CN115856150B

CN115856150B - Fingerprint construction method and identification method of traditional Chinese medicine and traditional Chinese medicine preparation

Info

Publication number: CN115856150B
Application number: CN202211680710.4A
Authority: CN
Inventors: 张志鹏; 刘燎原; 覃柳莹; 黄瑶; 梁丽金; 钟志奎; 邓怡芳; 周颖欣; 王文丽
Original assignee: Guangdong Yifang Pharmaceutical Co Ltd
Current assignee: Guangdong Yifang Pharmaceutical Co Ltd
Priority date: 2022-12-27
Filing date: 2022-12-27
Publication date: 2024-04-30
Anticipated expiration: 2042-12-27
Also published as: CN115856150A

Abstract

The application relates to the technical field of traditional Chinese medicine detection and analysis chemistry, in particular to a fingerprint construction method and an identification method of traditional Chinese medicine materials and traditional Chinese medicine preparations, and particularly relates to a fingerprint construction method of angelica sinensis or preparations thereof, angelica sinensis charcoal or preparations thereof, an identification method of angelica sinensis and angelica sinensis charcoal, and an identification method of angelica sinensis preparations and angelica sinensis charcoal preparations. The fingerprint construction method comprises the steps of adopting ultra-high performance liquid chromatography to analyze, wherein the chromatographic condition is that acetonitrile is used as a mobile phase A, and formic acid aqueous solution with the volume concentration of 0.08-0.12% is used as a mobile phase B. The fingerprint of the application has characteristics, reproducibility and operability, and can clearly determine the common components and the differential components of the angelica and the angelica charcoal. The identification method of the Chinese medicinal preparation can effectively identify the Chinese medicinal preparation and the Chinese medicinal charcoal preparation, and overcomes the defect that the Chinese medicinal preparation and the Chinese medicinal charcoal preparation cannot be checked and identified from microscopic and character identification features.

Description

Fingerprint construction method and identification method of traditional Chinese medicine and traditional Chinese medicine preparation

Technical Field

The application relates to the technical field of traditional Chinese medicine detection and analysis chemistry, in particular to a fingerprint construction method and an identification method of traditional Chinese medicine materials and traditional Chinese medicine preparations, and particularly relates to a fingerprint construction method of angelica sinensis or preparations thereof, angelica sinensis charcoal or preparations thereof, an identification method of angelica sinensis and angelica sinensis charcoal, and an identification method of angelica sinensis preparations and angelica sinensis charcoal preparations.

Background

Angelica sinensis is derived from dried roots of Angelica sinensis ANGELICA SINENSIS (Oliv.) Diels, an Umbelliferae plant. Sweet and pungent in flavor, warm in nature, enter liver, heart and spleen meridians. Has effects of replenishing blood, promoting blood circulation, regulating menstruation, relieving pain, loosening bowel, and relieving constipation. Mainly contains volatile oil, organic acid, amino acid, saccharide, etc. The hemostatic effect of stir-baked Chinese angelica is enhanced, and is mainly used for treating metrorrhagia, metrostaxis, menorrhagia and the like. The clinical effects and the main chemical components of the two medicines are different to a certain extent, so that the chemical component difference of the Chinese angelica before and after the charcoal is fried is clear, and the medicine has an important effect on researching the medicine property of decoction pieces.

A method for constructing the fingerprint of angelica sinensis is reported, but the method has less common peak calibration and identification number, and cannot realize the spectrum analysis of all components.

The Chinese medicine preparation is prepared with Chinese medicine product as basic material and through extraction, separation and purification to obtain semi-product, and other steps. Compared with decoction pieces, the traditional shape and characteristics of the decoction pieces are lost, and after extraction, the decoction pieces have certain differences with chemical components of medicinal materials. At present, identification methods of angelica sinensis or angelica sinensis charcoal preparations are not reported.

Therefore, it is necessary to establish a scientific method for detecting and identifying angelica or its preparation, angelica charcoal or its preparation.

Disclosure of Invention

In view of this, the application provides a method for constructing fingerprint of Chinese medicinal materials or Chinese medicinal preparations, which can objectively, scientifically and comprehensively reflect the intrinsic chemical characteristic information of Chinese medicinal materials and/or Chinese medicinal preparations as the basis for quality detection. The Chinese medicinal materials comprise angelica and angelica charcoal, and the Chinese medicinal preparation comprises an angelica preparation and an angelica charcoal preparation.

Specifically, the fingerprint construction method comprises the following steps:

extracting the traditional Chinese medicinal materials or the traditional Chinese medicinal preparation with an extraction solvent to obtain a sample solution;

taking the sample solution for ultra-high performance liquid chromatography analysis to obtain the fingerprint of the traditional Chinese medicine or the traditional Chinese medicine preparation;

wherein the performing of the ultra performance liquid chromatography analysis comprises eluting according to mobile phase conditions as follows: acetonitrile is taken as a mobile phase A, formic acid aqueous solution with the volume concentration of 0.08-0.12% is taken as a mobile phase B, and the sum of the volume percentages of the mobile phase A and the mobile phase B is 100%;

The elution procedure includes:

0-12 min, wherein the volume percentage of the mobile phase A is increased from 5% to 23%;

12-13 min, wherein the volume percentage of the mobile phase A is increased from 23% to 43%;

13-18 min, wherein the volume percentage of the mobile phase A is increased from 43% to 44%;

18-20 min, wherein the volume percentage of the mobile phase A is increased from 44% to 46%;

20-22 min, wherein the volume percentage of the mobile phase A is increased from 46% to 47%;

22-23 min, wherein the volume percentage of the mobile phase A is increased from 47% to 58%;

23-29 min, wherein the volume percentage of the mobile phase A is increased from 58% to 59%;

29-30 min, wherein the volume percentage of the mobile phase A is increased from 59% to 80%;

And (3) keeping the volume percentage of the mobile phase A at 80% for 30-34 min.

In some embodiments of the application, in the fingerprint construction method, the conditions of the ultra-high performance liquid chromatography include one or more of the following features:

(1) The chromatographic column is octadecylsilane chemically bonded silica chromatographic column;

(2) The model number of the chromatographic column is Waters ACQUITY UPLC BEH Shield RP;

(3) The column temperature is 20-25 ℃;

(4) The detection wavelength is 260 nm-270 nm;

(5) The flow rate of the mobile phase is 0.28 mL/min-0.32 mL/min;

(6) The sample injection amount is 0.5 mu L-1.5 mu L.

In some embodiments of the application, in the fingerprint construction method, the extraction solvent is methanol.

In some embodiments of the application, in the fingerprint construction method, the extraction method is ultrasound.

In some embodiments of the application, in the fingerprint construction method, the fingerprint of the Chinese medicinal material or the Chinese medicinal preparation is as follows:

the fingerprint of the angelica sinensis comprises 19 common peaks which are respectively a peak 1, a peak 2, a peak 3, a peak 4, a peak 5, a peak 6, a peak 7, a peak 8, a peak 9, a peak 10, a peak 11, a peak 12, a peak 13, a peak 14, a peak 15, a peak 16, a peak 17, a peak 18 and a peak 19;

The fingerprint of the angelica sinensis charcoal comprises 10 common peaks, namely a peak 6, a peak 7, a peak 10, a peak 11, a peak 12, a peak 19, a peak 20, a peak 21, a peak 22 and a peak 23;

The fingerprint of the angelica preparation comprises 8 common peaks, namely a peak 1, a peak 3, a peak 5, a peak 6, a peak 7, a peak 12, a peak 24 and a peak 25;

the fingerprint of the angelica sinensis charcoal preparation comprises 4 common peaks, namely a peak 3, a peak 6, a peak 7 and a peak 20;

wherein, peak 1 is tryptophan, peak 3 is chlorogenic acid, peak 5 is ferulic acid, peak 6 is senkyunolide I, peak 7 is senkyunolide H, peak 12 is ligustilide, and peak 20 is 5-hydroxymethylfurfural;

With peak 6 as a reference, each characteristic peak and its relative retention time are as follows: peak 1: 0.23+ -10%; peak 2: 0.57+ -10%; peak 3: 0.60+ -10%; peak 4: 0.91+ -10%; peak 5: 0.93+ -10%; peak 7: 1.07.+ -. 10%; peak 8: 1.30+ -10%; peak 9: 1.63+ -10%; peak 10: 1.67.+ -. 10%; peak 11: 1.74.+ -. 10%; peak 12: 1.77.+ -. 10%; peak 13: 1.83+ -10%; peak 14: 2.31+ -10%; peak 15: 2.33+ -10%; peak 16: 2.35+/-10%; peak 17: 2.37+ -10%; peak 18: 2.41.+ -. 10%; peak 19:2.43±10%; peak 20:0.16±10%; peak 21:0.21±10%; peak 22:2.82±10%; peak 23: 2.84.+ -. 10%; peak 24: 0.64+ -10%; peak 25: 0.67.+ -. 10%.

