CN117129602A

CN117129602A - Identification and identification construction method of same-family medicines in traditional Chinese medicine compound preparation

Info

Publication number: CN117129602A
Application number: CN202311179253.5A
Authority: CN
Inventors: 张志强; 梁勇满; 李纳纳; 韩红梅; 周永康; 吉艳慧
Original assignee: Beijing Tcmages Pharmaceutical Co Ltd
Current assignee: Beijing Tcmages Pharmaceutical Co Ltd
Priority date: 2023-09-13
Filing date: 2023-09-13
Publication date: 2023-11-28

Abstract

The invention provides a method for identifying and constructing a same kind of medicines in a traditional Chinese medicine compound preparation. The identification construction method of the same class of drugs in the traditional Chinese medicine compound preparation comprises the following steps: obtaining a differentiation component: obtaining the types of the medicines in the same family in the traditional Chinese medicine compound preparation, and obtaining common components and differential components based on the types of the medicines; the method for establishing the authentication comprises the following steps: and (3) obtaining a characteristic spectrum method for effectively identifying the same class of medicines in the traditional Chinese medicine compound preparation based on the differential components, or further constructing a thin-layer chromatography method based on the obtained characteristic spectrum method. The invention can scientifically and reasonably identify the medicinal taste of the same class in the Chinese medicinal compound preparation.

Description

Identification and identification construction method of same-family medicines in traditional Chinese medicine compound preparation

Technical Field

The invention relates to the field of traditional Chinese medicine detection, in particular to identification and identification construction methods of a same class of medicines in a traditional Chinese medicine compound preparation.

Background

The compound Chinese medicine preparation is one kind of crystal for the development of Chinese medicine, and has recipe comprising two or more kinds of medicine, dialectical treatment process, proper medicine selection, medicine dosage and compounding principle. However, because the components of the medicine are complex and the mechanism of action of the medicine is difficult to determine, how to scientifically and reasonably identify the medicine taste of the Chinese herbal medicine compound is a difficult problem, and particularly the identification difficulty is increased when the Chinese herbal medicine compound preparation contains the medicine taste of the same class. For example: a Chinese medicinal compound preparation, WENSHEN granule, comprises the following components: chinese medicinal compound granule is prepared from radix Angelicae sinensis (1 part), rhizoma Ligustici Chuanxiong (1 part), radix Paeoniae alba (1 part), cortex Cinnamomi (1 part), cortex moutan (1 part), curcumae rhizoma (1 part), ginseng radix (2 parts), achyranthis radix (2 parts), and Glycyrrhrizae radix (2 parts) by extracting, concentrating, drying, and granulating. Wherein, the Chinese angelica and the Szechuan lovage rhizome, the moutan bark and the white paeony root belong to the same category of medicinal herbs. In addition, in other traditional Chinese medicine compound preparations, for example: bai Zhu and Bai Zhu, ren Shen and Sanchi also belong to the same category of herbs. The existence of the medicinal herbs in the same family greatly increases the difficulty in qualitative and quantitative identification of the Chinese herbal compound preparation.

The qualitative identification method of the traditional Chinese medicine compound preparation comprises the following steps: chemical reaction method, microscopic chemical method, spectrometry, chromatography, fingerprint/characteristic spectrum identification method, etc., and whether the quality control index of the traditional Chinese medicine compound preparation has representativeness and specificity needs to be considered in selecting the quality control index of the traditional Chinese medicine compound preparation, and whether the prior art conditions are proper or not. The traditional Chinese medicine compound preparation has complex components, needs to be researched according to the characteristics of products, in particular to the traditional Chinese medicine compound preparation which is subjected to the processes of extraction, concentration, drying and the like, changes the original properties of medicinal herbs and needs to establish a special analysis method. In the content measurement item of the traditional Chinese medicine compound, the original single component quality control is changed into the quality control of a plurality of components, which is favorable for more comprehensively representing the quality of the medicine, but has great difficulty. Especially for the traditional Chinese medicine compound containing the medicinal herbs of the same family, most of the ingredients among the medicinal herbs of the same family are overlapped, so that the difficulty of identifying the specificity of the qualitative and quantitative characteristics of the medicinal herbs is higher. The prior art does not disclose how the identification of the medicinal taste of the same class contained in the Chinese medicinal compound can be realized.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to overcome the problem that how to identify the medicines containing the same class in the traditional Chinese medicine compound is not disclosed in the prior art; thereby providing a method for identifying and constructing the same class of medicines in the traditional Chinese medicine compound preparation.

In order to solve the problems, the technical scheme provided by the invention is as follows:

a method for identifying and constructing the same class of medicines in a traditional Chinese medicine compound preparation comprises the following steps:

obtaining a differentiation component: obtaining the types of the medicines in the same family in the traditional Chinese medicine compound preparation, and obtaining common components and differential components based on the types of the medicines;

the method for establishing the authentication comprises the following steps: the characteristic spectrum detection method for effectively identifying the same class of medicines in the traditional Chinese medicine compound preparation is obtained based on the differential components, or a thin layer chromatography method is further constructed based on the obtained characteristic spectrum detection method.

The medicines of the same family are angelica and Ligusticum wallichii, or tree peony bark and white peony root, or white atractylodes rhizome and rhizoma atractylodis, or ginseng and pseudo-ginseng.

The method for obtaining the common component and the differential component comprises the following steps: screening out common chemical components and differential components of different medicines in the same family according to the literature of chemical components of the medicines in the same family;

or firstly, establishing a characteristic spectrum detection method of the same class of different medicines of the traditional Chinese medicine compound preparation, detecting the different medicines of the same class according to the same characteristic spectrum detection method, and constructing a comparison characteristic spectrum of the different medicines of the same class; the same characteristic peak and different characteristic peaks are found out by comparing different medicinal herbs in the same family, the same characteristic peak is corresponding to the common component, and the different characteristic peak is corresponding to the differential component.

The Chinese herbal compound preparation is a meridian warming granule.

The identification method of the same kind of medicine in the Chinese medicine compound preparation comprises the steps of adopting a characteristic spectrum detection method to identify the same kind of medicine in the Chinese medicine compound preparation, wherein the Chinese medicine compound preparation is a meridian warming granule, and when the same kind of medicine is wine angelica and ligusticum wallichii, the chromatographic condition of the characteristic spectrum detection method is as follows:

chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m; acetonitrile is taken as a mobile phase A, and a 0.1% phosphoric acid aqueous solution is taken as a mobile phase B; gradient elution was performed as specified in the following table:

in the chromatographic conditions, the column temperature is 33-37 ℃, the flow rate is 0.95-1.05ml per minute, the detection wavelength is 254nm, the theoretical plate number is not lower than 5000 according to the ferulic acid peak, and the chromatographic column is UItimate LP C18.

When the test sample is wine angelica, the chromatogram of the test sample should show 12 characteristic peaks, wherein peak 3, peak 4, peak 5 and peak 12 should correspond to the reference chlorogenic acid, ferulic acid, senkyunolide I and ligustilide peak, the peak corresponding to the ferulic acid reference is S peak, the relative retention time of peak 1, peak 2, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11 and S peak is calculated, the relative retention time is within 10% of the specified value, and the specified value is: 0.41, 0.57, 1.22, 1.37, 1.60, 1.75, 1.98, 2.00;

And/or, when the sample is ligusticum wallichii, the chromatogram of the sample should show 12 characteristic peaks, wherein peak 1, peak 3, peak 5, peak 6, peak 11 and peak 12 should correspond to chlorogenic acid, ferulic acid, senkyunolide I, senkyunolide H, senkyunolide A and ligustilide peak, the peak corresponding to ferulic acid is S peak, and the relative retention time of peak 2, peak 4, peak 7, peak 8, peak 9, peak 10 and S peak is calculated, the relative retention time is within 10% of the specified value, and the specified value is: 0.80, 1.11, 1.38, 1.59, 1.71, 1.76.

