Construction method of characteristic chromatogram of amaranthaceous traditional Chinese medicine material or preparation and identification method thereof

Abstract

The application relates to the technical field of traditional Chinese medicine analysis, in particular to a construction method of a characteristic chromatogram of Amaranthaceae traditional Chinese medicine materials or preparations thereof and a discrimination method thereof, wherein the Amaranthaceae traditional Chinese medicine materials or preparations thereof include: cockscomb flower medicinal materials, cockscomb flower standard decoction, cockscomb flower traditional Chinese medicine dispensing granules, green amaranth flower medicinal materials, green amaranth flower standard decoction, green amaranth flower traditional Chinese medicine dispensing granules. In the technical scheme, the conditions of high performance liquid chromatography analysis include: gradient elution is carried out by taking methanol as a mobile phase A and taking a 0.08-0.12% phosphoric acid aqueous solution in volume concentration as a mobile phase B, the analysis time is short, the obtained characteristic peak has good separation degree and a stable baseline, and the chemical information of the test object can be comprehensively reflected and used for the discrimination of the variety, quality, authenticity and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine analysis, and particularly relates to a construction method of a characteristic chromatogram of a traditional Chinese medicine material of Amaranthaceae and a method for identifying the same, wherein the traditional Chinese medicine material of Amaranthaceae comprises cockscomb flower, cockscomb flower standard decoction, cockscomb flower traditional Chinese medicine dispensing granules, celosia argentea flower, celosia argentea flower standard decoction, and celosia argentea flower traditional Chinese medicine dispensing granules. BACKGROUND

[0002] Amaranthaceae plants are widely used in traditional Chinese medicine, and the pharmacopoeia clearly records that cockscomb flower is the dried inflorescence of cockscomb flower (Cetera cristoto L.) of Amaranthaceae, which is collected in autumn when the flowers are in full bloom, and is dried, sweet and astringent in nature, and is cold in flavor, and is related to the liver and large intestine channels, and has the effects of astringency, hemostasis, and pain relief, and is used for treating hemoptysis, metrorrhagia, hematochezia, hemorrhoids, leukorrhagia, and persistent pain; celosia argentea seed is the dried mature seed of celosia argentea (Celosia argentea L.) of Amaranthaceae, which is harvested in autumn when the fruits are mature, and is dried, and the seeds are collected and impurities are removed, and is bitter in nature and cold in flavor, and is related to the liver channel, and has the effects of clearing liver heat, drenching, and removing nebula, and is used for treating liver heat, red eye, nebula, blurred vision, and liver fire dizziness. Researching and constructing the characteristic chromatogram of the traditional Chinese medicine material of Amaranthaceae is conducive to fully understanding the chemical composition of traditional Chinese medicine and providing a theoretical basis for its clinical use.

[0003] Cockscomb flower was first recorded in “Dian Nan Ben Cao” as “cockcomb flower for treating intestinal wind, metrorrhagia, leukorrhagia, and dysentery”, and the original plant morphology of cockscomb flower was described in “Imperially-Commissioned Ben Cao Pin Hui Jing Yao”, “Ben Cao Gang Mu”, and “Ben Cao Yuan Shu”, which is consistent with the description in “Chinese Medicinal Plant Flora”. Celosia argentea, which is also a plant of Amaranthaceae, has a plant morphology very similar to that of cockscomb flower, and most people are confused about the understanding of cockscomb flower and celosia argentea flower, such as in Taizhou and Qingjiang, Jiangsu, the inflorescence of celosia argentea is used as cockscomb flower. According to the records in “Ben Cao Gang Mu”, “celosia argentea grows in fields, and the tender grass is edible like amaranth, and the long one is three to four feet high. The leaves, flowers, and fruits are the same as cockscomb flower and cannot be distinguished. But the cockscomb flower spike has large and flat or round ones. This one has flower spikes on the tips, which are four to five inches long and look like rabbit tails, and are water red, and there are also yellow and white ones. The seeds are in the spikes, and are the same as cockscomb seeds and amaranth seeds and cannot be distinguished. Su Gong said that it has angles, which is a mistake”. It can be seen that celosia argentea and cockscomb flower are different medicinal materials, and attention should be paid to the distinction.

[0004] Traditional Chinese medicine materials generally refer to pure medicinal materials and traditional Chinese medicine decoction pieces prepared by further cutting and processing the pure medicinal materials. The traditional Chinese medicine decoction pieces are important medicinal materials for traditional Chinese medicine to exert clinical efficacy, and their safety and effectiveness have been widely recognized. The traditional Chinese medicine decoction pieces are mainly used in the form of decoction. The traditional Chinese medicine dispensing granules are prepared by extracting, separating, concentrating, drying and granulating single traditional Chinese medicine decoction pieces with water heating. The traditional Chinese medicine dispensing granules lose the identification characteristics of the pure medicinal materials and decoction pieces, and cannot be inspected and identified from the original shapes, colors, textures, cross sections and odors of the medicinal materials. In order to enable the traditional Chinese medicine dispensing granules to carry the safety and effectiveness of the traditional Chinese medicine decoction pieces, a standard decoction is needed as a bridge. The standard decoction is a material reference for measuring whether the single traditional Chinese medicine dispensing granule is basically consistent with the single traditional Chinese medicine decoction piece. The "standard" in the standard decoction mainly covers the genuineness of the medicinal materials, the uniformity of the extraction process and the rigor of the quality control.

[0005] There is a reported method for detecting Flos Celosiae Cristatae. The method uses kaempferol as a reference substance, uses acetonitrile-0.2% phosphoric acid solution (containing 0.2% triethylamine) as the mobile phase for liquid chromatograph analysis, and constructs a characteristic chromatogram of Flos Celosiae Cristatae. In the preparation of the test solution, hydrochloric acid hydrolysis is used. The obtained characteristic chromatogram is the information of the compounds after acid hydrolysis, and cannot reflect the compound information of the sample itself. The method lacks research on the original compound components, and is insufficient to comprehensively reflect the quality characteristics of Flos Celosiae Cristatae.

[0006] There is a reported method for quality control of Flos Celosiae Cristatae. The method uses luteolin, quercetin and kaempferol as reference substances, uses acetonitrile-0.1% phosphoric acid solution as the mobile phase for liquid chromatograph analysis, and constructs an HPLC characteristic chromatogram of Flos Celosiae Cristatae. The method is time-consuming, and the separation degree between the characteristic peaks is low. The baseline is unstable, and it is difficult to accurately measure the quality of Flos Celosiae Cristatae.

[0007] Therefore, the technical content of the present application is proposed. SUMMARY

[0008] Based on this, one of the purposes of the present application is to provide a method for constructing a characteristic chromatogram of Amaranthaceae traditional Chinese medicine materials or preparations thereof. The method is short in detection time, good in separation degree of characteristic peaks, and can quickly and comprehensively reflect the chemical characteristics of the test sample. Another purpose of the present application is to provide a method for identifying Amaranthaceae traditional Chinese medicine materials or preparations thereof, which is suitable for detecting and identifying Flos Celosiae Cristatae, standard decoction of Flos Celosiae Cristatae, traditional Chinese medicine dispensing granules of Flos Celosiae Cristatae, Flos Celosiae Argenteae, standard decoction of Flos Celosiae Argenteae, traditional Chinese medicine dispensing granules of Flos Celosiae Argenteae, etc.