The application also relates to a method for identifying the traditional Chinese medicinal materials, which comprises the following steps:

Extracting the traditional Chinese medicinal materials with an extraction solvent to obtain a solution I to be detected;

carrying out high performance liquid chromatography analysis on the solution I to be detected to generate a sample map I to be detected;

the fingerprint of the traditional Chinese medicine established by the fingerprint construction method is used as a reference spectrum, and the similarity between the sample spectrum I to be detected and the fingerprint of the traditional Chinese medicine is calculated, and the quality is qualified when the similarity is more than 0.8;

The elution procedure includes:

In some embodiments of the present application, in the method for identifying a Chinese medicinal material, the conditions of the ultra-high performance liquid chromatography include one or more of the following characteristics:

(3) The column temperature is 20-25 ℃;

(4) The detection wavelength is 260 nm-270 nm;

(5) The flow rate of the mobile phase is 0.28 mL/min-0.32 mL/min;

(6) The sample injection amount is 0.5 mu L-1.5 mu L;

(7) The sample injection concentration is 6 mg/mL-10 mg/mL.

In some embodiments of the application, in the method for identifying Chinese medicinal materials, the extraction solvent is methanol.

In some embodiments of the application, in the method for identifying the Chinese medicinal materials, the method for extracting is ultrasonic.

The application also relates to a method for identifying the traditional Chinese medicine preparation, which comprises the following steps:

extracting the traditional Chinese medicine preparation with an extraction solvent to obtain a solution II to be detected;

carrying out high performance liquid chromatography analysis on the solution II to be detected to generate a sample map II to be detected;

Calculating the similarity between the sample spectrum II to be detected and the fingerprint spectrum of the traditional Chinese medicine preparation by taking the fingerprint spectrum of the traditional Chinese medicine preparation established by the fingerprint spectrum construction method as a control spectrum, wherein the similarity is more than 0.8, and the quality is qualified;

The elution procedure includes:

In some embodiments of the present application, in the method for identifying a Chinese medicinal preparation, the conditions of the ultra performance liquid chromatography include one or more of the following characteristics:

(3) The column temperature is 20-25 ℃;

(4) The detection wavelength is 260 nm-270 nm;

(5) The flow rate of the mobile phase is 0.28 mL/min-0.32 mL/min;

(6) The sample injection amount is 0.5 mu L-1.5 mu L.

In some embodiments of the application, in the method for identifying a Chinese medicinal preparation, the extraction solvent is methanol.

In some embodiments of the application, in the method for identifying the Chinese medicinal preparation, the method for extracting is ultrasound.

According to the characteristic that the components contained in the angelica sinensis and the angelica sinensis charcoal are different, the components of the object to be detected can be comprehensively detected by adopting proper chromatographic conditions, and the method is stable, precise and reliable.

The fingerprint prepared by the construction method has characteristics, reproducibility and operability, can provide more abundant characteristic peak information, thereby reflecting the quality of the sample more comprehensively and providing a quick, sensitive, objective and accurate detection means for the quality of angelica sinensis, angelica sinensis charcoal, angelica sinensis preparations and angelica sinensis charcoal preparations.

The construction method is based on the interaction between specific traditional Chinese medicine raw materials and chemical components in the preparation process, the retention time of each characteristic peak is fixed in a certain range, and the raw materials are changed, for example, the difference is generated when fake medicinal materials are used. Therefore, the quality of the medicinal materials or the preparation thereof can be well identified, and the effect of identifying the authenticity can be achieved.

The fingerprint spectrum of the traditional Chinese medicine prepared by the construction method can be used for determining the common components and the differential components of the angelica and the angelica charcoal, and provides references for quality evaluation, pharmacological and pharmacodynamic research and identification of the two components.

The fingerprint spectrum of the Chinese medicinal preparation prepared by the construction method can effectively identify the Chinese medicinal preparation and the Chinese angelica charcoal preparation, and overcomes the defect that the Chinese medicinal preparation and the Chinese angelica charcoal preparation cannot be inspected and identified from microscopic and character identification features.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing comparison of the extraction results of Angelica sinensis under the condition of different concentrations of methanol in 1.2.1 of the present application;

FIG. 2 is a graph showing comparison of the extraction results of angelica sinensis charcoal under the condition of different concentrations of methanol in the method 1.2.1 of the application;

FIG. 3 is a graph showing the comparison of the extraction results of angelica formula particles under the condition of different concentrations of methanol in 1.2.1 of the application;

FIG. 4 is a graph showing the comparison of the extraction results of angelica charcoal formula particles under the condition of different concentrations of methanol in the application 1.2.1;

FIG. 5 is a graph showing the comparison of the extraction results of Angelica sinensis under different extraction conditions in 1.2.2 of the present application;

FIG. 6 is a graph comparing the results of different mobile phase condition chromatograms of the present application 1.3.1;

FIG. 7 is a graph comparing the results of different flow rate condition chromatograms of the present application 1.3.2;

FIG. 8 is a graph comparing the results of different column temperature conditions in accordance with the present application 1.3.3;

FIG. 9 is a graph showing comparison of chromatographic results of different detection wavelength conditions in the present application 1.3.4;

FIG. 10 is a graph showing the chromatographic peak assignment of the control in example 1;

FIG. 11 is a superimposed chromatogram of 17 batches of Angelica sinensis in example 1;

FIG. 12 is a superimposed chromatogram of 17 batches of angelica sinensis charcoal in example 1;

FIG. 13 is a superimposed chromatogram of 17 batches of angelica formula particles in example 1;

FIG. 14 is a superimposed chromatograph of 10 batches of angelica sinensis charcoal formula granules in example 1;

FIG. 15 is a comparison fingerprint of 17 batches of radix Angelicae sinensis in example 1;

FIG. 16 is a comparison fingerprint of 17 batches of angelica sinensis charcoal in example 1;

FIG. 17 is a control fingerprint of 17 batches of angelica formula particles in example 1;

FIG. 18 is a control fingerprint of 10 batches of angelica sinensis charcoal formulas in example 1;

FIG. 19 is a graph showing the results of a cluster analysis of 34 batches of Angelica sinensis and Angelica sinensis charcoal raw materials in example 2;

FIG. 20 shows the PCA results for 34 batches of angelica and angelica char feedstock in example 2;

FIG. 21 shows the PLS-DA results for 34 batches of Angelica sinensis and Angelica sinensis char starting materials in example 2;

FIG. 22 is a graph showing the results of cluster analysis of 27 batches of angelica formula particles and angelica char formula particles in example 3;

FIG. 23 shows the PCA results for the 27 batches of angelica formula particles, angelica char formula particles in example 3;

FIG. 24 shows the results of 27 batches of angelica formula particles, angelica char formula particles PLS-DA in example 3.

Detailed Description

The application is further illustrated below in conjunction with the embodiments, examples and figures. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Furthermore, it is to be understood that various changes and modifications may be made by one skilled in the art after reading the teachings of the application, and such equivalents are intended to fall within the scope of the appended claims.

