Method for constructing characteristic spectrum of Jianqu or stir-fried Jianqu or its water extract, and detection method thereof

Abstract

The present application relates to the technical field of traditional Chinese medicine detection and analytical chemistry, and specifically to a method for constructing a characteristic spectrum of Jianqu or stir-fried Jianqu or its aqueous extract, and a detection method thereof. In the technical solution of the present application, ultra-high performance liquid chromatography is used for analysis, and the mobile phase conditions include: mobile phase A, which is a mixed solvent of methanol and acetonitrile with a volume ratio of (0.5 to 1.5): 1; mobile phase B, which is an aqueous formic acid solution with a volume concentration of 0.08% to 0.12%. The characteristic spectrum of the present application is characteristic, reproducible and operable, and can clearly identify the common components and differential components of Jianqu or stir-fried Jianqu or its aqueous extract, and has better specificity and accuracy.

Description

Technical Field

[0001] The present application relates to the technical field of traditional Chinese medicine detection and analytical chemistry, and specifically to a method for constructing a characteristic spectrum of Jianqu or stir-fried Jianqu or their water extracts, and a detection method thereof. Background Art

[0002] Jianqu (Jianqu) is pungent and sweet in flavor, warm in nature. It enters the spleen and stomach meridians, and has the functions of digesting food, dispelling wind and cold, and strengthening the spleen and stomach. According to the Qing Dynasty book "Compendium of Materia Medica Benefits," written by Ling Xiaowang, Jianshenqu can be traced back to Baicaoqu, created by Fan Zhi and Wu Yifei in Quanzhou, Fujian. The "Supplement to Compendium of Materia Medica" records Jianshenqu as also known as Fanzhiqu and Baicaoqu, and also states that Baicaoqu is the current Jianqu. Jianqu now mostly refers to Jianqu produced in Sichuan, included in the "Ministry of Health's Standards for Traditional Chinese Medicine Formulas" (Volume 17). It is made by grinding 21 herbs—Polygonum hydropiper, Xanthium sibiricum, Artemisia annua, bitter almond, red bean, malt, hawthorn (fried), tangerine peel, patchouli, Atractylodes lancea, Magnolia officinalis, Costus root, Angelica dahurica, Citrus aurantium (fried with bran), Areca catechu, Perilla frutescens, mint, barley sprout, cinnamon bark, Cyperus rotundus, and licorice—into a powder, which is then mixed with wheat bran, flour, and other auxiliary materials for fermentation. It mainly has the function of dispelling exterior pathogens and tonifying the middle pathogens, and is mainly used to treat symptoms such as cold and heat headaches, food stagnation, vomiting and bloating. After being stir-fried, Jianqu enhances its spleen-strengthening and diarrhea-relieving effects and moderates its medicinal properties, making it more suitable for the elderly, children and those with weak spleen and stomach.

[0003] Currently, there is a paucity of research literature on Jianqu or its processed products. Most studies focus on the historical evolution, processing, and clinical research of Jianqu, with fewer studies examining its quality. Zheng Yanping et al. reported "Determination of the Contents of Five Components in Sichuan-Produced Jianqu and Preliminary Establishment of an HPLC Fingerprint," Huang Yongliang et al. reported "Study on Improving the Quality Standards of Sichuan-Produced Jianqu," and Sun Mengmei et al. reported "Study on the Changes in Components in Jianqu at Different Fermentation Degrees Based on Fingerprinting and Chemometrics." However, these technical solutions generally suffer from lengthy analysis times and limited detection of specific components, often exceeding 100 minutes. The components detected are mostly uncharacteristic components such as chlorogenic acid and gallic acid. Sun Mengmei et al. studied the fingerprint spectra of Jianqu at different fermentation degrees and identified the characteristic components of medicinal flavors such as dried tangerine peel, bitter orange peel, Magnolia bark, and patchouli. However, their experimental samples only included a batch of laboratory-made Jianqu, and the fingerprint spectra had a large number of chromatographic peaks and poor peak separation. Whether this method is applicable to the detection of multiple batches of Jianqu, stir-fried Jianqu, their standard decoctions, and Chinese medicine formula granules from different origins needs further study. Summary of the Invention

[0004] In view of this, the technical content of this application is specially proposed.

[0005] The present application relates to a method for constructing a characteristic spectrum of Jianqu or stir-fried Jianqu or their aqueous extracts and a detection method thereof, which can more comprehensively and objectively reflect the chemical characteristic information of Jianqu or stir-fried Jianqu or their aqueous extracts as a basis for their quality detection.

[0006] The specific technical solutions are described below:

[0007] The method for constructing the characteristic spectrum of Jianqu or stir-fried Jianqu or its water extract comprises the following steps:

[0008] Take Jianqu or stir-fried Jianqu or its aqueous extract, extract with extraction solvent to prepare the test solution;

[0009] A reference solution was prepared by mixing scopoletin reference substance, liquiritigenin reference substance, hydrated oxypeucedanum reference substance, naringenin reference substance, honokiol reference substance and magnolol reference substance, and dissolving them in a solvent;

[0010] Taking the test solution and the reference solution for ultra-high performance liquid chromatography analysis respectively to obtain the characteristic spectrum of the Jianqu or stir-fried Jianqu or their water extracts;

[0011] Wherein, the mobile phase conditions of the ultra-high performance liquid chromatography analysis include:

[0012] Mobile phase A, which is a mixed solvent of methanol and acetonitrile in a volume ratio of (0.5-1.5):1;

[0013] Mobile phase B, which is an aqueous formic acid solution with a volume concentration of 0.08% to 0.12%;

[0014] The total volume percentage of the mobile phase A and the mobile phase B is 100%.

[0015] The method for detecting Jianqu or stir-fried Jianqu or their aqueous extracts comprises the following steps:

[0016] Take the Jianqu or stir-fried Jianqu or its aqueous extract to be tested, extract it with an extraction solvent to prepare a solution of the test product;

[0017] Take a standard sample of Jianqu or stir-fried Jianqu or its aqueous extract, extract it with an extraction solvent, and prepare a standard solution;

[0018] Performing high performance liquid chromatography analysis on the test product solution and the standard solution respectively to obtain a characteristic spectrum of the test product and a characteristic spectrum of the standard, and comparing the characteristic spectrum of the test product and the characteristic spectrum of the standard;

[0019] Wherein, the mobile phase conditions of the ultra-high performance liquid chromatography analysis include:

[0020] Mobile phase A, which is a mixed solvent of methanol and acetonitrile in a volume ratio of (0.5-1.5):1;

[0021] Mobile phase B, which is an aqueous formic acid solution with a volume concentration of 0.08% to 0.12%;

[0022] The total volume percentage of the mobile phase A and the mobile phase B is 100%.

[0023] This application conducts extensive research on the characteristic components of Jianqu or stir-fried Jianqu and their aqueous extracts. By establishing appropriate chromatographic conditions, 8 common characteristic peaks are obtained, and the characteristic components of 6 medicinal herbs, including Artemisia annua, Glycyrrhiza uralensis, Angelica dahurica, Tangerine peel, Fructus Aurantii Immaturus, and Magnolia officinalis, are identified (peak 1 is scopoletin peak, peak 3 is liquiritigenin peak, peak 4 is hydrated oxidized peucedanum peak, peak 6 is naringenin peak, peak 7 is honokiol peak, and peak 8 is magnolia officinalis peak). It can well characterize the material transfer from raw materials to decoctions, fully demonstrate the material basis characteristics of the clinical decoctions of Jianqu or stir-fried Jianqu, and provide a rapid and comprehensive detection method for the quality of Jianqu or stir-fried Jianqu and their aqueous extracts.