[0009] The following is a specific description of the technical solution of the present application:

[0010] A method for constructing a characteristic chromatogram of Amaranthaceae traditional Chinese medicine materials or preparations thereof, comprising the following steps:

[0011] Take the standard sample of ferulic acid, dissolve it with solvent to prepare a reference solution,

[0012] Take the test sample, add extraction solvent to extract it to prepare a test sample solution, the test sample being selected from Amaranthaceae Chinese medicinal material or preparation thereof,

[0013] Take the reference solution and the test sample solution to perform high performance liquid chromatography analysis,

[0014] The conditions of the high performance liquid chromatography analysis include:

[0015] Methanol is used as mobile phase A, and 0.08-0.12% phosphoric acid aqueous solution is used as mobile phase B to perform gradient elution, the program of the gradient elution including:

[0016] 0-7 min, the volume percentage of the mobile phase A is maintained at 20%,

[0017] 7 min-20 min, the volume percentage of the mobile phase A is increased from 20% to 24%,

[0018] 20 min-30 min, the volume percentage of the mobile phase A is increased from 24% to 28%,

[0019] 30 min-50 min, the volume percentage of the mobile phase A is increased from 28% to 43%,

[0020] 50 min-53 min, the volume percentage of the mobile phase A is maintained at 43%,

[0021] 53 min-58 min, the volume percentage of the mobile phase A is increased from 43% to 65%.

[0022] A method for identifying Amaranthaceae Chinese medicinal material or preparation thereof, comprising the following steps:

[0023] Take Amaranthaceae Chinese medicinal material or preparation thereof, and construct the characteristic pattern of the standard sample according to the method for constructing characteristic pattern described in any of the technical solutions above,

[0024] Take the test sample, add extraction solvent to extract it to prepare a test sample solution, the test sample being selected from Amaranthaceae Chinese medicinal material or preparation thereof,

[0025] Take the test sample solution to perform high performance liquid chromatography analysis to obtain the characteristic pattern of the test sample,

[0026] Compare the characteristic pattern of the standard sample with the characteristic pattern of the test sample,

[0027] The conditions of the high performance liquid chromatography analysis include:

[0028] Methanol as mobile phase A, and 0.08-0.12% phosphoric acid aqueous solution as mobile phase B for gradient elution,

[0029] The procedure of the gradient elution comprises:

[0030] 0-7 min, the volume percentage of the mobile phase A is maintained at 20%,

[0031] 7 min-20 min, the volume percentage of the mobile phase A is increased from 20% to 24%,

[0032] 20 min-30 min, the volume percentage of the mobile phase A is increased from 24% to 28%,

[0033] 30 min-50 min, the volume percentage of the mobile phase A is increased from 28% to 43%,

[0034] 50 min-53 min, the volume percentage of the mobile phase A is maintained at 43%,

[0035] 53 min-58 min, the volume percentage of the mobile phase A is increased from 43% to 65%. BRIEF DESCRIPTION OF DRAWINGS

[0036] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0037] Figure 1 The full-wave scan profile of the standard decoction of the cockscomb flower in Example 1 (A) and the 3D spectrum (B) are shown in the following figures. Figure 1 Figure 1

[0038] Figure 2 The chromatographic results of the standard decoction of the cockscomb flower in Example 1 under different detection wavelengths are shown in the following figures.

[0039] Figure 3 The chromatographic results of the standard decoction of the cockscomb flower in Example 1 under different gradient elution conditions are shown in the following figures.

[0040] Figure 4 The chromatographic results of the cockscomb flower medicinal material in Example 2 under different extraction solvent conditions are shown in the following figures.

[0041] Figure 5 The chromatographic results of the cockscomb flower medicinal material in Example 2 under different extraction methods are shown in the following figures. ​​

[0042] Figure 6 Figure for comparison of chromatographic results of the safflower herb in Example 2 at different extraction times;

[0043] Figure 7 Figure for comparison of chromatographic results of the safflower herb in Example 3 for specificity investigation;

[0044] Figure 8 Figure for comparison of chromatographic results of the safflower herb in Example 3 for intermediate precision investigation;

[0045] Figure 9 Figure for comparison of chromatographic results of the safflower herb in Example 3 for durability investigation under different column temperature conditions;

[0046] Figure 10 Figure for comparison of chromatographic results of the safflower herb in Example 3 for durability investigation under different flow rate conditions;

[0047] Figure 11 Figure for comparison of chromatographic results of the safflower herb in Example 3 for durability investigation under different column conditions;

[0048] Figure 12 Figure for comparison of chromatographic results of the safflower herb in Example 3 for durability investigation under different chromatograph conditions;

[0049] Figure 13 Figure for superimposed chromatograms of 16 batches of safflower herb in Example 4;

[0050] Figure 14 Figure for control characteristic chromatogram of safflower herb for control in Example 4;

[0051] Figure 15 Figure for superimposed chromatograms of 16 batches of safflower standard decoction in Example 4;

[0052] Figure 16 Figure for control characteristic chromatogram of safflower standard decoction for control in Example 4;

[0053] Figure 17 Figure for superimposed chromatograms of 3 batches of safflower traditional Chinese medicine formula granules in Example 4;

[0054] Figure 18 Figure for control characteristic chromatogram of safflower traditional Chinese medicine formula granules for control in Example 4;

[0055] Figure 19 Figure for superimposed chromatograms of 5 batches of carthamus tinctorius L. herb in Example 4;

[0056] Figure 20 Figure for control characteristic chromatogram of carthamus tinctorius L. herb for control in Example 4;

[0057] Figure 21 The chromatograms are superimposed for the five batches of standard decoction of Amaranthus australis flowers in Example 4;

[0058] Figure 22 This is the control characteristic spectrum of the standard decoction of Amaranthus chinensis flowers used for control in Example 4;

[0059] Figure 23 This is a comparison diagram of the characteristic spectra of the medicinal material Celosia cristata and the medicinal material Amaranthus chinensis in Example 4;

[0060] Figure 24 This is a comparison chart of the characteristic spectra of the standard decoction of Celosia cristata and the standard decoction of Amaranthus chinensis in Example 4;

[0061] Figure 25 This is the superimposed chromatogram of the Celosia cristata medicinal material prepared according to Method 1 in Comparative Example 1 ( Figure 25 JGH09, JGH13, JGH14) and the control chart ( Figure 25 Middle R);

[0062] Figure 26 This is the superimposed chromatogram of the Celosia cristata medicinal material prepared according to method 2 in comparative example 1 ( Figure 26 JGH09, JGH13, JGH14) and the control chart ( Figure 26 Middle R);

[0063] Figure 27 This is the superimposed chromatogram of the Celosia cristata medicinal material prepared according to method 3 in comparative example 1 ( Figure 27 JGH09, JGH13, JGH14) and the control chart ( Figure 27 Middle R);

[0064] Figure 28 This is a comparison chart of the characteristic spectrum of the Celosia cristata medicinal material prepared in Example 4 and the characteristic spectrum of the Celosia cristata medicinal material prepared by methods 1 to 3 of Comparative Example 1. DETAILED DESCRIPTION

[0065] 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.

[0066] 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.

[0067] Terms

[0068] Unless otherwise defined, or the context dictates otherwise, the terms or phrases used herein have the meanings provided below. The meaning of a term can depend on the context in which it is used, on the terminology used previously or subsequently, or on the usages common in the field.

[0069] As used herein, the terms "and / or", "or / and", "and / or" are intended to cover any and all combinations of two or more of the associated listed items, and can be used in various portions of this disclosure. For example, "and / or" shall be construed to cover any and all combinations of two or more of the associated listed items, and can be used in various portions of this disclosure.

[0070] As used herein, "preferably", "more preferably", "even more preferably", and the like are merely descriptive and not limiting, unless otherwise stated.

[0071] As used herein, "further", "even further", "particularly", and the like are used to describe a difference in content, and should not be construed as limiting the scope of protection of the present application.

[0072] As used herein, the technical features described in an open-ended manner include both closed technical solutions consisting of listed features and open technical solutions containing listed features.