Unless defined otherwise, 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 herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

Terminology

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

the term "and/or" as used herein includes the scope of selection of any one of two or more of the items listed in relation to each other and also includes any and all combinations of the items listed in relation to each other, any two or more of the items listed in relation to each other, or all combinations of the items listed in relation to each other. For example, "a and/or B" includes three parallel schemes A, B and a+b.

Herein, "preferred", "better", etc. are merely embodiments or examples that describe better results, and it should be understood that they do not limit the scope of the application.

In the present application, "further", "still further", "particularly" and the like are used for descriptive purposes to indicate differences in content but should not be construed as limiting the scope of the application.

In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present application, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. When a numerical range merely points to integers within the numerical range, both end integers of the numerical range are included, as well as each integer between the two ends, unless expressly stated otherwise. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

The temperature parameter in the present application is not particularly limited, and may be a constant temperature treatment or may vary within a predetermined temperature range. It should be appreciated that the constant temperature process described allows the temperature to fluctuate within the accuracy of the instrument control. Allows for fluctuations within a range such as + -0.5 ℃, + -0.4 ℃, + -0.3 ℃, + -0.2 ℃, + -0.1 ℃.

In the present application, the weight may be a mass unit known in the chemical industry field such as mu g, mg, g, kg.

In a first aspect of the present application, a method for constructing a fingerprint of a Chinese medicinal material or a Chinese medicinal preparation is provided. The fingerprint construction method can comprehensively reflect the quality of angelica sinensis, angelica sinensis charcoal, angelica sinensis preparation and angelica sinensis charcoal preparation by adopting proper chromatographic conditions, has stable method, good precision and reproducibility and provides more objective basis for quality detection and sample identification.

The Chinese medicinal materials comprise angelica and angelica charcoal, preferably angelica or angelica charcoal, and the Chinese medicinal preparation comprises an angelica preparation and an angelica charcoal preparation, preferably an angelica preparation or an angelica charcoal preparation.

The Chinese medicine preparation is prepared with Chinese medicine decoction pieces as material and through extraction, separation and purification to obtain semi-product, and other steps. Traditional Chinese medicine preparations include, but are not limited to, traditional Chinese medicine formula granules. Preferably a traditional Chinese medicine formula granule.

In the application, the angelica sinensis charcoal is prepared according to the standard of the charcoal stir-frying method under the general rule of the processing of the 2020 edition of Chinese pharmacopoeia. By way of example, the preparation method comprises the following steps: heating radix Angelicae sinensis decoction pieces in a hot pot, parching with strong fire to brown surface, brown interior or to a specified degree, spraying a little clear water, extinguishing Mars, taking out, and air drying.

In the application, the angelica formula particles and the angelica charcoal formula particles are prepared by adopting a water extraction method, and specifically comprise the following steps: extracting, concentrating, drying, and granulating. By way of example: the angelica formula particles can be prepared by the following steps: taking a proper amount of angelica decoction pieces, adding a proper amount of water, extracting for 2 times each for 30-60 minutes, filtering, concentrating the filtrate at 50-80 ℃ under reduced pressure to obtain clear paste with the relative density of 1.04-1.08 (80 ℃), adding a proper amount of auxiliary materials, stirring uniformly, spray drying, controlling the air inlet temperature to 170-190 ℃, the air outlet temperature to 85-95 ℃, spraying dry powder, adding a proper amount of auxiliary materials, mixing uniformly, and granulating by a dry method to obtain the Chinese angelica decoction pieces; the angelica sinensis charcoal formula granule can be prepared by the following steps: taking a proper amount of angelica sinensis decoction pieces, adding a proper amount of water, extracting for 2 times, each time for 1 hour, filtering, concentrating the filtrate under reduced pressure at 65-80 ℃ to obtain clear paste with the relative density of 1.08-1.10 (80 ℃), adding a proper amount of auxiliary materials, stirring uniformly, spray drying, controlling the air inlet temperature to 170-190 ℃, the air outlet temperature to 85-95 ℃, spraying dry powder, adding a proper amount of auxiliary materials, mixing uniformly, and granulating by a dry method to obtain the Chinese angelica decoction pieces.

In some embodiments of the present application, the fingerprint construction method includes the steps of:

Further, the eluting procedure comprises:

In some embodiments, the fingerprint construction method comprises the steps of:

Precisely weighing Chinese medicinal materials or Chinese medicinal preparation, and extracting with extraction solvent to obtain sample solution;

precisely weighing standard substances of tryptophan, chlorogenic acid, ferulic acid, senkyunolide I, senkyunolide H, ligustilide and 5-hydroxymethylfurfural, and dissolving with solvent to obtain reference substance solution;

Precisely sucking the sample solution and the reference solution, respectively performing ultra-high performance liquid chromatography to obtain sample fingerprint and reference chromatographic, and obtaining the fingerprint of the Chinese medicinal material or Chinese medicinal preparation according to the sample fingerprint and reference chromatographic.

In some embodiments, in the fingerprint construction method, the conditions of the ultra performance liquid chromatography include one or more of the following features:

(3) The column temperature is 20-25 ℃;

(4) The detection wavelength is 260 nm-270 nm;

(5) The flow rate of the mobile phase is 0.28 mL/min-0.32 mL/min;

(6) The sample injection amount is 0.5 mu L-1.5 mu L.

In some embodiments, the fingerprint construction method, the extraction solvent is methanol.

In some embodiments, the fingerprint construction method, the method of extraction is ultrasound.

In some embodiments of the present application, the fingerprint of the Chinese medicinal material or Chinese medicinal preparation is as follows:

The fingerprint of angelica comprises 19 common peaks, namely, a peak 1, a peak 2, a peak 3, a peak 4, a peak 5, a peak 6, a peak 7, a peak 8, a peak 9, a peak 10, a peak 11, a peak 12, a peak 13, a peak 14, a peak 15, a peak 16, a peak 17, a peak 18 and a peak 19;

Further, in each of the above characteristic peaks: with peak 6 (senkyunolide I) as a reference, each characteristic peak and its relative retention time are as follows: peak 1: 0.23+ -10%; peak 2: 0.57+ -10%; peak 3: 0.60+ -10%; peak 4: 0.91+ -10%; peak 5: 0.93+ -10%; peak 7: 1.07.+ -. 10%; peak 8: 1.30+ -10%; peak 9: 1.63+ -10%; peak 10: 1.67.+ -. 10%; peak 11: 1.74.+ -. 10%; peak 12: 1.77.+ -. 10%; peak 13: 1.83+ -10%; peak 14: 2.31+ -10%; peak 15: 2.33+ -10%; peak 16: 2.35+/-10%; peak 17: 2.37+ -10%; peak 18: 2.41.+ -. 10%; peak 19:2.43±10%; peak 20:0.16±10%; peak 21:0.21±10%; peak 22:2.82±10%; peak 23: 2.84.+ -. 10%; peak 24: 0.64+ -10%; peak 25: 0.67.+ -. 10%.

The application also relates to a method for identifying the traditional Chinese medicinal materials, which is based on the fingerprint spectrum of the traditional Chinese medicinal materials provided by the first aspect of the application, and the fingerprint spectrum of the medicinal materials to be detected is compared with the standard fingerprint spectrum, so that the attribution and the quality of the medicinal materials can be identified, and a reference is provided for the comprehensive quality evaluation and pharmacological efficacy research of the medicinal materials to be detected.