When the medicines of the same family are cortex moutan and radix paeoniae alba, the chromatographic conditions of the characteristic spectrum detection method are as follows:

chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 150mm, the inner diameter is 2.1mm, and the particle size is 1.6 mu m; acetonitrile is taken as a mobile phase A, and a 0.1% phosphoric acid aqueous solution is taken as a mobile phase B; gradient elution was performed as specified in the following table:

in the chromatographic conditions, the flow rate is 0.3ml per minute, the detection wavelength is 230nm, and the theoretical plate number is not lower than 8000 according to paeoniflorin peak.

And/or, when the sample is cortex moutan, the sample chromatograph should show 5 characteristic peaks, wherein peak 1, peak 2 and peak 4 should correspond to the peak of gallic acid, paeoniflorin and paeonol, the peak corresponding to paeoniflorin is S peak, the relative retention time of peak 3, peak 5 and S peak is calculated, the relative retention time is within 10% of the specified value, and the specified value is: 2.01, 3.17;

And/or, when the sample is white peony root, the sample chromatograph should present 4 characteristic peaks, wherein peak 1, peak 2 and peak 3 should correspond to the peak of gallic acid, paeoniflorin and paeoniflorin, the peak corresponding to paeoniflorin is S peak, the relative retention time of peak 4 and S peak is calculated, the relative retention time is within 10% of the specified value, and the specified value is: 3.17.

a method for identifying the same kind of medicine in a Chinese medicine compound preparation comprises the steps of identifying the same kind of medicine in the Chinese medicine compound preparation by adopting a thin layer chromatography method, wherein the Chinese medicine compound preparation is a meridian warming granule, and when the same kind of medicine is wine angelica and ligusticum wallichii, the detection process of the thin layer chromatography method is as follows:

respectively obtaining a sample solution, a control medicinal material solution of control medicinal materials of angelica and ligustilide, and a control solution of ligustilide A;

respectively sucking 2-4 μl of the above solutions, and spotting on the same silica gel GF ₂₅₄ Lamina plate or HSGF ₂₅₄ The thin layer plate is prepared by taking n-hexane-dichloromethane-ethyl acetate-formic acid with mass ratio of (8-9) 3 (1-2) 0.2 as developing agent, developing, taking out, air drying, and inspecting under ultraviolet lamp.

The technical scheme of the invention has the following advantages:

1. The identification construction method of the same-class drugs in the traditional Chinese medicine compound preparation provided by the invention can effectively obtain the characteristic spectrum detection method (namely high performance liquid chromatography) or the thin-layer chromatography method for identifying the same-class drugs in the traditional Chinese medicine compound preparation, thereby providing preconditions for scientifically and reasonably identifying the same-class drugs of the traditional Chinese medicine compound.

2. The identification method of the medicines of the same class in the traditional Chinese medicine compound preparation provided by the invention can effectively realize identification of the medicines of the same class in the meridian warming particles, and has accurate and stable detection result and obvious effect.

Drawings

In order to more clearly illustrate the embodiments of the present invention 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 invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a characteristic spectrum of Angelica sinensis Diels in example 1 of the present invention;

FIG. 2 is a characteristic map of Ligusticum wallichii in example 1 of the present invention;

FIG. 3 is a differential component comparison characteristic map of wine angelica sinensis and Ligusticum wallichii in example 1 of the present invention;

FIG. 4 is a thin layer identification chromatogram of the different solutions of example 1 under a 254nm light source;

FIG. 5 is a thin layer identification chromatogram of the different solutions of example 1 under a 365nm light source;

FIG. 6 is a thin layer identification chromatogram of 15 batches of temperature-sensitive particles in example 1 of the present invention;

FIG. 7 is a comparative chromatogram of Angelica sinensis with different chromatographic columns in example 2 of the invention;

FIG. 8 is a comparative chromatogram of Ligusticum wallichii for the different columns of example 2;

FIG. 9 is a thin layer chromatogram of Wenjing decoction at various concentrations and spotting amounts in example 3 of the present invention;

FIG. 10 is a thin layer chromatogram of a control drug at various concentrations and spotting amounts in example 3 of the present invention;

FIG. 11 is a thin layer chromatogram of the control at various concentrations and spotting amounts in example 3 of the present invention;

FIG. 12 is a thin layer chromatogram of the specificity verification in example 3 of the present invention;

FIG. 13 is a thin layer chromatogram of reproducibility verification in example 3 of the present invention;

FIG. 14 is a thin layer chromatogram of durability for different spotting amounts in example 3 of the present invention;

FIG. 15 is a thin layer chromatogram at normal temperature and humidity in example 3 of the present invention;

FIG. 16 is a thin layer chromatogram at low temperature under normal humidity in example 3 of the present invention;

FIG. 17 is a thin layer chromatogram at room temperature and low humidity in example 3 of the present invention;

FIG. 18 is a thin layer chromatogram at normal temperature and high humidity in example 3 of the present invention;

FIG. 19 is a chromatogram under a thin layer of Qingdao marine silica gel GF254 in example 3 of the invention;

FIG. 20 is a chromatogram under a thin layer of chemical silver Long Guijiao HSGF254 in example 3 of the present invention;

FIG. 21 is a chromatogram under a thin layer of Nicotiana tabacum-friendly silica gel HSGF254 in example 3 of the invention;

FIG. 22 is a thin layer chromatogram of the developing agent of example 1 in example 3 of the present invention;

FIG. 23 is a thin layer chromatogram of the developing agent of example 2 in example 3 of the present invention;

FIG. 24 is a comparative characteristic map of moutan bark in example 4 of the present invention;

FIG. 25 is a comparative characteristic spectrum of white peony root in example 4 of the present invention;

FIG. 26 is a graph showing the differential component comparison characteristics of moutan bark and white peony root in example 1 of the present invention.

Detailed Description

The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

Instrument: waters e2695 high performance liquid chromatograph, PDA detector; ML204T electronic balance (d=0.0001 g, meltrehler tolido instruments (Shanghai); msa6.6s-0 CE-DM electronic balance (d=0.001 mg, certolischen instruments (beijing) company limited); JA1002 electronic balance (Shanghai Pu Chun metering instruments limited); KQ-100DE ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); thin layer imaging system: goodLook-1000, shanghai family philosophy Biochemical technology Co., ltd;

Silica gel HSGF ₂₅₄ A plate: the institute of chemical industry in the tobacco stand market; smoke table city Chi Huang Wu silica gel development test plant;

chromatographic column: UItimate LP C18 (4.6X250 mm,5 μm); waters Brige C18 (4.6X105 mm,5 μm); waters Symmetry C18 (4.6X250 mm,5 μm);

reagent: acetonitrile is chromatographic purity; formic acid includes chromatographic purity and analytical purity; the water is distilled water (Chen's); the other reagents were all analytically pure.

Reagent: chlorogenic acid (lot number: 110753-202018, national food and drug verification institute); ferulic acid (lot number: 110773-201915, national food and drug verification institute); ligustilide (lot number: 111737-201910, national food and drug verification institute); senkyunolide I (lot number: PS2169-0025MG, manufactured by Dupusi Biotech Co., ltd.); senkyunolide H (lot number: 250058-202110, shanghai Kai Biotech Co., ltd.); senkyunolide A (lot number: AZ22011451, manufactured by Chengdu Biotechnology Co., ltd.); chinese angelica control medicine (lot number 121173-201805, chinese food and drug verification institute), 15 batches of wine Chinese angelica; ligusticum wallichii control medicinal material (lot number: 120918-201813, chinese food and drug verification institute), 15 batches of Ligusticum wallichii medicinal material; channel warming particles; chinese angelica single negative freeze-dried powder (batch number: JF-WJT-DG 2022041); ligusticum wallichii single negative freeze-dried powder (batch number: JF-WJT-DG 2022046); double negative lyophilized powder of radix Angelicae sinensis and rhizoma Ligustici Chuanxiong (batch number: JF-WJT-DG 2022047); angelica single positive freeze-dried powder (batch number: JF-WJT-DG 2022032); ligusticum wallichii single positive freeze-dried powder (batch number: JF-WJT-DG 2022037).

