Construction method of characteristic chromatogram of radix cortex euonymi or water extract thereof and identification method thereof

Abstract

The application relates to the technical field of traditional Chinese medicine detection and analytical chemistry, in particular to a characteristic chromatogram construction method of Radix Cynanchi Paniculati or water extract thereof and a discrimination method thereof. In the technical scheme, the test sample is extracted by water and diethyl ether, the prepared test sample is analyzed by using ultra-high performance liquid chromatography, and the mobile phase conditions include: gradient elution is carried out by taking methanol as mobile phase A and taking 0.04%-0.06% (volume concentration) phosphoric acid aqueous solution as mobile phase B. The characteristic chromatogram has better specificity, can well reflect the characteristic components of Radix Cynanchi Paniculati or water extract thereof, and provides a basis for quality and authenticity discrimination of Radix Cynanchi Paniculati or water extract thereof.

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 Cynanchum chinense or its aqueous extract and a method for identifying the same. Background Art

[0002] Cynanchum paniculatum (Bge.) Kitag., a plant of the Asclepiadaceae family, is harvested in autumn, freed of impurities, and dried in the shade. It has a pungent flavor and warm properties. It enters the Liver and Stomach meridians. It dispels wind, eliminates dampness, relieves pain, and relieves itching. It is used for rheumatic pain, stomach ache, toothache, back pain, pain from falls, rubella, and eczema. Common counterfeit and adulterated Cynanchum paniculatum products include Baiqian, Baiwei, and Asarum. A plant root called "Laoguatou" appeared several years ago, severely impacting the Cynanchum market and posing a significant safety risk to consumers.

[0003] Traditional Chinese medicine fingerprints are a comprehensive, quantifiable identification method based on systematic research into the chemical components of traditional Chinese medicines. They are primarily used to evaluate the authenticity, quality, and stability of Chinese medicinal materials and semi-finished Chinese medicine preparations. While methods for constructing HPLC fingerprints for Cynanchum indicum have been reported, the resulting fingerprints often suffer from a limited number of characteristic peaks and a limited identification of components such as paeonol. Furthermore, the peak separation, discernibility, and specificity of these fingerprints require improvement.

[0004] At present, the provincial local standards for the characteristic spectrum of Xu Changqing Chinese medicine formula granules are mainly divided into three categories: the first is the Zhejiang provincial standard, which stipulates that its characteristic spectrum includes 6 characteristic peaks and identifies taxifolin and paeonol, but there is no report that Xu Changqing medicinal materials contain taxifolin; the second is the Shanghai local standard, which stipulates that its characteristic spectrum includes 6 characteristic peaks and identifies paeonol; the third is the provincial local standard, which stipulates that its characteristic spectrum includes 4 characteristic peaks and identifies paeonol.

[0005] Paeonol is an active ingredient extracted and separated from the dried roots and rhizomes of Cynanchum chinense, a plant of the Asclepiadaceae family, or the root bark of Paeonia lactiflora, a plant of the genus ...

[0006] The water extract of Cynanchum chinense is one of the important raw materials for Chinese medicine preparations such as Chinese patent medicines and Chinese medicine formula granules. Since it has lost the characteristics of the medicinal material, it cannot be effectively identified. Therefore, there is an urgent need to provide a characteristic / fingerprint method that can effectively identify Cynanchum chinense and the water extract of Cynanchum chinense that has lost the characteristics of the medicinal material, so as to ensure the safety of the use of Cynanchum chinense and its related preparations. Summary of the Invention

[0007] In view of this, the technical content of this application is specially proposed.

[0008] One of the purposes of the present application is to provide a method for constructing a characteristic spectrum of Cynanchum chinense medicinal material or its aqueous extract, which fully considers the main volatile or semi-volatile components in Cynanchum chinense, improves the specificity of the method, and can more objectively reflect the chemical characteristic information of Cynanchum chinense medicinal material or its aqueous extract; another purpose of the present application is to provide a method for identifying the characteristic spectrum of Cynanchum chinense medicinal material or its aqueous extract, which can be used to identify the source, quality, authenticity, etc. of Cynanchum chinense medicinal material or its aqueous extract.

[0009] The specific technical solutions are described below:

[0010] A method for constructing a characteristic spectrum of Cynanchum chinense or its aqueous extract comprises the following steps:

[0011] Providing a test solution, the step of providing the test solution comprising: taking a test sample, adding a first extracting solution to perform a first extraction, solid-liquid separation to obtain a liquid, adding a second extracting solution to the liquid to perform a second extraction,

[0012] Wherein, the test sample is selected from Cynanchum chinense and its water extract.

[0013] The first extraction liquid includes water, the first extraction method includes ultrasound,

[0014] The second extracting solution includes diethyl ether, and the second extraction method includes extraction;

[0015] Providing a reference solution, wherein the reference substance in the reference solution includes a p-hydroxyacetophenone standard;

[0016] The test solution and the reference solution were analyzed by high performance liquid chromatography.

[0017] The conditions for the HPLC analysis include: using methanol as mobile phase A and using a phosphoric acid aqueous solution with a volume concentration of 0.04% to 0.06% as mobile phase B for gradient elution.

[0018] The gradient elution procedure includes: from 0 to 5 minutes, maintaining the volume percentage of the mobile phase A at 14%, from 5 minutes to 10 minutes, increasing the volume percentage of the mobile phase A from 14% to 20%, from 10 minutes to 60 minutes, maintaining the volume percentage of the mobile phase A at 20%, and from 60 minutes to 65 minutes, increasing the volume percentage of the mobile phase A from 20% to 70%.

[0019] A method for identifying Cynanchum chinense medicinal material or its aqueous extract comprises the following steps:

[0020] Providing a test solution, wherein the step of providing the test solution comprises: taking the test substance, adding a first extracting solution to perform a first extraction, performing solid-liquid separation to obtain a liquid, and adding a second extracting solution to the liquid to perform a second extraction,

[0021] Wherein, the analyte is selected from Cynanchum chinense and its water extract.

[0022] The first extraction liquid includes water, the first extraction method includes ultrasound,

[0023] The second extracting solution includes diethyl ether, and the second extraction method includes extraction;

[0024] Take a standard of Cynanchum chinense or its aqueous extract, prepare a characteristic spectrum of the standard and a characteristic spectrum of the analyte according to the method for constructing a characteristic spectrum according to any one of claims 1 to 6, compare the characteristic spectrum of the standard with the characteristic spectrum of the analyte,

[0025] The conditions for the HPLC analysis include: using methanol as mobile phase A and using a phosphoric acid aqueous solution with a volume concentration of 0.04% to 0.06% as mobile phase B for gradient elution.