In some embodiments, the method for identifying the traditional Chinese medicinal materials comprises the following steps:

Carrying out high performance liquid chromatography on the solution I to be detected to generate a sample map I to be detected;

The fingerprint of the traditional Chinese medicine established by the fingerprint construction method provided by the first aspect of the application is used as a control spectrum, and the similarity between the sample spectrum I to be detected and the sample spectrum I to be detected is calculated, and the similarity is more than 0.8, so that the quality is qualified;

Wherein performing the ultra performance liquid chromatography analysis comprises eluting according to mobile phase conditions as follows: acetonitrile is taken as a mobile phase A, formic acid aqueous solution with the volume concentration of 0.08-0.12% is taken as a mobile phase B, and the sum of the volume percentages of the mobile phase A and the mobile phase B is 100%;

Further, the procedure of eluting includes:

0-12 min, the volume percentage of the mobile phase A is increased from 5% to 23%;

The volume percentage of the mobile phase A is increased from 23% to 43% after 12-13 min;

13-18 min, the volume percentage of the mobile phase A is increased from 43% to 44%;

The volume percentage of the mobile phase A is increased from 44% to 46% after 18 min-20 min;

22-23 min, the volume percentage of the mobile phase A is increased from 47% to 58%;

23-29 min, the volume percentage of the mobile phase A is increased from 58% to 59%;

In some embodiments of the present application, in the method for identifying a Chinese medicinal material, the conditions for the ultra-high performance liquid chromatography include one or more of the following characteristics:

(3) The column temperature is 20-25 ℃;

(4) The detection wavelength is 260 nm-270 nm;

(5) The flow rate of the mobile phase is 0.28 mL/min-0.32 mL/min;

(6) The sample injection amount is 0.5 mu L-1.5 mu L.

In some embodiments of the application, the extraction solvent is methanol.

In some embodiments of the application, the method of identifying the Chinese medicinal material is ultrasound.

The application also relates to a method for identifying the Chinese medicinal preparation, which is based on the fingerprint of the Chinese medicinal preparation provided by the first aspect of the application, and the fingerprint of the preparation to be detected is compared with a standard fingerprint, so that the Chinese medicinal preparation and the Chinese angelica charcoal preparation can be effectively identified, and the defect that the Chinese medicinal preparation and the Chinese angelica charcoal preparation cannot be checked and identified from microscopic and character identification features is overcome.

In some embodiments of the present application, the method for identifying a Chinese medicinal preparation comprises the steps of:

the fingerprint of the traditional Chinese medicine preparation established by the fingerprint construction method provided by the first aspect of the application is used as a reference spectrum, and the similarity between the sample spectrum II to be detected and the fingerprint of the traditional Chinese medicine preparation is calculated, and the similarity is more than 0.8, namely the quality is qualified;

Further, the procedure of eluting includes:

In some embodiments of the present application, in the method for identifying a Chinese medicinal preparation, the conditions for the ultra performance liquid chromatography include one or more of the following features:

(3) The column temperature is 20-25 ℃;

(4) The detection wavelength is 260 nm-270 nm;

(5) The flow rate of the mobile phase is 0.28 mL/min-0.32 mL/min;

(6) The sample injection amount is 0.5 mu L-1.5 mu L.

In some embodiments of the application, in the method of identifying a chinese medicinal preparation, the extraction solvent is methanol.

In some embodiments of the application, in the method of identifying a Chinese medicinal preparation, the method of extraction is ultrasound.

The following are some specific examples.

The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, auxiliary materials, reagents and the like used in the following examples are all commercial products unless specified. Wherein:

The instrument and model are as follows:

Waters ACQUITY ultra-high performance liquid chromatography System (Waters Co., USA); ME204 type E analytical balance (mertrel-tolidol); KQ-500DE type numerical control ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.);

The materials and reagents are as follows:

The angelica is purchased and provided by the following manufacturers: the detailed sources of the Fingered mountain Long De pharmaceutical industry Co., ltd, west of Gansu Province von Feng sex herbal pieces Co., ltd, harbin Fushun Tang herbal Co., ltd, gansu Zhongping storage service Co., ltd, and Anhui province Yongxiang herbal pieces Co., ltd are shown in Table 1; the angelica sinensis charcoal is self-made and the preparation method is as follows: parching radix Angelicae sinensis decoction pieces with strong fire to brown surface, brown interior or to a specified degree, spraying a little clear water, extinguishing Mars, taking out, and air drying; the angelica and angelica charcoal formula granules are provided by Guangdong party pharmaceutical company, and the detailed batch numbers are shown in Table 2.

TABLE 1 Angelica sinensis Source lot number

TABLE 2 batch number of Angelica sinensis and Angelica sinensis charcoal formula granule

Tryptophan (lot number: 140686-201904, purity: 99.9%, national food and drug verification institute); chlorogenic acid (lot number: 110753-202119, purity: 96.3%, national food and drug verification institute); ferulic acid (lot number: 110773-201915, purity: 99.4%, national food and drug verification institute); senkyunolide I (lot No. 112071-202101, purity 99.2%, national food and drug verification institute); senkyunolide H (lot number: DSTDY018201, purity: 99.6%, lemeitian medicine); ligustilide (lot number: 20092403, purity: 98.77%, gendupofford biotechnology Co., ltd.); 5-hydroxymethylfurfural (lot number: 111626-202215, purity: 99.5%, national institute of food and drug testing);

Methanol (analytically pure, the company of the sciences, the company of the ridge); methanol, acetonitrile (chromatographic purity, merck limited); formic acid (chromatographic purity, division of the dense euler chemical reagent, the division of the Tianjin city); the water was ultrapure water (from the Milli-Q ultrapure water System, merck Co., ltd.).

The following tests involved chromatographic detection, all using the following assays: precisely sucking 1 μl of the solution to be measured (reference solution or sample solution), injecting into ultra-high performance liquid chromatograph, and recording chromatogram.

Example 1 construction of finger print

1.1 Preparation of reference

Precisely weighing appropriate amounts of tryptophan, chlorogenic acid, ferulic acid, senkyunolide I, senkyunolide H, ligustilide and 5-hydroxymethylfurfural reference substances, and adding methanol to prepare a mixed reference substance solution containing 1.5 mug tryptophan, 2 mug chlorogenic acid, 5 mug ferulic acid, 6 mug senkyunolide I, 1.5 mug senkyunolide H, 250 mug ligustilide and 20 mug 5-hydroxymethylfurfural per 1 mL.

1.2 Examination of sample preparation method

1.2.1 Investigation of extraction solvent

Taking about 0.2g of angelica sinensis, angelica sinensis charcoal, angelica sinensis prescription granule and angelica sinensis charcoal prescription granule respectively, precisely weighing, respectively adding 30% of methanol, 70% of methanol and 25mL of methanol, weighing, carrying out ultrasonic treatment for 30min, weighing again, adding methanol to supplement weight loss, filtering, taking the subsequent filtrate, obtaining the sample solution of the angelica sinensis, the angelica sinensis charcoal, the angelica sinensis prescription granule and the angelica sinensis charcoal prescription granule, carrying out sample injection measurement under the same chromatographic condition, and observing the extraction condition of different concentrations of methanol on a sample. As shown in fig. 1 to 4, comparing the extraction conditions of each extraction solvent, when methanol is used as the extraction solvent, the obtained chromatograms of the raw materials and preparations of angelica and angelica charcoal have more chromatographic peaks and larger peak areas, and can more comprehensively reflect the internal components of the raw materials and preparations of angelica and angelica charcoal, and finally, the extraction solvent is determined to be methanol.

1.2.2 Extraction method investigation

About 0.2g of angelica powder is taken, precisely weighed, 25mL of methanol is added, the weighed weight is respectively extracted at 30 minutes of reflux, 30 minutes of ultrasonic treatment, 45 minutes of ultrasonic treatment and 60 minutes of ultrasonic treatment, the cooled product is placed, the weighed weight is then weighed, the methanol is added to supplement the weight loss, and the subsequent filtrate is filtered, thus obtaining the Chinese angelica powder. And (5) carrying out sample injection measurement under the same chromatographic condition, and examining the extraction conditions of different extraction modes. The result shows that when the ultrasonic and reflux extraction is adopted, the number and the peak area of each chromatographic peak are not obviously increased, the ultrasonic extraction can be completed within 30 minutes, and the final extraction mode is determined to be ultrasonic for 30 minutes from the aspects of extraction efficiency and economic energy conservation.

1.2.3 Method for preparing defined test sample

Based on the above-mentioned investigation result, the preparation method is determined as follows: taking about 0.2g of angelica medicinal material and angelica charcoal decoction piece powder (sieving with a third sieve), precisely weighing, placing into a conical bottle with a plug, precisely adding 25mL of methanol, weighing, carrying out ultrasonic treatment for 30min, weighing again, adding methanol to supplement weight loss, filtering, and taking the subsequent filtrate to obtain the angelica and angelica charcoal sample solution.

