the following steps:

randomly selecting 20 schizonepeta samples of different batches, measuring the characteristic chromatogram of the volatile active ingredients of the schizonepeta according to the characteristic chromatogram detection method for the volatile active ingredients of the schizonepeta described in example 1, and obtaining a schizonepeta volatile ingredient comparison characteristic chromatogram by using a traditional Chinese medicine chromatogram fingerprint similarity evaluation system of the national pharmacopoeia committee, wherein the characteristic chromatogram is shown in fig. 6;

as can be seen from FIG. 6, the characteristic spectrum of the volatile components of the schizonepeta comprises 3 characteristic peaks, and the common characteristic peaks are as follows: the No. 1 peak is menthone, the No. 2 peak is pulegone, and the No. 3 peak is a common characteristic peak; taking the peak No. 2 as a reference peak, and the relative retention time of each peak is respectively as follows: peak No. 1: 0.67; peak No. 2: 1.00; peak No. 3: 1.50; wherein peaks No. 1 and 2 are consistent with the relative retention times of the reference peaks in the profile of reference solution II in example 1.

Example 5

The embodiment provides a method for detecting the quality of schizonepeta, which comprises the step of measuring the content of schizonepeta and comprises the following steps:

test solution III: preparing herba Schizonepetae decoction pieces to be tested from Anhui into powder (sieved with No. two sieve) 0.5g, placing in triangular flask with plug, adding methanol 25ml, sealing, weighing, refluxing for 30min, cooling, supplementing lost weight with methanol, shaking, collecting supernatant, filtering, and collecting filtrate;

control solution: weighing hesperidin (C)₂₈H₃₄O₁₅) Dissolving a proper amount of reference substance by adding methanol to prepare a reference substance solution I containing 68.231 mu g of hesperidin per 1ml, and then continuously diluting to prepare hesperidin reference substance solutions phi-phi with the concentrations of 34.116 mu g/ml, 17.058 mu g/ml, 8.529 mu g/ml, 4.264 mu g/ml and 2.132 mu g/ml respectively;

chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, using octadecyl bonded silica gel as column filler, and selecting Waters column (250mm × 4.6mm, 5 μm); using acetonitrile as a mobile phase A, using a phosphoric acid aqueous solution with a volume concentration of 0.2% as a mobile phase B for gradient elution, and performing gradient elution according to the following procedures: 0-28min, mobile phase A: the volume ratio of the mobile phase B is 0-28min, and the volume ratio of the mobile phase A: the volume ratio of the mobile phase B is 18%: 82% → 18%: 82%; 28-29min, mobile phase A: the volume ratio of the mobile phase B is 18%: 82% → 85%: 15 percent; 29-39min, mobile phase A: the volume ratio of the mobile phase B is 85%: 15% → 85%: 15 percent; the column temperature is 30 ℃; the flow rate is 1 ml/min; the detection wavelength is 283 nm; the number of theoretical plates is 9000 counted by hesperidin;

the determination method comprises the following steps: precisely sucking the reference solution and the test solution III10ul respectively, injecting into a liquid chromatograph, and measuring; the chromatogram of the reference solution I is shown in FIG. 7, and the chromatogram of the test solution III is shown in FIG. 8;

and (3) calculating: detecting the reference substance solutions (i) - (sixth), calculating peak areas and average peak areas respectively twice, obtaining a linear relation between the hesperidin concentration and the average peak area as shown in table 2, fitting a regression equation of the test sample, wherein the linear relation of the hesperidin is good in a range of 2.132 mu g/ml-68.231 mu g/ml, the regression equation is that Y is 21954X, r is 0.9997, and a curve graph of the regression equation is shown in figure 7; the content of hesperidin is calculated by an external standard one-point method, the calculated hesperidin content is shown in Table 3, and the test sample contains hesperidin (C) calculated according to a dry product₂₈H₃₄O₁₅) Not less than 0.06.

TABLE 2 Linear relationship examination of Schizonepeta hesperidin

Concentration (μ g/ml)	Peak area 1	Peak area 2	Average peak area
				2.132	45689	47099	46394
4.264	94946	94734	94840
				8.529	190110	187972	189041
17.058	375568	347321	361444.5
				34.116	696680	755340	726010
68.231	1541295	1483502	1512399

TABLE 3 content of hesperidin in the samples

Sample name	Hesperidin (%)
		Sample 1	0.309

Example 6

test solution III: taking 1g of tested schizonepeta medicinal material powder (screened by a No. two sieve) produced in Anhui province, putting the powder into a triangular flask with a plug, adding 15ml of methanol, sealing the plug, weighing, refluxing for 60 minutes, cooling, complementing the lost weight with the methanol, shaking uniformly, taking supernatant, filtering, and taking subsequent filtrate;

control solution: prepared according to the hesperidin control solution preparation method described in example 5;

chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, using octadecyl bonded silica gel as column filler, and selecting Alchromobond-AQ column (250mm × 4.6mm, 5 μm); using acetonitrile as a mobile phase A, using phosphoric acid water solution with volume fraction of 0.2% as a mobile phase B for gradient elution, and performing gradient elution according to the following procedures: 0-28min, mobile phase A: volume ratio of mobile phase B is 17%: 83% → 17%: 83 percent; 28-29min, mobile phase A: volume ratio of mobile phase B is 17%: 83% → 84%: 16 percent; 29-39min, mobile phase A: volume ratio of mobile phase B is 84%: 16% → 84%: 16 percent; the column temperature was 35 ℃; the flow rate is 0.8 ml/min; the detection wavelength is 278 nm; the theoretical plate number is 11235 according to hesperidin;

measurement and calculation: the hesperidin content of the schizonepeta tested medicinal material in the example was obtained according to the measurement and calculation method described in example 5, and is shown in table 4.

TABLE 4 content of hesperidin in the samples

Sample name	Hesperidin (%)
		Sample (I)	0.187

Example 7

test solution III: taking 0.8g of tested herba schizonepetae medicinal material powder (screened by a No. two sieve) produced in Hebei, putting the powder into a triangular flask with a plug, adding 30ml of ethanol, sealing the plug, weighing, refluxing for 120 minutes, cooling, complementing the loss weight by ethanol, shaking uniformly, taking supernatant, filtering, and taking subsequent filtrate;

chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, using octadecyl bonded silica gel as column filler, and selecting Phenomex-Luna column (250mm × 4.6mm, 5 μm); gradient elution was performed with acetonitrile as mobile phase a and 0.2% phosphoric acid in water as mobile phase B, according to the following procedure: 0-28min, mobile phase A: the volume ratio of the mobile phase B is 19%: 81% → 19%: 81 percent; 28-29min, mobile phase A: the volume ratio of the mobile phase B is 19%: 81% → 86%: 14 percent; 29-39min, mobile phase A: the volume ratio of the mobile phase B is 86%: 14% → 86%: 14 percent; the column temperature was 33 ℃; the flow rate is 1.2 ml/min; the detection wavelength is 288 nm; the theoretical plate number is not less than 3000 calculated according to hesperidin;

measurement and calculation: the hesperidin content of the schizonepeta tested medicinal material in the example was obtained according to the measurement and calculation method described in example 5, and is shown in table 5.