[0024] The technical solution of this application is based on multiple batches of koji samples from different origins. Compared with the existing technology, it has stronger representativeness and specificity, good separation effect of characteristic peaks, shorter analysis time and is more suitable for quality detection and evaluation of koji. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0026] Figure 1 This is the full wavelength scanning spectrum created in Section 3.1 of Example 1;

[0027] Figure 2 This is a comparison chart of the chromatographic results under different detection wavelength conditions in Section 3.1 of Example 1;

[0028] Figure 3 This is a comparison chart of the chromatographic results under different elution conditions in Section 3.2 of Example 1;

[0029] Figure 4 This is a comparison chart of the chromatographic results under different mobile phase conditions in Section 3.3 of Example 1;

[0030] Figure 5 This is a comparison chart of the chromatographic results under different flow rate conditions in Section 3.4 of Example 1;

[0031] Figure 6 This is a comparison chart of the chromatographic results of the curves constructed under different column temperature conditions in 3.5 of Example 1;

[0032] Figure 7This is a comparison chart of the chromatographic results under different chromatographic column conditions in Section 3.6 of Example 1;

[0033] Figure 8 The superimposed chromatograms for the 30 batches of songs in Section 4 of Example 1 are shown;

[0034] Figure 9 The chromatograms are superimposed for the 30 batches of stir-fried yeast in Section 4 of Example 1;

[0035] Figure 10 This is the characteristic spectrum of the Jianqu medicinal material used for comparison in Section 4 of Example 1;

[0036] Figure 11 The chromatograms are superimposed for 30 batches of Jianqu standard decoctions in Section 4 of Example 1;

[0037] Figure 12 The chromatograms are superimposed for 30 batches of Chao Jianqu standard decoction in Section 4 of Example 1;

[0038] Figure 13 Construct the UV chromatogram and total ion current diagram for Section 5.1 of Example 1;

[0039] Figure 14 Confirm the chromatogram for the characteristic peaks constructed in Section 5.2 of Example 1;

[0040] Figure 15 This is the reference characteristic spectrum of the Jianqu medicinal materials in Example 3;

[0041] Figure 16 The reference characteristic spectrum of the stir-fried Jianqu decoction pieces in Example 3;

[0042] Figure 17 This is the reference characteristic spectrum of the standard decoction of the Jianqu medicinal material in Example 3;

[0043] Figure 18 This is the reference characteristic map of the standard decoction of the stir-fried Jianqu pieces in Example 3. DETAILED DESCRIPTION

[0044] The present application will be further described below in conjunction with the embodiments, examples and accompanying drawings. It should be understood that these examples are intended only to illustrate the present application and are not intended to limit the scope of the present application. In addition, it should be understood that after reading the content taught in this application, those skilled in the art may make various changes or modifications to the present application, and these equivalent forms also fall within the scope of protection of the claims appended hereto.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0046] the term

[0047] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0048] As used herein, the term "and / or" includes any one of two or more of the listed items, as well as any and all combinations of the listed items, including any combination of any two, any more, or all of the listed items. For example, "A and / or B" includes A, B, and A+B.

[0049] Herein, "preferred", "better", "better", etc. are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0050] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0051] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0052] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution within the above numerical interval is considered continuous and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0053] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within the ranges of ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, and ±0.1°C are permitted.

[0054] In this application, weight can be mass units known in the chemical industry, such as μg, mg, g, and kg.

[0055] Since the current testing standards for Jianqu are still imperfect and lack qualitative and quantitative standards, the Jianqu standards referred to in this application are selected from the control medicinal materials sold by the China Food and Drug Inspection Institute or the working control medicinal materials tested by provincial and municipal drug inspection institutes.

[0056] In some of the embodiments, the Jianqu standard used is obtained by screening from at least 30 batches of Jianqu medicinal materials and is used as a control in daily testing work.

[0057] The aqueous extracts of Chinese medicinal materials referred to in this application include their standard decoctions and Chinese medicinal formula granules.

[0058] The standard decoction in this application is a single-flavor Chinese herbal medicine decoction prepared by a standardized process with reference to modern extraction methods and guided by traditional Chinese medicine theory and based on clinical application. The Chinese medicine formula granules in this application are a series of pure Chinese medicine products that are extracted, concentrated, separated, dried, granulated, and packaged with traditional Chinese medicine slices that meet the processing specifications as raw materials through modern pharmaceutical technology. It can be understood that the above-mentioned standard decoction and Chinese medicine formula granules can basically guarantee all the characteristics of the original Chinese medicine slices. As an example, the preparation method of the standard decoction of Jianqu or stir-fried Jianqu is as follows: take 100g of Jianqu or stir-fried Jianqu slices, put them in an electric ceramic pot, add water and boil twice, filter the decoction through a 200-mesh screen, and then use a rotary evaporator to reduce pressure and concentrate into a fluid extract, which is transferred to a vacuum freeze dryer and freeze-dried.

[0059] As an example, the preparation method of the traditional Chinese medicine formula granules of Jianqu or fried Jianqu is as follows: take 1000g of Jianqu or fried Jianqu slices, add water to decoct, filter, concentrate the filtrate into a clear paste, add appropriate amount of auxiliary materials, dry (or dry and crush), then add appropriate amount of auxiliary materials, mix well, and granulate to obtain.

[0060] The present application relates to a method for constructing a characteristic spectrum of Jianqu or stir-fried Jianqu or its water extract, which constructs the characteristic spectrum by adopting appropriate chromatographic conditions. The method is stable, has good precision and reproducibility, and is more characteristic and specific.

[0061] In one of the embodiments, the present application used a UPLC liquid chromatograph to study 30 batches of Jianqu, stir-fried Jianqu and their standard decoctions from at least 3 different origins, established a characteristic spectrum of 8 common peaks and identified 6 of them, which can provide a basis for the quality control and evaluation of Jianqu, stir-fried Jianqu and their water extracts.

[0062] In one embodiment, the method for constructing a characteristic spectrum of Jianqu or Chaojianqu or its aqueous extract comprises the following steps:

[0063] Take Jianqu or stir-fried Jianqu or its aqueous extract, extract with extraction solvent to prepare the test solution;

[0064] A reference solution was prepared by mixing scopoletin reference substance, liquiritigenin reference substance, hydrated oxypeucedanum reference substance, naringenin reference substance, honokiol reference substance and magnolol reference substance, and dissolving them in a solvent;

[0065] The test solution and the reference solution were subjected to ultra-high performance liquid chromatography analysis to obtain characteristic spectra of Jianqu or stir-fried Jianqu or their aqueous extracts;

[0066] Among them, the mobile phase conditions for ultra-high performance liquid chromatography analysis include:

[0067] Mobile phase A, which is a mixed solvent of methanol and acetonitrile in a volume ratio of (0.5-1.5):1;

[0068] Mobile phase B, which is an aqueous formic acid solution with a volume concentration of 0.08% to 0.12%;

[0069] The total volume percentage of mobile phase A and mobile phase B is 100%.

[0070] In one embodiment, in the method for constructing a characteristic spectrum of Jianqu or Chao Jianqu or its aqueous extract, the extraction solvent is methanol.

[0071] In one embodiment, in the method for constructing a characteristic spectrum of Jianqu or stir-fried Jianqu or their aqueous extracts, the extraction method is ultrasound. Furthermore, the ultrasound power is 200W to 400W, and the frequency is 40kHz to 50kHz. Furthermore, the ultrasound duration is 20min to 40min.