[0026] The gradient elution procedure includes: from 0 to 5 minutes, maintaining the volume percentage of the mobile phase A at 14%, from 5 minutes to 10 minutes, increasing the volume percentage of the mobile phase A from 14% to 20%, from 10 minutes to 60 minutes, maintaining the volume percentage of the mobile phase A at 20%, and from 60 minutes to 65 minutes, increasing the volume percentage of the mobile phase A from 20% to 70%. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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.

[0028] Figure 1 This is a comparison of the chromatograms obtained by detecting the Cynanchum chinense medicinal material in different extraction methods in 2.1.1 of Example 1;

[0029] Figure 2 This is a comparison of the chromatograms obtained by testing the Cynanchum chinense medicinal material in 2.1.2 of Example 1 under different extraction solvents;

[0030] Figure 3 This is a comparison of the chromatograms obtained by detecting the Cynanchum chinense medicinal material under different mobile phase conditions in 3.1 of Example 1;

[0031] Figure 4 This is a comparison of the chromatograms obtained by detecting the Cynanchum chinense medicinal material under different elution gradient conditions in 3.2 of Example 1;

[0032] Figure 5 This is a comparison of the chromatograms obtained by detecting the Cynanchum chinense medicinal material under different chromatographic column conditions in 3.3 of Example 1;

[0033] Figure 6 It is an overlay of the chromatograms obtained by testing 15 batches of Radix Cynanchifoliae medicinal materials in 4 of Example 1;

[0034] Figure 7 The characteristic spectrum of the Radix Cynanchifolia medicinal material prepared in 4 of Example 1;

[0035] Figure 8 The characteristic spectrum of the standard product of Cynanchum chinense used for comparison in Example 4 of Example 1;

[0036] Figure 9 The figure is an overlay of the chromatograms obtained by detecting 15 batches of Cynanchum chinense aqueous extracts in 2.3 of Example 2;

[0037] Figure 10 This is the characteristic spectrum of the water extract of Cynanchum chinense obtained in 2.3 of Example 2;

[0038] Figure 11 The figure is a comparison of the total ion current and ultraviolet absorption chromatogram of the test solution of the Cynanchum chinense medicinal material in 2.2 of Example 3;

[0039] Figure 12 The chromatographic peaks of the characteristic spectrum of Cynanchum chinense in 2.3 of Example 3 are compared with the retention time of the reference substance;

[0040] Figure 13 The figure is a comparison of the characteristic chromatographic peak of Cynanchum chinense in 2.3 of Example 3 and the ultraviolet absorption curve of the reference substance;

[0041] Figure 14 This is a comparison chart of the characteristic spectra of the Radix Cynanchifolia and the counterfeit Radix Cynanchifolia in Example 4.

[0042] Figure 15 Figure 2 is a comparison chart of the characteristic spectrum of the aqueous extract of Radix Cynanchi Paniculati and counterfeit;

[0043] Figure 16 Figure 1 is a comparison chart of the characteristic spectrum measured under each comparison chromatographic condition of Comparative Example 1, wherein 16A is a comparison chart of the detection results under comparison chromatographic conditions 1-3, and 16B is a comparison chart of the detection results under comparison chromatographic conditions 4-7. DETAILED DESCRIPTION

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

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0046] Terminology

[0047] Unless otherwise indicated or unless the context clearly indicates otherwise, the terms or phrases used in the present application have the following meanings:

[0048] The Chinese medicinal materials referred to in the present application include raw medicinal materials and Chinese medicinal decoction pieces further processed from the raw medicinal materials.

[0049] The aqueous extract of Chinese medicinal materials referred to in the present application includes standard decoctions and Chinese medicinal formula granules.

[0050] The standard decoction in the present application is a single-herb Chinese medicinal decoction piece prepared by a standard process with the guidance of the theory of traditional Chinese medicine, based on clinical application, and referring to modern extraction methods. The Chinese medicinal formula granule in the present application is a pure Chinese medicinal product series prepared by modern pharmaceutical technology, using traditional Chinese medicinal decoction pieces that meet the processing specifications as raw materials, through extraction, concentration, separation, drying, granulation and packaging. Understandably, the standard decoction and the Chinese medicinal formula granule described above can basically guarantee the overall characteristics of the original Chinese medicinal decoction pieces.

[0051] The standard substance of the Chinese medicinal materials referred to in the present application is selected from the control medicinal materials sold by the China Institute for Drug Control or the working control medicinal materials tested by the drug testing institutes of each province and city.

[0052] The term "and / or" as used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, wherein the any and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND" and technical solutions connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] The temperature parameters in the present application, if not particularly limited, allow for constant temperature treatment, but also allow for variations within a certain temperature range. It should be understood that the constant temperature treatment allows for fluctuations within the accuracy range of the instrument control. Fluctuations within a range such as ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, ±0.1°C are allowed.

[0058] In the present application, the weight can be μg, mg, g, kg, etc. mass units well known in the chemical industry.

[0059] Construction of the characteristic spectrum of Radix Cynanchi Paniculati:

[0060] In one aspect of the present application, a method for constructing a characteristic spectrum of Radix Cynanchi Paniculati or its water extract is provided. The method has the advantages of stability, good precision, and good reproducibility. The method has good specificity and can more objectively reflect the characteristic information of Radix Cynanchi Paniculati or its water extract.

[0061] In one embodiment, the method for constructing a characteristic spectrum of Radix Cynanchi Paniculati or its water extract comprises the following steps:

[0062] The step of providing the test solution includes: taking the test sample, adding a first extraction liquid for first extraction, solid-liquid separation to obtain a liquid, adding a second extraction liquid to the obtained liquid for second extraction,

[0063] The test sample is selected from Radix Cynanchi Paniculati and water extract of Radix Cynanchi Paniculati,

[0064] The first extraction liquid includes water, and the first extraction method includes ultrasonic extraction,

[0065] The second extraction liquid includes diethyl ether, and the second extraction method includes extraction;

[0066] The reference solution is provided, and the reference in the reference solution includes a standard of p-hydroxyacetophenone;

[0067] The test solution and the reference solution are taken for high performance liquid chromatography analysis,

[0068] The conditions for high performance liquid chromatography analysis include: gradient elution with methanol as mobile phase A and 0.04% to 0.06% phosphoric acid aqueous solution as mobile phase B,

[0069] The gradient elution program includes: 0-5 min, maintaining the volume percentage of mobile phase A at 14%, 5 min-10 min, increasing the volume percentage of mobile phase A from 14% to 20%, 10 min-60 min, maintaining the volume percentage of mobile phase A at 20%, 60 min-65 min, increasing the volume percentage of mobile phase A from 20% to 70%.