TABLE 5 content of hesperidin in the samples

Sample name	Hesperidin (%)
		Sample 3	0.174

Example 8

The embodiment provides a method for detecting the quality of schizonepeta, which further comprises the step of carrying out thin-layer chromatography identification on non-volatile components of schizonepeta, and the method comprises the following specific steps:

taking 0.8G of each of tested schizonepeta primary medicinal material produced in Anhui province, schizonepeta decoction pieces produced in Hebei province and schizonepeta control medicinal material produced in Hebei province, adding 20ml of ethyl acetate, performing ultrasonic treatment with the power of 250W and the frequency of 40KHZ for 20 minutes, filtering, volatilizing the filtrate to 1ml to obtain 3 parts of filtrate, precisely absorbing 2ul of each filtrate, respectively dropping the filtrate on the same silica gel G plate, presaturating the plate in a developing cylinder for 20 minutes, developing the plate in a trichloromethane-ethyl acetate-glacial acetic acid mixed developing agent with the volume ratio of 10:1:0.5, taking out, drying, and placing the plate under ultraviolet light with the wavelength of 366nm, wherein the result is shown in figure 10, the schizonepeta primary medicinal material is numbered 1, the schizonepeta control medicinal material is numbered 2, and the schizonepeta decoction pieces are numbered 3.

Example 9

taking 0.5g of each of tested schizonepeta raw medicinal material produced from Anhui, schizonepeta decoction pieces produced from Hebei and schizonepeta control medicinal material, adding 15ml of ethyl acetate respectively, carrying out ultrasonic treatment for 15 minutes at the power of 250W and the frequency of 40KHZ, filtering, volatilizing the filtrate to 1ml, precisely absorbing 1ul of filtrate respectively, respectively dropping the filtrate on the same silica gel H plate, developing in a trichloromethane-ethyl acetate-glacial acetic acid mixed developing agent with the volume ratio of 9:1:0.3, taking out, airing, and viewing under ultraviolet light with the wavelength of 361 nm.

Example 10

taking 1g of each of tested schizonepeta raw medicinal material produced from Anhui, schizonepeta decoction pieces produced from Hebei and schizonepeta control medicinal material, adding 25ml of ethyl acetate respectively, carrying out ultrasonic treatment for 25 minutes, filtering, volatilizing the filtrate to 1ml, precisely absorbing 5ul of the filtrate, respectively dropping the filtrate on the same silica gel H plate, developing in a trichloromethane-ethyl acetate-glacial acetic acid mixed developing agent with the volume ratio of 11:1:0.8, taking out, airing, and viewing under ultraviolet light with the wavelength of 371 nm.

Example 11

The embodiment provides a method for detecting the quality of schizonepeta, which further comprises the step of identifying volatile components of schizonepeta by thin-layer chromatography, and the method comprises the following specific steps:

taking tested herba Schizonepetae raw medicinal materials and herba Schizonepetae decoction pieces produced in Anhui province, measuring according to volatile oil determination method (2015 th four parts general rule 2204) to obtain volatile oil 0.2ml, and diluting with ethyl acetate to 3ml to obtain 2 parts of test solution. Separately, (-) -menthone and pulegone control substances were added with ethyl acetate to prepare a solution containing 20mg per 1ml, and 2 control substance solutions were obtained. Sucking 1ul of the solutions, dropping the solutions on the same silica gel G plate, spreading the solutions in a petroleum ether (60-90 ℃) and ethyl acetate mixed developing agent with the volume ratio of 12:1, taking out the solutions, drying the solutions in the air, spraying 5% vanillin sulfuric acid ethanol solution, and heating the solutions at 105 ℃ until spots are clearly developed, wherein the results are shown in figure 11, wherein the serial number 1 is (-) -menthone, the serial number 2 is pulegone, the serial number 3 is schizonepeta medicinal material, and the serial number 4 is schizonepeta decoction piece.

Example 12

The embodiment provides a method for detecting the quality of schizonepeta, which further comprises the step of identifying volatile oil of schizonepeta by thin-layer chromatography, and the method comprises the following specific steps:

taking tested herba Schizonepetae raw medicinal materials and herba Schizonepetae decoction pieces produced in Anhui province, measuring according to volatile oil determination method (2015 th four parts general rule 2204) to obtain volatile oil 0.1ml, and diluting with ethyl acetate to 1.5ml to obtain 2 parts of test solution. Additionally, a control of (-) -menthone and pulegone was added with ethyl acetate to make a solution containing 30mg per 1ml to obtain 2 control solutions. Sucking 2ul of the solution, respectively dropping on the same silica gel G plate, developing in petroleum ether (60-90 ℃) and ethyl acetate mixed developing agent with the volume ratio of 11:1, taking out, airing, spraying 4% vanillin sulfuric acid ethanol solution, and heating at 105 ℃ until spots are clearly developed.

Example 13

taking tested herba Schizonepetae raw medicinal materials and herba Schizonepetae decoction pieces produced in Anhui province, measuring according to volatile oil determination method (2015 th four parts general rule 2204) to obtain volatile oil 0.3ml, and diluting with ethyl acetate to 4.5ml to obtain 2 parts of test solution. Separately, (-) -menthone and pulegone control substances were added with ethyl acetate to prepare a solution containing 10mg per 1ml, and 2 control substance solutions were obtained. Sucking 5ul of the solution, respectively dropping on the same silica gel G plate, developing in petroleum ether (60-90 ℃) and ethyl acetate mixed developing agent with the volume ratio of 13:1, taking out, airing, spraying 6% vanillin sulfuric acid ethanol solution, and heating at 105 ℃ until spots are clearly developed.

Example 14

The embodiment provides a method for detecting the quality of schizonepeta, which further comprises the step of detecting the extract of schizonepeta, and the method comprises the following specific steps:

taking 4g of coarse powder prepared from a tested schizonepeta raw medicinal material produced in Anhui province, precisely weighing, precisely adding 100ml of ethanol with the volume fraction of 70%, shaking for 6 hours, standing for 18 hours, filtering, precisely weighing 20ml of subsequent filtrate, placing the subsequent filtrate in an evaporation dish dried to constant weight, evaporating, drying at 105 ℃ for 3 hours, cooling in a dryer for 30 minutes, weighing, and calculating the content of extract; the measured content of the extract was found to be 8.03%.

Example 15

The embodiment provides a method for detecting the quality of schizonepeta, which further comprises the step of detecting the extract of the schizonepeta, and the method comprises the following specific steps:

2g of coarse powder of tested schizonepeta tenuifolia produced from Anhui province, precisely weighing, precisely adding 100ml of 50% ethanol by volume, shaking for 4 hours, standing for 16 hours, filtering, precisely weighing 10ml of subsequent filtrate, placing the subsequent filtrate in an evaporation dish dried to constant weight, evaporating, drying at 100 ℃ for 3 hours, cooling in a dryer for 30 minutes, weighing, and calculating the content of extract; the measured content of the extract was found to be 8.54%.

Example 16

taking 5g of coarse powder prepared from schizonepeta tenuifolia decoction pieces to be tested produced in Hebei, precisely weighing, precisely adding 100ml of 95% ethanol by volume fraction, shaking for 9 hours, standing for 36 hours, filtering, precisely taking 30ml of subsequent filtrate, placing the subsequent filtrate in an evaporation dish dried to constant weight, evaporating, drying at 120 ℃ for 5 hours, cooling in a dryer for 30 minutes, weighing, and calculating the content of extract; the measured content of the extract was measured to be 19.20%.

Example 17

The embodiment provides a method for detecting the quality of schizonepeta, which further comprises the determination of heavy metals and harmful elements, comprises the determination of the content of heavy metals of lead, cadmium, arsenic, mercury and copper, and adopts the following steps that the adopted test products are schizonepeta medicinal material to be tested (sample 1) produced in Anhui, schizonepeta medicinal material to be tested (sample 2) produced in Hebei and schizonepeta decoction pieces to be tested (sample 3) produced in Hebei:

measuring with lead, cadmium, arsenic, mercury and copper measuring method (2321 atomic absorption spectrophotometry or inductively coupled plasma mass spectrometry in Chinese pharmacopoeia), wherein lead is not more than 5 mg/kg; the cadmium can not exceed 0.3 mg/kg; arsenic is not more than 2 mg/kg; the mercury can not exceed 0.2 mg/kg; the copper content should not exceed 20 mg/kg.