[0073] As used herein, with respect to numerical intervals (i.e., numerical ranges), unless otherwise stated, it is intended that the optional numerical distribution within the numerical interval be considered continuous and include the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range and every numerical value between the two numerical endpoints. Unless otherwise stated, when a numerical interval refers only to integers within the numerical interval, it includes the two numerical endpoint integers and every integer between the two numerical endpoints. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges can be combined. In other words, unless otherwise indicated, ranges disclosed herein are to be understood to include any and all sub-ranges subsumed therein.

[0074] As used herein, unless otherwise specified, temperature parameters allow for constant temperature treatment and also allow for variations within a certain temperature interval. It should be understood that the constant temperature treatment allows for fluctuations within the accuracy of the instrument control. Fluctuations within a range of, for example, ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, ±0.1°C are allowed.

[0075] As used herein, weight can be μg, mg, g, kg, and other mass units commonly known in the chemical industry.

[0076] In one aspect of the present application, a method for constructing a characteristic map of Amaranthaceae Chinese medicinal material or preparation thereof is provided. The method can more quickly and comprehensively reflect the chemical characteristics of the test sample. The characteristic peaks in the characteristic map are well separated, the baseline is smooth, the detection time is short, the method is reliable, and the three requirements of characteristic, reproducibility and operability are met.

[0077] In one embodiment, the method for constructing a characteristic map of Amaranthaceae Chinese medicinal material or preparation thereof comprises the following steps:

[0078] S100: taking a reference material of ferulic acid, dissolving it with a solvent to prepare a reference material solution,

[0079] S200: taking a test sample, adding an extraction solvent to extract it to prepare a test sample solution, the test sample being selected from Amaranthaceae Chinese medicinal material or preparation thereof,

[0080] S300: taking the reference material solution and the test sample solution for high performance liquid chromatography analysis,

[0081] The conditions for high performance liquid chromatography analysis include:

[0082] Methanol is used as mobile phase A, and a 0.08-0.12% phosphoric acid aqueous solution is used as mobile phase B for gradient elution. The gradient elution program includes:

[0083] 0-7 min, the volume percentage of the mobile phase A is maintained at 20%,

[0084] 7 min-20 min, the volume percentage of the mobile phase A is increased from 20% to 24%,

[0085] 20 min-30 min, the volume percentage of the mobile phase A is increased from 24% to 28%,

[0086] 30 min-50 min, the volume percentage of the mobile phase A is increased from 28% to 43%,

[0087] 50 min-53 min, the volume percentage of the mobile phase A is maintained at 43%,

[0088] 53 min-58 min, the volume percentage of the mobile phase A is increased from 43% to 65%.

[0089] Step S100 for preparing the reference material solution:

[0090] The control substance as the reference substance is usually a main component in the test substance or a common peak with relatively large peak area and stability. At present, kaempferol, luteolin, quercetin, and kaempferol are often used as the reference substance in the chromatographic analysis of amaranthaceae plants or medicinal materials. In the development process of the atlas construction method, one of the main components in Celosia argentea is ferulic acid, which has a hemostatic pharmacological effect and is a key effective component for the astringent and hemostatic effect of Celosia argentea. Therefore, ferulic acid is selected as the reference substance.

[0091] In one embodiment, an appropriate amount of ferulic acid control substance is precisely weighed and dissolved in methanol to prepare a solution containing 5 μg per 1 ml as the reference solution of the control substance.

[0092] The test substance solution is prepared in step S200.

[0093] In step S200, the test substance is extracted with an extraction solvent to prepare a test substance solution. The test substance is selected from amaranthaceae Chinese medicinal materials or preparations thereof.

[0094] To ensure that the main chemical components or effective components in the test substance are reflected in the characteristic atlas, an alcohol aqueous solution is preferably used as the extraction solvent.

[0095] In one embodiment, the extraction solvent is an alcohol aqueous solution in which the volume fraction of alcohol is 60% to 80%; further, the alcohol is methanol.

[0096] In one embodiment, the extraction method is ultrasonic extraction; further, the power of ultrasonic extraction can be 200 W to 400 W, and the frequency of ultrasonic extraction can be 30 kHz to 50 kHz; further, the power of ultrasonic extraction can be 300 W, and the frequency of ultrasonic extraction can be 40 kHz.

[0097] In one embodiment, the extraction method is heating reflux extraction. In the present application, heating reflux extraction refers to heating at the boiling point of the extraction solvent ± 5 ℃.

[0098] In one embodiment, the extraction time is 20 to 40 min, and further can be 25 to 35 min, for example, 20 min, 25 min, 30 min, 35 min, 40 min, etc.

[0099] In one embodiment, the test substance solution is prepared by the following steps: taking the test substance, adding the extraction solvent for first extraction, solid-liquid separation to obtain a first liquid, evaporating the first liquid, adding the extraction solvent for second extraction, and solid-liquid separation to obtain a second liquid. Optionally, the solid-liquid separation method is centrifugation and / or filtration.

[0100] In one of the embodiments, in step S200, the test sample is the flower of Celosia argentea L. About 2.0 g of the powder of the flower of Celosia argentea L. (passed through a No. 3 sieve) is placed in a conical flask with a stopper, 70% methanol 50 ml is added, ultrasonic treatment (power 300 W, frequency 40 kHz) is conducted for 30 minutes, centrifugation is conducted, 25 ml of supernatant is taken, evaporation is conducted, the residue is dissolved in 70% methanol to make up to 10 ml in a volumetric flask, shaking is conducted, filtration is conducted, and the filtrate is obtained.

[0101] In one of the embodiments, in step S200, the test sample is the flower of Celosia argentea L. About 2.0 g of the powder of the flower of Celosia argentea L. (passed through a No. 3 sieve) is placed in a conical flask with a stopper, 70% methanol 50 ml is added, ultrasonic treatment (power 300 W, frequency 40 kHz) is conducted for 30 minutes, centrifugation is conducted, 25 ml of supernatant is taken, evaporation is conducted, the residue is dissolved in 70% methanol to make up to 10 ml in a volumetric flask, shaking is conducted, filtration is conducted, and the filtrate is obtained.

[0102] In one of the embodiments, in step S200, the test sample is the flower of Celosia argentea L. About 2.0 g of the powder of the flower of Celosia argentea L. (passed through a No. 3 sieve) is placed in a conical flask with a stopper, 70% methanol 50 ml is added, ultrasonic treatment (power 300 W, frequency 40 kHz) is conducted for 30 minutes, centrifugation is conducted, 25 ml of supernatant is taken, evaporation is conducted, the residue is dissolved in 70% methanol to make up to 10 ml in a volumetric flask, shaking is conducted, filtration is conducted, and the filtrate is obtained.

[0103] In one of the embodiments, in step S200, the test sample is the flower of Celosia argentea L. About 2.0 g of the powder of the flower of Celosia argentea L. (passed through a No. 3 sieve) is placed in a conical flask with a stopper, 70% methanol 50 ml is added, ultrasonic treatment (power 300 W, frequency 40 kHz) is conducted for 30 minutes, centrifugation is conducted, 25 ml of supernatant is taken, evaporation is conducted, the residue is dissolved in 70% methanol to make up to 10 ml in a volumetric flask, shaking is conducted, filtration is conducted, and the filtrate is obtained.

[0104] In one of the embodiments, in step S200, the test sample is the flower of Celosia argentea L. About 2.0 g of the powder of the flower of Celosia argentea L. (passed through a No. 3 sieve) is placed in a conical flask with a stopper, 70% methanol 50 ml is added, ultrasonic treatment (power 300 W, frequency 40 kHz) is conducted for 30 minutes, centrifugation is conducted, 25 ml of supernatant is taken, evaporation is conducted, the residue is dissolved in 70% methanol to make up to 10 ml in a volumetric flask, shaking is conducted, filtration is conducted, and the filtrate is obtained.