[0070] In one embodiment, the conditions for high performance liquid chromatography analysis further include at least one of the following features:

[0071] (1) The chromatographic column was filled with octadecylsilane bonded silica gel, (2) the column temperature was 28°C to 32°C, (3) the detection wavelength was 270 nm to 280 nm, (4) the flow rate of the mobile phase was 0.55 mL / min to 0.65 mL / min, and (5) the injection volume was 4 μl to 6 μl.

[0072] In one embodiment, in the step of providing the test solution, the power of ultrasound is 200W to 400W and the frequency is 30kHz to 50kHz.

[0073] In one embodiment, in the step of providing the test solution, the solid-liquid separation method includes centrifugation, and further, the centrifugal speed is 3000 to 5000 rpm.

[0074] In one embodiment, in the step of providing the test solution, the weight-to-volume ratio of the test solution to the first extract is selected from: 0.4-0.6 g / 25 mL or 2-4 g / 25 mL;

[0075] Furthermore, the test sample is selected from the aqueous extract of Chinese medicinal materials and the weight volume ratio of the test sample to the first extract is: 0.4-0.6g / 25mL, or,

[0076] The test sample is selected from traditional Chinese medicine and the weight volume ratio of the test sample to the first extract is 2-4 g / 25 mL.

[0077] In one embodiment, in the step of providing the test solution, the volume ratio of the liquid obtained by solid-liquid separation to the second extract is 25:(40-60).

[0078] In one embodiment, the aqueous extract of Cynanchum chinense medicinal material includes a standard decoction of Cynanchum chinense medicinal material and a traditional Chinese medicine formula granule of Cynanchum chinense medicinal material.

[0079] In one embodiment, the aqueous extract of Cynanchum chinense is prepared by the following preparation steps:

[0080] Taking a Radix Cynanchifolia medicinal material, soaking it in water, adding a first solvent to perform a first decoction, wherein the weight ratio of the first solvent to the Radix Cynanchifolia medicinal material is (10-12):1, filtering to obtain a first filtrate and a filter residue, adding a second solvent to the filter residue to perform a second decoction, wherein the weight ratio of the second solvent to the Radix Cynanchifolia medicinal material is (8-10):1, filtering to obtain a second filtrate, and combining the first filtrate and the second filtrate;

[0081] The first solvent is water, and the first decoction time is 20 to 30 minutes.

[0082] The second solvent is water, and the second decoction time is 10 to 20 minutes.

[0083] In one embodiment, the characteristic spectrum of the Cynanchum chinense medicinal material or its aqueous extract constructed includes 11 common peaks.

[0084] Among them, taking peak 7 as a reference, the characteristic peaks and their relative retention times are as follows: peak 1: 0.37±10%, peak 2: 0.59±10%, peak 3: 0.67±10%, peak 4: 0.76±10%, peak 5: 0.86±10%, peak 6: 0.91±10%, peak 8: 1.37±10%, peak 9: 1.53±10%, peak 10: 1.70±10%, peak 11: 1.85±10%,

[0085] Among them, peak 2 is p-hydroxybenzoic acid, peak 4 is vanillic acid, peak 7 is p-hydroxyacetophenone, peak 8 is isovanillic acetophenone, peak 9 is 2,3-dihydroxy-4-methoxyacetophenone, peak 10 is 2,4-dihydroxyacetophenone, and peak 11 is 2,5-dihydroxy-4-methoxyacetophenone.

[0086] Another aspect of the present application relates to a method for identifying Cynanchum chinense medicinal material or its aqueous extract, which is based on the characteristic spectrum construction method described in any of the above technical solutions, and establishes characteristic spectra of the analyte and the standard respectively, and the identification of the analyte is achieved by comparing the characteristic spectrum of the analyte with the characteristic spectrum of the standard.

[0087] In one embodiment, the method for identifying Cynanchum chinense medicinal material or its aqueous extract comprises the following steps:

[0088] Providing a test solution, the steps of providing the test solution include: taking the test object, adding a first extracting solution to perform a first extraction, solid-liquid separation to obtain a liquid, adding a second extracting solution to the obtained liquid to perform a second extraction,

[0089] Wherein, the analyte is selected from one or both of Cynanchum chinense medicinal material and the aqueous extract of Cynanchum chinense medicinal material.

[0090] The first extraction liquid includes water, the first extraction method includes ultrasound,

[0091] The second extracting solution includes ether, and the second extraction method includes extraction;

[0092] Take a standard of Cynanchum chinense or its aqueous extract, prepare a characteristic spectrum of the standard and a characteristic spectrum of the analyte according to the characteristic spectrum construction method described in any of the above technical solutions, and compare the characteristic spectrum of the standard with the characteristic spectrum of the analyte.

[0093] The conditions for HPLC analysis include: using methanol as mobile phase A and a phosphoric acid aqueous solution with a volume concentration of 0.04% to 0.06% as mobile phase B for gradient elution.

[0094] The gradient elution procedure includes: from 0 to 5 minutes, the volume percentage of mobile phase A is maintained at 14%, from 5 minutes to 10 minutes, the volume percentage of mobile phase A is increased from 14% to 20%, from 10 minutes to 60 minutes, the volume percentage of mobile phase A is maintained at 20%, and from 60 minutes to 65 minutes, the volume percentage of mobile phase A is increased from 20% to 70%.

[0095] It is understandable that the characteristic spectrum obtained by testing the test solution using ultra-high performance liquid chromatography can be analyzed as needed. For example, a single test sample is tested and compared with the characteristic spectrum established by the method for constructing the characteristic spectrum of the Radix Cynanchifolia or its aqueous extract described in any of the above technical solutions to obtain the quality of the single test sample; or multiple batches of test samples are tested, and the spectra of the multiple batches of test samples are compared with each other to obtain the similarity between the multiple batches of test samples.

[0096] It can be understood that the method for constructing the characteristic spectrum of the standard or the test object in the above-mentioned identification method is similar to the method for constructing the characteristic spectrum of the Xu Changqing medicinal material or its water extract described in any of the above-mentioned technical solutions, and the method for preparing the test solution is the same as the method for preparing the test solution described in any of the above-mentioned technical solutions, which will not be repeated here.