[0072] In one embodiment, Jianqu or stir-fried Jianqu or its aqueous extract is placed in a stoppered conical flask, an extraction solvent is added, ultrasonic treatment is performed, filtration is performed, evaporation is performed, water is added to dissolve it, and the mixture is placed in a separatory funnel. Extraction is performed with dichloromethane, the dichloromethane liquid is combined, and the mixture is evaporated to dryness. The residue is dissolved in the extraction solvent, filtered, and the filtrate is taken to prepare a test solution.

[0073] Furthermore, the mass volume ratio of Jianqu or stir-fried Jianqu or its water extract to the extraction solvent is 1g:(15-25)mL.

[0074] In one embodiment, appropriate amounts of scopoletin reference substance, liquiritigenin reference substance, hydrated oxypeucedanum reference substance, naringenin reference substance, honokiol reference substance, and magnolol reference substance are accurately weighed and dissolved in a solvent to prepare a reference solution. Furthermore, the solvent is methanol.

[0075] In one embodiment, in the method for constructing a characteristic spectrum of Jianqu or stir-fried Jianqu or its aqueous extract, the elution procedure of ultra-performance liquid chromatography analysis includes:

[0076] From 0 to 12 minutes, the volume percentage of the mobile phase A increased from 16% to 18%;

[0077] From 12 to 13 minutes, the volume percentage of the mobile phase A increased from 18% to 28%;

[0078] From 13 to 18 minutes, the volume percentage of the mobile phase A increased from 28% to 30%;

[0079] 18min~40min, maintain the volume percentage of the mobile phase A at 30%;

[0080] From 40 to 41 minutes, the volume percentage of the mobile phase A increased from 30% to 42%;

[0081] From 41 min to 60 min, the volume percentage of the mobile phase A increased from 42% to 56%;

[0082] From 60 to 65 minutes, the volume percentage of the mobile phase A increased from 56% to 90%;

[0083] 65 min to 70 min, the volume percentage of the mobile phase A was maintained at 90%.

[0084] In one embodiment, in the method for constructing a characteristic spectrum of Jianqu or stir-fried Jianqu or its aqueous extract, the conditions for ultra-high performance liquid chromatography analysis include one or more of the following:

[0085] (1) The chromatographic column model is Waters BEH C18; further, the column length is 100 mm, the inner diameter is 2.1 mm, and the filler particle size is 1.7 μm;

[0086] (2) Column temperature is 28°C to 32°C;

[0087] (3) The detection wavelength is 280nm to 320nm;

[0088] (4) The flow rate of the mobile phase is 0.28 mL / min to 0.32 mL / min;

[0089] (5) The injection volume is 1.5 μL to 2.5 μL.

[0090] In one embodiment, the method for constructing a characteristic spectrum of Jianqu or Chaojianqu or its aqueous extract further comprises the following steps:

[0091] After obtaining the characteristic spectrum of Jianqu or stir-fried Jianqu or its water extract, mass spectrometry is used to identify the characteristic peaks therein.

[0092] In one embodiment, the mass spectrometry method includes the following detection conditions: the ionization mode is heated electrospray ionization; the sheath gas flow rate is 30arb~40arb; the auxiliary gas flow rate is 5arb~15arb; the spray voltage is 3.5kV~4kV; the S-lens voltage is 40V~60V; the heating temperature is 300℃~400℃; the capillary temperature is 300℃~400℃; the scanning mode is positive ion mode; the scanning range is 100~1500; and the normalized collision energy is 35~45.

[0093] In one embodiment, the characteristic spectrum of Jianqu or stir-fried Jianqu or its water extract prepared by any of the above technical solutions is:

[0094] Taking peak 4 as a reference, the characteristic peaks and their relative retention times are as follows: peak 1: 0.33 ± 10%; peak 2: 0.37 ± 10%; peak 3: 0.90 ± 10%; peak 5: 1.07 ± 10%; peak 6: 1.20 ± 10%; peak 7: 2.95 ± 10%; peak 8: 3.12 ± 10%;

[0095] Among them, peak 1 is scopoletin, peak 3 is liquiritigenin, peak 4 is hydrated oxypeucedanum, peak 6 is naringenin, peak 7 is magnolol, and peak 8 is magnolol.

[0096] The present application also relates to a method for detecting Jianqu or stir-fried Jianqu or their aqueous extracts, comprising the following steps:

[0097] Take the Jianqu or stir-fried Jianqu or its aqueous extract to be tested, extract it with an extraction solvent to prepare a solution of the test product;

[0098] Take a standard sample of Jianqu or stir-fried Jianqu or its aqueous extract, extract it with an extraction solvent, and prepare a standard solution;

[0099] Performing high performance liquid chromatography analysis on the test sample solution and the standard solution respectively to obtain a characteristic spectrum of the test sample and a characteristic spectrum of the standard, and comparing the characteristic spectrum of the test sample and the characteristic spectrum of the standard;

[0100] Among them, the mobile phase conditions for ultra-high performance liquid chromatography analysis include:

[0101] Mobile phase A, which is a mixed solvent of methanol and acetonitrile in a volume ratio of (0.5-1.5):1;

[0102] Mobile phase B, which is an aqueous formic acid solution with a volume concentration of 0.08% to 0.12%;

[0103] The total volume percentage of mobile phase A and mobile phase B is 100%.

[0104] It can be understood that the test product can be Jianqu medicinal material or a standard decoction of Jianqu.

[0105] It is understood that the characteristic spectrum obtained by ultra-high performance liquid chromatography testing of the test sample solution can be analyzed as needed. For example, a single test sample can be tested and compared with the characteristic spectrum established by the method for constructing the characteristic spectrum of Jianqu or stir-fried Jianqu or their aqueous extracts described in any of the above technical solutions to determine the quality of the single test sample; or multiple batches of test samples can be tested and the spectrum of the multiple batches of test samples can be compared to determine the similarity between the multiple batches of test samples.

[0106] It can be understood that the technical solution of the above-mentioned detection method is similar to the characteristic spectrum construction method of Jianqu or Chao Jianqu or its water extract described in any of the above-mentioned technical solutions, wherein the preparation of the test solution is the same as the preparation of the test solution described in any of the above-mentioned technical solutions, which will not be repeated here.

[0107] The following are some specific examples.

[0108] Example 1 A method for establishing a characteristic spectrum of Jianqu medicinal materials and stir-fried Jianqu decoction pieces

[0109] 1. Instruments and test drugs

[0110] 1.1 Instrument

[0111] An Agilent ultra-high performance liquid chromatograph (1260, Agilent), a Shimadzu ultra-high performance liquid chromatograph (LC-40xds), a Thermo Scientific™ Q Exactive™ Focus quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA); mzVault mass spectral database (Thermo Fisher Scientific, USA); Waters BEH C18 (2.1 mm × 100 mm, particle size 1.7 μm), Waters CORTECS T3 (2.1 mm × 100 mm, particle size 1.6 μm), a ten-thousandth balance (ME204E, Mettler-Toledo), a one-millionth balance (XP26, Mettler-Toledo), an electric constant temperature water bath (HWS-28, Shanghai Yiheng Technology Co., Ltd.), a digitally controlled ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), and an ultrapure water system (Milli-Q Direct, Merck & Co., Ltd.) were used.

[0112] 1.2 Reagents

[0113] The methanol, acetonitrile, phosphoric acid, and formic acid used in the liquid phase were of chromatographic grade, the water was purified water, and the other reagents were of analytical grade.