Grinding 0.2g of angelica and angelica charcoal formula particles, precisely weighing, placing into a conical bottle with a plug, precisely adding 25mL of methanol, weighing, performing ultrasonic treatment for 30min, weighing again, adding methanol to supplement weight loss, filtering, and collecting the subsequent filtrate to obtain the angelica and angelica charcoal formula particle sample solution.

1.3 Establishing chromatographic conditions

1.3.1 Investigation of mobile phase composition

Taking a sample solution of angelica, respectively examining elution conditions when acetonitrile-water, methanol-0.1% formic acid water and acetonitrile-0.1% formic acid water are taken as mobile phases, and referring to fig. 6, the results show that when acetonitrile-0.1% formic acid is taken as the mobile phase, the number of chromatographic peaks is large, the separation condition is good, and finally the composition of the mobile phase is determined to be acetonitrile-0.1% formic acid water.

1.3.2 Flow Rate investigation

Taking the angelica sample solution, respectively examining the separation conditions when the flow rates are 0.2mL/min, 0.25mL/min and 0.3mL/min, and referring to FIG. 7, the results show that the overall separation conditions of chromatographic peaks under the condition of 3 flow rates are similar, the separation conditions of chromatographic peaks in the intervals of 5-9 min and 26-34 min under the condition of 0.3mL/min are better, and the flow rate of 0.3mL/min is fast in peak emergence, the time consuming time is short, and the final preferred flow rate is 0.3mL/min in the same time.

1.3.3 Column temperature investigation

Taking the sample solution of angelica, respectively examining the separation condition when the column temperature is 20 ℃ and 25 ℃ and 30 ℃, and referring to figure 8, the result shows that the chromatographic peak at 10-11 min can be completely separated when the column temperature is 20-25 ℃, the separation effect is poor when the column temperature is raised to 30 ℃, and the final preferred column temperature is 25 ℃.

1.3.4 Detection wavelength investigation

Taking a sample solution of angelica sinensis, carrying out spectral scanning at 210-400 nm by using a DAD detector, detecting more chromatographic peaks at 254-290 nm, comparing chromatograms at different detection wavelengths, and finally determining that the detection wavelength is 270nm according to the result, wherein the chromatographic peaks at 19-22 min have strong absorption at 260-270 nm, the number of the chromatographic peaks is more, the peak area is larger, individual chromatographic peaks disappear at 280-290 nm, and the overall situation is comprehensively considered, and the chromatographic peak number and the average peak area situation are compared.

1.3.5 Determination of chromatographic conditions

Based on the above examination results, the chromatographic conditions were determined as follows: octadecylsilane chemically bonded silica (Waters ACQUITY UPLC BEH Shield RP Column, 2.1X100 mm,1.7 μm) was used as filler; acetonitrile as mobile phase a and 0.1% formic acid aqueous solution as mobile phase B, gradient elution was performed as specified in table 3 below; the flow rate was 0.3mL per minute; the column temperature is 25 ℃; the detection wavelength was 270nm.

TABLE 3 gradient elution table

1.4 Methodology investigation

1.4.1 Precision test

Taking about 0.2g of each of the Chinese angelica, chinese angelica charcoal and formula particle samples, precisely weighing, preparing a sample solution according to the established conditions under the item "1.2.3", continuously sampling for 6 times according to the established chromatographic conditions under the item "1.3.5", and respectively calculating the retention time and the peak area of each common peak. The results show that the relative retention time RSD of each common peak is less than 0.28%, and the relative peak area RSD of each common peak is less than 2.85%, which shows that the precision of the instrument is good.

1.4.2 Repeatability test

Taking about 0.2g of each of the Chinese angelica, chinese angelica charcoal and the formula particle sample, precisely weighing, preparing a sample solution in parallel by 6 parts according to the established conditions under the item "1.2.3", and respectively calculating the retention time and the peak area of each common peak according to the established chromatographic condition sample injection measurement under the item "1.3.5". The results showed that the relative retention time RSD of each common peak was <0.35%, and the relative peak area RSD of each common peak was <3.93%, indicating good reproducibility of the method.

1.4.3 Stability test

Taking about 0.2g of each of the angelica sinensis, the angelica sinensis charcoal and the formula particle samples, precisely weighing, preparing a sample solution according to the condition of 1.2.3, and respectively carrying out sample injection measurement at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours, wherein the measurement method conditions are the same as the chromatographic conditions established under the condition of 1.3.5, and calculating the retention time and the peak area of each common peak. As a result, the relative retention time RSD of each common peak was <1.20%, and the relative peak area RSD of each common peak was <4.14%. Indicating that the test article was stable for 24 hours.

1.5 Establishment of characteristic Property and determination of common Peak

1.5.1 Construction of finger print

Respectively taking 17 batches of angelica medicinal materials, angelica carbon decoction pieces, 17 batches of angelica formula particles and 10 batches of angelica carbon formula particles, preparing a sample solution according to the condition under the item of 1.2.3, carrying out sample injection measurement according to the chromatographic condition established under the item of 1.3.5, and carrying out peak identification by adopting a reference substance map (figure 10), wherein the result shows that the peak 1 is tryptophan, the peak 3 is chlorogenic acid, the peak 5 is ferulic acid, the peak 6 is senkyunolide I, the peak 7 is senkyunolide H, the peak 12 is ligustilide and the peak 20 is 5-hydroxymethylfurfural.

Comparing 17 batches of angelica medicinal material chromatograms, determining 19 common peaks which are respectively 1-19, and the chromatographic data are shown in tables 4-7.

TABLE 4 retention time of common peaks for 17 batches of Angelica sinensis (Peak 1-10)

TABLE 5 common Peak retention time for each of the batches of angelica sinensis (Peak 11-19)

TABLE 6 common Peak areas (Peak 1-10) for each of the 17 batches of Angelica sinensis medicinal materials

TABLE 7 common Peak areas (Peak 11-19) for each of the Chinese angelica batches

Numbering device	Peak 11	Peak 12	Peak 13	Peak 14	Peak 15	Peak 16	Peak 17	Peak 18	Peak 19
										DG 01	6471	289732	33809	2717	3581	4436	15077	4765	21078
DG 02	5975	379179	27988	2862	3769	4847	16963	6038	26245
										DG 03	7865	320618	29529	3784	8790	8182	25734	5690	21951
DG 04	8102	477370	32531	3530	5814	7067	23890	8019	27137
										DG 05	6434	271048	24217	2836	6675	5562	18825	5416	16647
DG 06	12005	334206	47660	4491	9603	9796	26860	4245	25353
										DG 07	8618	228103	51464	3114	4475	5277	16526	3450	21854
DG 08	6706	298565	22990	2760	5582	4165	13266	2603	15169
										DG 09	8095	352228	32462	3286	5304	3941	14116	5267	17209
DG 10	7314	610870	27094	3138	4711	6183	22347	10509	35890
										DG 11	10111	562885	40205	3714	6722	6094	22132	8395	27429
DG 12	7980	365351	39395	4534	7535	7687	21720	6051	26475
										DG 13	6501	236950	27914	4564	5041	4315	13169	3780	16077
DG 14	9728	434752	38713	5298	9072	12041	34199	8030	33550
										DG 15	5540	335198	31915	3388	3022	3755	13447	6846	25159
DG 16	8579	472617	31134	3817	6886	8615	28748	7896	32690
										DG 17	5537	140888	40741	3425	3577	2949	7201	2152	7604

Comparing 17 batches of angelica carbon chromatograms, 10 common peaks, namely peak 6, peak 7, peak 10, peak 11, peak 12, peak 19, peak 20, peak 21, peak 22 and peak 23 are determined, and chromatographic data are shown in tables 8-9.