The lead, cadmium, arsenic, mercury, and copper contents of the samples are shown in Table 6 below.

TABLE 6 determination of Pb, Cd, As, Hg and Cu contents in the samples

Sample name	Lead (mg/kg)	Cadmium (mg/kg)	Arsenic (mg/kg)	Mercury (mg/kg)	Copper (mg/kg)
						Sample 1	0.29	0.022	0.12	0.010	1.48
Sample 2	0.79	0.067	0.26	0.018	5.71
						Sample 3	1.12	0.150	0.16	0.014	5.18

The determination is carried out according to a pesticide residue determination method (a determination method for determining the residue of organic chlorine pesticides-a first method in general 2341 of Chinese pharmacopoeia).

The total content of the hexahexaflumuron (the sum of alpha-BHC, beta-BHC, gamma-BHC and delta-BHC) is not more than 0.2 mg/kg; the total nasal drip (the sum of pp '-DDE, pp' -DDD, op '-DDT, pp' -DDT) is less than 0.2 mg/kg; the content of the pentachloronitrobenzene cannot exceed 0.1 mg/kg.

The results of the pesticide residues on the samples are shown in Table 7 below.

Table 7 measurement results of amount of pesticide residue in samples

Sample name	Six in total (mg/kg)	Total dichloro-diphenyl-trichloroethane (mg/kg)	Pentachloronitrobenzene (mg/kg)
				Sample 1	Not detected (< 0.01)	Not detected (< 0.01)	Not detected (< 0.01)
Sample 2	Not detected (< 0.01)	Not detected (< 0.01)	Not detected (< 0.01)
				Sample 3	Not detected (< 0.01)	Not detected (< 0.01)	Not detected (< 0.01)

Examples of the experiments

1. Herba schizonepetae thin-layer chromatography identification method

1.1 method optimization

The schizonepeta medicinal material is identified according to a schizonepeta item lower thin-layer identification method in the 2015 edition of pharmacopoeia of the people's republic of China, as follows:

taking 0.8g of coarse powder of schizonepeta, adding 20ml of petroleum ether (60-90 ℃), sealing, shaking constantly, standing overnight, filtering, and volatilizing the filtrate to 1ml to serve as a test solution. Preparing herba Schizonepetae reference material 0.8g, and making reference material solution by the same method; performing thin layer chromatography (Pharmacopeia sinensis (PRC) 2015 edition of general rules of four parts 0502), sucking 10 μ l of the above two solutions, respectively dropping on the same silica gel H thin layer plate, developing with n-hexane-ethyl acetate (17:3) as developing agent, taking out, air drying, spraying 5% ethanol solution of 5% vanillin, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution. The thin layer chromatogram obtained by pharmacopoeia method is shown in FIG. 12. In the figure, "control" -control chromatogram, from left to right No. 2, No. 8, No. 10, No. 11 and No. 12 represent the tested herba Schizonepetae materials of 5 different producing areas. As can be seen from the figure, the effect of identifying the white atractylodes rhizome by adopting a pharmacopoeia method is not good, so that the white atractylodes rhizome is optimized.

(1) Examination of extraction methods

The pharmacopoeia method comprises the following steps: taking 0.8g of coarse schizonepeta powder, adding 20ml of petroleum ether (60-90 ℃), sealing, shaking constantly, standing overnight, filtering, and volatilizing the filtrate to 1ml to serve as a test solution; in order to shorten the time, the extraction method is determined by different extraction methods and different extraction solvent comparison, and comprises the following steps: taking 0.8g of coarse powder of herba schizonepetae decoction pieces, adding 20ml of ethyl acetate, performing ultrasonic treatment for 20 minutes, filtering, and volatilizing the filtrate to 1ml to obtain a sample solution.

(2) Examination of color development method

The pharmacopoeia method comprises the following steps: spraying 5% vanillin in 5% ethanol sulfate solution, and heating at 105 deg.C until the spots are clearly developed. Researches show that the developed thin-layer plate has a plurality of obvious fluorescent spots under the ultraviolet lamp 366nm, has better effect than spraying a color developing agent and is more environment-friendly, so the thin-layer plate is selected to be inspected under the ultraviolet lamp 366 nm.

(3) Developer selection

The variety and the proportion of the developing agent are researched, petroleum ether-ethyl acetate, n-hexane-ethyl acetate, toluene-ethyl acetate, cyclohexane-ethyl acetate-isopropanol-formic acid, n-hexane-acetone-glacial acetic acid, dichloromethane-ethyl acetate-glacial acetic acid, dichloromethane-ethanol-glacial acetic acid, cyclohexane-dichloromethane-ethyl acetate-glacial acetic acid, trichloromethane-ethyl acetate-glacial acetic acid and the like are respectively selected as the developing agent, and the trichloromethane-ethyl acetate-glacial acetic acid is finally selected as the developing agent in a ratio of 10:1: 0.5.

(4) Thin layer plate inspection

And (3) determining that the silica gel G plate has the best separation effect through examination of silica gel G plates, H plates, GF254 plates and the like.

1.2 methodological investigation

(1) Low temperature investigation

Operating according to the scheme of the embodiment 8 of the invention, spotting, developing in a refrigerator at 4 ℃, taking out, airing, and placing under an ultraviolet lamp (366nm) for inspection, wherein the result shows that the developing effect is good in the environment at 4 ℃, as shown in fig. 13, wherein 1 represents a reference medicinal material; 2-schizonepeta medicinal material; 3-schizonepeta decoction pieces.

(2) Humidity investigation

Operating according to the scheme of the embodiment 8 of the invention, spotting, developing in 18% and 72% relative humidity, taking out, drying in the air, and inspecting under an ultraviolet lamp (366nm), wherein the results show that the developing effect is better in different humidity environments, as shown in fig. 14 (18%), fig. 15 (72%), and fig. 14-15, 1-reference medicinal materials; 2-schizonepeta medicinal material; 3-schizonepeta decoction pieces.

(3) Specialization inspection

Operating according to the scheme in the embodiment 8 of the invention, using an ethyl acetate reagent as a blank solution, spotting, developing, taking out, airing, and inspecting under an ultraviolet lamp (366nm), wherein the result shows that the ethyl acetate is free of interference, and the method is proved to have good specificity, as shown in fig. 16, in which 1-ethyl acetate is shown; 2-reference medicinal materials; 3-schizonepeta medicinal material; 4-schizonepeta decoction pieces.

(4) Durability examination

Operating according to the scheme in the embodiment 8 of the invention, respectively spotting and developing on silica gel G thin-layer plates of Qingdao ocean chemical industry branch works and Nicoti city chemical industry research institute, taking out, airing, and placing under an ultraviolet lamp (366nm) for inspection, wherein the results show that 2 plates have better separation effect, and the method is proved to have good durability as shown in figure 17 and figure 18, and in figures 17-18, 1-reference medicinal materials; 2-schizonepeta medicinal material; 3-schizonepeta decoction pieces.

(5) Reproducibility study

2 experimenters operate according to the scheme in the embodiment 8 of the invention at the same time, and the result proves that the method has good reproducibility, as shown in figures 19 and 20, and in figures 19 to 20, 1 is a reference medicinal material; 2-schizonepeta medicinal material; 3-schizonepeta decoction pieces.

2. Herba schizonepetae volatile oil thin-layer chromatography identification method

2.1 method optimization

(1) Developer selection

The types and the mixture ratio of the developing agents are researched, n-hexane-ethyl acetate, toluene-ethyl acetate, petroleum ether-ethyl acetate and the like are respectively selected as the developing agents, and finally petroleum ether (60-90 ℃) and ethyl acetate are selected as the developing agents, wherein the ratio of 12:1 is better.