[0105] Step S300: High performance liquid chromatography analysis:

[0106] In step S300, by establishing suitable chromatographic conditions, the analysis time can be saved, the analysis efficiency can be improved, the peak separation degree can be good, and the response value of the characteristic peak can be appropriate.

[0107] In one of the embodiments, methanol is used as the mobile phase A, and 0.08-0.12% phosphoric acid aqueous solution is used as the mobile phase B for gradient elution. Further, the mobile phase B is 0.1% phosphoric acid solution.

[0108] In one of the embodiments, the procedure of gradient elution comprises:

[0109] 0-7 min, the volume percentage of the mobile phase A is maintained at 20%,

[0110] 7 min-20 min, the volume percentage of the mobile phase A is increased from 20% to 24%,

[0111] 20 min-30 min, the volume percentage of the mobile phase A is increased from 24% to 28%,

[0112] 30 min-50 min, the volume percentage of the mobile phase A is increased from 28% to 43%,

[0113] 50 min-53 min, the volume percentage of the mobile phase A is maintained at 43%,

[0114] 53 min-58 min, the volume percentage of the mobile phase A is increased from 43% to 65%.

[0115] In one of the embodiments, the detection wavelength of the high performance liquid chromatography analysis is 280 nm-290 nm, and further can be 286 nm.

[0116] In one of the embodiments, the column temperature of the high performance liquid chromatography analysis is 35°C-45°C, and further can be 40°C.

[0117] In one of the embodiments, the flow rate of the high performance liquid chromatography analysis is 0.25 mL / min-0.35 mL / min, and further can be 0.3 mL / min.

[0118] In one of the embodiments, the injection amount of the high performance liquid chromatography analysis is 1 μL-3 μL, and further can be 2 μL.

[0119] In one of the embodiments, the chromatographic column of the high performance liquid chromatography analysis uses octadecylsilane-bonded silica gel as the filler. Further, the inner diameter of the chromatographic column is 2 mm-2.2 mm, the column length is 140 mm-160 mm, and the particle size is 1.7 μm-1.9 μm.

[0120] In one of the embodiments, the theoretical plate number of the high performance liquid chromatography analysis calculated according to the ferulic acid peak should not be less than 5000.

[0121] In still another aspect of the present application, a method for identifying Amaranthaceae Chinese medicinal material or preparation thereof is provided. The method comprises the following steps: subjecting a test sample to high performance liquid chromatography to obtain a characteristic spectrum of the test sample; and comparing the characteristic spectrum of the test sample with a characteristic spectrum of a standard sample of Amaranthaceae Chinese medicinal material or preparation thereof, so as to identify the type and quality of the test sample.

[0122] In one embodiment, the method for identifying Amaranthaceae Chinese medicinal material or preparation thereof comprises the following steps:

[0123] S010: taking Amaranthaceae Chinese medicinal material or preparation thereof, and constructing a characteristic spectrum of the standard sample according to the method for constructing a characteristic spectrum described in any of the above technical solutions,

[0124] S020: taking a test sample, adding an extraction solvent to extract the test sample to prepare a test sample solution, wherein the test sample is selected from Amaranthaceae Chinese medicinal material or preparation thereof,

[0125] S030: subjecting the test sample solution to high performance liquid chromatography to obtain a characteristic spectrum of the test sample,

[0126] S040: comparing the characteristic spectrum of the standard sample with the characteristic spectrum of the test sample,

[0127] wherein the conditions for high performance liquid chromatography comprise:

[0128] gradient elution is performed using methanol as mobile phase A and 0.08-0.12% phosphoric acid aqueous solution as mobile phase B,

[0129] the gradient elution program comprises:

[0130] 0-7 min, the volume percentage of mobile phase A is maintained at 20%,

[0131] 7 min-20 min, the volume percentage of mobile phase A is increased from 20% to 24%,

[0132] 20 min-30 min, the volume percentage of mobile phase A is increased from 24% to 28%,

[0133] 30 min-50 min, the volume percentage of mobile phase A is increased from 28% to 43%,

[0134] 50 min-53 min, the volume percentage of mobile phase A is maintained at 43%,

[0135] 53 min-58 min, the volume percentage of mobile phase A is increased from 43% to 65%.

[0136] It can be understood that the high performance liquid chromatography analysis process in the identification method of the present application is similar to the high performance liquid chromatography analysis process of the characteristic map construction method described in any of the above technical solutions. The preparation of the test solution is the same as the preparation of the test solution described in any of the above technical solutions, which will not be repeated here.

[0137] It can be understood that the characteristic map of the test substance and the characteristic map of the standard substance obtained by the identification method of the present application can be used for direct comparison, or can be analyzed according to the needs. For example, a single test substance is detected and compared with the characteristic maps of different standard substances respectively to obtain the attribution, or the authenticity, or the quality detection result of the single test substance; or a plurality of batches of test substances are detected, and the characteristic maps of the plurality of batches of test substances are compared with each other, and the common characteristics between the plurality of batches of test substances are extracted after data analysis or without data analysis.

[0138] The following are some specific embodiments.

[0139] The following are the instruments, reagents and reagents used in the specific embodiments:

[0140] Instruments: Waters ultra-high performance liquid chromatograph (Waters H-Class, Waters Technology (China) Co., Ltd.), Agilent high performance liquid chromatograph (1290, Agilent), Thermo ultra-high performance liquid chromatograph (Themo Vanquish, Thermo), Waters HSS T3 chromatographic column (2.1 mm x 150 mm, 1.8 μm), Agilent ZORBAX SB-C18 (2.1 mm x 150 mm, 1.8 μm); one-hundredth balance (ME204E, Mettler-Toledo), millionth balance (XP26, Mettler-Toledo), constant temperature water bath (HWS28 type, Shanghai Yiheng Scientific Instrument Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck).

[0141] Reagents: ethanol (Xilong Scientific Co., Ltd.), methanol (Xilong Scientific Co., Ltd.), hydrochloric acid (Xilong Scientific Co., Ltd.) analytical pure; liquid phase phosphoric acid (Tianjin Kemio Chemical Reagent Co., Ltd.), acetonitrile (Merck), methanol (Merck) are HPLC grade, and water is ultrapure water (laboratory self-made).

[0142] Reagents: Ferulic acid (Batch No.: 110773-201915; content: 99.4%; source: China Institute for Food and Drug Control), p-hydroxy cinnamic acid (Batch No.: 21101401; content: 99.94%; source: Chengdu Gelipu Biotechnology Co., Ltd.), tryptophan (Batch No.: 140686-201904; content: 99.9%; source: China Institute for Food and Drug Control).

[0143] The Chinese Pharmacopoeia 2020 Edition Part I stipulates that cockscomb is the dried inflorescences of Celosia cristata L. of Amaranthaceae. The medicinal materials used in this study were identified by the Institute of Chinese Materia Medica, Chinese Academy of Traditional Chinese Medicine, and the specific information is shown in Table 1. The sample information of cardamom flower is shown in Table 2.

[0144] Table 1 Information Table of Cockscomb Medicinal Materials

[0145]

[0146]

[0147] Table 2 Information Table of Cardamom Flower Medicinal Materials

[0148]

[0149] Example 1

[0150] In this example, the chromatographic conditions of the characteristic spectrum construction method were established, considering the absorption wavelength and elution gradient comprehensively.