TABLE 8 retention time of common peaks for 17 batches of Dang Gui charcoal decoction pieces

Numbering device	Peak 6	Peak 7	Peak 10	Peak 11	Peak 12	Peak 19	Peak 20	Peak 21	Peak 22	Peak 23
											DGT 01	11.54	12.29	19.27	20.06	20.43	27.98	1.85	2.40	32.59	32.84
DGT 02	11.55	12.31	19.27	20.07	20.43	27.98	1.86	2.41	32.59	32.84
											DGT 03	11.59	12.34	19.29	20.09	20.47	28.02	1.86	2.41	32.62	32.87
DGT 04	11.55	12.31	19.25	20.05	20.43	27.97	1.86	2.41	32.59	32.84
											DGT 05	11.59	12.34	19.32	20.11	20.49	28.03	1.86	2.41	32.63	32.88
DGT 06	11.58	12.34	19.29	20.11	20.46	28.00	1.87	2.41	32.60	32.85
											DGT 07	11.56	12.31	19.29	20.10	20.45	28.00	1.86	2.41	32.61	32.86
DGT 08	11.58	12.33	19.29	20.08	20.46	28.01	1.86	2.41	32.61	32.85
											DGT 09	11.57	12.33	19.27	20.06	20.44	27.99	1.85	2.40	32.62	32.86
DGT 10	11.54	12.30	19.28	20.07	20.44	27.99	1.85	2.40	32.60	32.85
											DGT 11	11.60	12.35	19.33	20.13	20.51	28.07	1.87	2.41	32.63	32.88
DGT 12	11.55	12.31	19.26	20.06	20.43	27.99	1.87	2.41	32.61	32.86
											DGT 13	11.57	12.31	19.27	20.07	20.44	28.00	1.86	2.41	32.61	32.85
DGT 14	11.59	12.35	19.33	20.12	20.50	28.04	1.86	2.41	32.63	32.87
											DGT 15	11.57	12.33	19.29	20.09	20.46	28.02	1.86	2.41	32.62	32.87
DGT 16	11.56	12.32	19.31	20.12	20.48	28.02	1.86	2.41	32.61	32.86
											DGT 17	11.57	12.33	19.31	20.10	20.48	28.01	1.86	2.41	32.62	32.86

TABLE 9 peak to peak area common to batches 17 of Dang Gui charcoal decoction pieces

Comparing 17 batches of radix Angelicae sinensis granule chromatograms, 8 common peaks were determined, peak 1, peak 3, peak 5, peak 6, peak 7, peak 12, peak 24, and peak 25 respectively. The chromatographic data are shown in tables 10 to 11.

TABLE 10 peak retention times common to batches 17 of Angelica sinensis formula particles

Numbering device	Peak 1	Peak 3	Peak 5	Peak 6	Peak 7	Peak 12	Peak 24	Peak 25
									DG K1	2.74	6.84	10.81	11.59	12.34	20.43	7.41	7.78
DG K2	2.74	6.84	10.80	11.57	12.33	20.42	7.41	7.77
									DG K3	2.74	6.84	10.81	11.59	12.35	20.45	7.41	7.77
DG K4	2.77	6.88	10.86	11.63	12.38	20.49	7.46	7.82
									DG K5	2.75	6.85	10.83	11.59	12.34	20.45	7.43	7.78
DG K6	2.75	6.86	10.84	11.59	12.34	20.45	7.44	7.79
									DG K7	2.77	6.87	10.82	11.61	12.36	20.45	7.42	7.79
DG K8	2.76	6.86	10.84	11.61	12.36	20.45	7.44	7.80
									DG K9	2.74	6.85	10.83	11.59	12.34	20.44	7.43	7.78
DG K10	2.74	6.83	10.82	11.59	12.35	20.45	7.40	7.77
									DG K11	2.77	6.88	10.85	11.62	12.37	20.47	7.45	7.81
DG K12	2.73	6.83	10.79	11.57	12.33	20.43	7.39	7.76
									DG K13	2.74	6.84	10.82	11.58	12.34	20.44	7.41	7.77
DG K14	2.79	6.89	10.88	11.65	12.40	20.48	7.47	7.83
									DG K15	2.80	6.92	10.91	11.68	12.43	20.51	7.50	7.86
DG K16	2.76	6.86	10.83	11.61	12.36	20.45	7.42	7.79
									DG K17	2.75	6.83	10.80	11.59	12.34	20.43	7.39	7.76

TABLE 11 peak to peak area common to batches 17 of Angelica sinensis formula particles

Comparing the chromatograms of 10 batches of angelica carbon formula particles, determining 4 common peaks which are respectively peak 3, peak 6, peak 7 and peak 20, and the chromatographic data are shown in tables 12-13.

TABLE 12 common Peak retention time for batches 10 of Angelica sinensis charcoal formula particles

Numbering device	Peak 3	Peak 6	Peak 7	Peak 20
					DGT K1	6.99	11.75	12.51	1.92
DGT K2	6.95	11.67	12.43	1.90
					DGT K3	6.94	11.68	12.43	1.90
DGT K4	6.95	11.70	12.46	1.90
					DGT K5	6.94	11.68	12.43	1.89
DGT K6	7.05	11.80	12.39	1.93
					DGT K7	6.95	11.67	12.43	1.89
DGT K8	6.93	11.66	12.41	1.89
					DGT K9	6.96	11.70	12.45	1.90
DGT K10	6.95	11.67	12.43	1.90

TABLE 13 common Peak area for batches 10 of Angelica sinensis charcoal formula particles

Numbering device	Peak 3	Peak 6	Peak 7	Peak 20
					DGT K1	5462	27191	5869	75978
DGT K2	5835	26046	5968	75045
					DGT K3	6302	27043	6130	63207
DGT K4	6073	28460	6063	68216
					DGT K5	6131	28554	6673	64164
DGT K6	4257	25031	1922	68827
					DGT K7	3140	39361	7980	503503
DGT K8	3505	41069	8706	503274
					DGT K9	5638	23209	4195	354451
DGT K10	5592	19666	4005	327123

1.5.2 Similarity evaluation

Introducing 17 batches of Chinese angelica medicinal materials, 17 batches of Chinese angelica charcoal decoction pieces, 17 batches of Chinese angelica prescription granules and 10 batches of Chinese angelica charcoal prescription granule chromatograms obtained in 1.5.1 into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system respectively, and generating respective superposition maps (see fig. 11-14) and control maps (see fig. 15-18) by adopting a median method.

The similarity of the chromatograms of the 17 batches of Chinese angelica medicinal materials and the reference chromatogram is 0.998、0.997、0.998、0.997、0.996、0.993、0.962、0.999、0.999、0.991、0.999、0.995、0.998、0.999、0.998、0.996、0.943; in sequence and is more than 0.9, which indicates that each chemical component between the 17 batches of Chinese angelica medicinal materials is stable;

The similarity of the 17 batches of Chinese angelica charcoal decoction pieces sample chromatograms and the contrast map is that 15 batches of Chinese angelica charcoal decoction pieces in 0.848、0.996、0.989、0.989、0.990、0.996、0.999、0.898、0.990、0.993、0.989、0.997、0.998、0.994、0.991、0.996、0.996;17 batches are respectively more than 0.9, and only 2 batches of Chinese angelica charcoal decoction pieces have the similarity of 0.8-0.9, which indicates that the overall similarity among 17 batches of Chinese angelica charcoal decoction pieces is higher, and certain differences exist in part of batches, and the speculation possibly relates to the processing degree;

The similarity of the sample chromatograms of 17 batches of angelica formula particles and the comparison map is 0.999、0.999、0.999、1.000、0.996、1.000、1.000、0.996、1.000、0.998、0.997、0.998、0.995、0.985、0.996、0.996、0.989; in sequence and is more than 0.9, which indicates that each chemical component among 17 batches of angelica formula particles is stable;

The similarity between 10 batches of angelica carbon formula particles and a control map is 0.976, 0.978, 0.960, 0.963, 0.956, 0.977, 0.998, 0.997 and 0.997 in sequence. Similarity is more than 0.9, which indicates that each chemical component among 10 batches of angelica carbon formula particles is stable.