Example 1

The method for identifying and constructing the same class of medicines in the traditional Chinese medicine compound preparation comprises the steps of preparing a traditional Chinese medicine compound preparation with a classical name formula, wherein the traditional Chinese medicine compound preparation is a meridian warming granule, the meridian warming granule is a traditional Chinese medicine compound preparation with an ancient classical name formula, and is prepared by the national Chinese medicine administration and the national drug administration together with the national drug administration, and the prescription composition and the proportion are as follows: chinese medicinal compound granule is prepared from radix Angelicae sinensis (1 part), rhizoma Ligustici Chuanxiong (1 part), radix Paeoniae alba (1 part), cortex Cinnamomi (1 part), cortex moutan (1 part), curcumae rhizoma (1 part), ginseng radix (2 parts), achyranthis radix (2 parts), and Glycyrrhrizae radix (2 parts) by extracting, concentrating, drying, and granulating. The medicines of the same family in this embodiment are wine angelica and rhizoma ligustici wallichii.

The specific process of the authentication construction method in this embodiment is as follows:

1. screening of differential Components

The chemical compositions of the wine angelica and the ligusticum wallichii are shown in the following table 1 after the summary of the literature.

TABLE 1 summary of chemical ingredients of Angelica sinensis and Ligusticum chuanxiong

As can be seen from the above table, the components identified by screening radix Angelicae sinensis and rhizoma Ligustici Chuanxiong as characteristic patterns and thin layer chromatography mainly concentrate on volatile components (ligustilide is a thin layer control index) and organic acids (ferulic acid is a pharmacopoeia content measurement index).

2. Establishment of characteristic spectrum method of wine angelica sinensis

Chromatography conditions and System suitability test octadecylsilane chemically bonded silica was used as a filler (UItimate LP C18, column length of 250mm, inner diameter of 4.6mm, particle size of 5 μm) for a chromatography column, acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed as specified in Table 2 below; the flow rate was 1.0ml per minute and the detection wavelength was 254nm. The theoretical plate number should be not less than 5000 calculated according to ferulic acid peak.

TABLE 2

Preparation of a control solution: weighing appropriate amount of ferulic acid, precisely weighing, and adding 70% methanol to obtain 50 μg solution containing ferulic acid per 1 ml.

Preparation of test solution: weighing about 0.2g of radix Angelicae sinensis powder (sieved by a third sieve), precisely weighing, placing into a conical flask with a plug, precisely adding 70% methanol 10ml, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHZ) for 60 min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and collecting the filtrate as sample.

Assay: precisely sucking 10 μl of each of the control solution and the sample solution, and injecting into a liquid chromatograph for measurement.

The chromatogram of the sample is shown in figure 1, wherein peak 3, peak 4, peak 5, and peak 12 should correspond to chlorogenic acid, ferulic acid, senkyunolide I, and ligustilide peak, peak corresponding to ferulic acid reference is S peak, and relative retention time of peak 1, peak 2, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, and S peak should be within 10% of the specified value, which is: 0.41, 0.57, 1.22, 1.37, 1.60, 1.75, 1.98, 2.00.

3. Establishment of characteristic spectrum method of ligusticum wallichii

The conditions of the characteristic spectrum method of the wine angelica are the same, the difference is that the preparation of the reference substance solution and the preparation of the test substance solution are different, and the specific steps are as follows:

Preparation of test solution: about 0.2g of Ligusticum wallichii powder (sieved by a third sieve) is weighed, precisely weighed, placed in a conical bottle with a plug, precisely added with 10ml of 70% methanol, weighed, subjected to ultrasonic treatment (power 250W, frequency 40 kHZ) for 60 minutes, cooled, weighed again, and subjected to 70% methanol to supplement the lost weight, uniformly shaken, filtered, and the subsequent filtrate is taken as a sample.

The chromatogram of the sample is shown in figure 2, and 12 characteristic peaks are shown, wherein peak 1, peak 3, peak 5, peak 6, peak 11, and peak 12 correspond to the retention time of chlorogenic acid, ferulic acid, senkyunolide I, senkyunolide H, senkyunolide A, and ligustilide peak, the peak corresponding to ferulic acid is S peak, and the relative retention time of peak 2, peak 4, peak 7, peak 8, peak 9, peak 10, and S peak is calculated, and the relative retention time is within 10% of the specified value, which is: 0.80 (Peak 2), 1.11 (Peak 4), 1.38 (Peak 7), 1.59 (Peak 8), 1.71 (Peak 9), 1.76 (Peak 10).

4. Determination of the differentiation component

The characteristic patterns of different medicinal flavors of the same family of the Chinese angelica and the Szechuan lovage rhizome of the meridian warming granule are analyzed, and the common chemical components in the Chinese angelica and the Szechuan lovage rhizome are determined as follows: chlorogenic acid, ferulic acid, senkyunolide I, ligustilide, etc., there is no senkyunolide A in radix Angelicae sinensis, and senkyunolide A in rhizoma Ligustici Chuanxiong, therefore senkyunolide A is a differential component of radix Angelicae sinensis and rhizoma Ligustici Chuanxiong, as shown in figure 3.

5. Establishment of authentication method

The identification method in the invention can be a characteristic spectrum method or a thin layer chromatography method. In this embodiment, the condition of the feature pattern method of the wine angelica and the hemlock parsley is the same, so the feature pattern method for detecting the feature pattern of the wine angelica and the hemlock parsley can be adopted as the feature pattern method for identifying the same kind of medicines of the traditional Chinese medicine compound preparation. Meanwhile, the thin layer chromatography method can be further obtained by utilizing the characteristic spectrum method.

In this embodiment, the process of further obtaining the thin layer chromatography method by using the feature spectrum method is as follows:

5.1, chinese angelica root, ligusticum wallichii thin layer identification method

Preparation of test solution: taking 2g of the product powder, adding 30ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate, dissolving the residue with 20ml of hot water, filtering, extracting the filtrate with diethyl ether for 2 times by shaking, 10ml each time, combining diethyl ether solutions, volatilizing, and dissolving the residue with 1ml of acetone to obtain a sample solution.

Preparation of control medicinal material solution: weighing radix Angelicae sinensis control medicinal material and rhizoma Ligustici Chuanxiong control medicinal material 0.5g each, and making into control medicinal material solution with sample preparation method.

Preparation of a control solution: taking ligustilide reference substance, adding methanol to prepare reference substance solution containing 1mg per 1 ml; adding methanol into senkyunolide A to obtain reference solution containing 1mg per 1 ml.

Preparation of negative control solution: taking 2.0g of angelica single-negative freeze-dried powder, ligusticum chuanxiong single-negative freeze-dried powder, and angelica and Ligusticum chuanxiong double-negative freeze-dried powder respectively, and preparing angelica single-negative control solution, ligusticum chuanxiong single-negative control solution and angelica Ligusticum chuanxiong double-negative control solution by the same test solution preparation method.

Chromatographic conditions: respectively sucking the control medicinal material solution, the control substance solution, the test sample solution and the negative control solution 10 μl, and respectively spotting on the same silica gel HSGF ₂₅₄ On the thin layer plate, n-hexane-dichloromethane-ethyl acetate-formic acid (8:3:1:0.2) is used as developing agent, and the thin layer plate is developed, taken out, dried, observed under white light source, 254 light source and 365 light source respectively, and the color development of spots is recorded.

(1) White light source: no obvious spots and no discrimination significance are observed under a white light source;

(2) 254nm light source: the detection result is shown in fig. 4;

(3) 365nm light source: the detection result is shown in fig. 5;

in fig. 4 and 5, reference numeral 1 is a angelica control medicinal solution, reference numeral 2 is a ligusticum chuanxiong control medicinal solution, reference numeral 3 is a sample solution, reference numeral 4 is a ligustilide control solution, reference numeral 5 is a senkyunolide a control solution, reference numeral 6 is a euonylide a control solution, reference numeral 7 is a angelica single negative control solution, reference numeral 8 is a ligusticum chuanxiong single negative control solution, and reference numeral 9 is a angelica ligusticum chuanxiong double negative control solution.