[0151] 1.1 Determination of Absorption Wavelength

[0152] Take the cockscomb standard decoction, grind it finely, take about 0.5 g, accurately weigh it, put it in a conical flask with a plug, accurately add 25 ml of 70% methanol, weigh it, ultrasonic treat (power 300 W, frequency 40 kHz) for 30 minutes, weigh it again, make up the weight loss with 70% methanol, shake well, centrifuge, take 20 ml, evaporate to dryness, dilute to 10 ml with 70% methanol, shake well, filter, and take the filtrate, which is obtained.

[0153] Chromatographic conditions: Waters HSS T3 chromatographic column (2.1 mm x 150 mm, 1.8 μm) chromatographic column; methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to Table 3; flow rate is 0.30 ml per minute; column temperature is 40℃.

[0154] Table 3 Gradient Elution Table

[0155]

[0156] The detection results are as follows Figures 1-2 , according toFigures 1-2 It can be found that when 285nm is selected as the detection wavelength, there is more chromatographic peak information, so 285m is selected as the detection wavelength of the characteristic spectrum of Celosia cristata.

[0157] 1.2 Establishment of elution conditions

[0158] A Waters HSS T3 chromatographic column (2.1 mm×150 mm, 1.8 μm) was used; methanol was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the requirements in Table 4.

[0159] Table 4 Gradient elution table

[0160]

[0161]

[0162] Test results such as Figure 3 ,according to Figure 3 Different elution gradients significantly affected the separation of the chromatographic peaks in the characteristic spectrum of Celosia cristata. Using Condition 8, the peaks were well resolved and more evenly distributed. Therefore, the chromatographic conditions were determined as follows: a Waters HSS T3 column (2.1 mm × 150 mm, 1.8 μm); methanol as mobile phase A and 0.1% phosphoric acid as mobile phase B, with gradient elution according to Condition 8; a flow rate of 0.30 ml / min; a column temperature of 40°C; a detection wavelength of 285 nm; and an injection volume of 2 μl.

[0163] Example 2

[0164] In this example, the preparation method of the test solution was studied. Through single factor analysis, the effects of extraction solvent, extraction method and extraction time on the characteristic spectrum of Celosia cristata were investigated to determine the optimal sample pretreatment method.

[0165] 2.1 Investigation of extraction solvent

[0166] The effects of different extraction solvents on the characteristic spectrum of Celosia cristata medicinal materials were investigated respectively. Methanol, 70% methanol, 50% methanol, 30% methanol, ethanol, 70% ethanol, 50% ethanol, and 30% ethanol were used as extraction solvents. The peak shape and separation of 9 characteristic peaks consistent with the retention time of the characteristic peaks of the standard decoction were observed, and the "total peak area / sample weight" of the 9 characteristic peaks was calculated to compare the effects of different extraction solvents on the characteristic spectrum of Celosia cristata medicinal materials, and the optimal extraction solvent was selected.

[0167] Take the powder of Flos Genkwa (pass three screen, batch number: JGH06) about 2.0g, accurately weighed, parallel 8 groups, 2 copies in each group, placed in the conical flask with plug, respectively accurately added methanol, 70% methanol, 50% methanol, 30% methanol, ethanol, 70% ethanol, 50% ethanol, 30% ethanol 50ml, weighed, ultrasonic treatment (power 300W, frequency 40kHz) 30 minutes, cool, reweigh, respectively with the corresponding solvent to make up the weight loss, shake, centrifuge, take 25ml, dry, respectively with the corresponding solvent to 10ml, filter, take the filtrate sample. The results of Flos Genkwa characteristic map of different extraction solvents are shown in Table 5, Figure 4 .

[0168] Table 5 The results of Flos Genkwa characteristic map of different extraction solvents

[0169]

[0170] The results show that the extraction effect of 8 kinds of extraction solvents is compared by "total characteristic peak area / sample weight", and the results show that the extraction effect of 70% methanol is better, and the peak shape is better, so 70% methanol is selected as the extraction solvent.

[0171] 2.2 Extraction method investigation

[0172] This experiment investigates the influence of different extraction methods on the characteristic map of Flos Genkwa, and selects ultrasonic treatment and heating reflux as the extraction method. The total peak area of 9 temporarily determined chromatographic peaks / sample weight and the chromatogram comparison of characteristic map results of different extraction methods are compared.

[0173] Take the powder of Flos Genkwa (pass three screen, batch number: JGH06) about 2.0g, accurately weighed, 2 groups in total, 2 copies in each group, placed in the conical flask with plug, accurately added 70% ethanol 50ml respectively, weighed, heated reflux for 30 minutes, ultrasonic treatment (power 300W, frequency 40kHz) 30 minutes, cool, reweigh, make up the weight loss with 70% methanol, shake, centrifuge, take 25ml, dry, with 70% methanol to 10ml, shake, filter, take the filtrate sample. The results of Flos Genkwa characteristic map of different extraction methods are shown in Table 6, Figure 5 .

[0174] Table 6 The results of Flos Genkwa characteristic map of different extraction methods

[0175]

[0176] By comparing ultrasonic treatment and heating reflux extraction, it is found that different extraction methods have little effect on the characteristic map of Flos Genkwa. Considering the simplicity of operation, ultrasonic treatment is selected as the extraction method.

[0177] 2.3 Extraction time investigation

[0178] The experiment investigated the influence of different extraction times on the characteristic chromatogram of the flower of Crocus sativus L. Four different extraction times, 15 minutes, 30 minutes, 45 minutes and 60 minutes, were selected. The results of the characteristic chromatogram of the flower of Crocus sativus L. at different extraction times were compared by the total peak area / sample weight of the temporarily determined 9 chromatographic peaks and the chromatograms.

[0179] About 2.0 g of the powder of the flower of Crocus sativus L. (pass through No. 3 sieve, batch No. JGH06) was precisely weighed and placed in a conical flask with a stopper. 50 ml of 70% methanol was precisely added, the weight was determined, and the sample was ultrasonically treated (power 300 W, frequency 40 kHz) for 15 minutes, 30 minutes, 45 minutes and 60 minutes, respectively. The sample was cooled, the weight was determined again, the lost weight was made up with 70% methanol, and the sample was shaken, centrifuged, 25 ml was taken, evaporated to dryness, dissolved in 10 ml of 70% methanol, shaken, filtered, and the filtrate was taken for determination. The results of the investigation of the characteristic chromatogram of the flower of Crocus sativus L. at different extraction times are shown in Table 7. Figure 6 .

[0180] Table 7 Investigation results of the characteristic chromatogram of the flower of Crocus sativus L. at different extraction times

[0181]

[0182] By comparing the influence of different extraction times on the characteristic chromatogram of the standard decoction of the flower of Crocus sativus L., it was found that different extraction times had little influence on the characteristic chromatogram of the flower of Crocus sativus L. To ensure complete extraction, the extraction time of 30 minutes was selected.

[0183] 2.4 Determination of the preparation method of the test solution

[0184] According to the above experimental results, the sample pretreatment method of the characteristic chromatogram of the flower of Crocus sativus L. can be determined as follows: about 2.0 g of the powder of the flower of Crocus sativus L. (pass through No. 3 sieve) was precisely weighed, placed in a conical flask with a stopper, 25 ml of 70% methanol was added, and the sample was ultrasonically treated (power 300 W, frequency 40 kHz) for 30 minutes. The sample was cooled, the weight was determined again, the lost weight was made up with 70% methanol, centrifuged, 25 ml of the supernatant was taken, evaporated to dryness, dissolved in 10 ml of 70% methanol in a volumetric flask, shaken, filtered, and the filtrate was taken, which was the test solution.