(2) Thin layer plate inspection

2.2 methodological investigation

(1) Low temperature investigation

The procedure was performed according to the protocol of example 11 of the present invention, spotting, developing in a refrigerator at 4 ℃, taking out, air drying, spraying with 5% vanillin sulfuric acid ethanol solution, heating at 105 ℃ until the spots developed clearly, and the results showed that the development effect was good in an environment at 4 ℃, see fig. 21, in which 1- (-) -menthone; 2-pulegone; 3-schizonepeta medicinal material; 4-schizonepeta decoction pieces.

(2) Humidity investigation

Operating according to the scheme of the embodiment 11 of the invention, spotting, developing in 18% and 72% relative humidity, taking out, airing, spraying 5% vanillin-sulfuric acid ethanol solution, heating at 105 ℃ until the spots are clearly developed, and the results show that the developing effect is better in different humidity environments, as shown in fig. 22 (18%), fig. 23 (72%), and fig. 22-23, 1- (-) -menthone; 2-pulegone; 3-schizonepeta medicinal material; 4-schizonepeta decoction pieces.

(3) Specialization inspection

Operating according to the scheme in the embodiment 11 of the invention, using ethyl acetate reagent as a blank solution, spotting, developing, taking out, airing, spraying 5% vanillin sulfuric acid ethanol solution, heating at 105 ℃ until the spots are clearly developed, and the result shows that ethyl acetate is not interfered, thereby proving that the method has good specificity, as shown in figure 24, in which 1- (-) -menthone; 2-pulegone; 3-schizonepeta medicinal material; 4-schizonepeta decoction pieces; 5-Ethyl acetate.

(4) Durability examination

Operating according to the scheme in the embodiment 11 of the invention, respectively spotting and spreading on silica gel G thin-layer plates of Qingdao ocean chemical industry branch works and Nicoti city chemical industry research institute, developing, taking out, airing, spraying with 5% vanillin sulfuric acid ethanol solution, heating at 105 ℃ until the spots are clearly developed, and the result shows that the method has better separation effect on 2 plates, and proves that the method has good durability, as shown in figures 25 and 26, and 1- (-) -menthone in figures 25-26; 2-pulegone; 3-schizonepeta medicinal material; 4-schizonepeta decoction pieces.

(5) Reproducibility study

The procedure was carried out by 2 experimenters simultaneously according to the protocol of the invention in example 11, and the results demonstrated good reproducibility of the method, see FIGS. 27 and 28, FIGS. 27 to 28, 1- (-) -menthone; 2-pulegone; 3-schizonepeta medicinal material; 4-schizonepeta decoction pieces.

3. Determination of heavy metal and harmful elements in schizonepeta

3.1.1 methods

The atomic absorption spectrophotometry is adopted to measure lead, cadmium, arsenic, mercury and copper in the schizonepeta medicinal material. The specific method is a method for measuring lead, cadmium, arsenic, mercury and copper according to the general rule 2321 of the four departments of the 2015 edition of pharmacopoeia of the people's republic of China.

3.1.2 results

The measurement of lead, cadmium, arsenic, mercury and copper is carried out on 20 batches of schizonepeta medicinal materials, and the measurement results are shown in table 8.

TABLE 8 determination of heavy metals and harmful elements in Schizonepeta herbs (ppm, mg/kg)

3.2 determination of pesticide residue of herba Schizonepetae medicinal material

3.2.1 methods

The total hexachloro-hexa (the sum of alpha-BHC, beta-BHC, gamma-BHC and delta-BHC), the total dichlorodiphenyl-trichloroethane (the sum of pp '-DDE, pp' -DDD, op '-DDT and pp' -DDT) and the pentachloronitrobenzene in the schizonepeta medicinal material are measured by adopting a gas chromatography. The specific method is 2341 organic chlorine pesticide residue determination method-first method in the four ministry of communications of pharmacopoeia of the people's republic of China 2015 edition.

3.2.2 results

Total hexachloro-hexa (alpha-BHC, beta-BHC, gamma-BHC and delta-BHC), total dichlorodiphenyl trichloroethane (pp '-DDE, pp' -DDD, op '-DDT and pp' -DDT) and quintozene measurements are carried out on 20 batches of schizonepeta medicinal materials, and the measurement results are shown in Table 9.

TABLE 9 Fineleaf Schizonepeta medicinal materials pesticide residue amount measurement results (ppm, mg/kg)

4. Herba schizonepetae medicinal material extract determination

4.1 method examination

Taking 4g of schizonepeta, respectively adding 100ml of solvents (dilute ethanol, ethanol solution with the volume fraction of 70 percent and ethanol in the table), shaking for 6 hours, soaking for 18 hours, filtering, sucking 20ml of filtrate, placing the filtrate in an evaporation dish dried to constant weight, drying by distillation, placing the filtrate in an evaporation dish dried to be constant weight for 3 hours at 105 ℃, and weighing. The extract content (%) was calculated and the results are shown in Table 10.

TABLE 10 examination of extracts

Solvent(s)	Dilute ethanol	70% ethanol	Ethanol
				Extract (%)	8.09	10.07	8.64

From the results of examination, 70% ethanol was selected as the extract solvent because the extraction efficiency was the best with 70% ethanol.

4.2 results

The results of the measurement of the herba Schizonepetae extract in each batch are shown in Table 11.

TABLE 11 measurement results of extract of Schizonepeta herb

5. Content determination of schizonepeta medicinal material

5.1 examination of chromatographic conditions

The procedure of example 5 was followed, wherein the gradient elution procedure was performed according to the gradient elution method shown in tables 12 to 15, and the chromatograms are shown in fig. 29 to 32; according to results, the hesperidin chromatogram obtained by the gradient elution method 4 has a good peak shape, and purity analysis shows that the peak purity is good, the separation degree is 2.11, the USP tailing factor is 1.02, the symmetry factor is 1.02, and the number of USP theoretical plates is 14032.

TABLE 12 gradient elution method 1

Time (min)	Methanol	5% acetic acid water
			0～45	30％	70％

TABLE 13 gradient elution method 2

Time (min)	Acetonitrile	0.2% phosphoric acid water
			0～15	10％～14％	90％～86％
15～40	14％～26％	86％～74％
			40～55	26％～60％	74％～40％
55～60	60％～85％	40％～15％
			60～65	85％	15％

TABLE 14 gradient elution method 3

Time (min)	Acetonitrile	0.2% phosphoric acid water
			0～28	20％	80％
28～29	20％～85％	80％～15％
			29～39	85％	15％

TABLE 15 gradient elution method 4

Time (min)	Acetonitrile	0.2% phosphoric acid water
			0～28	18％	82％
28～29	18％～85％	82％～15％
			29～39	85％	15％

5.2 examination of extraction methods

5.2.1 examination of extraction solvent

In the procedure of example 5, 25ml of the extraction solvent in the following table was added and the reflux treatment was carried out for 30min, respectively, in the preparation of the test solutions, and the results are shown in the following table.

TABLE 16 examination of extraction solvent

Solvent(s)	Methanol	Ethanol	Water (W)
				Content (mg/g)	0.947	0.573	0.700

As can be seen from the data in the table, the extraction rate is the highest when methanol is used as the extraction solvent, which is obviously superior to ethanol and water, so the extraction solvent is selected to be methanol.

5.2.2 examination of extraction methods

The extraction method in the following table was selected to treat for 30min in the preparation of the test solution according to the protocol of example 5 of the present invention, and the results are shown in the following table.

TABLE 17 examination of extraction methods

Extraction method	Ultrasound	Refluxing	Soaking overnight
				Content (mg/g)	0.947	1.225	0.974

As can be seen from the data in the table, the reflux extraction rate is the highest, which is obviously superior to the ultrasonic extraction and the immersion overnight, so the extraction mode is selected as reflux extraction.