The results show that spots with the same color appear on the corresponding positions in the chromatogram of the sample and the chromatograms of the angelica control medicine, the ligustilide control medicine and the senkyunolide A control medicine, and the dual negative control chromatograms of the angelica and the ligustilide A are not interfered; at 365nm, ligustilide exists in the chromatograms of radix Angelicae sinensis and rhizoma Ligustici Chuanxiong, and can be used as common component of radix Angelicae sinensis and rhizoma Ligustici Chuanxiong for identification; under 254nm, senkyunolide A only has clear dark spots in the chromatogram of rhizoma Ligustici Chuanxiong, and can be used for distinguishing wine radix Angelicae sinensis and rhizoma Ligustici Chuanxiong, and proving that senkyunolide A is really a differential component of both; the Rf values of the control spots are moderate; the separation degree of each spot is good, so 254nm can be adopted as the thin layer chromatography identification method of angelica sinensis and ligusticum wallichii in the freeze-dried powder of the Wenzhen decoction.

5.2 methodological research on thin-layer identification method of angelica and ligusticum wallichii

The preparation method of the same sample solution, control medicinal material solution and negative control solution in 5.1 is adopted;

preparation of positive control solution: preparing radix Angelicae sinensis single positive lyophilized powder and rhizoma Ligustici Chuanxiong single positive lyophilized powder 1.0g each, and preparing radix Angelicae sinensis single positive control solution and rhizoma Ligustici Chuanxiong single positive control solution with the sample solution preparation method;

chromatographic conditions: respectively sucking 3 μl of the control medicinal material solution, control solution, test sample solution, negative control solution and positive control solution, and respectively spotting on the same silica gel GF ₂₅₄ Lamina plate or HSGF ₂₅₄ On the thin layer plate, n-hexane-dichloromethane-ethyl acetate-formic acid (8:1:3:0.2) is used as the raw materialDeveloping agent, spreading, taking out, air drying, and inspecting under ultraviolet lamp (254 nm). In the chromatogram of the sample, spots with the same color appear at the positions corresponding to the chromatogram of the control material and the chromatogram of the control material, and the double negative control of the angelica sinensis and the ligusticum wallichii is not interfered.

5.3 sample measurement

15 batches of warm warp particles were tested according to the thin layer method determined by the above study and the results are shown in figure 6. In figure 6, reference numeral 1 denotes ligustilide; reference numeral 2 is a angelica control medicinal material; the reference numerals 3 to 17 are respectively: the warm channel granules of JF-WJT-1, JF-WJT-2, JF-WJT-3, JF-WJT-4, JF-WJT-5, JF-WJT-6, JF-WJT-7, JF-WJT-8, JF-WJT-9, JF-WJT-10, JF-WJT-11, JF-WJT-12, JF-WJT-13, JF-WJT-14 and JF-WJT-15; reference numeral 18 is a rhizoma Ligustici Chuanxiong control medicinal material; reference numeral 19 indicates senkyunolide A.

The results can be effectively verified, and the liquid chromatography method and the thin layer method obtained by the identification construction method in the embodiment can effectively realize identification of the same class of medicines in the traditional Chinese medicine compound preparation.

Example 2

This example was used to perform methodological validation of the liquid chromatography method constructed in example 1, and the specific procedure is as follows:

1. liquid chromatography condition investigation of wine angelica sinensis

1.1 investigation of extraction solvent species

About 0.2g of wine angelica powder (sieving with a third sieve) is respectively weighed, precisely weighed, placed in a conical flask with a plug, respectively and precisely added with 10ml of proper solvents (methanol, 70% methanol, 50% methanol, 30% methanol, absolute ethanol, 70% ethanol, 50% ethanol, 30% ethanol and water), weighed by weight, subjected to ultrasonic treatment (power 250W and frequency 40 kHZ) for 60 minutes, cooled, weighed by weight again, respectively supplemented with the corresponding solvents to reduce the weight, shaken uniformly, filtered, and taken as a test sample. 10. Mu.l of each sample was precisely aspirated, and the mixture was injected into a high performance liquid chromatograph, and the peak areas were measured by the liquid chromatography method of Angelica sinensis in example 1.

As a result, it was found that there was a loss of chromatographic peaks when the extraction solvent was methanol, 30% ethanol, ethanol or water, preferably 50% or more methanol or ethanol was used, the chromatographic peak areas (about 37 min) of 70% methanol, 70% ethanol or 50% ethanol were larger than 50% methanol, the extraction solvent was 70% ethanol, the 50% ethanol solvent peak was larger than 70% methanol, and 70% methanol was selected as the extraction solvent in consideration of the total point.

1.2 investigation of extraction solvent volume

The powder (sieving with a third sieve) of about 0.2g is respectively weighed, precisely weighed, placed in a conical flask with a plug, respectively precisely weighed with 5ml, 10ml and 20ml of 70% methanol, subjected to ultrasonic treatment (power 250W and frequency 40 kHZ) for 60 minutes, cooled, weighed again, and subjected to shaking and filtering to obtain the subsequent filtrate as a test sample, wherein the weight loss is complemented by 70% methanol. 10. Mu.l of each sample was precisely aspirated, and the sample was injected into a high performance liquid chromatograph, and the relative retention time and the relative peak area of the characteristic peaks thus prepared were calculated by measuring the sample by the liquid chromatography method of Angelica sinensis in example 1, and the results were shown in Table 3 below.

TABLE 3 investigation of different extraction solvent volumes

From the table, it can be seen that: when the volumes (5 ml, 10ml and 20 ml) of the extraction solvents are inspected, the phenomenon of lack of peaks is avoided, the relative retention time RSD of each characteristic peak is less than 3%, the relative peak area is less than 10% except for the peak 6, and the volumes of the extraction solvents are selected to be 10ml by comprehensively considering the convenience and economy principle of operation.

1.3 investigation of extraction time

About 0.2g of the product powder (sieving with a third sieve) is respectively weighed, precisely weighed, placed in a conical flask with a plug, respectively and precisely added with 10ml of 70% methanol, weighed, subjected to ultrasonic treatment (power of 250W and frequency of 40 kHZ) for 30 minutes, 60 minutes and 90 minutes, cooled, weighed again, and subjected to shaking and filtering to obtain a subsequent filtrate as a sample, wherein the weight of the subsequent filtrate is reduced by supplementing the reduced weight with 70% methanol. 10 μl of each sample was precisely sucked, injected into a high performance liquid chromatograph, measured by the liquid chromatography method of Angelica sinensis in example 1, and calculated to obtain the relative retention time and relative peak area of the characteristic peak. The results show that: when the extraction time (30 minutes, 60 minutes and 90 minutes) is examined, each characteristic peak has no peak deficiency phenomenon, the relative retention time RSD of each characteristic peak is less than 3 percent, the relative peak area RSD is less than 10 percent, no large difference exists, and the extraction time is selected to be 60 minutes by comprehensively considering the convenience and economy principle of operation.

1.4 investigation of extraction modes

About 0.2g of the product powder (sieving with a third sieve) is respectively weighed, precisely weighed, placed in conical flasks with plugs, respectively and precisely added with 10ml of 70% methanol, weighed, subjected to ultrasonic treatment (power 250W, frequency 40 kHZ) for 60 minutes, subjected to reflux extraction for 60 minutes, cooled, weighed again, and subjected to shaking and filtering to obtain subsequent filtrate as a sample. 10 μl of each sample was precisely sucked, injected into a high performance liquid chromatograph, measured by the liquid chromatography method of Angelica sinensis in example 1, and calculated to obtain the relative retention time and relative peak area of the characteristic peak. The results show that: when the extraction modes (ultrasonic and reflux) are examined, the phenomenon of peak deficiency is avoided among the characteristic peaks, the relative average deviation of the relative retention time of each characteristic peak is less than 3 percent, the relative average deviation of the relative peak areas except for the peak 9 and the peak 11 is less than 10 percent, and when the ultrasonic extraction is carried out for 60 minutes, the relative peak areas of the peak 9 and the peak 11 are larger, so the extraction mode is selected as ultrasonic.