[0185] About 2.0 g of the powder of the flower of Crocus sativus L. (pass through No. 3 sieve) was precisely weighed, placed in a conical flask with a stopper, 25 ml of 70% methanol was added, and the sample was ultrasonically treated (power 300 W, frequency 40 kHz) for 30 minutes. The sample was cooled, the weight was determined again, the lost weight was made up with 70% methanol, centrifuged, 25 ml of the supernatant was taken, evaporated to dryness, dissolved in 10 ml of 70% methanol in a volumetric flask, shaken, filtered, and the filtrate was taken, which was the test solution.

[0186] Example 3

[0187] This embodiment is a methodology validation of the characteristic spectrum construction method, which is investigated from the aspects of specificity, precision, repeatability, stability, intermediate density, and durability.

[0188] 3.1 Specificity Investigation

[0189] 2 μl of the precision sample solution of JGH06, the reference material solution, and the blank solvent were injected into the liquid chromatograph for determination. The results are shown in Figure 7 .

[0190] The results show that the test sample chromatogram has the same chromatographic peak at the corresponding retention time as the control sample chromatogram, and the negative has no interference, indicating that the method has good specificity.

[0191] 3.2 Precision Investigation

[0192] An appropriate amount of JGH06 was finely ground, about 2.0 g was accurately weighed, the test sample solution was prepared according to the determined test sample solution preparation method, and the sample was analyzed. The same test sample solution was continuously injected 6 times, with ferulic acid peak as the reference peak, the relative retention time and relative peak area of each characteristic peak to S peak were calculated, and the RSD value was calculated. The experimental results are shown in Tables 8 and 9.

[0193] Table 8: Results of characteristic spectrum precision investigation of JGH06 (relative retention time)

[0194]

[0195] Table 9: Results of characteristic spectrum precision investigation of JGH06 (relative peak area)

[0196]

[0197] The results show that the same test sample solution is continuously injected 6 times, with ferulic acid peak as the reference peak S, the relative retention time RSD value of each characteristic peak to S peak is in the range of 0.02% to 0.05%, the relative peak area RSD value is in the range of 0.27% to 1.56%, the RSD value is less than 5%, indicating that the instrument precision is good.

[0198] 3.3 Repeatability Investigation

[0199] An appropriate amount of JGH06 was finely ground, about 2.0 g was accurately weighed, the test sample solution was prepared according to the determined test sample solution preparation method, and the sample was analyzed. The same test sample solution was continuously injected 6 times, with ferulic acid peak as the reference peak, the relative retention time and relative peak area of each characteristic peak to S peak were calculated, and the RSD value was calculated. The experimental results are shown in Tables 10 and 11.

[0200] Table 10. Results of repeatability test of characteristic chromatogram of Flos Genkwa (relative retention time)

[0201]

[0202]

[0203] Table 11. Results of repeatability test of characteristic chromatogram of Flos Genkwa (relative peak area)

[0204]

[0205] The results showed that the relative retention time RSD values of each characteristic peak to S peak were in the range of 0.01% to 0.09% and the relative peak area RSD values were in the range of 1.60% to 4.89% with the reference peak of ferulic acid, and the RSD values were less than 5% after repeated determination of the same sample for 6 times, indicating that the method had good repeatability.

[0206] 3.4 Stability test

[0207] An appropriate amount of Flos Genkwa (JGH06) was finely ground, about 2.0 g was accurately weighed, and the test sample solution was prepared according to the determined test sample solution preparation method. The sample was analyzed at 0, 3, 7, 10, 14, and 24 hours, respectively, with the reference peak of ferulic acid, and the relative retention time and relative peak area of each characteristic peak to S peak were calculated, and the RSD values were calculated. The experimental results are shown in Tables 12 and 13.

[0208] Table 12. Results of stability test of characteristic chromatogram of Flos Genkwa (relative retention time)

[0209]

[0210] Table 13. Results of stability test of characteristic chromatogram of Flos Genkwa (relative peak area)

[0211]

[0212] The experimental results showed that the RSD of relative retention time and relative peak area of each chromatographic peak was less than 5% within 24 hours, and the test sample solution was stable within 24 hours.

[0213] 3.5 Intermediate precision test

[0214] Different analysts operated at different times on different instruments, and an appropriate amount of Flos Genkwa (JGH06) was finely ground, about 2.0 g was accurately weighed, and 6 test sample solutions were prepared according to the determined test sample solution preparation method. The sample was analyzed, with the reference peak of ferulic acid, and the relative retention time and relative peak area of each characteristic peak to S peak were calculated, and the RSD values were calculated. The experimental results are shown in Tables 14 and 15, and the chromatogram of intermediate precision test is shown inFigure 8 .

[0215] Table 14 The results of the intermediate precision investigation of the cockscomb flower medicinal material (relative retention time)

[0216]

[0217] Table 15 The results of the intermediate precision investigation of the cockscomb flower medicinal material (relative peak area)

[0218]

[0219] The results show that, by different analysts at different times on different instruments, the same batch of samples were repeatedly determined for 6 times, with the ferulic acid peak as the reference peak S, the relative retention time RSD value of each characteristic peak was in the range of 0.04% to 0.16%, the relative peak area RSD value was in the range of 1.40% to 2.50%, the relative retention time and the repeatability test 6 data RSD value was in the range of 0.13% to 1.50%, the relative peak area and the repeatability test 6 data RSD value was in the range of 7.29% to 19.28%, the relative peak area RSD value was larger, and the reason was that the sample response value was smaller, therefore, the relative peak area limit of the cockscomb flower medicinal material was not specified.

[0220] 3.6 Durability investigation

[0221] 3.6.1 Investigation of different column temperatures

[0222] The effects of different column temperatures, 38°C, 40°C and 42°C, on the characteristic chromatogram of the cockscomb flower medicinal material were compared.

[0223] An appropriate amount of cockscomb flower medicinal material (JGH06) was finely ground, about 2.0 g was accurately weighed, and a test sample solution was prepared according to the determined test sample solution preparation method. Except that the column temperature was 38°C, 40°C and 42°C respectively, the other chromatographic conditions were the same as specified in the chromatographic condition item. The sample was analyzed, with the ferulic acid peak as the reference peak, the relative retention time and relative peak area of each characteristic peak to the S peak were calculated, and the RSD value was calculated. The experimental results are shown in Tables 16, 17, Figure 9 .

[0224] Table 16 The results of the column temperature investigation of the cockscomb flower medicinal material characteristic chromatogram (relative retention time)

[0225]

[0226] Table 17 The results of the column temperature investigation of the cockscomb flower medicinal material characteristic chromatogram (relative peak area)

[0227]

[0228] The experimental results show that at different column temperatures, the RSD values of the relative retention time of each chromatographic peak are in the range of 0.18% to 2.75%, and the RSD values of the relative peak area are in the range of 2.69% to 4.93%. The RSD values are all less than 5%, which indicates that the analysis method has good durability at different column temperatures. The small change in column temperature can meet the system applicability requirements.

[0229] 3.6.2 Investigation of different flow rates

[0230] The effects of different flow rates, 0.28 ml / min, 0.30 ml / min and 0.32 ml / min, on the characteristic chromatogram of the cockscomb medicinal material were compared.

[0231] An appropriate amount of cockscomb medicinal material (JGH06) was finely ground, about 2.0 g was accurately weighed, and a test solution was prepared according to the determined test solution preparation method. Except that the flow rates were 0.28 ml / min, 0.30 ml / min and 0.32 ml / min, respectively, the other chromatographic conditions were the same as specified in the chromatographic condition item. The sample was analyzed, the ferulic acid peak was taken as the reference peak, the relative retention time and relative peak area of each characteristic peak to the S peak were calculated, and the RSD values were calculated. The experimental results are shown in Tables 18 and 19, Figure 10 .