[0114] 1.3 Drug testing

[0115] A total of 30 batches of Jianqu medicinal materials were used in this study, 15 batches of Jianqu were homemade in the laboratory of Guangdong Yifang Co., Ltd., and 15 batches of Jianqu were purchased from companies in Sichuan Province with relevant qualifications for the preparation and processing of Jianqu, including 9 batches of Jianqu purchased from Sichuan Qianfang Chinese Medicine Co., Ltd., 4 batches of Jianqu purchased from Neijiang Lianghui Pharmaceutical Co., Ltd., 1 batch of Jianqu purchased from Sichuan Yum Pharmaceutical Co., Ltd., and 1 batch of Jianqu purchased from Sichuan Kexing Pharmaceutical Co., Ltd. They were stir-fried according to the stir-frying method (General Chapter 0213 of the 2020 edition of the Chinese Pharmacopoeia) until the surface was dark yellow and had an aroma, and the stir-fried Jianqu slices were obtained.

[0116] Reference substances: scopoletin (China Food and Drug Inspection Institute, 110768-202105), glycyrrhizin (Chengdu Gelipu Biotechnology Co., Ltd., 19072503), hydrated oxydeconvolved peucedanum (China Food and Drug Inspection Institute, 112082-202101), naringenin (Sichuan Weikeqi Biotechnology Co., Ltd., wkq20030401), magnolol (China Food and Drug Inspection Institute, 110730-201915), magnolol (China Food and Drug Inspection Institute, 110729-202015).

[0117] Table 1 Sample information table

[0118]

[0119] 2. Sample Preparation

[0120] 2.1 Preparation of reference solution

[0121] Take 1g of Jianqu reference medicinal material, place it in a stoppered conical flask, accurately add 20ml of 70% ethanol, ultrasonically treat (power 300W, frequency 45kHz) for 30 minutes, filter, evaporate to dryness, add 20ml of water to dissolve, place in a separatory funnel, extract twice with dichloromethane, 20ml each time, combine the dichloromethane liquid, recover the solvent under reduced pressure to dryness, add methanol to dissolve the residue, transfer to a 5ml volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate. Separately, take appropriate amounts of scopoletin reference substance, licorice reference substance, hydrated oxapeutin reference substance, naringenin reference substance, honokiol reference substance, and magnolol reference substance, accurately weigh them, add methanol to make a mixed solution containing 100μg of each per 1ml, which is used as the reference substance solution.

[0122] 2.2 Preparation of test solution

[0123] Take 1 g of the powder (passed through No. 3 sieve) of this product, place it in a stoppered conical flask, add 20 ml of methanol, ultrasonically treat (power 300 W, frequency 45 kHz) for 30 minutes, filter, evaporate to dryness, add 20 ml of water to dissolve it, place it in a separatory funnel, shake and extract with dichloromethane twice, 20 ml each time, combine the dichloromethane liquid, evaporate to dryness, add 5 ml of methanol to dissolve the residue, filter, and take the filtrate to obtain the product.

[0124] 3. Determination of chromatographic conditions

[0125] 3.1 Determination of the optimal wavelength

[0126] A Waters BEH C18 column (2.1 mm × 100 mm, particle size 1.7 μm) was used, with methanol:acetonitrile (1:1) as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, using a gradient elution according to Table 4. The column temperature was 30°C, the flow rate was 0.30 ml / min, and the detection wavelength was 300 nm. The injection volume was 2 μl. The characteristic spectrum of the curve was constructed under different wavelengths, and the optimal wavelength was selected. The results are shown in Figure 4. Figures 1 and 2 .

[0127] The investigation results show that when the wavelength is 300nm, the baseline is stable, the chromatographic peaks have good responses, and the peak information is more complete, so 300nm is selected as the wavelength for constructing the characteristic spectrum.

[0128] 3.2 Determination of optimal gradient conditions

[0129] A Waters BEH C18 column (2.1 mm × 100 mm, particle size 1.7 μm) was used, with methanol:acetonitrile (1:1) as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B. Gradient elution was performed according to Tables 2, 3, and 4. The column temperature was 30°C, the flow rate was 0.30 ml / min, and the detection wavelength was 300 nm. The injection volume was 2 μl, and the chromatographic peak separation of the three characteristic curves under different gradients was investigated. The results are shown in Figure 2. Figure 3 .

[0130] Table 2 Gradient elution program 1

[0131]

[0132] Table 3 Gradient elution program 2

[0133]

[0134] Table 4 Gradient elution program 3

[0135]

[0136] The results showed that the comparison of three different gradient conditions revealed that the chromatogram obtained under gradient condition 3 in Table 4 had a relatively uniform distribution of chromatographic peaks, good peak shapes, and a stable baseline, so it was selected as the analytical condition for constructing the characteristic spectrum.

[0137] 3.3 Determination of the optimal mobile phase

[0138] On the basis of the determined gradient conditions, a Waters BEH C18 (2.1 mm × 100 mm, particle size of 1.7 μm) column was used, with methanol and methanol:acetonitrile (1:1) as mobile phase A, 0.1% formic acid solution and 0.1% phosphoric acid solution as mobile phase B, and gradient elution according to Table 4; the column temperature was 30°C, the flow rate was 0.30 ml per minute, the detection wavelength was 300 nm, and the injection volume was 2 μl. The effects of different mobile phases on the separation of chromatographic peaks of the characteristic curve were investigated. The results are shown in Table 4. Figure 4 .

[0139] The investigation results showed that: when comparing four different mobile phases, it was found that when methanol: acetonitrile (1:1) was used as mobile phase A and 0.1% formic acid solution was used as mobile phase B, the chromatographic peaks in the chromatogram were distributed more evenly, and the separation and peak shape of each chromatographic peak were better. Therefore, methanol: acetonitrile (1:1) was selected as mobile phase A and 0.1% formic acid solution was selected as mobile phase B as the mobile phase for constructing the characteristic spectrum.

[0140] 3.4 Determination of optimal flow rate

[0141] Based on the determined mobile phase and gradient conditions, a Waters BEH C18 column (2.1mm×100mm, particle size 1.7μm) was used, with methanol:acetonitrile (1:1) as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, using a gradient elution according to Table 4; the column temperature was 30°C, the flow rates were 0.28ml / min, 0.30ml / min, and 0.32ml / min, respectively, and the detection wavelength was 300nm. The injection volume was 2μl, and the effect of different flow rates on the separation of the chromatographic peaks in the Jianqu characteristic spectrum was investigated. The results are shown in Figure 4. Figure 5 .

[0142] The results of the investigation showed that when the flow rate was 0.30 ml / min, the separation and peak shape of each characteristic peak were better and the retention time was moderate. Therefore, the flow rate of 0.30 ml / min was selected for constructing the characteristic spectrum.

[0143] 3.5 Optimal column temperature

[0144] Based on the determined mobile phase and gradient conditions, a Waters BEH C18 column (2.1 mm × 100 mm, particle size 1.7 μm) was used, with methanol:acetonitrile (1:1) as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, using a gradient elution according to Table 4; column temperatures were 28°C, 30°C, and 32°C, respectively, with a flow rate of 0.30 ml per minute and a detection wavelength of 300 nm. The injection volume was 2 μl, and the effect of different column temperatures on the separation of the chromatographic peaks in the Jianqu characteristic spectrum was investigated. The results are shown in Table 4. Figure 6 .

[0145] The results of the investigation showed that when the column temperature was 30℃, the separation and peak shape of each characteristic peak were better, so 30℃ was selected for constructing the characteristic spectrum.