[0097] The following are some specific examples.

[0098] Example 1

[0099] This embodiment is about the exploration and establishment of the construction method of the characteristic spectrum of the medicinal material Cynanchum chinense, and the specific steps are as follows:

[0100] 1. Instruments and reagents for drug testing

[0101] 1.1. Instruments

[0102] High performance liquid chromatography (Arc, Waters); Welch A C18 chromatographic column (4.6 × 250 mm, 2.7 μm) was used; a microbalance (ME204E, Mettler-Toledo), a microbalance (XP26, Mettler-Toledo), a digitally controlled ultrasonic cleaner (KQ500D, Kunshan Ultrasonic Instrument Co., Ltd.), a constant temperature water bath (HWS28, Shanghai Yiheng Technology Co., Ltd.), and an ultrapure water system (Milli-Q Direct, Merck KGaA).

[0103] Reagents

[0104] Ethanol (Tianjin Yongda Chemical Reagent Co., Ltd.) and methanol (Guangdong Guanghua Science and Technology Co., Ltd.) were of analytical grade, methanol and acetonitrile (Merck Co., Ltd.) were of chromatographic grade, and water was ultrapure water (prepared in the laboratory).

[0105] 1.3. Drug testing

[0106] Reference herbal medicine Cynanchum chinense (batch number: 121514-201202, China Food and Drug Administration); p-hydroxyacetophenone (batch number: 111897-201602, content: 99.9%, China Food and Drug Administration). The herbal medicine information of Cynanchum chinense is shown in Table 1:

[0107] Table 1 Source information of 15 batches of Cynanchum chinense

[0108]

[0109] 2. Sample Preparation

[0110] 2.1. Preparation of test solution

[0111] 2.1.1. Investigation of extraction methods

[0112] This experiment investigated the effects of different extraction methods on the characteristic spectrum of Cynanchum chinense medicinal materials. Two extraction methods, direct aqueous solution ultrasound and extraction, were investigated. The effects of different extraction methods on the characteristic spectrum of Cynanchum chinense medicinal materials were compared by comparing the peak shapes and separation degrees of 11 characteristic peaks.

[0113] ① Take about 3 g of crude powder of Cynanchum chinense (G1901091), place it in a stoppered conical flask, add 25 mL of water, and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes. Let it cool, shake well, filter, and take the filtrate to obtain the product.

[0114] ② Take about 3g of crude powder of Cynanchum indica (G1901091), place it in a stoppered conical flask, add 25mL of water, and ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes. Let it cool, centrifuge (4000 rpm) for 5 minutes, and extract the supernatant twice with ether, each time 25mL. Combine the ether solutions, evaporate to dryness, dissolve the residue in methanol, dilute to 5mL, shake well, filter, and take the filtrate. Figure 1 .

[0115] The results showed that the peak shape and separation effect of each characteristic peak were significantly different when using different extraction methods. The use of aqueous solution ultrasonic extraction resulted in more impurities, poor chromatographic peak separation, and an uneven baseline. After ultrasonic extraction and purification, ether was used. After purification, the chromatographic peak response was high, the separation was good, and the impurities were few. Therefore, in order to ensure the overall effect of the characteristic spectrum, aqueous solution ultrasonic extraction followed by ether extraction was selected as the extraction method for the characteristic spectrum of Cynanchum indica.

[0116] 2.1.2. Extraction solvent investigation

[0117] This experiment investigated the effects of different extraction solvents on the characteristic spectrum of Cynanchum chinense. Dichloromethane, ether and ethyl acetate were used as extraction solvents. The peak shapes and separations of 11 characteristic peaks were used to compare the effects of different extraction solvents on the characteristic spectrum of Cynanchum chinense.

[0118] Take about 3g of crude powder of Cynanchum indicum (G1901091) in three parallel groups, place in a stoppered conical flask, add 25mL of water, ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes, let cool, centrifuge (4000 rpm) for 5 minutes, extract the supernatant twice with dichloromethane, ether, and ethyl acetate, each time 25mL, combine the extracts, evaporate to dryness, dissolve the residue in methanol, dilute to 5mL, shake well, filter, and take the filtrate. Figure 2 .

[0119] The results showed that among the three extraction solvents, dichloromethane extraction lacked some features in the characteristic spectrum and had a low overall response, so dichloromethane was not selected as the extraction solvent for the time being; the chromatograms, responses, and separations obtained by ethyl acetate and ether extraction were not much different, and both had 11 characteristic peaks. However, since the evaporation time of ethyl acetate was too long, heating and evaporation would destroy the volatile oil compounds in Cynanchum cyrtonema, including acetophenones and phenolic compounds, which was not conducive to the experiment. Therefore, ether was selected as the extraction solvent.

[0120] 2.1.3 Establishment of the test solution preparation method

[0121] Take about 3 g of crude powder of this product, place it in a stoppered conical flask, add 25 mL of water, and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes. Let it cool, centrifuge (4000 rpm) for 5 minutes, and extract the supernatant twice with ether, 25 mL each time. Combine the ether solutions, evaporate to dryness, dissolve the residue in methanol, make up to 5 mL, shake well, filter, and take the filtrate to obtain the product.

[0122] 2.2. Preparation of reference solution and reference substance solution

[0123] Take 3 g of Cynanchum chinense reference medicinal material, place it in a stoppered conical flask, add 25 ml of water, and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes. Take it out, let it cool, and centrifuge it (4000 rpm) for 5 minutes. The supernatant was extracted twice with ether, 25 mL each time. The ether solutions were combined and evaporated. The residue was dissolved in methanol, the volume was adjusted to 5 mL, shaken well, filtered, and the filtrate was taken as the reference solution of Chinese medicinal materials.

[0124] Take an appropriate amount of para-hydroxyacetophenone reference substance, accurately weigh it, and add methanol to make a solution containing 10 μg per 1 ml, which will serve as the reference solution of the standard substance.

[0125] 3. Determination of chromatographic conditions

[0126] This section examines the effects of different chromatographic conditions (mobile phase, chromatographic column, column temperature, elution procedure, etc.) on the characteristic spectrum of Cynanchum chinense, and establishes chromatographic conditions based on the investigation results.