In summary, the preparation method of the sample for determining the characteristic spectrum of the angelica decoction pieces in the embodiment comprises the following steps: taking about 0.2g of the powder (sieving with a third sieve), precisely weighing, placing into a conical flask, precisely adding 10ml of 70% methanol, sealing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 60 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.

1.5 methodological validation

(1) System applicability

The system suitability results were calculated as ferulic acid peak (S), see table 4 below:

table 4 results of System suitability test

According to the results, the peak area, tailing factor and theoretical plate number all meet the analysis requirements.

(2) Precision of

Repeatability: the retention time of each characteristic peak and the RSD of the relative peak area were both less than 3.0%, indicating that the analytical method repeatability was consistent with the regulations.

Intermediate precision: relative retention time RSD of each characteristic peak, and relative retention time calculation RSD of repeatability ₁₂ The precision of the method is considered to be good when the content of the active ingredients is less than 1%.

(3) Stability of

The relative retention time RSD of each characteristic peak was less than 3%. Indicating that each component in the sample solution is stable within 24 hours under the sample injection condition.

(4) Durability of

Different column temperatures: the relative retention time RSD of the characteristic peak is less than 3% when the column temperature (33 ℃, 35 ℃ and 37 ℃) is examined, which shows that the relative retention time of the characteristic peak has better durability to the column temperature.

Different flow rates: the relative retention time RSD of the characteristic peaks is less than 3% when the flow rates (0.95 ml/min, 1.00ml/min and 1.05 ml/min) are examined, which shows that the relative retention time of the characteristic peaks has better durability for different flow rates.

Different chromatographic columns: taking wine angelica powder, preparing the wine angelica powder into a test solution according to a text test solution preparation method, measuring by adopting chromatographic columns of different types according to a text method, and examining the durability of an experimental method on the different chromatographic columns of the same type. The relative retention time of the proposed characteristic peaks was calculated and the results were as follows.

Chromatographic column 1: UItimate LP C18 (4.6X250 mm,5 μm)

Chromatographic column 2: waters Brige C18 (4.6X105 mm,5 μm)

Chromatographic column 3: waters Symmetry C18 (4.6X105 mm,5 μm)

The different chromatographic column contrast chromatograms are shown in fig. 7, and in fig. 7, the chromatographic column 1, the chromatographic column 2 and the chromatographic column 3 are arranged in sequence from top to bottom. As can be seen from fig. 7, the 12 characteristic peaks of different chromatographic columns are greatly different and the individual chromatographic peaks are not separated, which indicates that the characteristic peaks do not have better durability to different chromatographic columns under the method, and the chromatographic columns are recommended to be: UItimate LP C18 (4.6X250 mm,5 μm) column.

2. Inspection of liquid chromatography conditions of Ligusticum chuanxiong

2.1 investigation of extraction solvent species

About 0.2g of Ligusticum wallichii powder (sieving with a third sieve) is weighed, precisely weighed, placed in conical flasks with plugs, 10ml of proper solvents (methanol, 70% methanol, 50% methanol, 30% methanol, absolute ethanol, 70% ethanol, 50% ethanol, 30% ethanol and water) are precisely added, weighed, subjected to ultrasonic treatment (power 250W, frequency 40 kHZ) for 60 minutes, cooled, weighed again, and the reduced weight is complemented with corresponding solvents respectively, shaken uniformly, filtered, and the subsequent filtrate is taken as a sample. 10. Mu.l of each sample was precisely sucked, and the mixture was poured into a high performance liquid chromatograph, and the peak areas were measured by the liquid chromatography method of Ligusticum wallichii in example 1.

As a result, the extraction solvent was found to have a missing chromatographic peak except 70% methanol and 70% ethanol, and the extraction solvent was preferably 70% methanol or 70% ethanol, and the extraction solvent was 70% ethanol and the peak was larger than 70% methanol, and the extraction solvent was preferably 70% methanol in consideration of the total.

2.2 investigation of extraction solvent volume

The powder (sieving with a third sieve) of about 0.2g is respectively weighed, precisely weighed, placed in a conical flask with a plug, respectively precisely weighed with 5ml, 10ml and 20ml of 70% methanol, subjected to ultrasonic treatment (power 250W and frequency 40 kHZ) for 60 minutes, cooled, weighed again, and subjected to shaking and filtering to obtain the subsequent filtrate as a test sample, wherein the weight loss is complemented by 70% methanol. 10. Mu.l of each sample was precisely sucked, injected into a high performance liquid chromatograph, measured by the liquid chromatography method of Ligusticum wallichii in example 1, and the relative retention time and relative peak area of the characteristic peaks were calculated as shown in Table 5.

TABLE 5 investigation of different extraction solvent volumes

From the table, it can be seen that: when the volumes (5 ml, 10ml and 20 ml) of the extraction solvents are examined, the phenomenon of lack of peaks is avoided, the relative retention time RSD of each characteristic peak is less than 3%, the relative peak area is less than 10% except for peak 1, and the volumes of the extraction solvents are selected to be 10ml comprehensively considered.

2.3 investigation of extraction time

About 0.2g of the product powder (sieving with a third sieve) is respectively weighed, precisely weighed, placed in a conical flask with a plug, respectively and precisely added with 10ml of 70% methanol, weighed, subjected to ultrasonic treatment (power of 250W and frequency of 40 kHZ) for 30 minutes, 60 minutes and 90 minutes, cooled, weighed again, and subjected to shaking and filtering to obtain a subsequent filtrate as a sample, wherein the weight of the subsequent filtrate is reduced by supplementing the reduced weight with 70% methanol. Precisely sucking 10 μl of each sample, injecting into high performance liquid chromatograph, measuring by liquid chromatography method of rhizoma Ligustici Chuanxiong in example 1, and calculating relative retention time and relative peak area of the characteristic peak. The results show that: when the extraction time (30 minutes, 60 minutes and 90 minutes) is examined, the phenomenon of lack of peak is avoided in each characteristic peak, the relative retention time RSD of each characteristic peak is less than 3%, the relative peak area RSD is less than 10%, and the extraction time is set to be 60 minutes comprehensively.

2.4 investigation of extraction modes

About 0.2g of the product powder (sieving with a third sieve) is respectively weighed, precisely weighed, placed in conical flasks with plugs, respectively and precisely added with 10ml of 70% methanol, weighed, subjected to ultrasonic treatment (power 250W, frequency 40 kHZ) for 60 minutes, subjected to reflux extraction for 60 minutes, cooled, weighed again, and subjected to shaking and filtering to obtain subsequent filtrate as a sample. Precisely sucking 10 μl of each sample, injecting into high performance liquid chromatograph, measuring by liquid chromatography method of rhizoma Ligustici Chuanxiong in example 1, and calculating relative retention time and relative peak area of the characteristic peak. The results show that: when the extraction modes (ultrasonic and reflux) are examined, the phenomenon of peak deficiency does not occur in each characteristic peak, the relative average deviation of the relative retention time of each characteristic peak is less than 3%, the relative average deviation of the relative peak areas are remarkably different, and the relative peak areas are generally larger when the ultrasonic extraction is carried out for 60 minutes, so the extraction mode is selected as ultrasonic.

In summary, the preparation method of the sample for determining the characteristic spectrum of the ligusticum wallichii medicinal material in the research comprises the following steps: taking about 0.2g of the powder (sieving with a third sieve), precisely weighing, placing into a conical flask, precisely adding 10ml of 70% methanol, sealing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 60 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.