[0146] 3.6 Column selection

[0147] Based on the determined mobile phase and gradient conditions, Waters BEH C18 (2.1 mm × 100 mm, particle size 1.7 μm) and Waters CORTECS (2.1 mm × 100 mm, particle size 1.6 μm) columns were used, with methanol:acetonitrile (1:1) as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, with gradient elution according to Table 5; the column temperature was 30°C, the flow rate was 0.30 ml / min, and the detection wavelength was 300 nm. The injection volume was 2 μl, and the results are shown in Table 5. Figure 7 .

[0148] The investigation results show that when comparing three chromatographic columns of two different specifications, it was found that when the chromatographic column was Waters BEH C18 (2.1mm×100mm, particle size of 1.7μm), the separation and peak shape of each characteristic peak were better. Therefore, Waters BEH C18 (2.1mm×100mm, particle size of 1.7μm) was selected as the chromatographic column for constructing the characteristic spectrum.

[0149] 3.7 Determination of chromatographic conditions

[0150] Based on the above investigation, the chromatographic conditions for the characteristic spectrum of the curve were determined as follows: a Waters BEH C18 (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.7 μm) was used as the chromatographic column; methanol:acetonitrile (1:1) was used as the mobile phase A, and 0.1% formic acid solution was used as the mobile phase B, and gradient elution was performed according to the requirements in Table 5; the flow rate was 0.3 ml per minute; the column temperature was 30°C; the detection wavelength was 300 nm, and the injection volume was 2 μl.

[0151] Table 5 Gradient elution table

[0152]

[0153] 4. Characteristic Peak Determination

[0154] Take 30 batches of Jianqu medicinal materials and 30 batches of stir-fried Jianqu slices and prepare test solution I according to the method under "2.2". Take the control medicinal material solution and test solution I and measure them according to the chromatographic conditions determined under "3.7". The characteristic spectra of 30 batches of Jianqu medicinal materials are obtained. The results are shown in Figure 8 , 30 batches of fried Jianqu decoction pieces characteristic spectrum, the results are shown in Figure 9 , Jianqu reference medicinal material characteristic spectrum see Figure 10 .

[0155] Take 30 batches of Jianqu and stir-fried Jianqu standard decoctions and prepare test solution II according to the method under "2.2". Take test solution II and measure it according to the chromatographic conditions determined under "3.7". The characteristic spectra of 30 batches of Jianqu standard decoction are obtained. The results are shown in Figure 11 , 30 batches of stir-fried Jianqu standard decoction characteristic spectrum, the results are shown in Figure 12 .

[0156] The results showed that 8 common peaks could be identified by combining the characteristic spectra of 30 batches of Jianqu medicinal materials, 30 batches of stir-fried Jianqu slices and their standard decoctions. The above 8 common peaks could be stably transferred from the medicinal materials and slices to the standard decoctions. Therefore, these 8 common peaks were set as the characteristic peaks of the characteristic spectra of Jianqu and stir-fried Jianqu, and the 8 characteristic peaks were further identified and confirmed to clarify their chemical components.

[0157] 5. Characteristic Peak Identification and Confirmation

[0158] 5.1 Characteristic Peak Identification

[0159] UPLC-QE-Orbitrap-MS was used to identify the chemical components of the characteristic peaks on the Jianqu characteristic spectrum.

[0160] The chromatographic conditions are the same as those specified under "3.7", and the mass spectrometry conditions are shown in Table 6.

[0161] Table 6 Mass spectrometry parameters

[0162]

[0163] Accurately draw 1 μl of the Jianqu medicinal material test solution and inject it into the LC-MS chromatograph. The results are shown in the table. Figure 13 The collected data were imported into the mzVault database for analysis. The accurate mass number and secondary mass spectrum information obtained by the high-resolution system were combined with the literature to analyze its elemental composition and chemical structure, and the composition information of the 8 characteristic peaks of Jianqu were inferred.

[0164] 5.2 Characteristic Peak Confirmation

[0165] According to the identification information in Table 7, the corresponding reference solution was prepared, 2 μl of the test solution and reference solution were accurately aspirated and injected into the ultra-high performance liquid chromatography instrument. The results were shown in Table 7. Figure 14 By comparing the retention times and UV 3D spectra of the two, it was confirmed that characteristic peak 1 in the Jianqu characteristic spectrum is scopoletin, characteristic peak 3 is liquiritigenin, characteristic peak 4 is hydrated oxypeucedanum, characteristic peak 6 is naringenin, characteristic peak 7 is honokiol, and characteristic peak 8 is magnolol. Characteristic peak 2 had an extremely low response on the mass spectrometer detector, and no relevant mass spectral information was obtained. Characteristic peak 5, based on the primary and secondary information obtained from the mass spectrum, was compared with databases and literature, and the resulting compound types were numerous, so its chemical composition could not be confirmed at this time.

[0166] Table 7 Mass spectrometry identification information of characteristic peaks of Jianqu medicinal materials

[0167]

[0168]

[0169] 6. Methodological Investigation

[0170] 6.1 Precision test

[0171] Take one sample of Jianqu medicinal material and stir-fried Jianqu decoction piece (S1), prepare the test solution according to the method in "2.2", and inject the sample six times according to the chromatographic conditions in "3.7". Record the chromatogram. Using the hydrated oxidized praeruptosine peak as the reference peak S, calculate the relative retention time and peak area of ​​each characteristic peak relative to the S peak. The precision test results show that the RSD values ​​of the relative retention time and peak area of ​​each characteristic peak are all less than 3.0%, indicating good instrument precision.

[0172] 6.2 Repeatability test

[0173] Six parallel sample solutions of Jianqu herbal medicine and stir-fried Jianqu slices (S1) were prepared according to the method in "2.2." Six consecutive injections were made according to the chromatographic conditions in "3.7." The chromatograms were recorded. Using the hydrated oxidized praeruptosine peak as the reference peak, S, the relative retention times and peak areas of each characteristic peak were calculated relative to the S peak. Repeatability test results showed that the RSD values ​​for the relative retention times and peak areas of each characteristic peak were less than 3%, indicating good repeatability of this method.

[0174] 6.3 Stability test

[0175] Take one sample of Jianqu medicinal material and stir-fried Jianqu decoction piece (S1) and prepare the test solution according to the method in "2.2". Samples were injected at 0, 2, 4, 8, 12, and 24 hours, respectively. Six consecutive injections were performed according to the chromatographic conditions in "3.7". The chromatogram was recorded. Using the hydrated oxidized praeruptosine peak as the reference peak S, the relative retention time and peak area of ​​each characteristic peak were calculated relative to the S peak. Repeatability test results showed that the RSD values ​​of the relative retention time and peak area of ​​each characteristic peak were less than 3%, indicating good repeatability of this method.

[0176] 6.4 Durability

[0177] 6.4.1. Investigation of different flow rates

[0178] Using Jianqu and stir-fried Jianqu sample solutions, the chromatographic conditions under "3.7" were followed. Chromatograms were obtained at flow rates of 0.28 ml / min, 0.30 ml / min, and 0.32 ml / min. The hydrated oxidized praeruptosine peak was used as the reference peak, S, and the relative retention times of the characteristic peaks were calculated relative to the S peak. The results showed that the RSD values ​​for the relative retention times of the characteristic peaks were all less than 5%, demonstrating the robustness of the flow rate used in this method. A flow rate fluctuation of ±0.02 ml / min had minimal impact on the characteristic chromatograms.