Example 2: identification application of angelica sinensis and angelica sinensis charcoal fingerprint

2.1 Analysis of fingerprint similarity of Angelica sinensis and Angelica sinensis charcoal

Taking the control fingerprints (respectively marked as DG-R, DGT-R) of the angelica medicinal materials and the angelica charcoal decoction pieces obtained in the example 1 as reference fingerprints, respectively introducing the chromatograms of 17 batches of the angelica medicinal materials and 17 batches of the angelica charcoal decoction pieces in the example 1 into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, performing full spectrum peak matching with the control fingerprints, wherein the similarity range of 17 batches of angelica samples and the angelica control fingerprint ranges from 0.938 to 1.000, and the similarity range of 17 batches of angelica samples and the angelica charcoal control fingerprint ranges from 0.150 to 0.158;

17 batches of angelica sinensis charcoal samples have the similarity range of 0.804-0.999 with the angelica sinensis charcoal reference fingerprint spectrum and the similarity range of 0.080-0.686 with the angelica sinensis reference fingerprint spectrum;

The result shows that the corresponding raw material sources can be roughly judged through comparing the measured fingerprints of the angelica sinensis and the angelica sinensis charcoal and the interval where the similarity value is located.

TABLE 14 evaluation results of similarity of Angelica sinensis and Angelica sinensis charcoal samples

2.2. Analysis of differences of components of angelica sinensis and angelica sinensis charcoal fingerprint

The fingerprint of the angelica and the angelica charcoal are compared and analyzed, and the newly generated peaks 20, 21, 22 and 23 of the processed angelica charcoal can be directly used as important differences of the angelica and the angelica charcoal raw materials; the data normalization test is carried out on the peak areas (the undetected peak areas are calculated as 0) of the fingerprint chromatogram peaks 1-23 of 17 batches of angelica and angelica charcoal respectively, most of the chromatographic peak areas are in non-normal distribution, the non-parametric Mann-Whitney U test is adopted, and the test results are shown in tables 15-16. The results show that the Chinese angelica and Chinese angelica charcoal peak 1-peak 23 have significant difference (P < 0.05), and the raw material sources of Chinese angelica and Chinese angelica charcoal can be distinguished based on the fingerprint spectrum chromatographic peak.

TABLE 15 statistical results of peak area inspection of Angelica sinensis and Angelica sinensis charcoal raw materials (Peak 1-12)

TABLE 16 statistical results of peak area inspection of Angelica sinensis and Angelica sinensis charcoal raw materials (Peak 12-23)

2.3. Chinese angelica and Chinese angelica charcoal fingerprint pattern recognition research

① Fingerprint spectrum cluster analysis of angelica and angelica charcoal raw materials

The cluster analysis is carried out by taking 23 chromatographic peak areas of 34 batches of angelica and angelica charcoal as variables, and the results are obviously divided into two main types, namely 17 batches of angelica are gathered into one type and 17 batches of angelica charcoal are gathered into the other type, so that the large difference of the integral component compositions of the angelica and the angelica charcoal raw materials can be known.

② Fingerprint analysis (PCA) of angelica and angelica charcoal raw material

The peak area data (the undetected peak area is calculated as 0) of 34 batches of angelica sinensis and angelica sinensis charcoal are subjected to SPSS treatment for principal component analysis, 3 principal components are extracted in total according to characteristic value of >1, and the analysis results are shown in Table 17. The cumulative variance contribution rate is 90.975%, and the integral information of 34 batches of angelica sinensis and angelica sinensis charcoal can be reflected. The data is imported into SIMCA to obtain a principal component score map, see FIG. 20, which shows that 34 batches of samples can be divided into two major categories, wherein 17 batches of angelica samples are divided into one category, and 17 batches of angelica charcoal samples are divided into another category, which indicates that the fingerprint of the angelica and the angelica charcoal have larger difference in each component.

TABLE 17 Total variance interpretation Table

③ Partial least squares regression analysis (PLS-DA) of fingerprint spectrum of radix Angelicae sinensis and radix Angelicae sinensis charcoal raw material

Based on the PCA analysis result, processing the peak area data of 34 batches of angelica and angelica sinensis charcoal, introducing the processed peak area data into SIMCA for PLS-DA analysis, carrying out subjective classification on 34 batches of samples according to known categories, and obtaining results, wherein the results show that the angelica sinensis and the angelica sinensis charcoal raw materials are obviously distributed on two sides of a score chart respectively, the fitting parameters R2 (Y) =0.961 in a PLS-DA model are consistent with the PCA result, the model prediction parameters Q2=0.946 are all larger than 0.5, the modeling type prediction capability is higher, the model variable projection (VIP) value is larger than 1 and is used as an index to screen the differential components of the angelica sinensis and the angelica sinensis charcoal raw materials, 10 important characteristic indexes are extracted, the importance levels are respectively 23, 8, 22, 14, 21, 20, 5, 1, 2 and 13, and the differences among the angelica sinensis and the angelica sinensis charcoal raw materials are finally determined to be 23, 8, 22, 14, 21, 20, 1, 2 and 13 in sequence.

Example 3: identification application of angelica formula granule and angelica charcoal formula granule fingerprint

3.1. Fingerprint similarity analysis of angelica formula particles and angelica charcoal formula particles

The chromatograms of the 17 batches of angelica formula particles and the 10 batches of angelica carbon formula particles in example 1 are respectively introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system by taking the control fingerprints (respectively marked as DG-KR and DGT-KR) of the angelica formula particles and the angelica carbon formula particles obtained in example 1 as reference maps, and full spectrum peak matching is carried out with the control fingerprints, and the similarity is shown in Table 18 by virtue of the results.

As can be seen from Table 18, the similarity between 17 batches of angelica formula particle samples and the comparison fingerprint of the angelica formula particles ranges from 0.972 to 0.992, and the similarity between 17 batches of angelica formula particle samples and the comparison fingerprint of the angelica carbon formula particles ranges from 0.083 to 0.112; the similarity between 10 batches of angelica carbon formula particle samples and the comparison fingerprint of the angelica carbon formula particle samples ranges from 0.934 to 0.998, and the similarity between 10 batches of angelica carbon formula particle samples and the comparison fingerprint of the angelica formula particle samples ranges from 0.052 to 0.422; the result shows that the corresponding formula particle sources can be roughly judged through comparing the fingerprint spectrums of the determined angelica and angelica carbon formula particles and the interval where the similarity value is located.

Table 18 results of similarity evaluation of samples of angelica formula particles and angelica char formula particles

3.2. Fingerprint spectrum component difference analysis of angelica formula granule and angelica charcoal formula granule

The fingerprint spectra of the angelica formula particles and the angelica charcoal formula particles are compared and analyzed, the peak 12 and the peak 25 are detected only in the angelica formula particles, and the peak 20 is detected only in the angelica charcoal formula particles, so that the method can be directly used as the important difference between the angelica formula particles and the angelica charcoal formula particles; respectively carrying out data normalization test on the peak areas (not detected peak areas are counted as 0) of fingerprint chromatogram peak 1, peak 3, peak 5, peak 6, peak 7, peak 12, peak 20, peak 24 and peak 25 of 17 batches of angelica formula particles and 10 batches of angelica charcoal formula particles, wherein the normal distribution is in accordance with T test, the non-normal distribution is in accordance with non-parameter Mann-Whitney U test, and the test results are shown in tables 19-20.

The results show that the chromatographic peaks of the angelica formula particle and the angelica carbon formula particle have significant differences (P < 0.05), and the angelica formula particle and the angelica carbon formula particle can be distinguished based on the chromatographic peaks of the fingerprint.