2.5 methodological validation

(1) System applicability

The system suitability results were calculated as ferulic acid peak (S), see table 6 below:

table 6 results of System suitability test

(2) Precision of

(3) Stability of

(4) Durability of

Different chromatographic columns: the method comprises the steps of taking Ligusticum chuanxiong Hort powder (batch number: JF-CX-202005), preparing into test solution according to the text test solution preparation method, measuring by adopting chromatographic columns of different types according to the text method, and examining the durability of the experimental method on different chromatographic columns of the same type. The relative retention time of the proposed characteristic peaks was calculated and the results are shown in figure 8.

Chromatographic column 1: UItimate LP C18 (4.6X250 mm,5 μm)

Chromatographic column 2: waters Brige C18 (4.6X105 mm,5 μm)

Chromatographic column 3: waters Symmetry C18 (4.6X250 mm,5 μm);

the different chromatographic column contrast chromatograms are shown in fig. 8, and the chromatographic column 1, the chromatographic column 2 and the chromatographic column 3 are sequentially arranged from top to bottom in fig. 8. As can be seen from fig. 8, the 12 characteristic peaks of different chromatographic columns have large differences and individual chromatographic peaks are not separated, which indicates that the characteristic peaks do not have good durability for different chromatographic columns in the method, and the chromatographic columns are recommended to be: UItimate LP C18 (4.6X250 mm,5 μm) column.

Example 3

This example was used to methodologically verify the thin layer method constructed in example 1, and the specific procedure is as follows:

1. examination of sample solution concentration and sample application amount

Preparing a reference sample solution of the Wenying decoction according to the method under the identification item, respectively weighing 1.0g and 2.0g of the reference sample solution of the Wenying decoction, and preparing the sample solution according to the preparation method of the sample. According to thin layer chromatography (rule 0502 of 2020 edition of Chinese pharmacopoeia), the above sample solutions (1 μl, 3 μl, 5 μl, 7 μl, 10 μl) are respectively spottedIdentical silica gel HSGF ₂₅₄ The thin layer plate is developed according to the chromatographic condition, developed and inspected. The results are shown in FIG. 9. In FIG. 9, reference numerals 1 to 5 denote 1 to 5. Mu.l of a sample solution (2.0 g); reference numerals 6 to 10 denote 1 to 5. Mu.l of a sample solution (1.0 g).

The results show that the spots in the chromatogram of the test sample can be developed, and the separation effect is good. From the aspect of convenience, 2.0g of the sample preparation method which is the same as the cortex moutan is selected as the sample preparation weighing amount of the sample solution; the sample was clearly spotted in a 3. Mu.l sample amount in a 2.0 g-weighted chromatograph. In summary, the sample preparation was 2.0g and the sample application was 3. Mu.l.

2. Inspection of sample application amount of reference substance solution and reference medicinal material

Preparing radix Angelicae sinensis control solution, rhizoma Ligustici Chuanxiong control solution, senkyunolide A control solution and ligustilide control solution according to the method under the identification item, and respectively sucking the above control solutions (1 μl, 3 μl, 5 μl, 7 μl, 10 μl) onto the same silica gel HSGF according to thin layer chromatography (0502 of the rule of 2020 edition of Chinese pharmacopoeia) ₂₅₄ A thin layer plate; the ligustilide control solution (1. Mu.l, 2. Mu.l, 3. Mu.l, 4. Mu.l, 5. Mu.l) was pipetted and the senkyunolide A control solution (1. Mu.l, 3. Mu.l, 5. Mu.l, 7. Mu.l, 10. Mu.l) was spotted onto the same silica gel HSGF ₂₅₄ The results of the development and inspection of the thin layer plates under chromatographic conditions are shown in FIGS. 10-11 below.

In FIG. 10, reference numerals 1 to 5 refer to 1. Mu.l, 3. Mu.l, 5. Mu.l, 7. Mu.l, and 10. Mu.l of a control Chinese angelica medicinal material solution; the reference numerals 6-10 are 1 μl, 3 μl, 5 μl, 7 μl and 10 μl of rhizoma Ligustici Chuanxiong control medicinal solution. In FIG. 11, reference numerals 1 to 5 are 1 to 5. Mu.l of ligustilide control solution; the reference number is 6-10, and the reference solution of senkyunolide A is 1-5. Mu.l.

The results show that when the sample application amounts of the angelica control medicinal material solution, the Ligusticum wallichii control medicinal material solution, the angelica, the senkyunolide A control substance solution and the ligustilide control substance solution are all 3 mul, the thin layer chromatography spot color development effect and the separation degree are optimal, so that the sample application amounts of the angelica control medicinal material solution, the Ligusticum wallichii control medicinal material solution, the angelica, the senkyunolide A control substance solution and the ligustilide control substance solution are all 3 mul.

3. Methodological verification

3.1 specificity

The angelica control medicinal material solution, the ligusticum chuanxiong hort control medicinal material solution, the angelica sinensis, the senkyunolide A control solution, the ligustilide control solution, the substance standard test sample solution, the angelica single negative control solution, the ligusticum chuanxiong hort single negative control solution, the angelica chuanxiong hort double negative control solution, the angelica single positive control solution, the ligusticum chuanxiong hort single positive control solution and the blank solvent (acetone) are prepared according to the method under the identification item, the sample application amount is 3 mul, and the result is shown in figure 12. In fig. 12, reference numeral 1 is a angelica control medicinal solution, reference numeral 2 is a ligusticum chuanxiong control medicinal solution, reference numeral 3 is a sample solution, reference numeral 4 is a ligustilide control solution, reference numeral 5 is a senkyunolide a control solution, reference numeral 6 is a angelica single negative control solution, reference numeral 7 is a ligusticum chuanxiong single negative control solution, reference numeral 8 is a angelica ligusticum chuanxiong double negative control solution, reference numeral 9 is a angelica single positive control solution, and reference numeral 10 is a ligusticum chuanxiong single positive control solution.

The result shows that spots with the same color appear on the corresponding positions of the chromatogram of the angelica control medicine, the ligustilide control medicine and the senkyunolide control medicine in the chromatogram of the sample to be tested, the separation degree is good, and the wine angelica and ligustilide double negative control solution is free from interference and meets the requirement of specificity.

3.2 repeatability

The control solution, the control medicinal material solution and the test sample solution are prepared by different experimenters according to the method under the identification item, the sample application amount is 3 μl, and the result is shown in fig. 13. The reference number 1 is a angelica control medicinal material solution, the reference number 2 is a ligusticum chuanxiong control medicinal material solution, the reference number 3 is a sample solution, the reference number 4 is a ligustilide control solution, the reference number 5 is a senkyunolide A control solution, the reference number 6 is a angelica single negative control solution, the reference number 7 is a ligusticum chuanxiong single negative control solution, the reference number 8 is a angelica ligusticum chuanxiong double negative control solution, the reference number 9 is a angelica single positive control solution, the reference number 10 is a ligusticum chuanxiong single positive control solution, and the reference number 11 is a blank solvent (methanol).

The result shows that the sample chromatogram has the same color spots as the corresponding positions of the chromatogram of the Chinese angelica control medicine, the ligustilide control medicine and the senkyunolide control medicine, has good separation degree, has no interference with the Chinese angelica and ligustilide double negative control solution, and has good repeatability.

3.3 durability of

3.3.1, verification of different spotting amounts

Comparing the durability of different sample application amounts, preparing radix Angelicae sinensis control medicinal material solution, rhizoma Ligustici Chuanxiong control medicinal material solution, radix Angelicae sinensis, senkyunolide A control solution, ligustilide control solution and sample solution according to selected method, respectively sucking (2 μl, 3 μl, 4 μl) to obtain the same silica gel HSGF ₂₅₄ The results of the development and inspection of the thin layer plates under chromatographic conditions are shown in FIG. 14.