[0127] Mobile phase conditions

[0128] High-performance liquid chromatography (Arc, Waters Corporation); A C18 column (4.6×250 mm, 2.7 μm) was used; the methanol-0.05% phosphoric acid system and elution procedure in Example 1 were used to compare the acetonitrile-0.05% phosphoric acid, methanol-0.05% triethylamine, and methanol-water systems, respectively. Elution was performed according to the same elution procedure in Table 2; the column temperature was 30°C, the flow rate was 0.60 ml / min, and the detection wavelength was 274 nm. Figure 3 .

[0129] Table 2 Gradient elution table

[0130]

[0131]

[0132] The results show that compared with the mobile phase system of the present application (methanol-0.05% phosphoric acid), the number of common chromatographic peaks using the methanol-water system is reduced, some components are not reflected, and the representativeness is insufficient; using the acetonitrile-0.05% phosphoric acid system, since acetonitrile has a stronger elution ability than methanol, the retention time of most components is short, and they are eluted before they are completely separated, which does not meet the characteristic spectrum requirements; using the methanol-0.05% triethylamine system, there are even fewer chromatographic peaks, so it is not selected for the time being.

[0133] 3.2. Different elution gradients

[0134] High performance liquid chromatography (Arc, Waters); Welch C18 (4.6 × 250 mm, 2.7 μm); using the methanol-0.05% phosphoric acid system and elution gradient in Example 1, the characteristic chromatograms obtained by different elution procedures were compared. The column temperature was 30°C, the flow rate was 0.60 ml per minute, and the detection wavelength was 274 nm. Figure 4 .

[0135] Table 3 Gradient elution table ①

[0136]

[0137] Table 4 Gradient elution table ②

[0138]

[0139] Table 5 Gradient elution table ③

[0140]

[0141] The results showed that compared with the gradient elution program (gradient ③) of this application, using the other two gradient elution programs (gradients ① and ②), the chromatographic peaks obtained in the first 30 minutes of elution were fewer, and in the next 30 minutes, the elution gradient was faster, which affected the separation of the chromatographic peaks. The overall effect was not as good as gradient ③. The method established in this application eluted 11 chromatographic peaks within 1 hour, and the overall distribution of retention time was more appropriate. Each chromatographic peak achieved baseline separation. Therefore, gradient ③ was selected as the elution program for the characteristic spectrum of Xu Changqing.

[0142] 3.3. Chromatographic columns

[0143] The XCQ11 test solution in Example 1 was taken and the characteristic spectrum of the Cynanchum indicum was determined using different chromatographic columns according to the chromatographic conditions in 3.1.

[0144] High performance liquid chromatography (Arc, Waters); Agilent ZORBAX SB-C18 column (4.6×250 mm, 5 μm), Thermo Acclaim 120C18 column (4.6×250 mm, 5 μm), Waters Xselect HSS T3 column (4.6×250 mm, 5 μm); using the same methanol-0.05% phosphoric acid system and elution program, column temperature, flow rate, detection wavelength and other chromatographic conditions as in Example 1, the separation effects of different chromatographic columns were compared, see Figure 2. Figure 5 .

[0145] The results showed that, with Welch Compared with C18 columns, Agilent ZORBAX SB-C18 columns, Thermo Acclaim 120C18 columns, and Waters Xselect HSS T3 columns all failed to achieve ideal chromatographic peak separation and had less chromatographic peak information. Therefore, Welch The characteristic spectrum of Cynanchum indicum was detected by C18 chromatographic column.

[0146] 3.4. Established chromatographic conditions

[0147] Welch Use a C18 column (250 mm length, 4.6 mm inner diameter, 2.7 μm particle size) with methanol as mobile phase A and 0.05% phosphoric acid solution as mobile phase B, using a gradient elution according to Table 5; the column temperature is 30°C, the flow rate is 0.60 ml / min, and the detection wavelength is 274 nm. The number of theoretical plates based on the p-hydroxyacetophenone peak should be no less than 5000.

[0148] 4. Construct feature maps

[0149] The “Chinese Herbal Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012.0 Version)” was used to process the data of 15 batches of Cynanchum indica medicinal materials samples and generate superimposed spectra. Figure 6 , select 11 common peaks with relatively stable relative retention time and relative peak area RSD as characteristic peaks, and generate the control characteristic spectrum. Figure 7 .

[0150] Table 6 Characteristic spectra of 15 batches of Cynanchum indicum (relative retention time)

[0151]

[0152] Table 7 Characteristic spectra of 15 batches of Cynanchum indicum (relative peak area)

[0153]

[0154]

[0155] Table 8 Calculation results of similarity of characteristic spectra of 15 batches of Cynanchum indica

[0156]

[0157] The results showed that the characteristic spectra of 15 batches of Cynanchum indicum had 11 common peaks, and the similarity was greater than 0.99 (Table 8), which was consistent with the chromatographic peaks of Cynanchum indicum reference material provided by the China Food and Drug Administration (see Figure 8 ), indicating that the 11 common peaks established by this method all belong to components of Cynanchum chinense. Taking peak 7 (p-hydroxyacetophenone) as the reference peak, the relative retention time RSD values ​​of the other 10 characteristic peaks in the characteristic spectra of 15 batches of Cynanchum chinense medicinal materials ranged from 0.03% to 0.12%, meeting the standard requirements for the characteristic spectra of Cynanchum chinense medicinal materials. The relative peak area RSDs of the 10 characteristic peaks in the characteristic spectra of 15 batches of Cynanchum chinense medicinal materials ranged from 11.81% to 43.94%. These results indicate that there are certain differences in the corresponding components of the characteristic peaks of Cynanchum chinense medicinal materials from different origins. Therefore, no regulations on relative peak areas are currently being made.

[0158] Therefore, the characteristic spectrum standard of Cynanchum indicum is stipulated as follows: the test sample chromatogram should show 11 characteristic peaks, and the retention times should correspond to the 11 characteristic peaks in the chromatogram of the reference medicinal material. Among them, Peak 7 should be consistent with the retention time of the reference peak. The peak corresponding to the peak of the para-hydroxyacetophenone reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.37 (peak 1), 0.59 (peak 2), 0.67 (peak 3), 0.76 (peak 4), 0.86 (peak 5), 0.91 (peak 6), 1.37 (peak 8), 1.53 (peak 9), 1.70 (peak 10), and 1.85 (peak 11).

[0159] Example 2

[0160] This example is the construction of a characteristic spectrum of the aqueous extract of Cynanchum chinense. In the construction method of this application, the conditions such as instruments, materials, chromatographic conditions, and preparation of reference solutions are referred to in Example 1. The remaining steps are detailed below.