5.2.3 examination of sample weighing

In the step of preparing the test solution, the test samples were weighed according to the weighing methods in the following table, and the results are shown in the following table.

TABLE 18 results of sample weighing examination

Sample weighing	0.5g	1g	2g
				Content (mg/g)	1.299	1.225	1.198

As is clear from the data in the table, the extraction yield was the highest when the sample amount was 0.5g, so that the sample amount was selected to be 0.5 g.

5.2.4 examination of extraction time

The procedure of example 5 was followed, and the test solutions were refluxed for 30min, 60min, and 120min, respectively, during the preparation of the test solutions, and the results are shown in the following table.

TABLE 19 extraction of time finding results

Time of reflux	30min	60min	120min
				Content mg/g	1.299	1.248	1.198

As can be seen from the data in the table, the extraction time was selected to be 30min because the extraction rate was the highest when the extraction time was 30 min.

5.3 methodological considerations

5.3.1 Linear relationship investigation

Weighing hesperidin control 14.2mg (96.1%), placing in a 25ml measuring flask, adding methanol for dissolving, adding methanol for diluting to scale, and shaking. Precisely sucking 5ml of the solution, putting the solution into a 10ml measuring flask, adding methanol to dilute the solution to a scale, shaking the solution uniformly to obtain 272.924 mu g/ml reference substance solution, and sequentially diluting the solution step by step to obtain reference substance solutions with the concentrations of 136.462 mu g/ml, 68.231 mu g/ml, 34.116 mu g/ml, 17.058 mu g/ml, 8.529 mu g/ml, 4.264 mu g/ml and 2.132 mu g/ml respectively.

Drawing of standard curve

The control solutions were pipetted at concentrations of 68.231. mu.g/ml, 34.116. mu.g/ml, 17.058. mu.g/ml, 8.529. mu.g/ml, 4.264. mu.g/ml and 2.132. mu.g/ml, respectively, 10. mu.l thereof were subjected to measurement under the chromatographic conditions and assay method in example 7 and injected into a liquid chromatograph, and the results are shown in Table 20.

TABLE 20 Linear relationship examination

The result shows that the linear regression equation of hesperidin is Y (21954X), r (0.9997) and has good linear relation in the range of 2.132 mu g/ml-68.231 mu g/ml, the graph of the regression equation is shown in figure 10, and the linear regression equation can be calculated by an external standard point method.

5.3.2 precision investigation

A sample of Schizonepeta tenuifolia powder (sieved through sieve II) was weighed at 0.5g and processed according to the protocol of the present invention as described in example 5. Sample introduction is carried out for 6 times continuously, and the RSD value is calculated to be 0.47% according to the peak area, which shows that the precision is good.

TABLE 21 precision investigation

5.3.3 stability Studies

A sample of Schizonepeta tenuifolia powder (sieved through sieve II) was weighed at 0.5g and processed according to the protocol of the present invention as described in example 5. Injecting samples at 0, 2, 4, 8, 12 and 24 hours respectively, calculating RSD values according to peak areas, and inspecting the stability of the test sample. The results show that hesperidin in the test solution is substantially stable within 24 hours.

TABLE 22 stability study

5.3.4 repeatability test

A total of 6 parts of herba Schizonepetae sample powder (sieved through sieve II) 0.5g was weighed and processed according to the protocol of the invention in example 5. And (3) sampling, calculating the content according to the peak area, and displaying that the content is 1.30mg/g (0.130%) and the RSD value is 1.98%, which shows that the method has good repeatability.

TABLE 23 repeatability test

5.3.5 accuracy survey

Taking 0.25g of schizonepeta powder (screened by a No. two sieve), precisely adding 0.6ml of prepared hesperidin reference solution of 545.848 mu g/ml, precisely adding 24.4ml of methanol, sealing, weighing, refluxing for 30min, cooling, adding methanol to supplement the lost weight, shaking up, taking a subsequent filtrate to obtain a test solution, and then operating according to the scheme in the embodiment 5 of the invention. And (4) continuously preparing 6 samples, injecting samples, and calculating the sample recovery rate. The result shows that the sample adding recovery rate of the method is good.

Table 24 accuracy survey

5.3.6 durability examination

A sample of Schizonepeta tenuifolia powder (sieved through sieve II) was weighed at 0.5g and processed according to the protocol of the present invention as described in example 5. The content and RSD values were calculated by 3 different chromatographic columns. The results show that the method is very durable.

TABLE 25 determination of sample content by different chromatographic columns

Column chromatography manufacturers	Waters	Agilent	Kromasil	RSD(％)
					Content (mg/g)	1.30	1.23	1.26	2.78

The research establishes a detection method for measuring the content of hesperidin in schizonepeta by using a high performance liquid chromatography. Through investigation on the elution conditions of the mobile phase, the optimal chromatographic conditions are determined, and the methodology is comprehensively investigated; the pretreatment conditions of the sample are optimized by observing the influence of the extraction solvent, the extraction mode, the extraction time and the sample weighing amount on the extraction effect of the hesperidin in the schizonepeta. The method is simple, the measurement result is accurate and reliable, the repeatability is good, the practicability is strong, and the method can be used for evaluating the quality of the schizonepeta decoction pieces.

6. Research on HPLC (high performance liquid chromatography) characteristic spectrum of nonvolatile components of schizonepeta medicinal materials

6.1.1 selection of wavelength

The schizonepeta DAD full-wavelength scanning spectrum is shown in fig. 33, and as can be seen from the graph, the spectral peaks of the sample at the wavelength of 283nm are relatively balanced, so that 283nm is selected as the detection wavelength of the characteristic spectrum of the schizonepeta medicinal material and the decoction pieces.

6.1.2 examination of chromatographic conditions

The procedure of example 1 was followed, wherein the gradient elution methods were performed according to the gradient elution methods shown in tables 26 to 28, respectively, and the chromatograms are shown in fig. 34 to 36 in sequence, and it can be seen from the results that the chromatograms obtained by the gradient elution method 3 had good peak shapes and good degrees of separation, and thus, this method was selected as the characteristic pattern identification method.

TABLE 26 gradient elution method 1

Time (min)	Acetonitrile	0.2% phosphoric acid water
			0～20	20％	80％
20～30	25％	75％
			30～50	30％	70％
50～60	50％	50％
			60～70	80％	20％

TABLE 27 gradient elution method 2

Time (min)	Acetonitrile	0.05% trifluoroacetic acid water
			0～40	5％～40％	95％～60％
40～60	40％～60％	60％～40％
			60～65	60％～5％	40％～95％
65～90	5％	95％

TABLE 28 gradient elution method 3

6.2 methodological investigation

6.2.1 precision investigation

According to the scheme operation in the embodiment 1 of the invention, the same schizonepeta test sample solution is precisely absorbed, sample introduction is carried out for 7 times continuously, the No. 1 peak is taken as a reference peak (S), the RSD of each characteristic peak relative to the retention time is less than 3.0%, and the RSD of the relative peak area is less than 5.0%, which indicates that the instrument precision is good.