In FIG. 14, reference numerals 1-3 are respectively 2. Mu.l, 3. Mu.l and 4. Mu.l of radix Angelicae sinensis control medicinal material solutions, reference numerals 4-6 are respectively 2. Mu.l, 3. Mu.l and 4. Mu.l of rhizoma Ligustici Chuanxiong control medicinal material solutions, reference numerals 7-9 are respectively 2. Mu.l, 3. Mu.l and 4. Mu.l of sample solutions, reference numerals 10-12 are respectively 2. Mu.l, 3. Mu.l and 4. Mu.l of ligustilide control solutions, and reference numerals 13-15 are respectively 2. Mu.l, 3. Mu.l and 4. Mu.l of ligustilide A control solutions.

The results show that the method has better durability for different sample application amounts.

3.3.2 different ambient temperatures

Investigation of different temperatures: the control solution, control medicinal solution and test solution are prepared according to the selected method, and are respectively spotted on the same thin layer plate, and developed at 2 temperatures under 5-35deg.C, and the results are shown in fig. 15-16. Fig. 15 shows a normal temperature and humidity (T: 22 ℃ RH: 45%), FIG. 16 is a low temperature constant humidity (T: 6 ℃ RH: 68%), FIG. 15 and FIG. 16, wherein reference numeral 1 is a angelica sinensis control medicinal solution, reference numeral 2 is a Ligusticum wallichii control medicinal solution, reference numeral 3 is a sample solution, reference numeral 4 is a ligustilide control solution, reference numeral 5 is a senkyunolide A control solution, reference numeral 6 is a angelica sinensis single negative control solution, reference numeral 7 is a Ligusticum wallichii single negative control solution, reference numeral 8 is a angelica sinensis Ligusticum wallichii double negative control solution, reference numeral 9 is a angelica sinensis single positive control solution, and reference numeral 10 is a Ligusticum wallichii single positive control solution.

The result shows that the method has better adaptability to different temperatures.

3.4, different environmental humidity

Investigation of different humidity: the control solution, control solution and test solution were prepared according to the selected method and spotted on the same thin layer plate, and developed under 15-30% and 75-90% humidity respectively, and the results are shown in fig. 17-18. Fig. 17 is a normal temperature and low humidity (T: 21 ℃ RH: 21%), fig. 18 is a normal temperature and high humidity (T: 21 ℃ RH: 83%), in which reference numeral 1 is a angelica sinensis control medicinal solution, reference numeral 2 is a ligustilide control medicinal solution, reference numeral 3 is a sample solution, reference numeral 4 is a ligustilide control solution, reference numeral 5 is a senkyunolide a control solution, reference numeral 6 is a angelica sinensis single negative control solution, reference numeral 7 is a ligusticum wallichii single negative control solution, reference numeral 8 is a angelica sinensis ligusticum wallichii double negative control solution, reference numeral 9 is a angelica sinensis single positive control solution, and reference numeral 10 is a ligusticum wallichii single positive control solution.

The result shows that the method has good adaptability to different humidity.

3.5 verification of different lamina plates

Examination of different lamina plates: the control solution, the control solution and the test solution were prepared according to the selected method, and the spotted different thin-layer plates were spread, and the results are shown in fig. 19 to 21 below. FIG. 19 shows Qingdao marine silica gel GF ₂₅₄ Thin layer plates (manufactured by Qingdao ocean chemical Co., ltd., 20210108); FIG. 20 is a diagram of chemical silver Long Guijiao HSGF ₂₅₄ Thin-layer panels (tabacco market chemical industry institute, 20191022); FIG. 21 Nicotiana river friend silica gel HSGF ₂₅₄ Thin layer plate (Chii 32600; huang Wu silica gel development test Co., 2016, 10/07). In the figure: the reference number 1 is a angelica control medicinal material solution, the reference number 2 is a ligusticum chuanxiong control medicinal material solution, the reference number 3 is a sample solution, the reference number 4 is a ligustilide control solution, the reference number 5 is a senkyunolide A control solution, the reference number 6 is a angelica single negative control solution, the reference number 7 is a ligusticum chuanxiong single negative control solution, the reference number 8 is a angelica ligusticum chuanxiong double negative control solution, the reference number 9 is a angelica single positive control solution, and the reference number 10 is a ligusticum chuanxiong single positive control solution.

The results show that the method has better adaptability to different thin-layer plates.

3.6, different developing agent ratios

Investigation of different developer ratios: each solution was prepared and spotted according to the selected method, and the spotted thin-layer plates were spread under different proportions of spreading agents. The results show that the method has better adaptability to different developer ratios, and the results are shown in figures 22-23 below. FIG. 22 is a graph of ratio 1, n-hexane-dichloromethane-ethyl acetate-formic acid (9:3:1:0.2); FIG. 23 is a ratio of 2, n-hexane-dichloromethane-ethyl acetate-formic acid (8:3:2:0.2); in the figure, reference numeral 1 is a angelica control medicinal material solution, reference numeral 2 is a ligusticum chuanxiong control medicinal material solution, reference numeral 3 is a sample solution, reference numeral 4 is a ligustilide control solution, reference numeral 5 is a senkyunolide A control solution, reference numeral 6 is a angelica single negative control solution, reference numeral 7 is a ligusticum chuanxiong single negative control solution, reference numeral 8 is a angelica chuanxiong double negative control solution, reference numeral 9 is a angelica single positive control solution, and reference numeral 10 is a ligusticum chuanxiong single positive control solution.

In summary, the thin layer identification method of the meridian warming granule wine angelica sinensis and ligusticum wallichii comprises the following steps: taking 2.0g of the product powder, adding 30ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, dissolving the residue with 20ml of hot water, filtering, extracting the filtrate with diethyl ether for 2 times by shaking, 10ml each time, combining diethyl ether solutions, volatilizing, and dissolving the residue with 1ml of acetone to obtain a sample solution. The Chinese angelica and the szechuan lovage rhizome are respectively 0.5g of control medicinal materials, and the prepared control medicinal material solution is prepared by the same method; respectively taking ligustilide control and senkyunolide A control, and respectively adding methanol to make into 1mg solution per 1 ml. According to thin layer chromatography (general rule 0502), respectively sucking 2-4 μl of the above solutions, respectively spotting on the same silica gel GF ₂₅₄ Lamina plate or HSGF ₂₅₄ On the thin layer plate, n-hexane-dichloromethane-ethyl acetate-formic acid (8:3:1:0.2) is used as developing agent, and the thin layer plate is developed, taken out, dried and inspected under an ultraviolet lamp (254 nm). In the sample chromatogram, spots with the same color appear at the positions corresponding to the control chromatogram and the control chromatogram.

Conclusion: the thin layer identification method of the warm meridian granule wine angelica and the szechuan lovage rhizome can effectively identify the wine angelica and the szechuan lovage rhizome in the other party.

Example 4

The method for identifying and constructing the same class of medicines in the traditional Chinese medicine compound preparation comprises the steps of preparing the traditional Chinese medicine compound preparation of the ancient classical prescription as the meridian warming granule in the embodiment, wherein the meridian warming granule is derived from the ancient classical prescription directory (first batch) formulated by the national Chinese medicine administration and the national drug administration together, and the prescription composition and the proportion are as follows: chinese medicinal compound granule is prepared from radix Angelicae sinensis (1 part), rhizoma Ligustici Chuanxiong (1 part), radix Paeoniae alba (1 part), cortex Cinnamomi (1 part), cortex moutan (1 part), curcumae rhizoma (1 part), ginseng radix (2 parts), achyranthis radix (2 parts), and Glycyrrhrizae radix (2 parts) by extracting, concentrating, drying, and granulating. The medicines of the same family in this embodiment are wine angelica and rhizoma ligustici wallichii.

1. screening of differential Components

The chemical compositions of the cortex moutan and radix Paeoniae alba are shown in Table 7 below.

TABLE 7 chemical composition summary table of cortex moutan and radix Paeoniae alba

As is clear from the above table, the components identified by screening cortex moutan and radix Paeoniae alba as characteristic patterns and thin layer chromatography mainly concentrate on terpenes (paeoniflorin and paeonol are thin layers and contain measurement indexes).