[0179] 6.4.2. Investigation of different column temperatures

[0180] Using Jianqu and stir-fried Jianqu sample solutions, the chromatographic conditions in section "3.7" were used to compare the chromatographic profiles at column temperatures of 28°C, 30°C, and 32°C. Using the hydrated oxidized praeruptosine peak as the reference peak, S, the relative retention times of the characteristic peaks were calculated. The results showed that the RSD values ​​for the relative retention times of the characteristic peaks were all less than 5%, demonstrating the good column temperature robustness of this method, with a ±2°C temperature fluctuation having minimal impact on the characteristic chromatograms.

[0181] 6.4.3. Inspection of different brands of chromatographs

[0182] Chromatographic chromatograms of Jianqu and stir-fried Jianqu sample solutions acquired using a Warters H-class and an Agilent 1290 were compared using the chromatographic conditions outlined in "3.7." The hydrated oxidized praeruptosine peak was used as the reference peak (S), and the relative retention times of the characteristic peaks relative to the S peak were calculated. The results demonstrated that the Jianqu characteristic profile was well reproducible on both instruments, with RSDs for the relative retention times of the characteristic peaks less than 5%, demonstrating the robustness of this method across different chromatograph brands.

[0183] Example 2 Determination of the characteristic spectrum of the song

[0184] The characteristic spectra of 30 batches of Jianqu medicinal materials, 30 batches of stir-fried Jianqu slices and their standard decoctions were analyzed. The hydrated oxidized peucedanum peak was used as the reference peak S. The relative retention time and relative peak area of ​​each characteristic peak and the S peak were calculated, and the RSD values ​​were calculated.

[0185] Table 8 Characteristic spectra of 30 batches of Jianqu medicinal materials (relative retention time)

[0186]

[0187]

[0188] Table 9 Characteristic spectra of 30 batches of Jianqu medicinal materials (relative peak area)

[0189]

[0190] Table 10 Characteristic spectra of 30 batches of stir-fried Jianqu slices (relative retention time)

[0191]

[0192]

[0193] Table 11 Characteristic spectra of 30 batches of stir-fried Jianqu slices (relative peak area)

[0194]

[0195]

[0196] Table 12 Characteristic spectra of 30 batches of Jianqu standard decoction (relative retention time)

[0197]

[0198] Table 13 Characteristic spectra of 30 batches of Jianqu standard decoction (relative peak area)

[0199]

[0200] Table 14 Characteristic spectra of 30 batches of stir-fried Jianqu standard decoction (relative retention time)

[0201]

[0202]

[0203] Table 15 Characteristic spectra (relative peak area) of 30 batches of stir-fried Jianqu standard decoction

[0204]

[0205]

[0206] The characteristic spectra of 30 batches of Jianqu medicinal materials, 30 batches of stir-fried Jianqu slices and their standard decoctions were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System" to generate the comparative characteristic spectra of Jianqu medicinal materials and stir-fried Jianqu slices respectively. The results are shown in the figure. Figures 15 and 16 , Jianqu and stir-fried Jianqu standard decoction comparison characteristic spectrum, the results are shown in Figures 17 and 18 .

[0207] Results and Discussion

[0208] The results showed that, with the hydrated oxidized praeruptosine peak as the reference peak S, the RSD values ​​of the relative retention times of the characteristic peaks of 30 batches of Jianqu medicinal materials and the S peak were in the range of 0.25% to 3.13%, the RSD values ​​of the relative retention times of the characteristic peaks of 30 batches of stir-fried Jianqu decoction pieces and the S peak were in the range of 0.38% to 2.57%, the RSD values ​​of the relative retention times of the characteristic peaks of 30 batches of Jianqu standard decoctions and the S peak were in the range of 0.44% to 4.09%, and the RSD values ​​of the relative retention times of the characteristic peaks of 30 batches of stir-fried Jianqu standard decoctions and the S peak were in the range of 0.06% to 2.31%. The RSD values ​​were all less than 5.0%, indicating that the relative retention times of the characteristic peaks were relatively stable. The RSD values ​​of the relative peak areas between the characteristic peaks and the S peak in the characteristic spectra of 30 batches of Jianqu medicinal materials were between 33.77% and 217.80%, the RSD values ​​of the relative peak areas between the characteristic peaks and the S peak in the characteristic spectra of 30 batches of stir-fried Jianqu medicinal pieces were between 38.57% and 218.50%, the RSD values ​​of the relative peak areas between the characteristic peaks and the S peak in 30 batches of Jianqu standard decoctions were between 32.40% and 228.38%, and the RSD values ​​of the relative peak areas between the characteristic peaks and the S peak in 30 batches of stir-fried Jianqu standard decoctions were between 27.68% and 228.93%, indicating that no obvious changes were found in the chemical composition before and after frying Jianqu, but the relative peak areas of samples from different batches differed greatly, indicating that there were large differences in the proportions of the chemical components represented by the characteristic peaks of different batches of Jianqu. This difference may be related to multiple factors such as the quality differences of prescribed medicinal materials, fermentation process, and processing process. Therefore, it is not appropriate to make regulations on the relative peak areas of the characteristic peaks for the time being.

[0209] In summary, the characteristic chromatogram standards for Jianqu medicinal materials, stir-fried Jianqu medicinal pieces and their standard decoctions can be determined as follows: 8 characteristic peaks should be present in the chromatogram of the test sample, and the retention times should correspond to the 8 characteristic peaks in the chromatogram peaks of the reference medicinal material reference substance, among which Peak 1, Peak 3, Peak 4, Peak 6, Peak 7 and Peak 8 should correspond to the retention times of the corresponding reference substance peaks respectively; the peak corresponding to the hydrated oxidized praeruptosin reference substance peak is the S peak, and the relative retention time of Peak 5 and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is 1.07 (Peak 5).

[0210] Example 3 A method for detecting Jianqu medicinal materials and stir-fried Jianqu decoction pieces

[0211] 1. Instrument

[0212] Agilent ultra-high performance liquid chromatograph (1260, Agilent), Shimadzu ultra-high performance liquid chromatograph (LC-40xds); Waters BEH C18 (2.1 mm × 100 mm, particle size 1.7 μm), 1 / 10,000 balance (ME204E, Mettler-Toledo), 1 / 10,000 balance (XP26, Mettler-Toledo), electric constant temperature water bath (HWS-28, Shanghai Yiheng Technology Co., Ltd.), CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck).

[0213] 2. Reagents

[0214] The methanol, acetonitrile, phosphoric acid, and formic acid used in the liquid phase were of chromatographic grade, the water was purified water, and the other reagents were of analytical grade.

[0215] 3. Drug testing

[0216] Reference substances: scopoletin (China Food and Drug Inspection Institute, 110768-202105), glycyrrhizin (Chengdu Gelipu Biotechnology Co., Ltd., 19072503), hydrated oxydeconvolved peucedanum (China Food and Drug Inspection Institute, 112082-202101), naringenin (Sichuan Weikeqi Biotechnology Co., Ltd., wkq20030401), magnolol (China Food and Drug Inspection Institute, 110730-201915), magnolol (China Food and Drug Inspection Institute, 110729-202015).

[0217] 4. Preparation of reference herbs and reference substances

[0218] Take 1g of Jianqu reference medicinal material and place it in a stoppered conical flask. Accurately add 20ml of 70% ethanol, ultrasonically treat (power 300W, frequency 45kHz) for 30 minutes, filter, evaporate to dryness, add 20ml of water to dissolve, place in a separatory funnel, extract twice with dichloromethane, 20ml each time, combine the dichloromethane solutions, recover the solvent under reduced pressure to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate. Separately, take appropriate amounts of scopoletin, glycyrrhizin, hydrated oxypeucedanum, naringenin, honokiol, and magnolol reference substances, accurately weigh them, and add methanol to make a mixed solution containing 100μg per 1ml. This is used as the reference substance solution.