TABLE 29 results of precision vs. Retention time calculations

	S1	S2	S3	S4	S5	S6
								1	1.00	1.07	1.09	1.20	1.25	1.64
2	1.00	1.07	1.09	1.21	1.25	1.64
							3	1.00	1.07	1.09	1.21	1.26	1.64
4	1.00	1.07	1.09	1.21	1.26	1.63
							5	1.00	1.07	1.09	1.21	1.26	1.63
6	1.00	1.07	1.09	1.21	1.26	1.63
							7	1.00	1.07	1.09	1.21	1.26	1.63
RSD％	0.00	0.25	0.17	0.19	0.10	0.18

TABLE 30 results of relative peak area calculation for precision

	S1	S2	S3	S4	S5	S6
								1	1.00	0.39	0.64	0.23	0.50	0.39
2	1.00	0.39	0.64	0.23	0.50	0.39
							3	1.00	0.39	0.64	0.23	0.49	0.38
4	1.00	0.39	0.64	0.23	0.49	0.38
							5	1.00	0.39	0.64	0.23	0.49	0.38
6	1.00	0.38	0.62	0.22	0.53	0.37
							7	1.00	0.39	0.63	0.23	0.54	0.38
RSD％	0.00	1.10	0.93	1.67	3.36	1.68

6.2.2 stability Studies

According to the scheme operation in the embodiment 1 of the invention, the same schizonepeta test solution is precisely sucked, sample injection is carried out respectively at 0h, 1h, 3h, 6h, 9h, 12h and 24h after the test solution is prepared, the No. 1 peak is taken as a reference peak (S), the RSD of each characteristic peak relative retention time is less than 3.0%, and the RSD of the relative peak area is less than 6.0%, which indicates that the test solution is stable within 24h after the preparation.

TABLE 31 results of stability versus Retention time calculation

	S1	S2	S3	S4	S5	S6
							0h	1.00	1.07	1.09	1.20	1.25	1.64
1h	1.00	1.07	1.09	1.21	1.25	1.64
							3h	1.00	1.07	1.09	1.21	1.26	1.64
6h	1.00	1.07	1.09	1.21	1.26	1.63
							9h	1.00	1.07	1.09	1.21	1.26	1.63
12h	1.00	1.08	1.10	1.21	1.26	1.63
							24h	1.00	1.07	1.09	1.21	1.26	1.64
RSD％	0.00	0.31	0.22	0.24	0.13	0.24

TABLE 32 results of calculation of stability relative Peak area

	S1	S2	S3	S4	S5	S6
							0h	1.00	0.39	0.64	0.23	0.50	0.39
1h	1.00	0.39	0.64	0.23	0.50	0.39
							3h	1.00	0.39	0.64	0.23	0.49	0.38
6h	1.00	0.39	0.64	0.23	0.49	0.38
							9h	1.00	0.39	0.63	0.23	0.54	0.38
12h	1.00	0.39	0.64	0.24	0.56	0.38
							24h	1.00	0.40	0.65	0.22	0.52	0.39
RSD％	0.00	0.96	0.90	2.40	5.20	1.36

6.2.3 repeatability test

According to the scheme of the embodiment 1 of the invention, about 0.5g of the same batch of schizonepeta powder is taken, 6 parts of the schizonepeta powder are precisely weighed and respectively injected, a characteristic spectrum is recorded, the peak 1 is taken as a reference peak (S), the RSD of each characteristic peak relative to the retention time is less than 3.0%, and the RSD of the relative peak area is less than 6.0%, which indicates that the method has good repeatability.

TABLE 33 repeatability vs. retention time calculations

TABLE 34 results of calculation of the repetitive relative peak areas

	S1	S2	S3	S4	S5	S6
								1	1.00	0.39	0.64	0.23	0.50	0.39
2	1.00	0.38	0.60	0.21	0.52	0.35
							3	1.00	0.38	0.63	0.23	0.52	0.36
4	1.00	0.39	0.62	0.23	0.50	0.39
							5	1.00	0.38	0.61	0.20	0.48	0.36
6	1.00	0.39	0.60	0.21	0.51	0.40
							RSD％	0.00	1.19	2.26	5.93	3.08	5.19

6.2.4 durability examination

According to the scheme operation in the embodiment 1 of the invention, chromatograms of different chromatographic columns are adopted for detection, and the separation degree of each peak is checked. The Waters column, the Alchromobond-AQ column and the Phenomex-Luna column have good peak separation degree.

TABLE 35 determination of relative sample retention times for different chromatographic columns

Column chromatography manufacturers	Waters	AlchromBond-AQ	Phenomex-Luna
				S1	1.00	1.00	1.00
S2	1.07	1.07	1.07
				S3	1.09	1.14	1.09
S4	1.20	1.20	1.20
				S5	1.25	1.23	1.25
S6	1.64	1.49	1.59

TABLE 36 relative peak areas of samples determined on different chromatographic columns

6.3 feature Pattern Collection

The detection results of every batch of schizonepeta are analyzed by adopting a traditional Chinese medicine chromatography fingerprint similarity evaluation system (V2.0) compiled by the State pharmacopoeia Committee. A median method is adopted, the time window parameter is 0.30, and a comparison characteristic spectrum (R fingerprint spectrum) is generated by software.

6.3.1 characteristic Peak calibration

According to the scheme in the embodiment 1 of the invention, the characteristic spectrum of a representative sample of schizonepeta medicinal materials is constructed and analyzed, and typical chromatographic peaks appearing in the characteristic spectrum of each batch of samples are taken as characteristic peaks.

At 283nm, 6 characteristic peaks are totally obtained, wherein the peak 1 is hesperidin, the peak 2 is rosmarinic acid, the

peaks

3, 4 and 5 are common characteristic peaks, the peak 6 is pulegone, and the spectrum peaks are respectively expressed by the serial numbers of 1-6.

6.3.2 determination of samples of batches and establishment of control profiles

The relative retention time and the relative peak area of each batch of samples are shown in tables 37-38, and the generated control characteristic map is shown in fig. 37, and 6 characteristic peaks should be presented in the characteristic map of each batch of samples, and are marked by S1, S2, S3, S4, S5 and S6.

TABLE 37 relative retention time of 6 characteristic peaks of each batch of herba Schizonepetae materials

Numbering	S1	S2	S3	S4	S5	S6
							J1	1.00	1.07	1.09	1.21	1.26	1.66
J2	1.00	1.07	1.09	1.21	1.26	1.63
							J3	1.00	1.08	1.10	1.20	1.25	1.63
J4	1.00	1.09	1.11	1.22	1.27	1.65
							J5	1.00	1.07	1.09	1.21	1.26	1.64
J6	1.00	1.06	1.09	1.20	1.25	1.65
							J7	1.00	1.07	1.10	1.21	1.26	1.63
J8	1.00	1.08	1.10	1.21	1.26	1.63

TABLE 38 relative peak areas of 6 characteristic peaks of each batch of Schizonepeta medicinal materials

Numbering	S1	S2	S3	S4	S5	S6
							J1	1.00	0.41	0.25	0.64	0.46	0.42
J2	1.00	0.26	0.18	0.18	0.26	0.19
							J3	1.00	2.71	0.62	0.02	0.02	0.42
J4	1.00	0.60	0.01	0.05	0.03	0.62
							J5	1.00	4.82	0.85	0.40	1.64	1.22
J6	1.00	0.75	0.21	0.17	0.38	0.23
							J7	1.00	0.70	0.18	0.27	0.77	0.16
J8	1.00	1.25	0.25	0.23	0.47	0.26

7. Research on GC characteristic spectrum of schizonepeta medicinal material volatile oil

7.1.1 chromatographic Condition selection

The procedure of example 4 was followed, wherein the column temperature was programmed according to the temperature profiles of tables 39 to 43 below; the characteristic maps obtained by the temperature raising programs 1 to 5 are shown in fig. 38 to 42, and the results show that the chromatogram peak shape obtained by the temperature raising program 3 is better, so the temperature raising program 3 is selected.