2. Method for establishing characteristic spectrum of moutan bark and white peony root

Instrument: waters acquisition H-class ultra-high performance liquid chromatograph, PDA detector; ML204T electronic balance (d=0.0001 g, meltrehler tolido instruments (Shanghai); msa6.6s-0 CE-DM electronic balance (d=0.001 mg, certolischen instruments (beijing) company limited); KQ-100DE ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.).

Chromatographic column: waters CORTECS O UPLC O R T3 (2.1X105 mm,1.6 μm)

Reagent: paeoniflorin (China food and drug inspection institute, batch No. 110736-202145); paeonol (national food and drug verification institute, 110708-201908); white peony root reference medicine (China food and drug inspection institute, lot number: 120905-202011); albiflorin (Chengdu Biotechnology Co., ltd., lot: AF 20041801); cortex moutan reference material (Chinese food and drug assay institute, batch number 121490-201603), acetonitrile and formic acid as chromatographic purity; the water is distilled water (Chen's); the other reagents were all analytically pure.

Characteristic spectrum chromatographic conditions:

the measurement is carried out by high performance liquid chromatography (four general rules 0512 in 2020 edition of Chinese pharmacopoeia).

Chromatographic conditions and System suitability test A Waters CORTECS UPLC T3 (column length 150mm, inner diameter 2.1mm, particle size 1.6 μm) column was used, acetonitrile as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, and gradient elution was performed as specified in Table 8 below; the column temperature was 30℃and the flow rate was 0.3ml per minute, and the detection wavelength was 230nm. The theoretical plate number is not lower than 8000 calculated according to paeoniflorin peak.

TABLE 8

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Preparation of control solution A proper amount of paeoniflorin and paeonol control is taken, and 50% methanol is added to prepare a mixed control solution containing 50 mug of paeoniflorin and 20 mug of paeonol per 1 ml.

The preparation of the sample solution comprises weighing about 0.3g of the material of the decoction, precisely weighing, placing in a 50ml conical flask with a plug, precisely adding 25ml of 50% methanol, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHZ) for 30 min, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking, filtering, and collecting the subsequent filtrate.

The measurement method was performed by precisely sucking 3. Mu.l of each of the control solution and the test solution, and injecting the solutions into a liquid chromatograph, as shown in FIG. 24 and FIG. 25.

Wherein, fig. 24 is a cortex moutan contrast characteristic map, in which, peak 1: gallic acid; peak 2: paeoniflorin; peak 4: paeonol; fig. 25 is a white peony control profile, wherein peak 1: gallic acid; peak 2: paeoniflorin; peak 3: paeoniflorin.

3. Determination of the differentiation component

By analyzing characteristic patterns of different medicinal flavors of the meridian warming particles of moutan bark and white paeony root in the same family, the common chemical components in the moutan bark and the white paeony root are determined as follows: gallic acid, paeoniflorin, etc., paeonol in cortex moutan has no paeoniflorin, and paeonia lactiflora has no paeonol, so that paeoniflorin and paeonol are differential components of cortex moutan and paeonia lactiflora, as shown in figure 26.

4. Establishment of authentication method

Therefore, the identification method can be used as the characteristic spectrum method for identifying the same kind of medicines of the traditional Chinese medicine compound preparation.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims

1. The identification construction method of the same class of drugs in the traditional Chinese medicine compound preparation is characterized by comprising the following steps:

2. The method according to claim 1, wherein the medicines of the same family are angelica sinensis and Ligusticum wallichii, or tree peony bark and white peony root, or white atractylodes rhizome and rhizoma atractylodis, or ginseng and pseudo-ginseng.

3. The authentication construction method according to claim 1 or 2, wherein the method for obtaining the common component and the differential component comprises: screening out common chemical components and differential components of different medicines in the same family according to the literature of chemical components of the medicines in the same family;

4. The identification construction method according to any one of claims 1 to 3, wherein the Chinese herbal compound preparation is a warming channel granule.

5. The identification method of the same kind of medicine in the Chinese medicine compound preparation is characterized by comprising the steps of adopting a characteristic spectrum detection method to identify the same kind of medicine in the Chinese medicine compound preparation, wherein the Chinese medicine compound preparation is a meridian warming granule;

when the medicines of the same family are wine angelica and ligusticum wallichii, the chromatographic conditions of the characteristic spectrum detection method are as follows:

6. the method according to claim 5, wherein the chromatographic column temperature is 33-37 ℃, the flow rate is 0.95-1.05 ml/min, the detection wavelength is 254nm, the theoretical plate number is not less than 5000 calculated as ferulic acid peak, and the chromatographic column is UItimate LP C18.

7. The method according to claim 5 or 6, wherein when the sample of the characteristic spectrum detection method is Angelica sinensis, the chromatogram of the sample should show 12 characteristic peaks, wherein peak 3, peak 4, peak 5, peak 12 should correspond to the reference chlorogenic acid, ferulic acid, senkyunolide I, ligustilide peak, peak corresponding to the reference ferulic acid is S peak, and the relative retention time of peak 1, peak 2, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11 and S peak should be within 10% of the specified value, and the specified value is: 0.41, 0.57, 1.22, 1.37, 1.60, 1.75, 1.98, 2.00;

and/or, when the sample in the feature map detection method is ligusticum wallichii, the chromatogram of the sample should show 12 feature peaks, wherein peak 1, peak 3, peak 5, peak 6, peak 11 and peak 12 should correspond to chlorogenic acid, ferulic acid, senkyunolide I, senkyunolide H, senkyunolide A and ligustilide peak, the peak corresponding to the ferulic acid is S peak, the relative retention time of peak 2, peak 4, peak 7, peak 8, peak 9, peak 10 and S peak is calculated, the relative retention time is within 10% of the specified value, and the specified value is: 0.80, 1.11, 1.38, 1.59, 1.71, 1.76.

8. The identification method according to claim 5, wherein when the same family of drugs is tree peony bark and white peony root, the chromatographic conditions of the characteristic spectrum detection method are:

9. the method according to claim 8, wherein the chromatographic condition has a flow rate of 0.3 ml/min, a detection wavelength of 230nm, and the theoretical plate number is not lower than 8000 as calculated by paeoniflorin peak;

and/or, when the sample in the feature map detection method is moutan bark, the chromatogram of the sample should show 5 feature peaks, wherein peak 1, peak 2 and peak 4 should correspond to the peak of gallic acid, paeoniflorin and paeonol, the peak corresponding to paeoniflorin is S peak, the relative retention time of peak 3, peak 5 and S peak is calculated, the relative retention time is within 10% of the specified value, and the specified value is: 2.01, 3.17;

and/or, when the sample of the characteristic spectrum detection method is white peony root, the chromatogram of the sample should show 4 characteristic peaks, wherein peak 1, peak 2 and peak 3 should correspond to the peak of gallic acid, paeoniflorin and paeoniflorin, the peak corresponding to paeoniflorin is S peak, the relative retention time of peak 4 and S peak is calculated, the relative retention time is within 10% of the specified value, and the specified value is: 3.17.

10. The identification method of the same kind of medicine in the Chinese medicine compound preparation is characterized by comprising the steps of adopting a thin layer chromatography method to identify the same kind of medicine in the Chinese medicine compound preparation, wherein the Chinese medicine compound preparation is a meridian warming granule;

when the medicines of the same family are wine angelica and ligusticum wallichii, the detection process of the thin layer chromatography method is as follows:

respectively sucking 2-4 μl of the above solutions, and spotting on the same silica gel GF ₂₅₄ Lamina plate or HSGF ₂₅₄ On the thin layer plate, the mass ratio of (8-9) 3 (1-2) 0.2 of n-hexylAnd (3) taking alkane-methylene dichloride-ethyl acetate-formic acid as a developing agent, developing, taking out, airing, and putting under an ultraviolet lamp for inspection.