[0219] 5. Preparation of Jianqu Herbal Materials and Stir-fried Jianqu Pieces

[0220] Take 1 g of the powder (passed through No. 3 sieve) of this product, place it in a stoppered conical flask, add 20 ml of methanol, ultrasonically treat (power 300 W, frequency 45 kHz) for 30 minutes, filter, evaporate to dryness, add 20 ml of water to dissolve it, place it in a separatory funnel, shake and extract with dichloromethane twice, 20 ml each time, combine the dichloromethane liquid, evaporate to dryness, add 5 ml of methanol to dissolve the residue, filter, and take the filtrate to obtain the product.

[0221] 6. Chromatographic conditions

[0222] A Waters BEH C18 column (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.7 μm) was used as the chromatographic column; methanol:acetonitrile (1:1) was used as the mobile phase A, and 0.1% formic acid solution was used as the mobile phase B. Gradient elution was performed according to the requirements in Table 5; the flow rate was 0.3 ml / min; the column temperature was 30°C; and the detection wavelength was 300 nm.

[0223] There should be 8 characteristic peaks in the chromatogram of the test sample, and the retention times of the 8 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. Among them, peak 1, peak 3, peak 4, peak 6, peak 7, and peak 8 should correspond to the retention times of the corresponding reference material peaks respectively; the peak corresponding to the hydrated oxidized praeruptosporin reference material peak is the S peak, and the relative retention time of peak 5 and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is 1.07 (peak 5).

[0224] Example 4 Detection Method for Jianqu and Chaojianqu Standard Decoction

[0225] 1. Instrument

[0226] Agilent ultra-high performance liquid chromatograph (1260, Agilent), Shimadzu ultra-high performance liquid chromatograph (LC-40xds); Waters BEH C18 (2.1 mm × 100 mm, particle size 1.7 μm), 1 / 10,000 balance (ME204E, Mettler-Toledo), 1 / 10,000 balance (XP26, Mettler-Toledo), electric constant temperature water bath (HWS-28, Shanghai Yiheng Technology Co., Ltd.), CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck).

[0227] 2. Reagents

[0228] The methanol, acetonitrile, phosphoric acid, and formic acid used in the liquid phase were of chromatographic grade, the water was purified water, and the other reagents were of analytical grade.

[0229] 3. Drug testing

[0230] Reference substances: scopoletin (China Food and Drug Inspection Institute, 110768-202105), glycyrrhizin (Chengdu Gelipu Biotechnology Co., Ltd., 19072503), hydrated oxydeconvolved peucedanum (China Food and Drug Inspection Institute, 112082-202101), naringenin (Sichuan Weikeqi Biotechnology Co., Ltd., wkq20030401), magnolol (China Food and Drug Inspection Institute, 110730-201915), magnolol (China Food and Drug Inspection Institute, 110729-202015).

[0231] 4. Preparation of reference herbs and reference substances

[0232] Take 0.5g of Jianqu reference medicinal material and place it in a stoppered conical flask. Accurately add 20ml of 70% ethanol, ultrasonicate (power 300W, frequency 45kHz) for 30 minutes, filter, evaporate to dryness, add 20ml of water to dissolve, place in a separatory funnel, extract twice with dichloromethane, 20ml each time, combine the dichloromethane solutions, recover the solvent under reduced pressure to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate. Separately, take appropriate amounts of scopoletin, glycyrrhizin, hydrated oxypeucedanum, naringenin, honokiol, and magnolol reference substances, accurately weigh them, and add methanol to make a mixed solution containing 100μg per 1ml to serve as the reference substance solution.

[0233] 5. Preparation of Jianqu and Chaojianqu Standard Decoctions

[0234] Take an appropriate amount of this product, grind it into powder, take 0.5g, place it in a stoppered conical flask, add 20ml of 70% ethanol, ultrasonically treat (power 300W, frequency 45kHz) for 30 minutes, filter, evaporate to dryness, add 20ml of water to the residue to dissolve it, place it in a separatory funnel, extract it twice with dichloromethane, 20ml each time, combine the dichloromethane liquid, evaporate to dryness, add 5ml of methanol to dissolve the residue, filter, and take the filtrate to obtain the product.

[0235] 6. Chromatographic conditions

[0236] A Waters BEH C18 column (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.7 μm) was used as the chromatographic column; methanol:acetonitrile (1:1) was used as the mobile phase A, and 0.1% formic acid solution was used as the mobile phase B. Gradient elution was performed according to the requirements in Table 5; the flow rate was 0.3 ml / min; the column temperature was 30°C; and the detection wavelength was 300 nm.

[0237] There should be 8 characteristic peaks in the chromatogram of the test sample, and the retention times of the 8 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. Among them, peak 1, peak 3, peak 4, peak 6, peak 7, and peak 8 should correspond to the retention times of the corresponding reference material peaks respectively; the peak corresponding to the hydrated oxidized praeruptosporin reference material peak is the S peak, and the relative retention time of peak 5 and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is 1.07 (peak 5).

[0238] Example 5: A method for detecting Jianqu and stir-fried Jianqu formula granules

[0239] 1. Instrument

[0240] Agilent ultra-high performance liquid chromatograph (1260, Agilent), Shimadzu ultra-high performance liquid chromatograph (LC-40xds); Waters BEH C18 (2.1 mm × 100 mm, particle size 1.7 μm), 1 / 10,000 balance (ME204E, Mettler-Toledo), 1 / 10,000 balance (XP26, Mettler-Toledo), electric constant temperature water bath (HWS-28, Shanghai Yiheng Technology Co., Ltd.), CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck).

[0241] 2. Reagents

[0242] The methanol, acetonitrile, phosphoric acid, and formic acid used in the liquid phase were of chromatographic grade, the water was purified water, and the other reagents were of analytical grade.

[0243] 3. Drug testing

[0244] Reference substances: scopoletin (China Food and Drug Inspection Institute, 110768-202105), glycyrrhizin (Chengdu Gelipu Biotechnology Co., Ltd., 19072503), hydrated oxydeconvolved peucedanum (China Food and Drug Inspection Institute, 112082-202101), naringenin (Sichuan Weikeqi Biotechnology Co., Ltd., wkq20030401), magnolol (China Food and Drug Inspection Institute, 110730-201915), magnolol (China Food and Drug Inspection Institute, 110729-202015).

[0245] 4. Preparation of reference herbs and reference substances

[0246] Take 1g of Jianqu reference medicinal material and place it in a stoppered conical flask. Accurately add 20ml of 70% ethanol, ultrasonically treat (power 300W, frequency 45kHz) for 30 minutes, filter, evaporate to dryness, add 20ml of water to dissolve, place in a separatory funnel, extract twice with dichloromethane, 20ml each time, combine the dichloromethane solutions, recover the solvent under reduced pressure until dry, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate. Separately, take appropriate amounts of scopoletin reference substance, hydrated oxypeucedanum reference substance, naringenin reference substance, honokiol reference substance, and magnolol reference substance, accurately weigh them, and add methanol to make a mixed solution containing 100μg of each per 1ml, which is used as the reference substance solution.