TABLE 39 temp. PROGRAM 1

Speed/min	Temperature of	Holding time min
			——	60	2
3	120	5
			3	130	10
3	160	0
			5	230	0

TABLE 40 temperature program 2

TABLE 41 temperature-raising program 3

Speed/min	Temperature of	Temperature of
			——	60	2
2	120	5
			3	160	0
5	220	0

TABLE 42 temp. UP PROGRAM 4

Speed/min	Temperature of	Speed/min
			——	60	2
2	140	5
			5	210	0
10	220	0

TABLE 43 temperature increasing program 5

Rate (. degree.C./min)	Temperature (. degree.C.)	Temperature (. degree.C.)
			——	60	2
2	120	5
			5	170	5
10	220	0

7.2 methodological investigation

7.2.1 precision investigation

According to the operation of the method in the embodiment 4 of the invention, the same schizonepeta test sample solution is precisely absorbed, sample introduction is carried out for 6 times continuously, the No. 2 peak is taken as a reference peak (S), the RSD of each characteristic peak relative to the retention time is less than 3.0 percent, and the RSD of the relative peak area is less than 3.0 percent, which indicates that the precision of the instrument is good.

TABLE 44 results of precision vs. retention time calculations

TABLE 45 results of relative peak area calculation for precision

	S1	S2	S3
					1	1.05	1.00	0.03
2	1.02	1.00	0.03
				3	1.05	1.00	0.03
4	1.04	1.00	0.03
				5	1.05	1.00	0.03
6	1.05	1.00	0.03
				7	1.03	1.00	0.03
RSD％	1.09	0.00	1.61

7.2.2 stability Studies

According to the operation of the method in the embodiment 4 of the invention, the same schizonepeta test solution is precisely sucked, sample injection is carried out respectively at 0h, 1h, 3h, 6h, 9h, 12h and 24h after the test solution is prepared, the No. 2 peak is taken as a reference peak (S), the RSD of each characteristic peak relative to the retention time is less than 3.0%, and the RSD of the relative peak area is less than 5.0%, which indicates that the test solution is stable within 24h after the preparation.

TABLE 46 calculation of stability versus Retention time

TABLE 47 calculation of stability relative Peak area

	S1	S2	S3
				0h	1.05	1.00	0.03
1h	1.02	1.00	0.03
				3h	1.04	1.00	0.03
6h	1.03	1.00	0.03
				9h	1.04	1.00	0.03
12h	1.04	1.00	0.03
				24h	1.07	1.00	0.03
RSD％	1.39	0.00	3.12

7.2.3 repeatability test

According to the operation of the method in the embodiment 4 of the invention, the same batch of schizonepeta powder is taken and 6 parts are taken in parallel, the gas chromatogram is recorded, the peak No. 2 is taken as a reference peak (S), the RSD of each characteristic peak relative to the retention time is less than 3.0%, and the RSD of the relative peak area is less than 3.0%, which shows that the method has good repeatability.

TABLE 48 repeatability vs. Retention time calculations

	S1	S2	S3
					1	0.67	1.00	1.51
2	0.67	1.00	1.51
				3	0.67	1.00	1.51
4	0.67	1.00	1.51
				5	0.67	1.00	1.50
6	0.67	1.00	1.50
				RSD％	0.08	0.00	0.10

TABLE 49 repeatability relative peak area calculation results

	S1	S2	S3
					1	1.04	1.00	0.03
2	1.04	1.00	0.03
				3	1.04	1.00	0.03
4	1.04	1.00	0.03
				5	1.06	1.00	0.03
6	1.04	1.00	0.03
				RSD％	0.88	0.00	2.65

7.3 feature Pattern Collection

The detection results of the gas phase spectra of each batch of schizonepeta are analyzed by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (V2.0) compiled by the State pharmacopoeia Committee. A median method is adopted, the time window parameter is 0.30, and a comparison characteristic spectrum (R fingerprint spectrum) is generated by software.

7.3.1 characteristic Peak calibration

According to the scheme in the embodiment 4 of the invention, the characteristic spectrum of a representative sample of schizonepeta medicinal materials is constructed and analyzed, and typical chromatographic peaks appearing in the characteristic spectrum of each batch of samples are taken as characteristic peaks.

The total number of the characteristic peaks is 3, and the common characteristic peaks are as follows: the No. 1 peak is menthone, the No. 2 peak is pulegone, the No. 3 peak, and each spectrum peak is represented by the serial numbers of 1-3.

7.3.2 determination of samples of various batches and establishment of control profiles

The relative retention time and the relative peak area of each batch of samples are shown in tables 50-51, the generated control characteristic map is shown in FIG. 43, and 3 characteristic peaks should be presented in the characteristic map of each batch of samples, which are marked by S1, S2 and S3.

TABLE 50 relative retention time of 3 characteristic peaks of each batch of Schizonepeta medicinal materials

TABLE 51 relative peak area of 3 characteristic peaks of each batch of Schizonepeta medicinal materials

Numbering	S1	S2	S3
				J1	0.44	1.00	0.05
J2	0.20	1.00	0.04
				J3	0.59	1.00	0.02
J4	0.71	1.00	0.03
				J5	0.27	1.00	0.02
J6	0.39	1.00	0.01
				J7	0.20	1.00	0.02
J8	0.46	1.00	0.01

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. A method for constructing a herba schizonepetae characteristic spectrum is characterized by comprising a method for constructing a non-volatile component characteristic spectrum and a method for constructing a volatile component characteristic spectrum, wherein the method comprises the following steps:

preparing a test solution I or a reference substance solution I of a reference medicinal material: preparing herba Schizonepetae sample or herba Schizonepetae reference material into powder, precisely weighing 0.5-1g, adding solvent 15-30ml, sealing, extracting, cooling, adding the solvent to compensate the lost weight, shaking, filtering the supernatant, and collecting the filtrate to obtain sample solution I or reference material solution I; the solvent is methanol, ethanol or water;

preparation of reference solution I: preparing a reference substance solution I of the hesperidin reference substance;

chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, and using octadecyl bonded silica gel as chromatographic column filler; taking acetonitrile as a mobile phase A, and taking a phosphoric acid aqueous solution with the volume fraction of 0.2% as a mobile phase B for elution; the elution mode is a gradient elution mode, and the gradient elution program comprises the following steps: 0-15min, mobile phase A: the volume ratio of the mobile phase B is 8-12%: 92-88% → 12-16%: 88 to 84 percent; 15-40min, mobile phase A: the volume ratio of the mobile phase B is 12-16%: 88-84% → 24-28%: 76 to 72 percent; 40-55min, mobile phase A: the volume ratio of the mobile phase B is 24-28%: 76-72% → 58-62%: 42 to 38 percent; 55-60min, mobile phase A: the volume ratio of the mobile phase B is 58-62%: 42-38% → 83-87%: 17 to 13 percent; 60-65min, mobile phase A: the volume ratio of the mobile phase B is 83-87%: 17-13% → 83-87%: 17 to 13 percent; the chromatographic conditions are as follows: the column temperature is 25-40 ℃; the flow rate is 0.8-1.2 ml/min; the detection wavelength is 278-288 nm;

the determination method comprises the following steps: precisely absorbing the reference substance solution I and the test solution I respectively, injecting into a liquid chromatograph, and measuring to obtain the final product;

the non-volatile component characteristic spectrum comprises 6 characteristic peaks, wherein the characteristic peaks are as follows: the No. 1 peak is hesperidin, the No. 2 peak is rosmarinic acid, the No. 3 peak, the No. 4 peak and the No. 5 peak are common characteristic peaks, and the No. 6 peak is pulegone; and

preparation of test solution II: taking a schizonepeta tenuifolia sample, preparing volatile oil by an A method for measuring 2204 volatile oil according to general rules of the four ministry of the pharmacopoeia of the people's republic of China 2015 edition, precisely absorbing 25-50ul of volatile oil, adding ethyl acetate to a constant volume of 2ml, removing water and drying to obtain the schizonepeta tenuifolia volatile oil;

preparation of reference solution II: preparing reference solution II of reference substance from pulegone and menthone, wherein the reference solution II is reference substance solution containing pulegone 3-6mg/ml and menthone 3-6mg/ml, and the solvent is ethyl acetate;