TABLE 19 peak area t-test statistics of Angelica sinensis and Angelica sinensis charcoal formula particles

/>

TABLE 20 non-parametric test of peak area for Dang Gui and Dang Gui charcoal formulas particles

3.3. Fingerprint pattern chemical pattern recognition research of angelica formula particles and angelica charcoal formula particles

① Fingerprint spectrum cluster analysis of angelica formula granule and angelica charcoal formula granule

The cluster analysis is carried out by taking the areas of 9 chromatographic peaks of 27 batches of angelica formula particles and 9 chromatographic peaks of the angelica charcoal formula particles as variables, and the results can be obviously divided into two main types, namely 17 batches of angelica formula particles are gathered into one type, and 10 batches of angelica charcoal formula particles are gathered into the other type, so that the obvious difference of the integral component compositions of the angelica formula particles and the angelica charcoal formula particles can be known.

② Fingerprint analysis of angelica and angelica charcoal formula granule main component

The peak area data (the undetected peak area is calculated as 0) of the 27 batches of angelica formula particles and angelica charcoal formula particles were subjected to SPSS treatment to conduct principal component analysis, 2 principal components were extracted in total with a characteristic value >1, and the analysis results are shown in Table 21. The cumulative variance contribution rate is 89.909%, and the overall information of 27 batches of angelica formula particles and angelica charcoal formula particles can be reflected. The data is imported into SIMCA to obtain a main component score chart, and the result shows that 27 batches of samples can be divided into two main types, see FIG. 23, wherein 17 batches of angelica formula particle samples are divided into one type, 10 batches of angelica carbon formula particle samples are divided into another type (10 batches of angelica carbon formula particles can be subdivided into three types again, wherein K9 and K10 batches can be subdivided into one type, K7 and K8 batches can be subdivided into one type, and the rest batches are divided into another type), which indicates that the fingerprint of the angelica formula particle and the angelica carbon formula particle have larger component difference; certain differences exist among batches of the angelica carbon formula particles, and presumably related to the raw material differences of different batches.

TABLE 21 Total variance interpretation Table

③ Fingerprint spectrum partial least squares regression analysis (PLS-DA) of angelica formula particles and angelica charcoal formula particles

Based on the PCA analysis result, the peak area data of 27 batches of angelica formula particles and angelica charcoal formula particles are processed and then are imported into SIMCA for PLS-DA analysis, and 27 batches of samples are subjectively classified according to known categories, and the classification result is shown in FIG. 24.

The result shows that the angelica and the angelica carbon formula particles are obviously distributed on two sides of a score chart respectively, the fitting parameter R2 (Y) =0.971 in a PLS-DA model, the model prediction parameter Q2=0.952, are both larger than 0.5, the modeling type prediction capability is strong, the model variable projection (VIP) value is larger than 1 and is used as an index to screen the differential components of the angelica and the angelica carbon formula particles, 5 important characteristic indexes are extracted, the importance degree is sequentially peak 1, peak 25, peak 5, peak 12 and peak 24, and the differential chromatographic peaks in the angelica and the angelica carbon formula particles are finally determined to be sequentially peak 1, peak 25, peak 5, peak 12, peak 24 and peak 20 according to the differential analysis result of each component.

The technical features of the above-described embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the above-described embodiments and examples are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be considered to be within the scope described in the present specification.

The above examples merely represent a few embodiments of the present application and are not to be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above teachings, and equivalents thereof are intended to fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims

1. A fingerprint construction method of Chinese medicinal materials or Chinese medicinal formula particles is characterized in that the Chinese medicinal materials are Chinese angelica or Chinese angelica charcoal, and the Chinese medicinal formula particles are Chinese angelica formula particles or Chinese angelica charcoal formula particles;

The fingerprint construction method comprises the following steps:

Extracting the traditional Chinese medicinal materials or the traditional Chinese medicinal formula particles with an extraction solvent to obtain a sample solution; the extraction solvent is methanol;

taking the sample solution for ultra-high performance liquid chromatography analysis to obtain the fingerprint of the traditional Chinese medicine or the traditional Chinese medicine formula particles;

Wherein the performing of the ultra performance liquid chromatography analysis comprises eluting according to mobile phase conditions as follows: acetonitrile is used as a mobile phase A, a formic acid aqueous solution with the volume concentration of 0.08% -0.12% is used as a mobile phase B, and the sum of the volume percentages of the mobile phase A and the mobile phase B is 100%;

The elution procedure includes:

30-34 min, and keeping the volume percentage of the mobile phase A to be 80%;

the conditions for the ultra performance liquid chromatography include the following features:

(1) The column temperature is 20-25 ℃;

(2) The detection wavelength is 260 nm-270 nm;

(3) The column model is Waters ACQUITY UPLC BEH Shield RP.

2. The fingerprint construction method according to claim 1, wherein the conditions of the ultra-high performance liquid chromatography further include one or more of the following features:

(1) The flow rate of the mobile phase is 0.28 mL/min-0.32 mL/min;

(2) The sample injection amount is 0.5 mu L-1.5 mu L.

3. The fingerprint construction method according to claim 1 or 2, wherein the extraction method is ultrasound.

4. The fingerprint construction method according to claim 1 or 2, wherein in the fingerprint of the Chinese medicinal material or the Chinese medicinal formula particle:

the fingerprint of the angelica formula particle comprises 8 common peaks, namely a peak 1, a peak 3, a peak 5, a peak 6, a peak 7, a peak 12, a peak 24 and a peak 25;

The fingerprint of the angelica sinensis charcoal formula particle comprises 4 common peaks, namely a peak 3, a peak 6, a peak 7 and a peak 20;

5. A method for identifying Chinese medicinal materials is characterized in that the Chinese medicinal materials are Chinese angelica or Chinese angelica charcoal;

The identification method comprises the following steps:

Extracting the traditional Chinese medicinal materials with an extraction solvent to obtain a solution I to be detected; the extraction solvent is methanol;

Carrying out ultra-high performance liquid chromatography analysis on the solution I to be detected to generate a sample map I to be detected;

Calculating the similarity between a sample map I to be detected and the fingerprint of the traditional Chinese medicine by taking the fingerprint of the traditional Chinese medicine formulated by the fingerprint construction method as a control map, wherein the similarity is more than 0.8, and the quality is qualified;

The elution procedure includes:

30-34 min, and keeping the volume percentage of the mobile phase A to be 80%;

(1) The column temperature is 20-25 ℃;

(2) The detection wavelength is 260 nm-270 nm;

(3) The column model is Waters ACQUITY UPLC BEH Shield RP.

6. The method according to claim 5, wherein the conditions of the ultra-high performance liquid chromatography further comprise one or more of the following characteristics:

(1) The flow rate of the mobile phase is 0.28 mL/min-0.32 mL/min;

(2) The sample injection amount is 0.5 mu L-1.5 mu L.

7. The method for identifying Chinese medicinal materials according to claim 5 or 6, wherein the extraction method is ultrasonic.

8. The identification method of the traditional Chinese medicine formula particles is characterized in that the traditional Chinese medicine formula particles are angelica formula particles or angelica charcoal formula particles;

the identification method comprises the following steps: extracting the traditional Chinese medicine formula particles with an extraction solvent to obtain a solution II to be detected; the extraction solvent is methanol;

Carrying out ultra-high performance liquid chromatography analysis on the solution II to be detected to generate a sample map II to be detected;

Calculating the similarity between a sample spectrum II to be detected and the fingerprint spectrum of the traditional Chinese medicine formula particles by taking the fingerprint spectrum of the traditional Chinese medicine formula particles formulated by the fingerprint spectrum construction method as a reference spectrum, wherein the similarity is more than 0.8, and the quality is qualified;

The elution procedure includes:

30-34 min, and keeping the volume percentage of the mobile phase A to be 80%;

(1) The column temperature is 20-25 ℃;

(2) The detection wavelength is 260 nm-270 nm;

(3) The column model is Waters ACQUITY UPLC BEH Shield RP.

9. The method of claim 8, wherein the conditions of the ultra-high performance liquid chromatography further comprise one or more of the following characteristics:

(1) The flow rate of the mobile phase is 0.28 mL/min-0.32 mL/min;

(2) The sample injection amount is 0.5 mu L-1.5 mu L.

10. The method of claim 8 or 9, wherein the method of extraction is ultrasound.