[0232] Table 18 Investigation results of cockscomb medicinal material characteristic chromatogram (relative retention time)

[0233]

[0234] Table 19 Investigation results of cockscomb medicinal material characteristic chromatogram (relative peak area)

[0235]

[0236] The experimental results show that at different flow rates, the RSD values of the relative retention time of each chromatographic peak are in the range of 0.76% to 4.05%, and the RSD values of the relative peak area are in the range of 0.40% to 4.36%. The RSD values are all less than 5%, which indicates that the analysis method has good durability at different flow rates. The small change in flow rate can meet the system applicability requirements.

[0237] 3.6.3 Investigation of different chromatographic columns

[0238] The effects of three Waters HSS T3 chromatographic columns (2.1 mm x 150 mm, 1.8 μm) on the durability of the cockscomb medicinal material characteristic chromatogram were compared. The column numbers were BH-313, BH-317 and BH-318, respectively.

[0239] Take JGH06, grind, take about 2.0g, accurately weigh, prepare the test solution according to the determined test solution preparation method, except that the chromatographic column is different, other chromatographic conditions are the same as the provisions in the chromatographic condition item, sample analysis, take the ferulic acid peak as the reference peak S, calculate the relative retention time and relative peak area of each characteristic peak to the S peak, and calculate the RSD value, the experimental results are shown in Table 20, Table 21, Figure 11 .

[0240] Table 20 Chromatographic column investigation results of JGH06 characteristic chromatogram (relative retention time)

[0241]

[0242] Table 21 Chromatographic column investigation results of JGH06 characteristic chromatogram (relative peak area)

[0243]

[0244] The experimental results show that using the same brand and different batches of chromatographic column, the relative retention time RSD value of each chromatographic peak is in the range of 0.10% to 1.53%; the relative peak area RSD value is in the range of 7.92% to 12.65%. It shows that the relative retention time of each characteristic peak is good in durability, while the relative peak area is greatly affected by different batches of chromatographic column. Therefore, the relative peak area range is not specified at present.

[0245] 3.6.4 Investigation of different chromatographs

[0246] Compare the effects of Waters, Agilent, Thermo different chromatographs on the durability of JGH06 characteristic chromatogram.

[0247] Take JGH06, grind, take about 2.0g, accurately weigh, prepare the test solution according to the determined test solution preparation method, respectively in Waters, Agilent, Thermo chromatograph sample analysis, take the ferulic acid peak as the reference peak S, calculate the relative retention time and relative peak area of each characteristic peak to the S peak, and calculate the RSD value, the experimental results are shown in Table 23, Table 24, Figure 12 .

[0248] Table 22 Flow rate investigation results of JGH06 characteristic chromatogram (relative retention time)

[0249]

[0250] Table 23 Flow rate investigation results of JGH06 characteristic chromatogram (relative peak area)

[0251]

[0252] The experimental results show that the RSD of relative retention time of each chromatographic peak is in the range of 0.07% to 1.81% and the RSD of relative peak area is in the range of 12.28% to 28.93% by using different chromatographs. It is indicated that the relative retention time of each characteristic peak can meet the system suitability requirement, while the relative peak area is slightly affected by the chromatograph. Therefore, the range of relative peak area is not regulated at present.

[0253] 3.6.5 Durability Summary

[0254] The specificity and integrity of the characteristic chromatogram analysis method of Flos Celosiae Cristatae are investigated. The characteristic peaks are not interfered by solvent peaks. The precision, stability and repeatability are investigated. The RSD of relative retention time of each chromatographic peak is less than 5%. The test solution is stable within 24 hours. The intermediate precision is investigated. The relative retention time of each chromatographic peak is within the range (±10%) by different analysts on different dates and different chromatographs. The durability is investigated. The relative retention time of each chromatographic peak is within the range (±10%) by different chromatographic columns, different flow rates and small range of column temperature variation, which can meet the system suitability requirement.

[0255] Example 4

[0256] In this example, the characteristic chromatograms of Flos Celosiae Cristatae, standard decoction of Flos Celosiae Cristatae, Flos Celosiae Cristatae dispensing granules, Flos Celosiae Cristatae, and standard decoction of Flos Celosiae Cristatae are constructed, which specifically include the following steps:

[0257] Step 1: Preparation of Reference Solution

[0258] A certain amount of ferulic acid reference substance is accurately weighed, methanol is added to prepare a solution containing 5 μg per 1 ml, which is used as the reference solution.

[0259] Step 2: Preparation of Test Solution

[0260] About 2.0 g of Flos Celosiae Cristatae powder (passed through a No. 3 sieve) is placed in a conical flask with a stopper, 70% methanol 50 ml is added, ultrasonic treatment (power 300 W, frequency 40 kHz) is performed for 30 minutes, centrifugation is performed, 25 ml of supernatant is taken, evaporation is performed, the residue is dissolved in 70% methanol to a 10 ml volumetric flask, shaken, filtered, and the filtrate is taken, which is obtained.

[0261] About 2.0 g of Flos Celosiae Cristatae powder (passed through a No. 3 sieve) is placed in a conical flask with a stopper, 70% methanol 50 ml is added, ultrasonic treatment (power 300 W, frequency 40 kHz) is performed for 30 minutes, centrifugation is performed, 25 ml of supernatant is taken, evaporation is performed, the residue is dissolved in 70% methanol to a 10 ml volumetric flask, shaken, filtered, and the filtrate is taken, which is obtained.

[0262] Take the standard decoction of Celosia argentea L. appropriate amount, grind finely, take about 0.5g, put in a conical flask with a plug, add 70% methanol 25ml, ultrasonic treatment (power 300W, frequency 40kHz) for 30 minutes, centrifugal, take supernatant 20ml, evaporate to dryness, add 70% methanol to constant volume to 10ml volumetric flask, shake well, filter, take the filtrate, that is, get.

[0263] Take the standard decoction of Celosia argentea L. appropriate amount, grind finely, take about 0.5g, put in a conical flask with a plug, add 70% methanol 25ml, ultrasonic treatment (power 300W, frequency 40kHz) for 30 minutes, centrifugal, take supernatant 20ml, evaporate to dryness, add 70% methanol to constant volume to 10ml volumetric flask, shake well, filter, take the filtrate, that is, get.

[0264] Take the standard decoction of Celosia argentea L. appropriate amount, grind finely, take about 0.5g, put in a conical flask with a plug, add 70% methanol 25ml, ultrasonic treatment (power 300W, frequency 40kHz) for 30 minutes, centrifugal, take supernatant 20ml, evaporate to dryness, add 70% methanol to constant volume to 10ml volumetric flask, shake well, filter, take the filtrate, that is, get.

[0265] Step three: sample determination

[0266] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as the filler (column length of 150mm, inner diameter of 2.1mm, particle size of 1.8μm); methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the provisions of table 24; flow rate of 0.3ml per minute; column temperature of 40℃; detection wavelength of 286nm. The theoretical plate number should not be less than 5000 according to the peak of ferulic acid.

[0267] Table 24 gradient elution table

[0268]

[0269] Respectively, 2μl of control solution and test solution were precisely sucked and injected into liquid chromatograph for determination;

[0270] Using "Chinese medicine chromatographic fingerprint similarity evaluation software", the chromatogram was generated by average method (or median method), and the characteristic chromatogram of Celosia argentea L. and the characteristic chromatogram of Celosia argentea L. were established, in which 9 common peaks were determined in Celosia argentea L. medicine, Celosia argentea L. standard decoction and Celosia argentea L. formula granules, and 3 chromatographic peaks were identified, peak 1 was tryptophan, peak 2 was p-hydroxy cinnamic acid, and peak 3 was ferulic acid; 5 common peaks were detected in Celosia argentea L. medicine and Celosia argentea L. standard decoction, which were consistent with peaks 1-4 and peak 8 in the chromatogram of Celosia argentea L. The chromatogram is as follows Figures 13-22 .