[0161] Preparation of aqueous extracts

[0162] Take Cynanchum chinense and prepare them into slices, soak them for 30 minutes, and decoct them twice with water. For the first decoction, add 11 times the amount of water and decoct them for 20 minutes. The decoction is filtered through a 350-mesh sieve while hot. For the second decoction, add 9 times the amount of water and decoct them for 15 minutes. The decoction is filtered through a 350-mesh sieve while hot. The filtrate is quickly cooled with cold water, the two decoctions are combined, transferred to a vacuum freeze dryer for freeze-drying, and taken out to obtain the product.

[0163] 2.2. Preparation of test solution

[0164] Take an appropriate amount of this product, grind it into powder, take about 0.5g, place it in a stoppered conical flask, add 25mL of water, ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes, let it cool, centrifuge (4000 rpm) for 5 minutes, extract the supernatant twice with ether, 25mL each time, combine the ether solutions, evaporate to dryness, dissolve the residue in methanol, make up to 5mL, shake well, filter, and take the filtrate to obtain the product.

[0165] 2.3. Establishment of feature maps

[0166] The “Chinese Herbal Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012.0 Version)” was used to process the data of 15 batches of Cynanchum chinense aqueous extract samples and generate superimposed spectra. Figure 9 , select 11 common peaks with relatively stable relative retention time and relative peak area RSD as characteristic peaks, and generate the control characteristic spectrum. Figure 10 .

[0167] The characteristic spectra of 15 batches of Cynanchum chinense aqueous extract were analyzed, and the chromatographic peak 7 corresponding to p-hydroxyacetophenone was used as the reference peak S. The relative retention times (Table 9) and relative peak areas (Table 10) of the other chromatographic peaks were calculated, and the RSD values ​​were calculated. The experimental results are shown below:

[0168] Table 9 Characteristic spectra of 15 batches of Cynanchum chinense aqueous extracts (relative retention time)

[0169]

[0170] Table 10 Characteristic spectra of 15 batches of Cynanchum chinense aqueous extracts (relative peak areas)

[0171]

[0172] The results showed that the characteristic spectra of 15 batches of Cynanchum chinense water extracts had 11 common peaks, which were consistent with the common peaks of the characteristic spectra of Cynanchum chinense medicinal materials.

[0173] Taking peak 7 p-hydroxyacetophenone as the reference peak, the RSD values ​​of the relative retention times of the other 10 characteristic peaks of the characteristic spectra of 15 batches of Cynanchum oxyphylla water extracts were between 0.06% and 0.12%, which met the standard requirements of the characteristic spectra of Cynanchum oxyphylla water extracts; the RSD values ​​of the relative peak areas of the 10 characteristic peaks of the characteristic spectra of 15 batches of Cynanchum oxyphylla water extracts were between 10.68% and 30.76%. The results showed that there were certain differences in the corresponding components of the characteristic peaks of the water extracts made from Cynanchum oxyphylla medicinal materials from different origins, so no regulations were made for the relative peak areas for the time being.

[0174] Therefore, the characteristic spectrum standard for the aqueous extract of Cynanchum sphenantherae is stipulated as follows: the test sample chromatogram should show 11 characteristic peaks, and the retention times should correspond to the 11 characteristic peaks in the chromatogram of the reference medicinal material. Peak 7 should be consistent with the retention time of the reference material peak. The peak corresponding to the peak of the para-hydroxyacetophenone reference material is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.37 (peak 1), 0.59 (peak 2), 0.67 (peak 3), 0.76 (peak 4), 0.86 (peak 5), 0.91 (peak 6), 1.37 (peak 8), 1.53 (peak 9), 1.70 (peak 10), and 1.85 (peak 11). This is consistent with the Cynanchum sphenantherae.

[0175] Example 3

[0176] This embodiment is the identification of the chromatographic peak components of the characteristic spectrum of Cynanchum serrata.

[0177] 1. Instruments and test drugs

[0178] 1.1. Instruments

[0179] Thermo Vanquish Flex ultra-high-performance liquid chromatography-mass spectrometry (Thermo Fisher QE) was used; a high-performance liquid chromatograph (Arc, Waters Corporation); an ultrapure water system (Milli-Q Direct, Merck & Co., Ltd.); a microbalance (ME204E, Mettler-Toledo); and a digitally controlled ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.) were used.

[0180] Reagents

[0181] Methanol (HPLC grade, Merck & Co., Ltd.); formic acid (LC-MS grade, ≥98.0%, batch number: A8310050, CNW); water was homemade ultrapure water; all other reagents were of analytical grade. p-Hydroxybenzoic acid (batch number: 2169, Shanghai Shidande Biotechnology Co., Ltd.); vanillic acid (batch number: 110776-201503, China National Institutes for Food and Drug Control); p-Hydroxyacetophenone (batch number: 111897-201602, China National Institutes for Food and Drug Control); isovanillyl acetophenone (batch number: 14738, Shanghai Shidande Biotechnology Co., Ltd.); 2,4-dihydroxyacetophenone (batch number: 12713, Shanghai Shidande Biotechnology Co., Ltd.); 2,3-dihydroxy-4-methoxyacetophenone (made in the laboratory); and 2,5-dihydroxy-4-methoxyacetophenone (made in the laboratory).

[0182] 1.3. Drug testing

[0183] Take the XCQ11 sample solution in Example 1.

[0184] 2. Mass spectrometry identification

[0185] 2.1. Liquid chromatography-mass spectrometry conditions

[0186] The chromatographic conditions were in accordance with the optimal chromatographic conditions established in Example 1. The mass spectrometry parameters are shown in Table 11.

[0187] Table 11 Mass spectrometry parameters

[0188]

[0189] 2.2. Identification of compounds

[0190] The test solution was detected by using the above liquid chromatography and mass spectrometry analysis conditions. By comparing the accurate mass spectrometry primary and secondary molecular weight, fragment ion comparison and analysis, and comparing with the local mass spectrometry database, 7 components in the characteristic chromatogram of Xuchangqing medicinal materials were confirmed, including p-hydroxybenzoic acid (peak 2), vanillic acid (peak 4), p-hydroxyacetophenone (peak 7), isovanillin (peak 8), 2,3-dihydroxy-4-methoxyacetophenone (peak 9), 2,4-dihydroxyacetophenone (peak 10), and 2,5-dihydroxy-4-methoxyacetophenone (peak 11). The total ion chromatogram and ultraviolet absorption chromatogram of the test solution are shown in Figure 11 , and the compound identification result information is shown in Table 12.