[0247] 5. Preparation of Jianqu and Stir-fried Jianqu Granules

[0248] Take an appropriate amount of this product, grind it into powder, take 1g, place it in a stoppered conical flask, add 20ml of 70% ethanol, ultrasonically treat it (power 300W, frequency 45kHz) for 30 minutes, filter, evaporate to dryness, add 20ml of water to the residue to dissolve it, place it in a separatory funnel, shake and extract with dichloromethane twice, 20ml each time, combine the dichloromethane liquid, evaporate to dryness, add 5ml of methanol to dissolve the residue, filter, and take the filtrate to obtain the product.

[0249] 6. Chromatographic conditions

[0250] A Waters BEH C18 column (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.7 μm) was used as the chromatographic column; methanol:acetonitrile (1:1) was used as the mobile phase A, and 0.1% formic acid solution was used as the mobile phase B. Gradient elution was performed according to the requirements in Table 5; the flow rate was 0.3 ml / min; the column temperature was 30°C; and the detection wavelength was 300 nm.

[0251] There should be 8 characteristic peaks in the chromatogram of the test sample, and the retention times of the 8 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. Among them, peak 1, peak 3, peak 4, peak 6, peak 7, and peak 8 should correspond to the retention times of the corresponding reference material peaks respectively; the peak corresponding to the hydrated oxidized praeruptosporin reference material peak is the S peak, and the relative retention time of peak 5 and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is 1.07 (peak 5).

[0252] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0253] The embodiments described above only express several implementation methods of the present application, but they should not be understood as limiting the scope of the patent application. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent in this application shall be based on the attached claims, and the description and drawings may be used to explain the contents of the claims.

Claims

1. A method for constructing a characteristic spectrum of Jianqu or stir-fried Jianqu or Jianqu water extract or stir-fried Jianqu water extract, characterized in that: The steps include: Take Jianqu or stir-fried Jianqu or Jianqu water extract or stir-fried Jianqu water extract, extract with an extraction solvent to prepare a test solution; the extraction solvent is methanol; A reference solution was prepared by mixing scopoletin reference substance, liquiritigenin reference substance, hydrated oxypeucedanum reference substance, naringenin reference substance, honokiol reference substance and magnolol reference substance, and dissolving them in a solvent; Taking the test solution and the reference solution for ultra-performance liquid chromatography analysis respectively, to obtain the characteristic spectrum of the Jianqu or stir-fried Jianqu or Jianqu water extract or stir-fried Jianqu water extract; Wherein, the mobile phase conditions of the ultra-high performance liquid chromatography analysis include: Mobile phase A, which is a mixed solvent of methanol and acetonitrile in a volume ratio of 1:1; Mobile phase B, which is an aqueous formic acid solution with a volume concentration of 0.08% to 0.12%; The sum of the volume percentages of the mobile phase A and the mobile phase B is 100%; The elution procedure of the ultra-high performance liquid chromatography analysis includes: From 0 to 12 minutes, the volume percentage of the mobile phase A increased from 16% to 18%; From 12 to 13 minutes, the volume percentage of the mobile phase A increased from 18% to 28%; From 13 to 18 minutes, the volume percentage of the mobile phase A increased from 28% to 30%; 18min~40min, maintain the volume percentage of the mobile phase A at 30%; From 40 to 41 minutes, the volume percentage of the mobile phase A increased from 30% to 42%; From 41 min to 60 min, the volume percentage of the mobile phase A increased from 42% to 56%; From 60 to 65 minutes, the volume percentage of the mobile phase A increased from 56% to 90%; 65min~70min, maintain the volume percentage of the mobile phase A at 90%; The chromatographic column model is Waters BEH C18, with a column length of 100 mm, an inner diameter of 2.1 mm, and a filler particle size of 1.7 μm; The detection wavelength is 300 nm.

2. The method for constructing a characteristic map according to claim 1, wherein: The conditions for the ultra-high performance liquid chromatography analysis include one or more of the following: (1) Column temperature is 28°C to 32°C; (2) The flow rate of the mobile phase is 0.28 mL / min to 0.32 mL / min; (3) The injection volume is 1.5 μL to 2.5 μL.

3. The method for constructing a characteristic map according to claim 1 or 2, characterized in that: The extraction method is ultrasound.

4. The method for constructing a characteristic map according to claim 1 or 2, wherein: In the characteristic spectrum of the Jianqu or stir-fried Jianqu or Jianqu water extract or stir-fried Jianqu water extract: Taking peak 4 as a reference, the characteristic peaks and their relative retention times are as follows: peak 1: 0.33 ± 10%; peak 2: 0.37 ± 10%; peak 3: 0.90 ± 10%; peak 5: 1.07 ± 10%; peak 6: 1.20 ± 10%; peak 7: 2.95 ± 10%; peak 8: 3.12 ± 10%; Among them, peak 1 is scopoletin, peak 3 is liquiritigenin, peak 4 is hydrated oxypeucedanum, peak 6 is naringenin, peak 7 is magnolol, and peak 8 is magnolol.

5. A method for detecting Jianqu or stir-fried Jianqu or Jianqu water extract or stir-fried Jianqu water extract, characterized in that: The steps include: Take the Jianqu or stir-fried Jianqu or Jianqu water extract or stir-fried Jianqu water extract to be tested, extract with an extraction solvent to prepare a test solution; the extraction solvent is methanol; Taking a standard of Jianqu or stir-fried Jianqu or Jianqu water extract or stir-fried Jianqu water extract, extracting with the extraction solvent to prepare a standard solution; Performing ultra-performance liquid chromatography analysis on the test product solution and the standard solution respectively to obtain a characteristic spectrum of the test product and a characteristic spectrum of the standard, and comparing the characteristic spectrum of the test product and the characteristic spectrum of the standard; Wherein, the mobile phase conditions of the ultra-high performance liquid chromatography analysis include: Mobile phase A, which is a mixed solvent of methanol and acetonitrile in a volume ratio of 1:1; Mobile phase B, which is an aqueous formic acid solution with a volume concentration of 0.08% to 0.12%; The sum of the volume percentages of the mobile phase A and the mobile phase B is 100%; The elution procedure of the ultra-high performance liquid chromatography analysis includes: From 0 to 12 minutes, the volume percentage of the mobile phase A increased from 16% to 18%; From 12 to 13 minutes, the volume percentage of the mobile phase A increased from 18% to 28%; From 13 to 18 minutes, the volume percentage of the mobile phase A increased from 28% to 30%; 18min~40min, maintain the volume percentage of the mobile phase A at 30%; From 40 to 41 minutes, the volume percentage of the mobile phase A increased from 30% to 42%; From 41 min to 60 min, the volume percentage of the mobile phase A increased from 42% to 56%; From 60 to 65 minutes, the volume percentage of the mobile phase A increased from 56% to 90%; 65min~70min, maintain the volume percentage of the mobile phase A at 90%; The chromatographic column model is Waters BEH C18, with a column length of 100 mm, an inner diameter of 2.1 mm, and a filler particle size of 1.7 μm; The detection wavelength is 300nm; The characteristic spectrum of the standard product has a total of 8 characteristic peaks, including the chromatographic peaks of scopoletin, liquiritigenin, hydrated oxypeucedanum, naringenin, magnolol and magnolol.

6. The detection method according to claim 5, characterized in that The conditions for the ultra-high performance liquid chromatography analysis include one or more of the following: (1) Column temperature is 28°C to 32°C; (2) The flow rate of the mobile phase is 0.28 mL / min to 0.32 mL / min; (3) The injection volume is 1.5 μL to 2.5 μL.

7. The detection method according to claim 5 or 6, characterized in that The extraction method is ultrasound.

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