[0271] According to the analysis results, the common peaks of cockscomb flower include: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9; the peak corresponding to the ferulic acid reference is S peak, the relative retention time of each characteristic peak and S peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified value is: 0.24 (peak 1), 0.77 (peak 2), 1.21 (peak 4), 1.50 (peak 5), 1.55 (peak 6), 1.60 (peak 7), 2.13 (peak 8), 2.53 (peak 9);

[0272] The common peaks of cockscomb flower include: peak 1, peak 2, peak 3, peak 4, peak 8; the peak corresponding to the ferulic acid reference is S peak, the relative retention time of each characteristic peak and S peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified value is: 0.24 (peak 1), 0.77 (peak 2), 1.21 (peak 4), 2.13 (peak 8);

[0273] The characteristic spectrum of cockscomb flower obtained ( Figure 23 ) and the characteristic spectrum of cockscomb flower obtained ( Figure 24 ) are compared, and it is found that the characteristic spectrum of cockscomb flower is obviously different from that of cockscomb flower, and cockscomb flower and cockscomb flower have 5 common peaks, among which cockscomb flower has characteristic peaks 5, 6, 7 and 9, which are not found in the characteristic spectrum of cockscomb flower, so the characteristic peaks can be used to distinguish the two.

[0274] Comparative example 1

[0275] Through literature research, it is found that some scholars have established the HPLC fingerprint of cockscomb flower, and the difference between this case and the literature is that the test solution is prepared by heating in an alcohol solution of hydrochloric acid or extracted with 95% ethanol or water, and the preparation method is as follows:

[0276] Method 1: Take cockscomb flower medicinal material powder (pass through No. 3 sieve) about 0.2g, accurately weigh, put into a conical flask with plug, accurately add 50ml of ethanol-water-hydrochloric acid (50:20:15) mixed solution, weigh, heat reflux for 60min, cool, weigh again, supplement the lost mass with ethanol-water-hydrochloric acid (50:20:15) mixed solution, shake well, filter, take the filtrate, and obtain it.

[0277] Method 2: Take cockscomb flower medicinal material powder (pass through 60 mesh sieve), weigh 5.0g of dried powder, add 95% ethanol 50ml, accurately weigh the mass, ultrasonic extraction for 60min, cool, weigh again, and supplement the lost mass with solvent, shake well, filter, take the filtrate, and obtain it.

[0278] Method 3: 2.5 g of the powder of the Flos Miliacae was precisely weighed, placed in a 50 ml conical flask with a stopper, 20 ml of water was precisely added, the weight was fixed, ultrasonic treatment (power 250 W, frequency 40 kHz) was performed for 40 min, the sample was cooled, the weight was fixed, the lost weight was made up with water, filtered, the filtrate was passed through a 0.45 μm microporous filter, and the test sample solution was obtained.

[0279] Comparing the preparation method of the test sample solution with the preparation method of the test sample solution of the present application, the test sample solution prepared was determined by the chromatographic conditions of the present application. From the comparison of the characteristic chromatograms obtained and the characteristic chromatograms of the standard sample of the present application, it can be seen that there are obvious differences, which indicates that the chemical components are different when the extraction methods are different, and the compound components separated and the response values are also different under the chromatographic conditions. See Figures 25-28 It can be seen that the compound information obtained by the method of the present application is more comprehensive, and the separation degree and response value of the chromatogram are better.

[0280] All the documents mentioned in the present application are incorporated by reference in the present application, as if each document is incorporated by reference individually. Unless and to the extent that the documents incorporated by reference conflict with the purpose and / or technical solution of the present application, the documents incorporated by reference are incorporated by reference in the present application in their entirety and for all purposes. When the present application refers to the documents incorporated by reference, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the documents incorporated by reference are also incorporated by reference. When the present application refers to the documents incorporated by reference, the examples and preferred modes of the relevant technical features incorporated by reference can also be incorporated by reference into the present application, but to the extent that the present application can be implemented. It should be understood that when the content incorporated by reference conflicts with the description in the present application, the present application is used as the reference or is modified according to the description in the present application.

[0281] The technical features of the above-mentioned embodiments and examples can be combined in any suitable manner. In order to make the description simple, all possible combinations of the technical features in the above-mentioned embodiments and examples are not described, however, as long as the combinations of the technical features do not exist contradictory, it should be considered that they are within the scope of the present description.

[0282] The above embodiments only express several implementation ways of the present application, but cannot be understood as limitation to the patent scope. It should be pointed out that, for ordinary skilled in the art, several variations and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. In addition, it should be understood that, after reading the above teaching of the present application, the skilled in the art can make various changes or modifications to the present application, and the equivalent forms also fall within the protection scope of the present application. It should also be understood that, the skilled in the art can obtain the technical solutions on the basis of the technical solutions provided by the present application through logical analysis, reasoning or limited test, which all fall within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for constructing a characteristic spectrum of a Chinese medicinal material of the Amaranthaceae family or its preparation, characterized in that: The Amaranthaceae Chinese medicinal material or its preparation is Cockscomb medicinal material, Cockscomb standard decoction or Cockscomb Chinese medicinal formula granules; The construction method comprises the following steps: Take the standard of ferulic acid, dissolve it in solvent, and prepare the reference solution. Taking a test sample, adding an extraction solvent for extraction to prepare a test sample solution, wherein the extraction solvent is an alcohol-water mixture with an alcohol volume ratio of 60% to 80%, and the alcohol-water mixture is a methanol-water solution or an ethanol-water solution; Take the reference solution and the test solution for liquid chromatography analysis, Wherein, the conditions of the liquid chromatography analysis include: The chromatographic column used was Waters HSS T3, with a length of 150 mm, an inner diameter of 2.1 mm, and a filler particle size of 1.8 μm; Gradient elution was performed using methanol as mobile phase A and a 0.08% to 0.12% volume concentration of phosphoric acid aqueous solution as mobile phase B. The gradient elution procedure included: 0-7min, the volume percentage of the mobile phase A is maintained at 20%, 7min~20min, the volume percentage of the mobile phase A increases from 20% to 24%, 20min~30min, the volume percentage of the mobile phase A increases from 24% to 28%, 30min~50min, the volume percentage of the mobile phase A increases from 28% to 43%, 50min~53min, the volume percentage of the mobile phase A is maintained at 43%, From 53 to 58 minutes, the volume percentage of the mobile phase A increased from 43% to 65%; The detection wavelength is 280nm~290nm; The constructed characteristic spectrum of Amaranthaceae Chinese medicinal materials or their preparations has a total of 9 characteristic peaks, among which peak 1 is tryptophan, peak 2 is p-hydroxycinnamic acid, and peak 3 is ferulic acid.

2. The method for constructing a characteristic map according to claim 1, wherein: The conditions for the liquid chromatography analysis also include at least one of the following: (1) Column temperature is 38°C to 42°C; (2) Flow rate is 0.28 mL / min to 0.32 mL / min, (3) The injection volume is 1 μL to 3 μL.

3. The method for constructing a characteristic map according to claim 1, wherein: The extraction method is ultrasound and / or heating reflux.

4. The method for constructing a characteristic map according to claim 3, wherein: The test solution is prepared by the following steps: The test sample is taken, the extraction solvent is added to perform a first extraction, solid-liquid separation is performed to obtain a first liquid, the first liquid is evaporated to dryness, the extraction solvent is added to perform a second extraction, solid-liquid separation is performed to obtain a second liquid, The solid-liquid separation method is centrifugation and / or filtration.

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