[0191] Table 12 Compound mass spectrometry identification results in Xuchangqing medicinal materials

[0192]

[0193]

[0194] 2.3. Identification of reference substances

[0195] The p-hydroxybenzoic acid reference substance, vanillic acid reference substance, p-hydroxyacetophenone reference substance, isovanillin reference substance, 2,4-dihydroxyacetophenone reference substance, 2,3-dihydroxy-4-methoxyacetophenone reference substance, and 2,5-dihydroxy-4-methoxyacetophenone reference substance were taken in appropriate amounts and dissolved in methanol to prepare reference substance solutions, which were analyzed by injection according to the chromatography conditions in Example 1, and the retention time ( Figure 12 ) and 3D ultraviolet absorption curve ( Figure 13 ) were compared.

[0196] The results showed that the retention time and 3D ultraviolet absorption curve of peak 2, peak 4, peak 7, peak 8, peak 9, peak 10, and peak 11 in the characteristic chromatogram of Xuchangqing were consistent with those of the p-hydroxybenzoic acid reference substance, vanillic acid reference substance, p-hydroxyacetophenone reference substance, isovanillin reference substance, 2,3-dihydroxy-4-methoxyacetophenone reference substance, 2,4-dihydroxyacetophenone reference substance, and 2,5-dihydroxy-4-methoxyacetophenone reference substance, respectively. The results were consistent with the mass spectrometry identification results.

[0197] Example 4

[0198] This example is to identify Cynanchum chinense and its counterfeit products (Baiwei, Baiqian, Asarum, Laoguatou), including the steps of performing chromatographic analysis on the above-mentioned test substances (medicinal materials or their water extracts) and establishing their characteristic spectra. Among them, the preparation of instruments and reagents, chromatographic conditions, and reference solution is the same as in "Example 1". The preparation of the test solution of the counterfeit medicinal materials (Baiwei, Baiqian, Asarum, Laoguatou) is the same as in "Example 1", and the preparation of the test solution of the water extract of the counterfeit products (Baiwei, Baiqian, Asarum, Laoguatou) is the same as in "Example 2". The source information of Baiwei, Baiqian, Asarum, Laoguatou medicinal materials is shown in Table 13.

[0199] Table 13 Information on the sources of the medicinal materials of Baiwei, Baiqian, Xixin and Laoguatou

[0200]

[0201]

[0202] 1. Comparison of characteristic spectra of Cynanchum chinense and different counterfeit medicinal materials

[0203] Take Radix Cynanchifolia and other counterfeit herbs listed in Table 13, prepare test solution and inject into the sample for determination. The test results are shown in Figure 14 .

[0204] The results showed that the characteristic spectra of the three batches of Cynanchum indicum had 11 common peaks. Five chromatographic peaks were shared by 14 batches of Cynanchum indicum, including Baiwei, Baiqian, Asarum, and Laoguatou: Peaks 2, 4, 5, 6, and 9. Different varieties can be distinguished based on the number of chromatographic peaks, for example, Baiqian and Asarum lack Peaks 3 and 8. However, Cynanchum indicum can be distinguished from other herbs based on Peak 11, which is a characteristic chromatographic peak of Cynanchum indicum.

[0205] 2. Comparison of characteristic spectra of water extracts of Cynanchum chinense and different counterfeit water extracts

[0206] Take the water extract of Cynanchum chinense and other counterfeit water extracts in Table 13, make the test solution and inject it into the sample for determination. The results are shown in Figure 15 .

[0207] The results showed that the characteristic chromatograms of the three batches of Cynanchum indicum aqueous extracts shared 11 peaks. After aqueous extraction of Cynanchum indicum, Baiwei, Baiqian, Asarum, and Laoguatou, the 14 batches of aqueous extracts shared three chromatographic peaks: Peak 2, Peak 4, and Peak 6. Compared to the results obtained for the herbal differentiation, the aqueous extracts of different varieties can also be distinguished based on the number of chromatographic peaks. The Cynanchum indicum aqueous extract can also be distinguished from other aqueous extracts based on Peak 11, which is the characteristic component of Cynanchum indicum.

[0208] In summary, the characteristic spectrum of Cynanchum wilfordii established by the method of the present application can effectively characterize its chemical composition, can effectively distinguish it from other counterfeits, and has a certain specificity.

[0209] Comparative Example 1

[0210] This experiment investigates the influence of different chromatographic conditions (mobile phase, chromatographic column, column temperature, elution program, etc.) on the characteristic chromatogram of Xu Changqing. The Xu Changqing 11 test solution in Example 1 was used to determine the characteristic chromatogram of Xu Changqing medicinal materials using different chromatographic conditions. The test results are shown in Figure 16 , and the annotations in the figure correspond to different comparative chromatographic conditions.

[0211] 1. Comparative chromatographic condition 1

[0212] High performance liquid chromatograph (Arc, Waters Corporation); YMC Triart C18 chromatographic column (4.6 x 250 mm, 5 μm); methanol as mobile phase A, water as mobile phase B, gradient elution according to Table 14; column temperature 25 °C, flow rate 0.60 ml / min, detection wavelength 274 nm.

[0213] Table 14 Gradient elution table

[0214]

[0215] 2. Comparative chromatographic condition 2

[0216] High performance liquid chromatograph (Arc, Waters Corporation); Agilent TC-C18 chromatographic column (4.6 x 250 mm, 5 μm), Agilent ZORBAX SB-C18 chromatographic column (4.6 x 250 mm, 5 μm), YMC Triart C18 chromatographic column (4.6 x 250 mm, 5 μm), Waters Xselect HSS T3 chromatographic column (4.6 x 250 mm, 5 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution according to Table 15; column temperature 40 °C, flow rate 1.0 ml / min, detection wavelength 274 nm.

[0217] Table 15 Gradient elution table

[0218]

[0219] 3. Comparative chromatographic condition 3

[0220] Ultra-high performance liquid chromatograph (H-class, Waters Corporation); Thermo Acclaim C18 chromatographic column (2.1 x 150 mm, 2.2 μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution according to Table 16; column temperature 25 °C, flow rate 0.25 ml / min, detection wavelength 274 nm. See Figure 1 .

[0221] Table 16 Gradient elution table

[0222]

[0223] 4. Comparative Chromatographic Conditions 4

[0224] Ultra-high performance liquid chromatography (H-class, Waters Corporation); ACE EXCEL Super C18 chromatographic column (2.1×100 mm, 1.7 μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution according to Table 17; column temperature 25°C, flow rate 0.30 ml / min, detection wavelength 274 nm.