chromatographic conditions and system applicability test: measured by gas chromatography, as N₂Taking PEG as a carrier gas, controlling the temperature of a sample inlet to be 245-class 255 ℃, the temperature of a detector to be 245-class 255 ℃, the total flow rate to be 3.5-3.9ml/min, controlling the temperature of the column to be in gradient temperature rise, wherein the gradient temperature rise procedure is as follows: the initial temperature is 55-65 deg.C, and is maintained for 1-3 min; the temperature is raised from 55-65 ℃ to 115-125 ℃, and then the temperature is kept for 4-6min, and the temperature raising rate is 1.5-2.5 ℃/min; the temperature is increased from 115 ℃ to 125 ℃ to 155 ℃ to 165 ℃, and the temperature increase rate is 2.5-3.5 ℃/min; the temperature is increased from 155 ℃ to 165 ℃ to 235 ℃ to 245 ℃, and the temperature increase rate is 4.5-5.5 ℃/min;

the determination method comprises the following steps: precisely absorbing the reference substance solution II and the test solution II respectively, injecting into a gas chromatograph, and measuring to obtain the final product;

the volatile component characteristic spectrum comprises 3 characteristic peaks, wherein the characteristic peaks are as follows: the peak 1 is menthone, the peak 2 is pulegone, and the peak 3 is a common characteristic peak.

2. The method for constructing the schizonepeta characteristic spectrum of claim 1, which is characterized by further comprising the step of constructing a comparison characteristic spectrum, wherein the characteristic spectrum of at least 15 batches of obtained test solution I is analyzed by utilizing a traditional Chinese medicine chromatography fingerprint similarity evaluation system of the national pharmacopoeia committee to generate a non-volatile component comparison characteristic spectrum; and/or

3. The method for constructing a nepeta characteristic map of claim 1 or 2, wherein in the method for constructing a non-volatile component characteristic map, the gradient elution procedure is: 0-15min, mobile phase A: the volume ratio of the mobile phase B is 10%: 90% → 14%: 86 percent; 15-40min, mobile phase A: the volume ratio of the mobile phase B is 14%: 86% → 26%: 74 percent; 40-55min, mobile phase A: the volume ratio of the mobile phase B is 26%: 74% → 60%: 40 percent; 55-60min, mobile phase A: the volume ratio of the mobile phase B is 60%: 40% → 85%: 15 percent; 60-65min, mobile phase A: the volume ratio of the mobile phase B is 85%: 15% → 85%: 15 percent.

4. A method according to claim 1 or 2, wherein the chromatographic conditions are as follows: the column temperature was 40 ℃; the flow rate is 1 ml/min; the detection wavelength was 283 nm.

5. The method for constructing the herba schizonepetae feature map of claim 1 or 2, wherein the gradient temperature rise program is as follows: the initial temperature is 60 ℃, and the temperature is kept for 2 min; the temperature is increased from 60 ℃ to 120 ℃, the temperature is kept for 5min, and the temperature increasing rate is 2 ℃/min; the temperature is increased from 120 ℃ to 160 ℃, and the temperature increasing rate is 3 ℃/min; the temperature is increased from 160 ℃ to 240 ℃, and the temperature increasing rate is 5 ℃/min.

6. The method of claim 1 or 2, wherein the catmint temperature is 250 ℃ at the inlet, 250 ℃ at the detector, and the total flow rate is 3.7 ml/min.

7. A method of constructing a nepeta characteristic map of claim 1 or 2, wherein,

the preparation method of the reference substance solution I of the reference substance comprises the following steps: dissolving hesperidin control in the solvent to obtain reference solution I containing 55-65 μ g/ml hesperidin.

8. A method of constructing a nepeta characteristic map of claim 1 or 2, wherein,

the test sample is selected from raw medicinal materials or decoction pieces of herba schizonepetae.

9. A method of constructing a nepeta characteristic map of claim 1 or 2, wherein,

the extraction treatment adopts ultrasonic treatment for 30-90min, reflux for 30-120min or dipping for 12-24 h.

10. The method for constructing the characteristic spectrum of schizonepeta according to claim 7, wherein,

the reference substance solution I of the control product is a reference substance solution I of the control product containing 60 mu g/ml hesperidin.

11. A method for detecting the quality of schizonepeta, which is characterized by comprising the step of detecting the quality of schizonepeta products by using the method for constructing the characteristic map of schizonepeta as defined in any one of claims 1 to 10.

12. The method for detecting the quality of the schizonepeta according to claim 11, wherein the schizonepeta product to be detected is detected according to the same method for constructing the schizonepeta characteristic map of any one of claims 1 to 10, and the obtained schizonepeta non-volatile component characteristic map and the schizonepeta volatile component characteristic map are obtained, the non-volatile component characteristic map presents 6 characteristic peaks and corresponds to 6 characteristic peaks in a reference substance characteristic map of a reference medicinal material, wherein the peaks 1 and 6 are consistent with the retention time of the reference substance peaks of the reference substance of the reference medicinal material; the volatile component characteristic spectrum presents 3 characteristic peaks, wherein the peak 1 and the peak 2 are consistent with the retention time of a reference substance peak of a reference substance.

13. The method for detecting the quality of the schizonepeta as claimed in claim 11 or 12, further comprising the step of measuring the content of the schizonepeta, and the method comprises the following steps:

14. The method for detecting the quality of the schizonepeta according to claim 11 or 12, further comprising the steps of thin layer chromatography identification, heavy metal and harmful element measurement and/or extract measurement:

the thin layer plate is a silica gel G plate or an H plate;

the developing solvent is chloroform-ethyl acetate-glacial acetic acid mixed solution, and the volume ratio of the developing solvent to the mixed solution is (9-11) to 1 (0.3-0.8); and/or

taking a proper amount of coarse powder of a schizonepeta test sample, measuring according to a volatile oil measuring method 2204 of the national pharmacopoeia 2015 edition of the people's republic of China, dividing and taking 0.1-0.3 ml of volatile oil, adding ethyl acetate to dilute the volatile oil to 1.5-4.5 ml, and taking the volatile oil as a test sample solution V; adding ethyl acetate into (-) -menthone and pulegone reference substances to prepare a solution containing 5-30 mg of the (-) -menthone and pulegone per 1ml as a reference substance solution; sucking 1-5ul of the raw materials respectively, dropping the raw materials on the same thin-layer plate, developing the raw materials in a developing agent, taking out the raw materials, drying the raw materials in the air, spraying 2-8% of vanillin sulfuric acid ethanol solution, and heating the raw materials at 105 ℃ until spots are clearly developed;

and/or;

F. The step of extract determination comprises:

the extract determination operation steps are as follows: 2-5g of coarse powder of a schizonepeta test medicinal material is taken, precisely weighed, placed in a 250-300 ml conical flask, precisely added with 100ml of a solvent, sealed, cold soaked, shaken all the time within the first 6 hours, then kept stand for 18 hours, rapidly filtered by a drying filter, precisely taken with 20ml of a subsequent filtrate, placed in an evaporation dish dried to constant weight, dried on a water bath, dried at 105 ℃ for 3 hours, placed in a dryer for cooling for 30 minutes, rapidly and precisely weighed, and the content of extract in the test sample is calculated by using the dried product; wherein the solvent is ethanol, and the volume ratio fraction is 50-90%.

15. The method for detecting the quality of the schizonepeta according to claim 14, wherein in the volatile component thin-layer chromatography identification step D, the thin-layer plate is a silica gel G plate or an H plate; and/or

The developing solvent is ethyl acetate-petroleum ether, wherein the petroleum ether is 60-90 ℃, and the volume ratio of the petroleum ether to the developing solvent is 1: (11-13).