[0225] Table 17 Gradient elution table

[0226]

[0227] 5. Comparison of chromatographic conditions 5

[0228] High performance liquid chromatography (Arc, Waters Corporation); Agilent ZORBAX Extend-C18 column (4.6×250 mm, 5 μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution according to Table 18; column temperature 25°C, flow rate 1.0 ml / min, detection wavelength 274 nm.

[0229] Table 18 Gradient elution table

[0230]

[0231] 6. Comparison of chromatographic conditions

[0232] High performance liquid chromatography (Arc, Waters Corporation); YMC Triart C18 chromatographic column (4.6×250 mm, 5 μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution according to Table 19; column temperature 25°C, flow rate 1.0 ml / min, detection wavelength 274 nm.

[0233] Table 19 Gradient elution table

[0234]

[0235] 7. Comparative Chromatographic Conditions

[0236] High performance liquid chromatography (Arc, Waters Corporation); YMC Triart C18 column (4.6×250 mm, 5 μm); acetonitrile as mobile phase A, 0.1% glacial acetic acid as mobile phase B, gradient elution according to Table 20; column temperature 25°C, flow rate 0.8 ml / min, detection wavelength 274 nm.

[0237] Table 20 Gradient elution table

[0238]

[0239] The results showed that, compared with the chromatographic conditions of the present application, the use of other different types of chromatographic columns, mobile phases and elution procedures, column temperatures, flow rates, etc., the chromatograms obtained did not achieve the results of the present application in terms of the number of chromatographic peaks or the separation of the main chromatographic peaks (see the relevant figures in Examples 1-2 for details, such as Figure 6 、 Figure 7 、 Figure 8 、 Figure 9 、 Figure 10 etc.). Use Welch A C18 column (4.6×250mm, 2.7μm) is more suitable for separating acetophenones and phenols in Cynanchum cyrtonema. Using a methanol-0.05% phosphoric acid mobile phase system improves the separation of chromatographic peaks because methanol has a lower elution capacity than acetonitrile, while the phosphoric acid solution improves the responsiveness of some chromatographic peaks and increases the number of major chromatographic peaks.

[0240] 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.

[0241] 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 Cynanchum chinense or its aqueous extract, characterized in that: The steps include: Providing a test solution, the step of providing the test solution comprising: taking a test sample, adding a first extracting solution to perform a first extraction, solid-liquid separation to obtain a liquid, adding a second extracting solution to the obtained liquid to perform a second extraction, The water extract of the Cynanchum chinense medicinal material includes a standard decoction of the Cynanchum chinense medicinal material and a traditional Chinese medicine formula granule of the Cynanchum chinense medicinal material. The first extraction liquid includes water, the first extraction method includes ultrasound, The second extracting solution includes diethyl ether, and the second extraction method includes extraction; Providing a reference solution, wherein the reference substance in the reference solution includes a p-hydroxyacetophenone standard; The test solution and the reference solution were analyzed by high performance liquid chromatography. The conditions for the HPLC analysis include: using methanol as mobile phase A and using a phosphoric acid aqueous solution with a volume concentration of 0.04% to 0.06% as mobile phase B for gradient elution. The gradient elution procedure includes: 0-5min, maintaining the volume percentage of the mobile phase A at 14%, 5min-10min, increasing the volume percentage of the mobile phase A from 14% to 20%, 10min-60min, maintaining the volume percentage of the mobile phase A at 20%, 60min-65min, increasing the volume percentage of the mobile phase A from 20% to 70%; the model of the chromatographic column is Welch The column length is 250 mm, the inner diameter is 4.6 mm, the particle size of the filler is 2.7 μm; the detection wavelength is 270 nm to 280 nm; The characteristic spectrum of the constructed Cynanchum chinense medicinal material or the water extract of the Cynanchum chinense medicinal material includes 11 common peaks, among which peak 2 is p-hydroxybenzoic acid, peak 4 is vanillic acid, peak 7 is p-hydroxyacetophenone, peak 8 is isovanillic acetonide, peak 9 is 2,3-dihydroxy-4-methoxyacetophenone, peak 10 is 2,4-dihydroxyacetophenone, and peak 11 is 2,5-dihydroxy-4-methoxyacetophenone.

2. The method for constructing a characteristic map according to claim 1, wherein: The conditions for the HPLC analysis also include at least one of the following features: (1) The column temperature was 28°C to 32°C, (2) the flow rate of the mobile phase was 0.55 mL / min to 0.65 mL / min, and (3) the injection volume was 4 μl to 6 μl.

3. The method for constructing a characteristic map according to claim 1, wherein: In the step of providing a test solution: The ultrasonic power is 200W-400W, the frequency is 30kHz-50kHz, and / or The solid-liquid separation method includes centrifugation, the centrifugal speed is 3000-5000 rpm, and / or The weight-to-volume ratio of the test sample to the first extract is selected from: 0.4-0.6 g / 25 mL or 2-4 g / 25 mL, and / or The volume ratio of the liquid obtained by the solid-liquid separation to the second extract is 25:(40-60).

4. The method for constructing a characteristic map according to claim 1, wherein: The water extract of the Cynanchum chinense medicinal material is prepared by the following preparation steps: Taking a Cynanchum chinense medicinal material, soaking it in water, adding a first solvent to perform a first decoction, wherein the weight ratio of the first solvent to the Cynanchum chinense medicinal material is (10-12):1, filtering to obtain a first filtrate and a filter residue, adding a second solvent to the filter residue to perform a second decoction, wherein the weight ratio of the second solvent to the Cynanchum chinense medicinal material is (8-10):1, filtering to obtain a second filtrate, and combining the first filtrate and the second filtrate; Wherein, the first solvent is water, and the first decoction time is 20 min to 30 min; The second solvent is water, and the second decoction time is 10 min to 20 min.

5. The method for constructing a characteristic map according to any one of claims 1 to 3, wherein: Among the 11 common peaks, taking peak 7 as a reference, the characteristic peaks and their relative retention times are as follows: peak 1: 0.37±10%, peak 2: 0.59±10%, peak 3: 0.67±10%, peak 4: 0.76±10%, peak 5: 0.86±10%, peak 6: 0.91±10%, peak 8: 1.37±10%, peak 9: 1.53±10%, peak 10: 1.70±10%, peak 11: 1.85±10%.

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